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Sample records for interaction regulates pluripotency

  1. The BET family member BRD4 interacts with OCT4 and regulates pluripotency gene expression.

    PubMed

    Wu, Tao; Pinto, Hugo Borges; Kamikawa, Yasunao F; Donohoe, Mary E

    2015-03-10

    Embryonic stem cell (ESC) pluripotency is controlled by defined transcription factors. During cellular differentiation, ESCs undergo a global epigenetic reprogramming. Female ESCs exemplify this process as one of the two X-chromosomes is globally silenced during X chromosome inactivation (XCI) to balance the X-linked gene disparity with XY males. The pluripotent factor OCT4 regulates XCI by triggering X chromosome pairing and counting. OCT4 directly binds Xite and Tsix, which encode two long noncoding RNAs (lncRNAs) that suppress the silencer lncRNA, Xist. To control its activity as a master regulator in pluripotency and XCI, OCT4 must have chromatin protein partners. Here we show that BRD4, a member of the BET protein subfamily, interacts with OCT4. BRD4 occupies the regulatory regions of pluripotent genes and the lncRNAs of XCI. BET inhibition or depletion of BRD4 reduces the expression of many pluripotent genes and shifts cellular fate showing that BRD4 is pivotal for transcription in ESCs.

  2. [The alchemy--epigenetic regulation of pluripotency].

    PubMed

    Bem, Joanna; Grabowska, Iwona

    2013-01-01

    Embryonic stem cells (ESCs) self renew their population, also they are pluripotent which means they can differentiate into any given cell type. In specific culture conditions they remain undifferentiated. On the cellular level pluripotency is determined by many transcription factors, e.g. Sox2, Nanog, Klf4, Oct4. Epigenetic regulation is also crucial for both self renewal and pluripotency. This review focuses on epigenetic mechanisms, among them DNA methylation, posttranslational histone modifications, ATP dependent chromatin remodeling and miRNAs interactions. These mechanisms affect embryonic stem cells functions keeping them poised for differentiation. PMID:24044279

  3. [The alchemy--epigenetic regulation of pluripotency].

    PubMed

    Bem, Joanna; Grabowska, Iwona

    2013-01-01

    Embryonic stem cells (ESCs) self renew their population, also they are pluripotent which means they can differentiate into any given cell type. In specific culture conditions they remain undifferentiated. On the cellular level pluripotency is determined by many transcription factors, e.g. Sox2, Nanog, Klf4, Oct4. Epigenetic regulation is also crucial for both self renewal and pluripotency. This review focuses on epigenetic mechanisms, among them DNA methylation, posttranslational histone modifications, ATP dependent chromatin remodeling and miRNAs interactions. These mechanisms affect embryonic stem cells functions keeping them poised for differentiation.

  4. Synergistic transcriptional and post-transcriptional regulation of ESC characteristics by core pluripotency transcription factors in protein-protein interaction networks.

    PubMed

    Li, Leijie; Zhang, Liangcai; Liu, Guiyou; Feng, Rennan; Jiang, Yongshuai; Yang, Lei; Zhang, Shihua; Liao, Mingzhi; Hua, Jinlian

    2014-01-01

    The molecular mechanism that maintains the pluripotency of embryonic stem cells (ESCs) is not well understood but may be reflected in complex biological networks. However, there have been few studies on the effects of transcriptional and post-transcriptional regulation during the development of ESCs from the perspective of computational systems biology. In this study, we analyzed the topological properties of the "core" pluripotency transcription factors (TFs) OCT4, SOX2 and NANOG in protein-protein interaction networks (PPINs). Further, we identified synergistic interactions between these TFs and microRNAs (miRNAs) in PPINs during ESC development. Results show that there were significant differences in centrality characters between TF-targets and non-TF-targets in PPINs. We also found that there was consistent regulation of multiple "core" pluripotency TFs. Based on the analysis of shortest path length, we found that the module properties were not only within the targets regulated by common or multiple "core" pluripotency TFs but also between the groups of targets regulated by different TFs. Finally, we identified synergistic regulation of these TFs and miRNAs. In summary, the synergistic effects of "core" pluripotency TFs and miRNAs were analyzed using computational methods in both human and mouse PPINs. PMID:25171496

  5. Maintenance of an undifferentiated state of human induced pluripotent stem cells through migration-dependent regulation of the balance between cell-cell and cell-substrate interactions.

    PubMed

    Kim, Mee-Hae; Kino-oka, Masahiro

    2015-06-01

    We present an outlook on the current strategies for maintaining and culturing human induced pluripotent stem cells (hiPSCs) in an undifferentiated state without affecting their pluripotency. First, cell structures and function are described in relation to interactions between hiPSCs and their surroundings. Second, the phenomenon of spontaneous deviation from undifferentiated hiPSCs in cultures with feeder cells is addressed, with a summary of current topics that are of particular interest to our studies. The key regulatory factors that can contribute to the growth and maintenance of undifferentiated hiPSCs are also discussed, with a summary of recent work toward a culture strategy to control hiPSC fate through balancing cell-cell and cell-substrate interactions. Finally, we discuss culture process design in our previous studies with respect to maintaining and expanding cultures of undifferentiated and pluripotent hiPSCs. We focus on the regulation of migration-dependent balance between cell-cell and cell-substrate interactions. This review offers novel insights into the regulation and processing of stem cells for research in regenerative medicine.

  6. Molecular coupling of Tsix regulation and pluripotency.

    PubMed

    Navarro, Pablo; Oldfield, Andrew; Legoupi, Julie; Festuccia, Nicola; Dubois, Agnès; Attia, Mikael; Schoorlemmer, Jon; Rougeulle, Claire; Chambers, Ian; Avner, Philip

    2010-11-18

    The reprogramming of X-chromosome inactivation during the acquisition of pluripotency in vivo and in vitro is accompanied by the repression of Xist, the trigger of X-inactivation, and the upregulation of its antisense counterpart Tsix. We have shown that key factors supporting pluripotency-Nanog, Oct4 and Sox2-bind within Xist intron 1 in undifferentiated embryonic stem cells (ESC) to repress Xist transcription. However, the relationship between transcription factors of the pluripotency network and Tsix regulation has remained unclear. Here we show that Tsix upregulation in embryonic stem cells depends on the recruitment of the pluripotent marker Rex1, and of the reprogramming-associated factors Klf4 and c-Myc, by the DXPas34 minisatellite associated with the Tsix promoter. Upon deletion of DXPas34, binding of the three factors is abrogated and the transcriptional machinery is no longer efficiently recruited to the Tsix promoter. Additional analyses including knockdown experiments further demonstrate that Rex1 is critically important for efficient transcription elongation of Tsix. Hence, distinct embryonic-stem-cell-specific complexes couple X-inactivation reprogramming and pluripotency, with Nanog, Oct4 and Sox2 repressing Xist to facilitate the reactivation of the inactive X, and Klf4, c-Myc and Rex1 activating Tsix to remodel Xist chromatin and ensure random X-inactivation upon differentiation. The holistic pattern of Xist/Tsix regulation by pluripotent factors that we have identified suggests a general direct governance of complex epigenetic processes by the machinery dedicated to pluripotency.

  7. The C-terminal pentapeptide of Nanog tryptophan repeat domain interacts with Nac1 and regulates stem cell proliferation but not pluripotency.

    PubMed

    Ma, Tianhua; Wang, Zhe; Guo, Yunqian; Pei, Duanqing

    2009-06-12

    Overexpression of Nanog in mouse embryonic stem (ES) cells has been shown to abrogate the requirement of leukemia inhibitory factor for self-renewal in culture. Little is known about the molecular mechanism of Nanog function. Here we describe the role of the tryptophan repeat (WR) domain, one of the two transactivators at its C terminus, in regulating stem cell proliferation as well as pluripotency. We first created a supertransactivator, W2W3x10, by duplicating repeats W2W3 10 times and discovered that it can functionally substitute for wild type WR at sustaining pluripotency, albeit with a significantly slower cell cycle, phenocopying Nanog(9W) with the C-terminal pentapeptide (WNAAP) of WR deleted. ES cells carrying both W2W3x10 and Nanog(9W) have a longer G1 phase, a shorter S phase in cell cycle distribution and progression analysis, and a lower level of pAkt(Ser473) compared with wild type Nanog, suggesting that both mutants impact the cell cycle machinery via the phosphatidylinositol 3-kinase/Akt pathway. Both mutants remain competent in dimerizing with Nanog but cannot form a complex with Nac1 efficiently, suggesting that WNAAP may be involved in Nac1 binding. By tagging Gal4DBD with WNAAP, we demonstrated that this pentapeptide is sufficient to confer Nac1 binding. Furthermore, we can rescue W2W3x10 by placing WNAAP at the corresponding locations. Finally, we found that Nanog and Nac1 synergistically up-regulate ERas expression and promote the proliferation of ES cells. These results suggest that Nanog interacts with Nac1 through WNAAP to regulate the cell cycle of ES cells via the ERas/phosphatidylinositol 3-kinase/Akt pathway, but not pluripotency, thus decoupling cell cycle control from pluripotency.

  8. Molecular coupling of Xist regulation and pluripotency.

    PubMed

    Navarro, Pablo; Chambers, Ian; Karwacki-Neisius, Violetta; Chureau, Corinne; Morey, Céline; Rougeulle, Claire; Avner, Philip

    2008-09-19

    During mouse embryogenesis, reversion of imprinted X chromosome inactivation in the pluripotent inner cell mass of the female blastocyst is initiated by the repression of Xist from the paternal X chromosome. Here we report that key factors supporting pluripotency-Nanog, Oct3/4, and Sox2-bind within Xist intron 1 in undifferentiated embryonic stem (ES) cells. Whereas Nanog null ES cells display a reversible and moderate up-regulation of Xist in the absence of any apparent modification of Oct3/4 and Sox2 binding, the drastic release of all three factors from Xist intron 1 triggers rapid ectopic accumulation of Xist RNA. We conclude that the three main genetic factors underlying pluripotency cooperate to repress Xist and thus couple X inactivation reprogramming to the control of pluripotency during embryogenesis.

  9. Long Noncoding RNA Regulation of Pluripotency.

    PubMed

    Rosa, Alessandro; Ballarino, Monica

    2016-01-01

    Pluripotent stem cells (PSCs) represent a unique kind of stem cell, as they are able to indefinitely self-renew and hold the potential to differentiate into any derivative of the three germ layers. As such, human Embryonic Stem Cells (hESCs) and human induced Pluripotent Stem Cells (hiPSCs) provide a unique opportunity for studying the earliest steps of human embryogenesis and, at the same time, are of great therapeutic interest. The molecular mechanisms underlying pluripotency represent a major field of research. Recent evidence suggests that a complex network of transcription factors, chromatin regulators, and noncoding RNAs exist in pluripotent cells to regulate the balance between self-renewal and multilineage differentiation. Regulatory noncoding RNAs come in two flavors: short and long. The first class includes microRNAs (miRNAs), which are involved in the posttranscriptional regulation of cell cycle and differentiation in PSCs. Instead, long noncoding RNAs (lncRNAs) represent a heterogeneous group of long transcripts that regulate gene expression at transcriptional and posttranscriptional levels. In this review, we focus on the role played by lncRNAs in the maintenance of pluripotency, emphasizing the interplay between lncRNAs and other pivotal regulators in PSCs.

  10. Long Noncoding RNA Regulation of Pluripotency

    PubMed Central

    Ballarino, Monica

    2016-01-01

    Pluripotent stem cells (PSCs) represent a unique kind of stem cell, as they are able to indefinitely self-renew and hold the potential to differentiate into any derivative of the three germ layers. As such, human Embryonic Stem Cells (hESCs) and human induced Pluripotent Stem Cells (hiPSCs) provide a unique opportunity for studying the earliest steps of human embryogenesis and, at the same time, are of great therapeutic interest. The molecular mechanisms underlying pluripotency represent a major field of research. Recent evidence suggests that a complex network of transcription factors, chromatin regulators, and noncoding RNAs exist in pluripotent cells to regulate the balance between self-renewal and multilineage differentiation. Regulatory noncoding RNAs come in two flavors: short and long. The first class includes microRNAs (miRNAs), which are involved in the posttranscriptional regulation of cell cycle and differentiation in PSCs. Instead, long noncoding RNAs (lncRNAs) represent a heterogeneous group of long transcripts that regulate gene expression at transcriptional and posttranscriptional levels. In this review, we focus on the role played by lncRNAs in the maintenance of pluripotency, emphasizing the interplay between lncRNAs and other pivotal regulators in PSCs. PMID:26697072

  11. Epigenetic regulation of open chromatin in pluripotent stem cells.

    PubMed

    Kobayashi, Hiroshi; Kikyo, Nobuaki

    2015-01-01

    The recent progress in pluripotent stem cell research has opened new avenues of disease modeling, drug screening, and transplantation of patient-specific tissues unimaginable until a decade ago. The central mechanism underlying pluripotency is epigenetic gene regulation; the majority of cell signaling pathways, both extracellular and cytoplasmic, alter, eventually, the epigenetic status of their target genes during the process of activating or suppressing the genes to acquire or maintain pluripotency. It has long been thought that the chromatin of pluripotent stem cells is open globally to enable the timely activation of essentially all genes in the genome during differentiation into multiple lineages. The current article reviews descriptive observations and the epigenetic machinery relevant to what is supposed to be globally open chromatin in pluripotent stem cells, including microscopic appearance, permissive gene transcription, chromatin remodeling complexes, histone modifications, DNA methylation, noncoding RNAs, dynamic movement of chromatin proteins, nucleosome accessibility and positioning, and long-range chromosomal interactions. Detailed analyses of each element, however, have revealed that the globally open chromatin hypothesis is not necessarily supported by some of the critical experimental evidence, such as genomewide nucleosome accessibility and nucleosome positioning. Greater understanding of epigenetic gene regulation is expected to determine the true nature of the so-called globally open chromatin in pluripotent stem cells.

  12. Regulation of Pluripotency by RNA Binding Proteins

    PubMed Central

    Ye, Julia; Blelloch, Robert

    2015-01-01

    Establishment, maintenance, and exit from pluripotency require precise coordination of a cell’s molecular machinery. Substantial headway has been made in deciphering many aspects of this elaborate system, particularly with respect to epigenetics, transcription, and noncoding RNAs. Less attention has been paid to posttranscriptional regulatory processes such as alternative splicing, RNA processing and modification, nuclear export, regulation of transcript stability, and translation. Here, we introduce the RNA binding proteins that enable the posttranscriptional regulation of gene expression, summarizing current and ongoing research on their roles at different regulatory points and discussing how they help script the fate of pluripotent stem cells. PMID:25192462

  13. Glycosyltransferase ST6GAL1 contributes to the regulation of pluripotency in human pluripotent stem cells

    PubMed Central

    Wang, Yu-Chieh; Stein, Jason W.; Lynch, Candace L.; Tran, Ha T.; Lee, Chia-Yao; Coleman, Ronald; Hatch, Adam; Antontsev, Victor G.; Chy, Hun S.; O’Brien, Carmel M.; Murthy, Shashi K.; Laslett, Andrew L.; Peterson, Suzanne E.; Loring, Jeanne F.

    2015-01-01

    Many studies have suggested the significance of glycosyltransferase-mediated macromolecule glycosylation in the regulation of pluripotent states in human pluripotent stem cells (hPSCs). Here, we observed that the sialyltransferase ST6GAL1 was preferentially expressed in undifferentiated hPSCs compared to non-pluripotent cells. A lectin which preferentially recognizes α-2,6 sialylated galactosides showed strong binding reactivity with undifferentiated hPSCs and their glycoproteins, and did so to a much lesser extent with differentiated cells. In addition, downregulation of ST6GAL1 in undifferentiated hPSCs led to a decrease in POU5F1 (also known as OCT4) protein and significantly altered the expression of many genes that orchestrate cell morphogenesis during differentiation. The induction of cellular pluripotency in somatic cells was substantially impeded by the shRNA-mediated suppression of ST6GAL1, partially through interference with the expression of endogenous POU5F1 and SOX2. Targeting ST6GAL1 activity with a sialyltransferase inhibitor during cell reprogramming resulted in a dose-dependent reduction in the generation of human induced pluripotent stem cells (hiPSCs). Collectively, our data indicate that ST6GAL1 plays an important role in the regulation of pluripotency and differentiation in hPSCs, and the pluripotent state in human cells can be modulated using pharmacological tools to target sialyltransferase activity. PMID:26304831

  14. Citrullination regulates pluripotency and histone H1 binding to chromatin

    NASA Astrophysics Data System (ADS)

    Christophorou, Maria A.; Castelo-Branco, Gonçalo; Halley-Stott, Richard P.; Oliveira, Clara Slade; Loos, Remco; Radzisheuskaya, Aliaksandra; Mowen, Kerri A.; Bertone, Paul; Silva, José C. R.; Zernicka-Goetz, Magdalena; Nielsen, Michael L.; Gurdon, John B.; Kouzarides, Tony

    2014-03-01

    Citrullination is the post-translational conversion of an arginine residue within a protein to the non-coded amino acid citrulline. This modification leads to the loss of a positive charge and reduction in hydrogen-bonding ability. It is carried out by a small family of tissue-specific vertebrate enzymes called peptidylarginine deiminases (PADIs) and is associated with the development of diverse pathological states such as autoimmunity, cancer, neurodegenerative disorders, prion diseases and thrombosis. Nevertheless, the physiological functions of citrullination remain ill-defined, although citrullination of core histones has been linked to transcriptional regulation and the DNA damage response. PADI4 (also called PAD4 or PADV), the only PADI with a nuclear localization signal, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune response to infection. Here we show that the expression and enzymatic activity of Padi4 are also induced under conditions of ground-state pluripotency and during reprogramming in mouse. Padi4 is part of the pluripotency transcriptional network, binding to regulatory elements of key stem-cell genes and activating their expression. Its inhibition lowers the percentage of pluripotent cells in the early mouse embryo and significantly reduces reprogramming efficiency. Using an unbiased proteomic approach we identify linker histone H1 variants, which are involved in the generation of compact chromatin, as novel PADI4 substrates. Citrullination of a single arginine residue within the DNA-binding site of H1 results in its displacement from chromatin and global chromatin decondensation. Together, these results uncover a role for citrullination in the regulation of pluripotency and provide new mechanistic insights into how citrullination regulates chromatin compaction.

  15. Glutamine Metabolism Regulates the Pluripotency Transcription Factor OCT4

    PubMed Central

    Marsboom, Glenn; Zhang, Guo-Fang; Pohl-Avila, Nicole; Zhang, Yanmin; Yuan, Yang; Kang, Hojin; Hao, Bo; Brunengraber, Henri; Malik, Asrar B.; Rehman, Jalees

    2016-01-01

    SUMMARY The molecular mechanisms underlying the regulation of pluripotency by cellular metabolism in human embryonic stem cells (hESCs) are not fully understood. We found that high levels of glutamine metabolism are essential to prevent degradation of OCT4, a key transcription factor regulating hESC pluripotency. Glutamine withdrawal depletes the endogenous anti-oxidant glutathione, which results in the oxidation of OCT4 cysteine residues required for its DNA binding and enhanced OCT4 degradation. The emergence of the OCT4lo cell population following glutamine withdrawal did not result in greater propensity for cell death. Instead, glutamine withdrawal during vascular differentiation of hESCs generated cells with greater angiogenic capacity, thus indicating that modulating glutamine metabolism enhances the differentiation and functional maturation of cells. These findings demonstrate that the pluripotency transcription factor OCT4 can serve as a metabolic-redox sensor in hESCs and that metabolic cues can act in concert with growth factor signaling to orchestrate stem cell differentiation. PMID:27346346

  16. Qualitative modeling identifies IL-11 as a novel regulator in maintaining self-renewal in human pluripotent stem cells

    PubMed Central

    Peterson, Hedi; Abu Dawud, Raed; Garg, Abhishek; Wang, Ying; Vilo, Jaak; Xenarios, Ioannis; Adjaye, James

    2013-01-01

    Pluripotency in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is regulated by three transcription factors—OCT3/4, SOX2, and NANOG. To fully exploit the therapeutic potential of these cells it is essential to have a good mechanistic understanding of the maintenance of self-renewal and pluripotency. In this study, we demonstrate a powerful systems biology approach in which we first expand literature-based network encompassing the core regulators of pluripotency by assessing the behavior of genes targeted by perturbation experiments. We focused our attention on highly regulated genes encoding cell surface and secreted proteins as these can be more easily manipulated by the use of inhibitors or recombinant proteins. Qualitative modeling based on combining boolean networks and in silico perturbation experiments were employed to identify novel pluripotency-regulating genes. We validated Interleukin-11 (IL-11) and demonstrate that this cytokine is a novel pluripotency-associated factor capable of supporting self-renewal in the absence of exogenously added bFGF in culture. To date, the various protocols for hESCs maintenance require supplementation with bFGF to activate the Activin/Nodal branch of the TGFβ signaling pathway. Additional evidence supporting our findings is that IL-11 belongs to the same protein family as LIF, which is known to be necessary for maintaining pluripotency in mouse but not in human ESCs. These cytokines operate through the same gp130 receptor which interacts with Janus kinases. Our finding might explain why mESCs are in a more naïve cell state compared to hESCs and how to convert primed hESCs back to the naïve state. Taken together, our integrative modeling approach has identified novel genes as putative candidates to be incorporated into the expansion of the current gene regulatory network responsible for inducing and maintaining pluripotency. PMID:24194720

  17. Cell-Cycle Control of Bivalent Epigenetic Domains Regulates the Exit from Pluripotency

    PubMed Central

    Singh, Amar M.; Sun, Yuhua; Li, Li; Zhang, Wenjuan; Wu, Tianming; Zhao, Shaying; Qin, Zhaohui; Dalton, Stephen

    2015-01-01

    Summary Here we show that bivalent domains and chromosome architecture for bivalent genes are dynamically regulated during the cell cycle in human pluripotent cells. Central to this is the transient increase in H3K4-trimethylation at developmental genes during G1, thereby creating a “window of opportunity” for cell-fate specification. This mechanism is controlled by CDK2-dependent phosphorylation of the MLL2 (KMT2B) histone methyl-transferase, which facilitates its recruitment to developmental genes in G1. MLL2 binding is required for changes in chromosome architecture around developmental genes and establishes promoter-enhancer looping interactions in a cell-cycle-dependent manner. These cell-cycle-regulated loops are shown to be essential for activation of bivalent genes and pluripotency exit. These findings demonstrate that bivalent domains are established to control the cell-cycle-dependent activation of developmental genes so that differentiation initiates from the G1 phase. PMID:26278042

  18. Cell-Cycle Control of Bivalent Epigenetic Domains Regulates the Exit from Pluripotency.

    PubMed

    Singh, Amar M; Sun, Yuhua; Li, Li; Zhang, Wenjuan; Wu, Tianming; Zhao, Shaying; Qin, Zhaohui; Dalton, Stephen

    2015-09-01

    Here we show that bivalent domains and chromosome architecture for bivalent genes are dynamically regulated during the cell cycle in human pluripotent cells. Central to this is the transient increase in H3K4-trimethylation at developmental genes during G1, thereby creating a "window of opportunity" for cell-fate specification. This mechanism is controlled by CDK2-dependent phosphorylation of the MLL2 (KMT2B) histone methyl-transferase, which facilitates its recruitment to developmental genes in G1. MLL2 binding is required for changes in chromosome architecture around developmental genes and establishes promoter-enhancer looping interactions in a cell-cycle-dependent manner. These cell-cycle-regulated loops are shown to be essential for activation of bivalent genes and pluripotency exit. These findings demonstrate that bivalent domains are established to control the cell-cycle-dependent activation of developmental genes so that differentiation initiates from the G1 phase. PMID:26278042

  19. Regulation of pluripotency and differentiation by deubiquitinating enzymes.

    PubMed

    Suresh, B; Lee, J; Kim, H; Ramakrishna, S

    2016-08-01

    Post-translational modifications (PTMs) of stemness-related proteins are essential for stem cell maintenance and differentiation. In stem cell self-renewal and differentiation, PTM of stemness-related proteins is tightly regulated because the modified proteins execute various stem cell fate choices. Ubiquitination and deubiquitination, which regulate protein turnover of several stemness-related proteins, must be carefully coordinated to ensure optimal embryonic stem cell maintenance and differentiation. Deubiquitinating enzymes (DUBs), which specifically disassemble ubiquitin chains, are a central component in the ubiquitin-proteasome pathway. These enzymes often control the balance between ubiquitination and deubiquitination. To maintain stemness and achieve efficient differentiation, the ubiquitination and deubiquitination molecular switches must operate in a balanced manner. Here we summarize the current information on DUBs, with a focus on their regulation of stem cell fate determination and deubiquitinase inhibition as a therapeutic strategy. Furthermore, we discuss the possibility of using DUBs with defined stem cell transcription factors to enhance cellular reprogramming efficiency and cell fate conversion. Our review provides new insight into DUB activity by emphasizing their cellular role in regulating stem cell fate. This role paves the way for future research focused on specific DUBs or deubiquitinated substrates as key regulators of pluripotency and stem cell differentiation.

  20. The primate-specific noncoding RNA HPAT5 regulates pluripotency during human preimplantation development and nuclear reprogramming.

    PubMed

    Durruthy-Durruthy, Jens; Sebastiano, Vittorio; Wossidlo, Mark; Cepeda, Diana; Cui, Jun; Grow, Edward J; Davila, Jonathan; Mall, Moritz; Wong, Wing H; Wysocka, Joanna; Au, Kin Fai; Reijo Pera, Renee A

    2016-01-01

    Long intergenic noncoding RNAs (lincRNAs) are derived from thousands of loci in mammalian genomes and are frequently enriched in transposable elements (TEs). Although families of TE-derived lincRNAs have recently been implicated in the regulation of pluripotency, little is known of the specific functions of individual family members. Here we characterize three new individual TE-derived human lincRNAs, human pluripotency-associated transcripts 2, 3 and 5 (HPAT2, HPAT3 and HPAT5). Loss-of-function experiments indicate that HPAT2, HPAT3 and HPAT5 function in preimplantation embryo development to modulate the acquisition of pluripotency and the formation of the inner cell mass. CRISPR-mediated disruption of the genes for these lincRNAs in pluripotent stem cells, followed by whole-transcriptome analysis, identifies HPAT5 as a key component of the pluripotency network. Protein binding and reporter-based assays further demonstrate that HPAT5 interacts with the let-7 microRNA family. Our results indicate that unique individual members of large primate-specific lincRNA families modulate gene expression during development and differentiation to reinforce cell fate.

  1. An epigenetic switch regulates de novo DNA methylation at a subset of pluripotency gene enhancers during embryonic stem cell differentiation

    PubMed Central

    Petell, Christopher J.; Alabdi, Lama; He, Ming; San Miguel, Phillip; Rose, Richard; Gowher, Humaira

    2016-01-01

    Coordinated regulation of gene expression that involves activation of lineage specific genes and repression of pluripotency genes drives differentiation of embryonic stem cells (ESC). For complete repression of pluripotency genes during ESC differentiation, chromatin at their enhancers is silenced by the activity of the Lsd1-Mi2/NuRD complex. The mechanism/s that regulate DNA methylation at these enhancers are largely unknown. Here, we investigated the affect of the Lsd1-Mi2/NuRD complex on the dynamic regulatory switch that induces the local interaction of histone tails with the Dnmt3 ATRX-DNMT3-DNMT3L (ADD) domain, thus promoting DNA methylation at the enhancers of a subset of pluripotency genes. This is supported by previous structural studies showing a specific interaction between Dnmt3-ADD domain with H3K4 unmethylated histone tails that is disrupted by histone H3K4 methylation and histone acetylation. Our data suggest that Dnmt3a activity is triggered by Lsd1-Mi2/NuRD-mediated histone deacetylation and demethylation at these pluripotency gene enhancers when they are inactivated during mouse ESC differentiation. Using Dnmt3 knockout ESCs and the inhibitors of Lsd1 and p300 histone modifying enzymes during differentiation of E14Tg2A and ZHBTc4 ESCs, our study systematically reveals this mechanism and establishes that Dnmt3a is both reader and effector of the epigenetic state at these target sites. PMID:27179026

  2. A Novel Role for miR-1305 in Regulation of Pluripotency-Differentiation Balance, Cell Cycle, and Apoptosis in Human Pluripotent Stem Cells.

    PubMed

    Jin, Shibo; Collin, Joseph; Zhu, Lili; Montaner, David; Armstrong, Lyle; Neganova, Irina; Lako, Majlinda

    2016-09-01

    Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are defined as pluripotent in view of their self-renewal ability and potential to differentiate to cells of all three germ layers. Recent studies have indicated that microRNAs (miRNAs) play an important role in the maintenance of pluripotency and cell cycle regulation. We used a microarray based approach to identify miRNAs that were enriched in hESCs when compared to differentiated cells and at the same time showed significant expression changes between different phases of cell cycle. We identified 34 candidate miRNAs and performed functional studies on one of these, miR-1305, which showed the highest expression change during cell cycle transition. Overexpression of miR-1305 induced differentiation of pluripotent stem cells, increased cell apoptosis and sped up G1/S transition, while its downregulation facilitated the maintenance of pluripotency and increased cell survival. Using target prediction software and luciferase based reporter assays we identified POLR3G as a downstream target by which miR-1305 regulates the fine balance between maintenance of pluripotency and onset of differentiation. Overexpression of POLR3G rescued pluripotent stem cell differentiation induced by miR-1305 overexpression. In contrast, knock-down of POLR3G expression abolished the miR-1305-knockdown mediated enhancement of pluripotency, thus validating its role as miR-1305 target in human pluripotent stem cells. Together our data point to an important role for miR-1305 as a novel regulator of pluripotency, cell survival and cell cycle and uncovers new mechanisms and networks by which these processes are intertwined in human pluripotent stem cells. Stem Cells 2016;34:2306-2317.

  3. The pluripotency factor Oct4 interacts with Ctcf and also controls X-chromosome pairing and counting.

    PubMed

    Donohoe, Mary E; Silva, Susana S; Pinter, Stefan F; Xu, Na; Lee, Jeannie T

    2009-07-01

    Pluripotency of embryonic stem (ES) cells is controlled by defined transcription factors. During differentiation, mouse ES cells undergo global epigenetic reprogramming, as exemplified by X-chromosome inactivation (XCI) in which one female X chromosome is silenced to achieve gene dosage parity between the sexes. Somatic XCI is regulated by homologous X-chromosome pairing and counting, and by the random choice of future active and inactive X chromosomes. XCI and cell differentiation are tightly coupled, as blocking one process compromises the other and dedifferentiation of somatic cells to induced pluripotent stem cells is accompanied by X chromosome reactivation. Recent evidence suggests coupling of Xist expression to pluripotency factors occurs, but how the two are interconnected remains unknown. Here we show that Oct4 (also known as Pou5f1) lies at the top of the XCI hierarchy, and regulates XCI by triggering X-chromosome pairing and counting. Oct4 directly binds Tsix and Xite, two regulatory noncoding RNA genes of the X-inactivation centre, and also complexes with XCI trans-factors, Ctcf and Yy1 (ref. 17), through protein-protein interactions. Depletion of Oct4 blocks homologous X-chromosome pairing and results in the inactivation of both X chromosomes in female cells. Thus, we have identified the first trans-factor that regulates counting, and ascribed new functions to Oct4 during X-chromosome reprogramming.

  4. MicroRNAs in regulation of pluripotency and somatic cell reprogramming

    PubMed Central

    Wang, Tian; Shi, San-bao; Sha, Hong-ying

    2013-01-01

    MicroRNAs (miRNAs), a group of small non-coding RNAs, have emerged as significant modulators in the establishment and generation of pluripotency, a developmental process that consists of complex cell-fate arrangements. The finding of embryonic stem cell (ESC) cycle-specific miRNAs reveals an important regulation scheme of pluripotency. Subsequent studies showed the ESC-enriched or ESC-depleted miRNAs can regulate induced pluripotent stem cells(iPSC). Moreover, miRNA profiling of iPSC and ESC may distinguish them from one another and facilitate the complex of regulatory network. The accumulative effects of miRNA action enable using miRNA alone to generate iPSCs. Despite the robustness of iPSC studies, further investigations are needed since miRNA may have more impact on induced pluripotency, and the roles of miRNAs in somatic cell nuclear transfer (SCNT), another approach toward cellular reprogramming, remains unclear. This point-of-view article will discuss miRNAs and their impact on the normal and induced pluripotency, as well as bring new insights on somatic cell reprogramming. PMID:23921205

  5. Protein post-translational modifications and regulation of pluripotency in human stem cells.

    PubMed

    Wang, Yu-Chieh; Peterson, Suzanne E; Loring, Jeanne F

    2014-02-01

    Post-translational modifications (PTMs) are known to be essential mechanisms used by eukaryotic cells to diversify their protein functions and dynamically coordinate their signaling networks. Defects in PTMs have been linked to numerous developmental disorders and human diseases, highlighting the importance of PTMs in maintaining normal cellular states. Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), are capable of self-renewal and differentiation into a variety of functional somatic cells; these cells hold a great promise for the advancement of biomedical research and clinical therapy. The mechanisms underlying cellular pluripotency in human cells have been extensively explored in the past decade. In addition to the vast amount of knowledge obtained from the genetic and transcriptional research in hPSCs, there is a rapidly growing interest in the stem cell biology field to examine pluripotency at the protein and PTM level. This review addresses recent progress toward understanding the role of PTMs (glycosylation, phosphorylation, acetylation and methylation) in the regulation of cellular pluripotency.

  6. Epigenetic regulation in pluripotent stem cells: a key to breaking the epigenetic barrier.

    PubMed

    Watanabe, Akira; Yamada, Yasuhiro; Yamanaka, Shinya

    2013-01-01

    The differentiation and reprogramming of cells are accompanied by drastic changes in the epigenetic profiles of cells. Waddington's classical model clearly describes how differentiating cells acquire their cell identity as the developmental potential of an individual cell population declines towards the terminally differentiated state. The recent discovery of induced pluripotent stem cells as well as of somatic cell nuclear transfer provided evidence that the process of differentiation can be reversed. The identity of somatic cells is strictly protected by an epigenetic barrier, and these cells acquire pluripotency by breaking the epigenetic barrier by reprogramming factors such as Oct3/4, Sox2, Klf4, Myc and LIN28. This review covers the current understanding of the spatio-temporal regulation of epigenetics in pluripotent and differentiated cells, and discusses how cells determine their identity and overcome the epigenetic barrier during the reprogramming process.

  7. Epigenetic regulation in pluripotent stem cells: a key to breaking the epigenetic barrier.

    PubMed

    Watanabe, Akira; Yamada, Yasuhiro; Yamanaka, Shinya

    2013-01-01

    The differentiation and reprogramming of cells are accompanied by drastic changes in the epigenetic profiles of cells. Waddington's classical model clearly describes how differentiating cells acquire their cell identity as the developmental potential of an individual cell population declines towards the terminally differentiated state. The recent discovery of induced pluripotent stem cells as well as of somatic cell nuclear transfer provided evidence that the process of differentiation can be reversed. The identity of somatic cells is strictly protected by an epigenetic barrier, and these cells acquire pluripotency by breaking the epigenetic barrier by reprogramming factors such as Oct3/4, Sox2, Klf4, Myc and LIN28. This review covers the current understanding of the spatio-temporal regulation of epigenetics in pluripotent and differentiated cells, and discusses how cells determine their identity and overcome the epigenetic barrier during the reprogramming process. PMID:23166402

  8. The THO Complex Regulates Pluripotency Gene mRNA Export and Controls Embryonic Stem Cell Self-renewal and Somatic Cell Reprogramming

    PubMed Central

    Wang, Li; Miao, Yi-liang; Zheng, Xiaofeng; Lackford, Brad; Zhou, Bingying; Han, Leng; Yao, Chengguo; Ward, James; Burkholder, Adam; Lipchina, Inna; Fargo, David C.; Hochedlinger, Konrad; Shi, Yongsheng; Williams, Carmen J.; Hu, Guang

    2014-01-01

    SUMMARY Embryonic stem cell (ESC) self-renewal and differentiation are governed by a broad-ranging regulatory network. Although the transcriptional regulatory mechanisms involved have been investigated extensively, post-transcriptional regulation is still poorly understood. Here we describe a critical role of the THO complex in ESC self-renewal and differentiation. We show that THO preferentially interacts with pluripotency gene transcripts through Thoc5, and is required for self-renewal at least in part by regulating their export and expression. During differentiation, THO loses its interaction with those transcripts due to reduced Thoc5 expression, leading to decreased expression of pluripotency proteins that facilitates exit from self-renewal. THO is also important for the establishment of pluripotency, as its depletion inhibits somatic cell reprogramming and blastocyst development. Together, our data indicate that THO regulates pluripotency gene mRNA export to control ESC self-renewal and differentiation, and therefore uncover a role for this aspect of post-transcriptional regulation in stem cell fate specification. PMID:24315442

  9. The pluripotency factor Nanog regulates pericentromeric heterochromatin organization in mouse embryonic stem cells.

    PubMed

    Novo, Clara Lopes; Tang, Calvin; Ahmed, Kashif; Djuric, Ugljesa; Fussner, Eden; Mullin, Nicholas P; Morgan, Natasha P; Hayre, Jasvinder; Sienerth, Arnold R; Elderkin, Sarah; Nishinakamura, Ryuichi; Chambers, Ian; Ellis, James; Bazett-Jones, David P; Rugg-Gunn, Peter J

    2016-05-01

    An open and decondensed chromatin organization is a defining property of pluripotency. Several epigenetic regulators have been implicated in maintaining an open chromatin organization, but how these processes are connected to the pluripotency network is unknown. Here, we identified a new role for the transcription factor NANOG as a key regulator connecting the pluripotency network with constitutive heterochromatin organization in mouse embryonic stem cells. Deletion of Nanog leads to chromatin compaction and the remodeling of heterochromatin domains. Forced expression of NANOG in epiblast stem cells is sufficient to decompact chromatin. NANOG associates with satellite repeats within heterochromatin domains, contributing to an architecture characterized by highly dispersed chromatin fibers, low levels of H3K9me3, and high major satellite transcription, and the strong transactivation domain of NANOG is required for this organization. The heterochromatin-associated protein SALL1 is a direct cofactor for NANOG, and loss of Sall1 recapitulates the Nanog-null phenotype, but the loss of Sall1 can be circumvented through direct recruitment of the NANOG transactivation domain to major satellites. These results establish a direct connection between the pluripotency network and chromatin organization and emphasize that maintaining an open heterochromatin architecture is a highly regulated process in embryonic stem cells. PMID:27125671

  10. Divergent lncRNAs Regulate Gene Expression and Lineage Differentiation in Pluripotent Cells.

    PubMed

    Luo, Sai; Lu, J Yuyang; Liu, Lichao; Yin, Yafei; Chen, Chunyan; Han, Xue; Wu, Bohou; Xu, Ronggang; Liu, Wei; Yan, Pixi; Shao, Wen; Lu, Zhi; Li, Haitao; Na, Jie; Tang, Fuchou; Wang, Jianlong; Zhang, Yong E; Shen, Xiaohua

    2016-05-01

    Divergent lncRNAs that are transcribed in the opposite direction to nearby protein-coding genes comprise a significant proportion (∼20%) of total lncRNAs in mammalian genomes. Through genome-wide analysis, we found that the distribution of this lncRNA class strongly correlates with essential developmental regulatory genes. In pluripotent cells, divergent lncRNAs regulate the transcription of nearby genes. As an example, the divergent lncRNA Evx1as promotes transcription of its neighbor gene, EVX1, and regulates mesendodermal differentiation. At a single-cell level, early broad expression of Evx1as is followed by a rapid, high-level transcription of EVX1, supporting the idea that Evx1as plays an upstream role to facilitate EVX1 transcription. Mechanistically, Evx1as RNA binds to regulatory sites on chromatin, promotes an active chromatin state, and interacts with Mediator. Based on our analyses, we propose that the biological function of thousands of uncharacterized lncRNAs of this class may be inferred from the role of their neighboring adjacent genes.

  11. Transcriptional regulation in pluripotent stem cells by methyl CpG-binding protein 2 (MeCP2).

    PubMed

    Tanaka, Yoshiaki; Kim, Kun-Yong; Zhong, Mei; Pan, Xinghua; Weissman, Sherman Morton; Park, In-Hyun

    2014-02-15

    Rett syndrome (RTT) is one of the most prevalent female mental disorders. De novo mutations in methyl CpG-binding protein 2 (MeCP2) are a major cause of RTT. MeCP2 regulates gene expression as a transcription regulator as well as through long-range chromatin interaction. Because MeCP2 is present on the X chromosome, RTT is manifested in an X-linked dominant manner. Investigation using murine MeCP2 null models and post-mortem human brain tissues has contributed to understanding the molecular and physiological function of MeCP2. In addition, RTT models using human induced pluripotent stem cells derived from RTT patients (RTT-iPSCs) provide novel resources to elucidate the regulatory mechanism of MeCP2. Previously, we obtained clones of female RTT-iPSCs that express either wild-type or mutant MECP2 due to the inactivation of one X chromosome. Reactivation of the X chromosome also allowed us to have RTT-iPSCs that express both wild-type and mutant MECP2. Using these unique pluripotent stem cells, we investigated the regulation of gene expression by MeCP2 in pluripotent stem cells by transcriptome analysis. We found that MeCP2 regulates genes encoding mitochondrial membrane proteins. In addition, loss of function in MeCP2 results in de-repression of genes on the inactive X chromosome. Furthermore, we showed that each mutation in MECP2 affects a partly different set of genes. These studies suggest that fundamental cellular physiology is affected by mutations in MECP2 from early development, and that a therapeutic approach targeting to unique forms of mutant MeCP2 is needed.

  12. Netrin-1 regulates somatic cell reprogramming and pluripotency maintenance

    PubMed Central

    Ozmadenci, Duygu; Féraud, Olivier; Markossian, Suzy; Kress, Elsa; Ducarouge, Benjamin; Gibert, Benjamin; Ge, Jian; Durand, Isabelle; Gadot, Nicolas; Plateroti, Michela; Bennaceur-Griscelli, Annelise; Scoazec, Jean-Yves; Gil, Jesus; Deng, Hongkui; Bernet, Agnes; Mehlen, Patrick; Lavial, Fabrice

    2015-01-01

    The generation of induced pluripotent stem (iPS) cells holds great promise in regenerative medicine. The use of the transcription factors Oct4, Sox2, Klf4 and c-Myc for reprogramming is extensively documented, but comparatively little is known about soluble molecules promoting reprogramming. Here we identify the secreted cue Netrin-1 and its receptor DCC, described for their respective survival/death functions in normal and oncogenic contexts, as reprogramming modulators. In various somatic cells, we found that reprogramming is accompanied by a transient transcriptional repression of Netrin-1 mediated by an Mbd3/Mta1/Chd4-containing NuRD complex. Mechanistically, Netrin-1 imbalance induces apoptosis mediated by the receptor DCC in a p53-independent manner. Correction of the Netrin-1/DCC equilibrium constrains apoptosis and improves reprogramming efficiency. Our work also sheds light on Netrin-1's function in protecting embryonic stem cells from apoptosis mediated by its receptor UNC5b, and shows that the treatment with recombinant Netrin-1 improves the generation of mouse and human iPS cells. PMID:26154507

  13. The Oct4 homologue PouV and Nanog regulate pluripotency in chicken embryonic stem cells.

    PubMed

    Lavial, Fabrice; Acloque, Hervé; Bertocchini, Federica; Macleod, David J; Boast, Sharon; Bachelard, Elodie; Montillet, Guillaume; Thenot, Sandrine; Sang, Helen M; Stern, Claudio D; Samarut, Jacques; Pain, Bertrand

    2007-10-01

    Embryonic stem cells (ESC) have been isolated from pregastrulation mammalian embryos. The maintenance of their pluripotency and ability to self-renew has been shown to be governed by the transcription factors Oct4 (Pou5f1) and Nanog. Oct4 appears to control cell-fate decisions of ESC in vitro and the choice between embryonic and trophectoderm cell fates in vivo. In non-mammalian vertebrates, the existence and functions of these factors are still under debate, although the identification of the zebrafish pou2 (spg; pou5f1) and Xenopus Pou91 (XlPou91) genes, which have important roles in maintaining uncommitted putative stem cell populations during early development, has suggested that these factors have common functions in all vertebrates. Using chicken ESC (cESC), which display similar properties of pluripotency and long-term self-renewal to mammalian ESC, we demonstrated the existence of an avian homologue of Oct4 that we call chicken PouV (cPouV). We established that cPouV and the chicken Nanog gene are required for the maintenance of pluripotency and self-renewal of cESC. These findings show that the mechanisms by which Oct4 and Nanog regulate pluripotency and self-renewal are not exclusive to mammals.

  14. Alternative splicing regulates mouse embryonic stem cell pluripotency and differentiation.

    PubMed

    Salomonis, Nathan; Schlieve, Christopher R; Pereira, Laura; Wahlquist, Christine; Colas, Alexandre; Zambon, Alexander C; Vranizan, Karen; Spindler, Matthew J; Pico, Alexander R; Cline, Melissa S; Clark, Tyson A; Williams, Alan; Blume, John E; Samal, Eva; Mercola, Mark; Merrill, Bradley J; Conklin, Bruce R

    2010-06-01

    Two major goals of regenerative medicine are to reproducibly transform adult somatic cells into a pluripotent state and to control their differentiation into specific cell fates. Progress toward these goals would be greatly helped by obtaining a complete picture of the RNA isoforms produced by these cells due to alternative splicing (AS) and alternative promoter selection (APS). To investigate the roles of AS and APS, reciprocal exon-exon junctions were interrogated on a genome-wide scale in differentiating mouse embryonic stem (ES) cells with a prototype Affymetrix microarray. Using a recently released open-source software package named AltAnalyze, we identified 144 genes for 170 putative isoform variants, the majority (67%) of which were predicted to alter protein sequence and domain composition. Verified alternative exons were largely associated with pathways of Wnt signaling and cell-cycle control, and most were conserved between mouse and human. To examine the functional impact of AS, we characterized isoforms for two genes. As predicted by AltAnalyze, we found that alternative isoforms of the gene Serca2 were targeted by distinct microRNAs (miRNA-200b, miRNA-214), suggesting a critical role for AS in cardiac development. Analysis of the Wnt transcription factor Tcf3, using selective knockdown of an ES cell-enriched and characterized isoform, revealed several distinct targets for transcriptional repression (Stmn2, Ccnd2, Atf3, Klf4, Nodal, and Jun) as well as distinct differentiation outcomes in ES cells. The findings herein illustrate a critical role for AS in the specification of ES cells with differentiation, and highlight the utility of global functional analyses of AS. PMID:20498046

  15. Epigenetic regulation leading to induced pluripotency drives cancer development in vivo

    SciTech Connect

    Ohnishi, Kotaro; Semi, Katsunori; Yamada, Yasuhiro

    2014-12-05

    Highlights: • Epigenetic regulation of failed reprogramming-associated cancer cells is discussed. • Similarity between pediatric cancer and reprogramming-associated cancer is discussed. • Concept for epigenetic cancer is discussed. - Abstract: Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by the transient expression of reprogramming factors. During the reprogramming process, somatic cells acquire the ability to undergo unlimited proliferation, which is also an important characteristic of cancer cells, while their underlying DNA sequence remains unchanged. Based on the characteristics shared between pluripotent stem cells and cancer cells, the potential involvement of the factors leading to reprogramming toward pluripotency in cancer development has been discussed. Recent in vivo reprogramming studies provided some clues to understanding the role of reprogramming-related epigenetic regulation in cancer development. It was shown that premature termination of the in vivo reprogramming result in the development of tumors that resemble pediatric cancers. Given that epigenetic modifications play a central role during reprogramming, failed reprogramming-associated cancer development may have provided a proof of concept for epigenetics-driven cancer development in vivo.

  16. Regulation of CpG methylation by Dnmt and Tet in pluripotent stem cells

    PubMed Central

    HORII, Takuro; HATADA, Izuho

    2016-01-01

    Vertebrate genomes are highly methylated at cytosine residues in CpG sequences. CpG methylation plays an important role in epigenetic gene silencing and genome stability. Compared with other epigenetic modifications, CpG methylation is thought to be relatively stable; however, it is sometimes affected by environmental changes, leading to epigenetic instability and disease. CpG methylation is reversible and regulated by DNA methyltransferases and demethylases including ten-eleven translocation. Here, we discuss CpG methylation instability and the regulation of CpG methylation by DNA methyltransferases and ten-eleven translocation in pluripotent stem cells. PMID:27151232

  17. Regulation of c-Myc Expression by Ahnak Promotes Induced Pluripotent Stem Cell Generation.

    PubMed

    Lim, Hee Jung; Kim, Jusong; Park, Chang-Hwan; Lee, Sang A; Lee, Man Ryul; Kim, Kye-Seong; Kim, Jaesang; Bae, Yun Soo

    2016-01-01

    We have previously reported that Ahnak-mediated TGFβ signaling leads to down-regulation of c-Myc expression. Here, we show that inhibition of Ahnak can promote generation of induced pluripotent stem cells (iPSC) via up-regulation of endogenous c-Myc. Consistent with the c-Myc inhibitory role of Ahnak, mouse embryonic fibroblasts from Ahnak-deficient mouse (Ahnak(-/-) MEF) show an increased level of c-Myc expression compared with wild type MEF. Generation of iPSC with just three of the four Yamanaka factors, Oct4, Sox2, and Klf4 (hereafter 3F), was significantly enhanced in Ahnak(-/-) MEF. Similar results were obtained when Ahnak-specific shRNA was applied to wild type MEF. Of note, expressionof Ahnak was significantly induced during the formation of embryoid bodies from embryonic stem cells, suggesting that Ahnak-mediated c-Myc inhibition is involved in embryoid body formation and the initial differentiation of pluripotent stem cells. The iPSC from 3F-infected Ahnak(-/-) MEF cells (Ahnak(-/-)-iPSC-3F) showed expression of all stem cell markers examined and the capability to form three primary germ layers. Moreover, injection of Ahnak(-/-)-iPSC-3F into athymic nude mice led to development of teratoma containing tissues from all three primary germ layers, indicating that iPSC from Ahnak(-/-) MEF are bona fide pluripotent stem cells. Taken together, these data provide evidence for a new role for Ahnak in cell fate determination during development and suggest that manipulation of Ahnak and the associated signaling pathway may provide a means to regulate iPSC generation.

  18. Expression of endogenous retroviruses is negatively regulated by the pluripotency marker Rex1/Zfp42

    PubMed Central

    Guallar, D.; Pérez-Palacios, R.; Climent, M.; Martínez-Abadía, I.; Larraga, A.; Fernández-Juan, M.; Vallejo, C.; Muniesa, P.; Schoorlemmer, J.

    2012-01-01

    Rex1/Zfp42 is a Yy1-related zinc-finger protein whose expression is frequently used to identify pluripotent stem cells. We show that depletion of Rex1 levels notably affected self-renewal of mouse embryonic stem (ES) cells in clonal assays, in the absence of evident differences in expression of marker genes for pluripotency or differentiation. By contrast, marked differences in expression of several endogenous retroviral elements (ERVs) were evident upon Rex1 depletion. We demonstrate association of REX1 to specific elements in chromatin-immunoprecipitation assays, most strongly to muERV-L and to a lower extent to IAP and musD elements. Rex1 regulates muERV-L expression in vivo, as we show altered levels upon transient gain-and-loss of Rex1 function in pre-implantation embryos. We also find REX1 can associate with the lysine-demethylase LSD1/KDM1A, suggesting they act in concert. Similar to REX1 binding to retrotransposable elements (REs) in ES cells, we also detected binding of the REX1 related proteins YY1 and YY2 to REs, although the binding preferences of the two proteins were slightly different. Altogether, we show that Rex1 regulates ERV expression in mouse ES cells and during pre-implantation development and suggest that Rex1 and its relatives have evolved as regulators of endogenous retroviral transcription. PMID:22844087

  19. Wnt-YAP interactions in the neural fate of human pluripotent stem cells and the implications for neural organoid formation.

    PubMed

    Bejoy, Julie; Song, Liqing; Li, Yan

    2016-01-01

    Human pluripotent stem cells (hPSCs) have shown the ability to self-organize into different types of neural organoids (e.g., whole brain organoids, cortical spheroids, midbrain organoids etc.) recently. The extrinsic and intrinsic signaling elicited by Wnt pathway, Hippo/Yes-associated protein (YAP) pathway, and extracellular microenvironment plays a critical role in brain tissue morphogenesis. This article highlights recent advances in neural tissue patterning from hPSCs, in particular the role of Wnt pathway and YAP activity in this process. Understanding the Wnt-YAP interactions should provide us the guidance to predict and modulate brain-like tissue structure through the regulation of extracellular microenvironment of hPSCs. PMID:26901039

  20. Nac1 Coordinates a Sub-network of Pluripotency Factors to Regulate Embryonic Stem Cell Differentiation.

    PubMed

    Malleshaiah, Mohan; Padi, Megha; Rué, Pau; Quackenbush, John; Martinez-Arias, Alfonso; Gunawardena, Jeremy

    2016-02-01

    Pluripotent cells give rise to distinct cell types during development and are regulated by often self-reinforcing molecular networks. How such networks allow cells to differentiate is less well understood. Here, we use integrative methods to show that external signals induce reorganization of the mouse embryonic stem cell pluripotency network and that a sub-network of four factors, Nac1, Oct4, Tcf3, and Sox2, regulates their differentiation into the alternative mesendodermal and neuroectodermal fates. In the mesendodermal fate, Nac1 and Oct4 were constrained within quantitative windows, whereas Sox2 and Tcf3 were repressed. In contrast, in the neuroectodermal fate, Sox2 and Tcf3 were constrained while Nac1 and Oct4 were repressed. In addition, we show that Nac1 coordinates differentiation by activating Oct4 and inhibiting both Sox2 and Tcf3. Reorganization of progenitor cell networks around shared factors might be a common differentiation strategy and our integrative approach provides a general methodology for delineating such networks.

  1. Nac1 Coordinates a Sub-network of Pluripotency Factors to Regulate Embryonic Stem Cell Differentiation

    PubMed Central

    Malleshaiah, Mohan; Padi, Megha; Rué, Pau; Quackenbush, John; Martinez-Arias, Alfonso; Gunawardena, Jeremy

    2015-01-01

    Pluripotent cells give rise to distinct cell types during development and are regulated by often self-reinforcing molecular networks. How such networks allow cells to differentiate is less well understood. Here, we use integrative methods to show that external signals induce reorganization of the mouse embryonic stem cell pluripotency network and that a sub-network of four factors - Nac1, Oct4, Tcf3 and Sox2 – regulates their differentiation into the alternative mesendodermal and neuroectodermal fates. In the mesendodermal fate, Nac1 and Oct4 were constrained within quantitative windows, while Sox2 and Tcf3 were repressed. In contrast, in the neuroectodermal fate, Sox2 and Tcf3 were constrained while Nac1 and Oct4 were repressed. In addition, we show that Nac1 coordinates differentiation by activating Oct4 and inhibiting both Sox2 and Tcf3. Reorganization of progenitor cell networks around shared factors might be a common differentiation strategy and our integrative approach provides a general methodology for delineating such networks. PMID:26832399

  2. Interaction of Salmonella Typhimurium with Dendritic Cells Derived from Pluripotent Embryonic Stem Cells

    PubMed Central

    Rossi, Raffaella; Hale, Christine; Goulding, David; Andrews, Robert; Abdellah, Zarah; Fairchild, Paul J.; Dougan, Gordon

    2012-01-01

    Using an in vitro differentiation protocol we isolated cells with the properties of dendritic cells (DCs) from immunologically refractive pluripotent murine embryonic stem cells (ESCs). These ES-derived dendritic cells (ESDCs) expressed cytokines and were able to present antigen to a T cell line. Infection of ESDCs with Salmonella Typhimurium stimulated the expression of immune cell markers and thousands of murine genes, many associated with the immune response. Consequently, this system provides a novel in vitro model, amenable to genetic modification, for monitoring host/pathogen interactions. PMID:23284947

  3. Advances in microfluidic platforms for analyzing and regulating human pluripotent stem cells.

    PubMed

    Qian, Tongcheng; Shusta, Eric V; Palecek, Sean P

    2015-10-01

    Microfluidic devices employ submillimeter length scale control of flow to achieve high-resolution spatial and temporal control over the microenvironment, providing powerful tools to elucidate mechanisms of human pluripotent stem cell (hPSC) regulation and to elicit desired hPSC fates. In addition, microfluidics allow control of paracrine and juxtracrine signaling, thereby enabling fabrication of microphysiological systems comprised of multiple cell types organized into organs-on-a-chip. Microfluidic cell culture systems can also be integrated with actuators and sensors, permitting construction of high-density arrays of cell-based biosensors for screening applications. This review describes recent advances in using microfluidics to understand mechanisms by which the microenvironment regulates hPSC fates and applications of microfluidics to realize the potential of hPSCs for in vitro modeling and screening applications.

  4. Interaction of Salmonella enterica Serovar Typhimurium with Intestinal Organoids Derived from Human Induced Pluripotent Stem Cells.

    PubMed

    Forbester, Jessica L; Goulding, David; Vallier, Ludovic; Hannan, Nicholas; Hale, Christine; Pickard, Derek; Mukhopadhyay, Subhankar; Dougan, Gordon

    2015-07-01

    The intestinal mucosa forms the first line of defense against infections mediated by enteric pathogens such as salmonellae. Here we exploited intestinal "organoids" (iHOs) generated from human induced pluripotent stem cells (hIPSCs) to explore the interaction of Salmonella enterica serovar Typhimurium with iHOs. Imaging and RNA sequencing were used to analyze these interactions, and clear changes in transcriptional signatures were detected, including altered patterns of cytokine expression after the exposure of iHOs to bacteria. S. Typhimurium microinjected into the lumen of iHOs was able to invade the epithelial barrier, with many bacteria residing within Salmonella-containing vacuoles. An S. Typhimurium invA mutant defective in the Salmonella pathogenicity island 1 invasion apparatus was less capable of invading the iHO epithelium. Hence, we provide evidence that hIPSC-derived organoids are a promising model of the intestinal epithelium for assessing interactions with enteric pathogens.

  5. Pluripotency Factors on Their Lineage Move

    PubMed Central

    Weidgang, Clair E.; Seufferlein, Thomas; Kleger, Alexander; Mueller, Martin

    2016-01-01

    Pluripotent stem cells are characterised by continuous self-renewal while maintaining the potential to differentiate into cells of all three germ layers. Regulatory networks of maintaining pluripotency have been described in great detail and, similarly, there is great knowledge on key players that regulate their differentiation. Interestingly, pluripotency has various shades with distinct developmental potential, an observation that coined the term of a ground state of pluripotency. A precise interplay of signalling axes regulates ground state conditions and acts in concert with a combination of key transcription factors. The balance between these transcription factors greatly influences the integrity of the pluripotency network and latest research suggests that minute changes in their expression can strengthen but also collapse the network. Moreover, recent studies reveal different facets of these core factors in balancing a controlled and directed exit from pluripotency. Thereby, subsets of pluripotency-maintaining factors have been shown to adopt new roles during lineage specification and have been globally defined towards neuroectodermal and mesendodermal sets of embryonic stem cell genes. However, detailed underlying insights into how these transcription factors orchestrate cell fate decisions remain largely elusive. Our group and others unravelled complex interactions in the regulation of this controlled exit. Herein, we summarise recent findings and discuss the potential mechanisms involved. PMID:26770212

  6. The extended pluripotency protein interactome and its links to reprogramming

    PubMed Central

    Huang, Xin; Wang, Jianlong

    2014-01-01

    A pluripotent state of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is maintained through the combinatorial activity of core transcriptional factors (TFs) such as Oct4, Sox2, and Nanog in conjunction with many other factors including epigenetic regulators. Proteins rarely act alone, and knowledge of protein-protein interaction network (interactome) provides an extraordinary resource about how pluripotency TFs collaborate and crosstalk with epigenetic regulators in ESCs. Recent advances in affinity purification coupled with mass spectrometry (AP-MS) allow for efficient, high-throughput identification of hundreds of interacting protein partners, which can be used to map the pluripotency landscape. Here we review recent publications employing AP-MS to investigate protein interaction networks in ESCs, discuss how protein-protein connections reveal novel pluripotency regulatory circuits and new factors for efficient reprogramming of somatic cells. PMID:25173149

  7. Identification of cisplatin-regulated metabolic pathways in pluripotent stem cells.

    PubMed

    von Stechow, Louise; Ruiz-Aracama, Ainhoa; van de Water, Bob; Peijnenburg, Ad; Danen, Erik; Lommen, Arjen

    2013-01-01

    The chemotherapeutic compound, cisplatin causes various kinds of DNA lesions but also triggers other pertubations, such as ER and oxidative stress. We and others have shown that treatment of pluripotent stem cells with cisplatin causes a plethora of transcriptional and post-translational alterations that, to a major extent, point to DNA damage response (DDR) signaling. The orchestrated DDR signaling network is important to arrest the cell cycle and repair the lesions or, in case of damage beyond repair, eliminate affected cells. Failure to properly balance the various aspects of the DDR in stem cells contributes to ageing and cancer. Here, we performed metabolic profiling by mass spectrometry of embryonic stem (ES) cells treated for different time periods with cisplatin. We then integrated metabolomics with transcriptomics analyses and connected cisplatin-regulated metabolites with regulated metabolic enzymes to identify enriched metabolic pathways. These included nucleotide metabolism, urea cycle and arginine and proline metabolism. Silencing of identified proline metabolic and catabolic enzymes indicated that altered proline metabolism serves as an adaptive, rather than a toxic response. A group of enriched metabolic pathways clustered around the metabolite S-adenosylmethionine, which is a hub for methylation and transsulfuration reactions and polyamine metabolism. Enzymes and metabolites with pro- or anti-oxidant functions were also enriched but enhanced levels of reactive oxygen species were not measured in cisplatin-treated ES cells. Lastly, a number of the differentially regulated metabolic enzymes were identified as target genes of the transcription factor p53, pointing to p53-mediated alterations in metabolism in response to genotoxic stress. Altogether, our findings reveal interconnecting metabolic pathways that are responsive to cisplatin and may serve as signaling modules in the DDR in pluripotent stem cells.

  8. Identification of Cisplatin-Regulated Metabolic Pathways in Pluripotent Stem Cells

    PubMed Central

    van de Water, Bob; Peijnenburg, Ad; Danen, Erik; Lommen, Arjen

    2013-01-01

    The chemotherapeutic compound, cisplatin causes various kinds of DNA lesions but also triggers other pertubations, such as ER and oxidative stress. We and others have shown that treatment of pluripotent stem cells with cisplatin causes a plethora of transcriptional and post-translational alterations that, to a major extent, point to DNA damage response (DDR) signaling. The orchestrated DDR signaling network is important to arrest the cell cycle and repair the lesions or, in case of damage beyond repair, eliminate affected cells. Failure to properly balance the various aspects of the DDR in stem cells contributes to ageing and cancer. Here, we performed metabolic profiling by mass spectrometry of embryonic stem (ES) cells treated for different time periods with cisplatin. We then integrated metabolomics with transcriptomics analyses and connected cisplatin-regulated metabolites with regulated metabolic enzymes to identify enriched metabolic pathways. These included nucleotide metabolism, urea cycle and arginine and proline metabolism. Silencing of identified proline metabolic and catabolic enzymes indicated that altered proline metabolism serves as an adaptive, rather than a toxic response. A group of enriched metabolic pathways clustered around the metabolite S-adenosylmethionine, which is a hub for methylation and transsulfuration reactions and polyamine metabolism. Enzymes and metabolites with pro- or anti-oxidant functions were also enriched but enhanced levels of reactive oxygen species were not measured in cisplatin-treated ES cells. Lastly, a number of the differentially regulated metabolic enzymes were identified as target genes of the transcription factor p53, pointing to p53-mediated alterations in metabolism in response to genotoxic stress. Altogether, our findings reveal interconnecting metabolic pathways that are responsive to cisplatin and may serve as signaling modules in the DDR in pluripotent stem cells. PMID:24146875

  9. Cell-Cycle Control of Developmentally Regulated Transcription Factors Accounts for Heterogeneity in Human Pluripotent Cells

    PubMed Central

    Singh, Amar M.; Chappell, James; Trost, Robert; Lin, Li; Wang, Tao; Tang, Jie; Wu, Hao; Zhao, Shaying; Jin, Peng; Dalton, Stephen

    2013-01-01

    Summary Heterogeneity within pluripotent stem cell (PSC) populations is indicative of dynamic changes that occur when cells drift between different states. Although the role of metastability in PSCs is unclear, it appears to reflect heterogeneity in cell signaling. Using the Fucci cell-cycle indicator system, we show that elevated expression of developmental regulators in G1 is a major determinant of heterogeneity in human embryonic stem cells. Although signaling pathways remain active throughout the cell cycle, their contribution to heterogeneous gene expression is restricted to G1. Surprisingly, we identify dramatic changes in the levels of global 5-hydroxymethylcytosine, an unanticipated source of epigenetic heterogeneity that is tightly linked to cell-cycle progression and the expression of developmental regulators. When we evaluated gene expression in differentiating cells, we found that cell-cycle regulation of developmental regulators was maintained during lineage specification. Cell-cycle regulation of developmentally regulated transcription factors is therefore an inherent feature of the mechanisms underpinning differentiation. PMID:24371808

  10. SOX2 O-GlcNAcylation alters its protein-protein interactions and genomic occupancy to modulate gene expression in pluripotent cells

    PubMed Central

    Myers, Samuel A; Peddada, Sailaja; Chatterjee, Nilanjana; Friedrich, Tara; Tomoda, Kiichrio; Krings, Gregor; Thomas, Sean; Maynard, Jason; Broeker, Michael; Thomson, Matthew; Pollard, Katherine; Yamanaka, Shinya; Burlingame, Alma L; Panning, Barbara

    2016-01-01

    The transcription factor SOX2 is central in establishing and maintaining pluripotency. The processes that modulate SOX2 activity to promote pluripotency are not well understood. Here, we show SOX2 is O-GlcNAc modified in its transactivation domain during reprogramming and in mouse embryonic stem cells (mESCs). Upon induction of differentiation SOX2 O-GlcNAcylation at serine 248 is decreased. Replacing wild type with an O-GlcNAc-deficient SOX2 (S248A) increases reprogramming efficiency. ESCs with O-GlcNAc-deficient SOX2 exhibit alterations in gene expression. This change correlates with altered protein-protein interactions and genomic occupancy of the O-GlcNAc-deficient SOX2 compared to wild type. In addition, SOX2 O-GlcNAcylation impairs the SOX2-PARP1 interaction, which has been shown to regulate ESC self-renewal. These findings show that SOX2 activity is modulated by O-GlcNAc, and provide a novel regulatory mechanism for this crucial pluripotency transcription factor. DOI: http://dx.doi.org/10.7554/eLife.10647.001 PMID:26949256

  11. SOX2 O-GlcNAcylation alters its protein-protein interactions and genomic occupancy to modulate gene expression in pluripotent cells.

    PubMed

    Myers, Samuel A; Peddada, Sailaja; Chatterjee, Nilanjana; Friedrich, Tara; Tomoda, Kiichrio; Krings, Gregor; Thomas, Sean; Maynard, Jason; Broeker, Michael; Thomson, Matthew; Pollard, Katherine; Yamanaka, Shinya; Burlingame, Alma L; Panning, Barbara

    2016-03-07

    The transcription factor SOX2 is central in establishing and maintaining pluripotency. The processes that modulate SOX2 activity to promote pluripotency are not well understood. Here, we show SOX2 is O-GlcNAc modified in its transactivation domain during reprogramming and in mouse embryonic stem cells (mESCs). Upon induction of differentiation SOX2 O-GlcNAcylation at serine 248 is decreased. Replacing wild type with an O-GlcNAc-deficient SOX2 (S248A) increases reprogramming efficiency. ESCs with O-GlcNAc-deficient SOX2 exhibit alterations in gene expression. This change correlates with altered protein-protein interactions and genomic occupancy of the O-GlcNAc-deficient SOX2 compared to wild type. In addition, SOX2 O-GlcNAcylation impairs the SOX2-PARP1 interaction, which has been shown to regulate ESC self-renewal. These findings show that SOX2 activity is modulated by O-GlcNAc, and provide a novel regulatory mechanism for this crucial pluripotency transcription factor.

  12. Impact of transient down-regulation of DREAM in human embryonic stem cell pluripotency: The role of DREAM in the maintenance of hESCs.

    PubMed

    Fontán-Lozano, A; Capilla-Gonzalez, V; Aguilera, Y; Mellado, N; Carrión, A M; Soria, B; Hmadcha, A

    2016-05-01

    Little is known about the functions of downstream regulatory element antagonist modulator (DREAM) in embryonic stem cells (ESCs). However, DREAM interacts with cAMP response element-binding protein (CREB) in a Ca(2+)-dependent manner, preventing CREB binding protein (CBP) recruitment. Furthermore, CREB and CBP are involved in maintaining ESC self-renewal and pluripotency. However, a previous knockout study revealed the protective function of DREAM depletion in brain aging degeneration and that aging is accompanied by a progressive decline in stem cells (SCs) function. Interestingly, we found that DREAM is expressed in different cell types, including human ESCs (hESCs), human adipose-derived stromal cells (hASCs), human bone marrow-derived stromal cells (hBMSCs), and human newborn foreskin fibroblasts (hFFs), and that transitory inhibition of DREAM in hESCs reduces their pluripotency, increasing differentiation. We stipulate that these changes are partly mediated by increased CREB transcriptional activity. Overall, our data indicates that DREAM acts in the regulation of hESC pluripotency and could be a target to promote or prevent differentiation in embryonic cells. PMID:26999760

  13. Impact of transient down-regulation of DREAM in human embryonic stem cell pluripotency: The role of DREAM in the maintenance of hESCs.

    PubMed

    Fontán-Lozano, A; Capilla-Gonzalez, V; Aguilera, Y; Mellado, N; Carrión, A M; Soria, B; Hmadcha, A

    2016-05-01

    Little is known about the functions of downstream regulatory element antagonist modulator (DREAM) in embryonic stem cells (ESCs). However, DREAM interacts with cAMP response element-binding protein (CREB) in a Ca(2+)-dependent manner, preventing CREB binding protein (CBP) recruitment. Furthermore, CREB and CBP are involved in maintaining ESC self-renewal and pluripotency. However, a previous knockout study revealed the protective function of DREAM depletion in brain aging degeneration and that aging is accompanied by a progressive decline in stem cells (SCs) function. Interestingly, we found that DREAM is expressed in different cell types, including human ESCs (hESCs), human adipose-derived stromal cells (hASCs), human bone marrow-derived stromal cells (hBMSCs), and human newborn foreskin fibroblasts (hFFs), and that transitory inhibition of DREAM in hESCs reduces their pluripotency, increasing differentiation. We stipulate that these changes are partly mediated by increased CREB transcriptional activity. Overall, our data indicates that DREAM acts in the regulation of hESC pluripotency and could be a target to promote or prevent differentiation in embryonic cells.

  14. A Novel Role for miR‐1305 in Regulation of Pluripotency‐Differentiation Balance, Cell Cycle, and Apoptosis in Human Pluripotent Stem Cells

    PubMed Central

    Jin, Shibo; Collin, Joseph; Zhu, Lili; Montaner, David; Armstrong, Lyle

    2016-01-01

    Abstract Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are defined as pluripotent in view of their self‐renewal ability and potential to differentiate to cells of all three germ layers. Recent studies have indicated that microRNAs (miRNAs) play an important role in the maintenance of pluripotency and cell cycle regulation. We used a microarray based approach to identify miRNAs that were enriched in hESCs when compared to differentiated cells and at the same time showed significant expression changes between different phases of cell cycle. We identified 34 candidate miRNAs and performed functional studies on one of these, miR‐1305, which showed the highest expression change during cell cycle transition. Overexpression of miR‐1305 induced differentiation of pluripotent stem cells, increased cell apoptosis and sped up G1/S transition, while its downregulation facilitated the maintenance of pluripotency and increased cell survival. Using target prediction software and luciferase based reporter assays we identified POLR3G as a downstream target by which miR‐1305 regulates the fine balance between maintenance of pluripotency and onset of differentiation. Overexpression of POLR3G rescued pluripotent stem cell differentiation induced by miR‐1305 overexpression. In contrast, knock‐down of POLR3G expression abolished the miR‐1305‐knockdown mediated enhancement of pluripotency, thus validating its role as miR‐1305 target in human pluripotent stem cells. Together our data point to an important role for miR‐1305 as a novel regulator of pluripotency, cell survival and cell cycle and uncovers new mechanisms and networks by which these processes are intertwined in human pluripotent stem cells. Stem Cells 2016;34:2306–2317 PMID:27339422

  15. Evolution of gene regulation of pluripotency - the case for wiki tracks at genome browsers

    PubMed Central

    2010-01-01

    Background Experimentally validated data on gene regulation are hard to obtain. In particular, information about transcription factor binding sites in regulatory regions are scattered around in the literature. This impedes their systematic in-context analysis, e.g. the inference of their conservation in evolutionary history. Results We demonstrate the power of integrative bioinformatics by including curated transcription factor binding site information into the UCSC genome browser, using wiki and custom tracks, which enable easy publication of annotation data. Data integration allows to investigate the evolution of gene regulation of the pluripotency-associated genes Oct4, Sox2 and Nanog. For the first time, experimentally validated transcription factor binding sites in the regulatory regions of all three genes were assembled together based on manual curation of data from 39 publications. Using the UCSC genome browser, these data were then visualized in the context of multi-species conservation based on genomic alignment. We confirm previous hypotheses regarding the evolutionary age of specific regulatory patterns, establishing their "deep homology". We also confirm some other principles of Carroll's "Genetic theory of Morphological Evolution", such as "mosaic pleiotropy", exemplified by the dual role of Sox2 reflected in its regulatory region. Conclusions We were able to elucidate some aspects of the evolution of gene regulation for three genes associated with pluripotency. Based on the expected return on investment for the community, we encourage other scientists to contribute experimental data on gene regulation (original work as well as data collected for reviews) to the UCSC system, to enable studies of the evolution of gene regulation on a large scale, and to report their findings. Reviewers This article was reviewed by Dr. Gustavo Glusman and Dr. Juan Caballero, Institute for Systems Biology, Seattle, USA (nominated by Dr. Doron Lancet, Department of

  16. Genetic regulators of a pluripotent adult stem cell system in planarians identified by RNAi and clonal analysis.

    PubMed

    Wagner, Daniel E; Ho, Jaclyn J; Reddien, Peter W

    2012-03-01

    Pluripotency is a central, well-studied feature of embryonic development, but the role of pluripotent cell regulation in somatic tissue regeneration remains poorly understood. In planarians, regeneration of entire animals from tissue fragments is promoted by the activity of adult pluripotent stem cells (cNeoblasts). We utilized transcriptional profiling to identify planarian genes expressed in adult proliferating, regenerative cells (neoblasts). We also developed quantitative clonal analysis methods for expansion and differentiation of cNeoblast descendants that, together with RNAi, revealed gene roles in stem cell biology. Genes encoding two zinc finger proteins, Vasa, a LIM domain protein, Sox and Jun-like transcription factors, two candidate RNA-binding proteins, a Setd8-like protein, and PRC2 (Polycomb) were required for proliferative expansion and/or differentiation of cNeoblast-derived clones. These findings suggest that planarian stem cells utilize molecular mechanisms found in germ cells and other pluripotent cell types and identify genetic regulators of the planarian stem cell system.

  17. Genetic regulators of a pluripotent adult stem cell system in planarians identified by RNAi and clonal analysis

    PubMed Central

    Wagner, Daniel E.; Ho, Jaclyn J.

    2012-01-01

    Summary Pluripotency is a central, well-studied feature of embryonic development, but the role of pluripotent cell regulation in somatic tissue regeneration remains poorly understood. In planarians, regeneration of entire animals from tissue fragments is promoted by the activity of adult pluripotent stem cells (cNeoblasts). We utilized transcriptional profiling to identify planarian genes expressed in adult proliferating, regenerative cells (neoblasts). We also developed quantitative clonal analysis methods for expansion and differentiation of cNeoblast descendants that, together with RNAi, revealed gene roles in stem cell biology. Genes encoding two zinc finger proteins, Vasa, a LIM domain protein, Sox and Jun-like transcription factors, two candidate RNA-binding proteins, a Setd8-like protein, and PRC2 (Polycomb) were required for proliferative expansion and/or differentiation of cNeoblast-derived clones. These findings suggest that planarian stem cells utilize molecular mechanisms found in germ cells and other pluripotent cell types, and identify novel genetic regulators of the planarian stem cell system. PMID:22385657

  18. Confined 3D microenvironment regulates early differentiation in human pluripotent stem cells.

    PubMed

    Giobbe, Giovanni G; Zagallo, Monica; Riello, Massimo; Serena, Elena; Masi, Giulia; Barzon, Luisa; Di Camillo, Barbara; Elvassore, Nicola

    2012-12-01

    The therapeutic potential of human pluripotent stem (hPS) cells is threatened, among various problems, by the difficulty to homogenously direct cell differentiation into specific lineages. The transition from hPSC into committed differentiated cells is accompanied by secretome activity, remodeling of extracellular matrix and self-organization into germ layers. In this work, we aimed to investigate how different three-dimensional microenvironments regulate the early differentiation of the three germ layers in human embryonic stem (hES) cells derived embryoid bodies. In particular, a permeable, biocompatible, hydrogel microwell array was specifically designed for recreating a confined niche in which EB secreted molecules accumulate in accordance with hydrogel diffusional cut-off. Fluorescence recovery after photobleaching technique was performed to accurately evaluate hydrogel permeability, mesh size and diffusional cutoff for soluble molecules. Three different culture conditions of EB culture were analyzed: suspension, confinement in microwells of width/depth ratio 1:1 and 1:2. Results show that EBs cultured in microwells are viable and have comparable average size after 8 days culture. Whole genome microarrays show that significative differential gene expression was observed between suspension and confined EBs culture. In particular, EBs culture in microwells promotes the expression of genes involved in pattern specification processes, brain development, ectoderm and endoderm differentiation. On the contrary, suspension EBs express instead genes involved in mesoderm specification and heart development. These results suggest that local accumulation of EBs secreted molecules drives differentiation patterns, as confirmed by immunofluorescence of germ layer markers, in hydrogel confined EB culture from both hES cells and human induced pluripotent stem (hiPS) cells. Our findings highlight an additional potential role of biomaterial in controlling hPSC differentiation

  19. The X-inactivation trans-activator Rnf12 is negatively regulated by pluripotency factors in embryonic stem cells.

    PubMed

    Navarro, Pablo; Moffat, Michael; Mullin, Nicholas P; Chambers, Ian

    2011-08-01

    X-inactivation, the molecular mechanism enabling dosage compensation in mammals, is tightly controlled during mouse early embryogenesis. In the morula, X-inactivation is imprinted with exclusive silencing of the paternally inherited X-chromosome. In contrast, in the post-implantation epiblast, X-inactivation affects randomly either the paternal or the maternal X-chromosome. The transition from imprinted to random X-inactivation takes place in the inner cell mass (ICM) of the blastocyst from which embryonic stem (ES) cells are derived. The trigger of X-inactivation, Xist, is specifically downregulated in the pluripotent cells of the ICM, thereby ensuring the reactivation of the inactive paternal X-chromosome and the transient presence of two active X-chromosomes. Moreover, Tsix, a critical cis-repressor of Xist, is upregulated in the ICM and in ES cells where it imposes a particular chromatin state at the Xist promoter that ensures the establishment of random X-inactivation upon differentiation. Recently, we have shown that key transcription factors supporting pluripotency directly repress Xist and activate Tsix and thus couple Xist/Tsix control to pluripotency. In this manuscript, we report that Rnf12, a third X-linked gene critical for the regulation of X-inactivation, is under the control of Nanog, Oct4 and Sox2, the three factors lying at the heart of the pluripotency network. We conclude that in mouse ES cells the pluripotency-associated machinery exerts an exhaustive control of X-inactivation by taking over the regulation of all three major regulators of X-inactivation: Xist, Tsix, and Rnf12.

  20. Epigenetic regulation of pluripotent genes mediates stem cell features in human hepatocellular carcinoma and cancer cell lines.

    PubMed

    Wang, Xiao Qi; Ng, Ray Kit; Ming, Xiaoyan; Zhang, Wu; Chen, Lin; Chu, Andrew C Y; Pang, Roberta; Lo, Chung Mau; Tsao, Sai Wah; Liu, Xuqing; Poon, Ronnie T P; Fan, Sheung Tat

    2013-01-01

    Activation of the stem cell transcriptional circuitry is an important event in cancer development. Although cancer cells demonstrate a stem cell-like gene expression signature, the epigenetic regulation of pluripotency-associated genes in cancers remains poorly understood. In this study, we characterized the epigenetic regulation of the pluripotency-associated genes NANOG, OCT4, c-MYC, KLF4, and SOX2 in a variety of cancer cell lines and in primary tumor samples, and investigated the re-activation of pluripotency regulatory circuits in cancer progression. Differential patterns of DNA methylation, histone modifications, and gene expression of pluripotent genes were demonstrated in different types of cancers, which may reflect their tissue origins. NANOG promoter hypomethylation and gene upregulation were found in metastatic human liver cancer cells and human hepatocellular carcinoma (HCC) primary tumor tissues. The upregulation of NANOG, together with p53 depletion, was significantly associated with clinical late stage of HCC. A pro-metastatic role of NANOG in colon cancer cells was also demonstrated, using a NANOG-overexpressing orthotopic tumor implantation mouse model. Demethylation of NANOG promoter was observed in CD133+(high) cancer cells. In accordance, overexpression of NANOG resulted in an increase in the population of CD133+(high) cells. In addition, we demonstrated a cross-regulation between OCT4 and NANOG in cancer cells via reprogramming of promoter methylation. Taken together, epigenetic reprogramming of NANOG can lead to the acquisition of stem cell-like properties. These results underscore the restoration of pluripotency circuits in cancer cells as a potential mechanism for cancer progression. PMID:24023739

  1. PAK4: a pluripotent kinase that regulates prostate cancer cell adhesion

    PubMed Central

    Wells, Claire M.; Whale, Andrew D.; Parsons, Maddy; Masters, John R. W.; Jones, Gareth E.

    2010-01-01

    Hepatocyte growth factor (HGF) is associated with tumour progression and increases the invasiveness of prostate carcinoma cells. Migration and invasion require coordinated reorganisation of the actin cytoskeleton and regulation of cell-adhesion dynamics. Rho-family GTPases orchestrate both of these cellular processes. p21-activated kinase 4 (PAK4), a specific effector of the Rho GTPase Cdc42, is activated by HGF, and we have previously shown that activated PAK4 induces a loss of both actin stress fibres and focal adhesions. We now report that DU145 human prostate cancer cells with reduced levels of PAK4 expression are unable to successfully migrate in response to HGF, have prominent actin stress fibres, and an increase in the size and number of focal adhesions. Moreover, these cells have a concomitant reduction in cell-adhesion turnover rates. We find that PAK4 is localised at focal adhesions, is immunoprecipitated with paxillin and phosphorylates paxillin on serine 272. Furthermore, we demonstrate that PAK4 can regulate RhoA activity via GEF-H1. Our results suggest that PAK4 is a pluripotent kinase that can regulate both actin cytoskeletal rearrangement and focal-adhesion dynamics. PMID:20406887

  2. Critical components of the pluripotency network are targets for the p300/CBP interacting protein (p/CIP) in embryonic stem cells.

    PubMed

    Chitilian, J M; Thillainadesan, G; Manias, J L; Chang, W Y; Walker, E; Isovic, M; Stanford, W L; Torchia, J

    2014-01-01

    p/CIP, also known as steroid receptor coactivator 3 (SRC-3)/Nuclear Receptor Coactivator 3 (NCoA3), is a transcriptional coactivator that binds liganded nuclear hormone receptors, as well as other transcription factors, and facilitates transcription through direct recruitment of accessory factors. We have found that p/CIP is highly expressed in undifferentiated mouse embryonic stem cells (mESCs) and is downregulated during differentiation. siRNA-mediated knockdown of p/CIP decreased transcript levels of Nanog, but not Oct4 or Sox2. Microarray expression analysis showed that Klf4, Tbx3, and Dax-1 are significantly downregulated in mESCs when p/CIP is knocked down. Subsequent chromatin immunoprecipitation (ChIP) analysis demonstrated that Tbx3, Klf4, and Dax-1 are direct transcriptional targets of p/CIP. Using the piggyBac transposition system, a mouse ESC line that expresses Flag-p/CIP in a doxycycline-dependent manner was generated. p/CIP overexpression increased the level of target genes and promoted the formation of undifferentiated colonies. Collectively, these results indicate that p/CIP contributes to the maintenance of ESC pluripotency through direct regulation of essential pluripotency genes. To better understand the mechanism by which p/CIP functions in ESC pluripotency, we integrated our ChIP and transcriptome data with published protein-protein interaction and promoter occupancy data to draft a p/CIP gene regulatory network. The p/CIP gene regulatory network identifies various feed-forward modules including one in which p/CIP activates members of the extended pluripotency network, demonstrating that p/CIP is a component of this extended network. PMID:24115386

  3. Epigenetic Mechanisms Regulate MHC and Antigen Processing Molecules in Human Embryonic and Induced Pluripotent Stem Cells

    PubMed Central

    Suárez-Álvarez, Beatriz; Rodriguez, Ramón M.; Calvanese, Vincenzo; Blanco-Gelaz, Miguel A.; Suhr, Steve T.; Ortega, Francisco; Otero, Jesus; Cibelli, Jose B.; Moore, Harry; Fraga, Mario F.; López-Larrea, Carlos

    2010-01-01

    Background Human embryonic stem cells (hESCs) are an attractive resource for new therapeutic approaches that involve tissue regeneration. hESCs have exhibited low immunogenicity due to low levels of Mayor Histocompatibility Complex (MHC) class-I and absence of MHC class-II expression. Nevertheless, the mechanisms regulating MHC expression in hESCs had not been explored. Methodology/Principal Findings We analyzed the expression levels of classical and non-classical MHC class-I, MHC class-II molecules, antigen-processing machinery (APM) components and NKG2D ligands (NKG2D-L) in hESCs, induced pluripotent stem cells (iPSCs) and NTera2 (NT2) teratocarcinoma cell line. Epigenetic mechanisms involved in the regulation of these genes were investigated by bisulfite sequencing and chromatin immunoprecipitation (ChIP) assays. We showed that low levels of MHC class-I molecules were associated with absent or reduced expression of the transporter associated with antigen processing 1 (TAP-1) and tapasin (TPN) components in hESCs and iPSCs, which are involved in the transport and load of peptides. Furthermore, lack of β2-microglobulin (β2m) light chain in these cells limited the expression of MHC class I trimeric molecule on the cell surface. NKG2D ligands (MICA, MICB) were observed in all pluripotent stem cells lines. Epigenetic analysis showed that H3K9me3 repressed the TPN gene in undifferentiated cells whilst HLA-B and β2m acquired the H3K4me3 modification during the differentiation to embryoid bodies (EBs). Absence of HLA-DR and HLA-G expression was regulated by DNA methylation. Conclusions/Significance Our data provide fundamental evidence for the epigenetic control of MHC in hESCs and iPSCs. Reduced MHC class I and class II expression in hESCs and iPSCs can limit their recognition by the immune response against these cells. The knowledge of these mechanisms will further allow the development of strategies to induce tolerance and improve stem cell allograft acceptance

  4. TRIM32 modulates pluripotency entry and exit by directly regulating Oct4 stability

    PubMed Central

    Bahnassawy, Lamia’a; Perumal, Thanneer M.; Gonzalez-Cano, Laura; Hillje, Anna-Lena; Taher, Leila; Makalowski, Wojciech; Suzuki, Yutaka; Fuellen, Georg; Sol, Antonio del; Schwamborn, Jens Christian

    2015-01-01

    Induced pluripotent stem cells (iPSCs) have revolutionized the world of regenerative medicine; nevertheless, the exact molecular mechanisms underlying their generation and differentiation remain elusive. Here, we investigated the role of the cell fate determinant TRIM32 in modulating such processes. TRIM32 is essential for the induction of neuronal differentiation of neural stem cells by poly-ubiquitinating cMyc to target it for degradation resulting in inhibition of cell proliferation. To elucidate the role of TRIM32 in regulating somatic cell reprogramming we analysed the capacity of TRIM32-knock-out mouse embryonic fibroblasts (MEFs) in generating iPSC colonies. TRIM32 knock-out MEFs produced a higher number of iPSC colonies indicating a role for TRIM32 in inhibiting this cellular transition. Further characterization of the generated iPSCs indicated that the TRIM32 knock-out iPSCs show perturbed differentiation kinetics. Additionally, mathematical modelling of global gene expression data revealed that during differentiation an Oct4 centred network in the wild-type cells is replaced by an E2F1 centred network in the TRIM32 deficient cells. We show here that this might be caused by a TRIM32-dependent downregulation of Oct4. In summary, the data presented here reveal that TRIM32 directly regulates at least two of the four Yamanaka Factors (cMyc and Oct4), to modulate cell fate transitions. PMID:26307407

  5. Myostatin acts as an autocrine/paracrine negative regulator in myoblast differentiation from human induced pluripotent stem cells

    SciTech Connect

    Gao, Fei; Kishida, Tsunao; Ejima, Akika; Gojo, Satoshi; Mazda, Osam

    2013-02-08

    Highlights: ► iPS-derived cells express myostatin and its receptor upon myoblast differentiation. ► Myostatin inhibits myoblast differentiation by inhibiting MyoD and Myo5a induction. ► Silencing of myostatin promotes differentiation of human iPS cells into myoblasts. -- Abstract: Myostatin, also known as growth differentiation factor (GDF-8), regulates proliferation of muscle satellite cells, and suppresses differentiation of myoblasts into myotubes via down-regulation of key myogenic differentiation factors including MyoD. Recent advances in stem cell biology have enabled generation of myoblasts from pluripotent stem cells, but it remains to be clarified whether myostatin is also involved in regulation of artificial differentiation of myoblasts from pluripotent stem cells. Here we show that the human induced pluripotent stem (iPS) cell-derived cells that were induced to differentiate into myoblasts expressed myostatin and its receptor during the differentiation. An addition of recombinant human myostatin (rhMyostatin) suppressed induction of MyoD and Myo5a, resulting in significant suppression of myoblast differentiation. The rhMyostatin treatment also inhibited proliferation of the cells at a later phase of differentiation. RNAi-mediated silencing of myostatin promoted differentiation of human iPS-derived embryoid body (EB) cells into myoblasts. These results strongly suggest that myostatin plays an important role in regulation of myoblast differentiation from iPS cells of human origin. The present findings also have significant implications for potential regenerative medicine for muscular diseases.

  6. Myostatin acts as an autocrine/paracrine negative regulator in myoblast differentiation from human induced pluripotent stem cells.

    PubMed

    Gao, Fei; Kishida, Tsunao; Ejima, Akika; Gojo, Satoshi; Mazda, Osam

    2013-02-01

    Myostatin, also known as growth differentiation factor (GDF-8), regulates proliferation of muscle satellite cells, and suppresses differentiation of myoblasts into myotubes via down-regulation of key myogenic differentiation factors including MyoD. Recent advances in stem cell biology have enabled generation of myoblasts from pluripotent stem cells, but it remains to be clarified whether myostatin is also involved in regulation of artificial differentiation of myoblasts from pluripotent stem cells. Here we show that the human induced pluripotent stem (iPS) cell-derived cells that were induced to differentiate into myoblasts expressed myostatin and its receptor during the differentiation. An addition of recombinant human myostatin (rhMyostatin) suppressed induction of MyoD and Myo5a, resulting in significant suppression of myoblast differentiation. The rhMyostatin treatment also inhibited proliferation of the cells at a later phase of differentiation. RNAi-mediated silencing of myostatin promoted differentiation of human iPS-derived embryoid body (EB) cells into myoblasts. These results strongly suggest that myostatin plays an important role in regulation of myoblast differentiation from iPS cells of human origin. The present findings also have significant implications for potential regenerative medicine for muscular diseases. PMID:23291166

  7. Genome-wide analysis reveals Sall4 to be a major regulator of pluripotency in murine-embryonic stem cells.

    PubMed

    Yang, Jianchang; Chai, Li; Fowles, Taylor C; Alipio, Zaida; Xu, Dan; Fink, Louis M; Ward, David C; Ma, Yupo

    2008-12-16

    Embryonic stem cells have potential utility in regenerative medicine because of their pluripotent characteristics. Sall4, a zinc-finger transcription factor, is expressed very early in embryonic development with Oct4 and Nanog, two well-characterized pluripotency regulators. Sall4 plays an important role in governing the fate of stem cells through transcriptional regulation of both Oct4 and Nanog. By using chromatin immunoprecipitation coupled to microarray hybridization (ChIP-on-chip), we have mapped global gene targets of Sall4 to further investigate regulatory processes in W4 mouse ES cells. A total of 3,223 genes were identified that were bound by the Sall4 protein on duplicate assays with high confidence, and many of these have major functions in developmental and regulatory pathways. Sall4 bound approximately twice as many annotated genes within promoter regions as Nanog and approximately four times as many as Oct4. Immunoprecipitation revealed a heteromeric protein complex(es) between Sall4, Oct4, and Nanog, consistent with binding site co-occupancies. Decreasing Sall4 expression in W4 ES cells decreases the expression levels of Oct4, Sox2, c-Myc, and Klf4, four proteins capable of reprogramming somatic cells to an induced pluripotent state. Further, Sall4 bound many genes that are regulated in part by chromatin-based epigenetic events mediated by polycomb-repressive complexes and bivalent domains. This suggests that Sall4 plays a diverse role in regulating stem cell pluripotency during early embryonic development through integration of transcriptional and epigenetic controls. PMID:19060217

  8. TGF-βI Regulates Cell Migration through Pluripotent Transcription Factor OCT4 in Endometriosis

    PubMed Central

    Au, Heng-Kien; Chang, Jui-Hung; Wu, Yu-Chih; Kuo, Yung-Che; Chen, Yu-Hsi; Lee, Wei-Chin; Chang, Te-Sheng; Lan, Pei-Chi; Kuo, Hung-Chih; Lee, Kha-Liang; Lee, Mei-Tsu; Tzeng, Chii-Ruey; Huang, Yen-Hua

    2015-01-01

    Transforming growth factor (TGF-β)/TGF-β receptor signal is known to promote cell migration. Up-regulation of TGF-β in serum/peritoneal fluid and increased levels of pluripotent transcription factor OCT4 in endometriotic tissues are frequently observed in patients with endometriosis. However, the mechanisms underlying how TGF-β/TGF-β receptor and OCT4 affect endometriotic cell migration still remain largely unknown. Therefore, endometriotic tissue with high cell migratory capacity were collected from patients with adenomyotic myometrium (n = 23) and chocolate cyst (n = 24); and endometrial tissue with low cell migratory capacity in normal endometrium or hyperplastic endometrium (n = 8) were collected as the controls. We found the mRNA levels of TGF-β receptor I (TGF-β RI) and OCT4 were significantly higher in the high-migratory ectopic endometriotic tissues than those of the low-migratory normal or hyperplastic endometrium. Positive correlations between TGF-β RI and OCT4, and either TGF-β RI or OCT4 with migration-related genes (SNAIL, SLUG and TWIST) regarding the mRNA levels were observed in human endometriotic tissues. TGF-βI dose-dependently increased the gene and protein levels of OCT4, SNAIL and N-Cadherin (N-CAD) and silencing of endogenous OCT4 significantly suppressed the TGF-βI-induced expressions of N-CAD and SNAIL in primary human endometriotic stromal cells and human endometrial carcinoma cell lines RL95-2 and HEC1A. Furthermore, TGF-βI significantly increased the migration ability of endometriotic cells and silencing of OCT4 dramatically suppressed the TGF-βI-induced cell migration activity evidenced by wound-closure assay, transwell assay, and confocal image of F-actin cellular distribution. In conclusion, the present findings demonstrate that the niche TGF-β plays a critical role in initiating expressions of pluripotent transcription factor OCT4 which may contribute to the ectopic endometrial growth by stimulating endometrial cell

  9. MYC/MAX control ERK signaling and pluripotency by regulation of dual-specificity phosphatases 2 and 7.

    PubMed

    Chappell, James; Sun, Yuhua; Singh, Amar; Dalton, Stephen

    2013-04-01

    Suppression of extracellular signal-regulated kinase (ERK) signaling is an absolute requirement for the maintenance of murine pluripotent stem cells (mPSCs) and requires the MYC-binding partner MAX. In this study, we define a mechanism for this by showing that MYC/MAX complexes suppress ERK activity by transcriptionally regulating two members of the dual-specificity phosphatase (DUSP) family. DUSPs function by binding and then inactivating ERK1,2 by dephosphorylating residues required for catalytic activity. MYC/MAX complexes achieve this by binding the promoters of DUSP2 and DUSP7, leading to their transcriptional activation, resulting in the attenuation of ERK activity. In the absence of MYC, ectopic DUSP2,7 expression severely delays differentiation, while loss of DUSP2,7 ectopically activates ERK, resulting in loss of pluripotency. These findings elucidate a novel regulatory role for MYC in PSC maintenance involving the stimulation of phosphatases that directly inhibit the MAPK/ERK signaling pathway. Moreover, it provides a mechanism for how leukemia inhibitory factor (LIF)/STAT3 signaling reaches across to the MAPK/ERK pathway through MYC and MAX to sustain pluripotency.

  10. [Two vital transcriptional factors Oct-4 and Nanog to keep the pluripotency and self-renewal of stem cells and related regulation network].

    PubMed

    Zhou, Yi-Ye; Zeng, Fan-Yi

    2008-05-01

    Oct-4 and Nanog are two critical transcriptional factors to keep pluripotency and self-renewal of stem cells in vivo and in vitro, and they usually express only in pluripotent cells and not in differentiated cells. They bind to the regulatory regions of targeted gene and often interact with other transcriptional factors and extracellular signal path components, such as Sox-2, FoxD3, LIF and BMP in specific tissues or developmental stages. So that all of these constitute a transcriptional crosstalk, and finally determine the cells destiny: keeping pluripotency or turning to differentiation. PMID:18487140

  11. JMJD5 regulates cell cycle and pluripotency in human embryonic stem cells.

    PubMed

    Zhu, Hui; Hu, Shijun; Baker, Julie

    2014-08-01

    In mammalian embryos, embryonic stem cells (ESCs) and induced pluripotent cells, a shortened G1 phase is correlated with the pluripotent state. To molecularly define this phase, we compared transcripts from the shortened G1 of human ESCs (hESCs) with those from the longer G1 of derived endoderm. We identified JMJD5, a JmjC (Jumonji C) domain containing protein that, when depleted in hESCs, causes the accumulation of cells in G1 phase, loss of pluripotency, and subsequent differentiation into multiple lineages, most prominently ectoderm and trophectoderm. Furthermore, we demonstrate that the JMJD5 phenotype is caused by the upregulation of CDKN1A (p21), as depleting both JMJD5 and CDKN1A (p21) in hESCs restores the rapid G1 phase and rescues the pluripotent state. Overall, we provide genetic and biochemical evidence that the JMJD5/CDKN1A (p21) axis is essential to maintaining the short G1 phase which is critical for pluripotency in hESCs.

  12. Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture

    PubMed Central

    Tan, Boon Siang Nicholas; Kwek, Joly; Wong, Chong Kum Edwin; Saner, Nicholas J.; Yap, Charlotte; Felquer, Fernando; Morris, Michael B.; Gardner, David K.; Rathjen, Peter D.; Rathjen, Joy

    2016-01-01

    Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL) cells from mouse embryonic stem (mES) cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation. PMID:27723793

  13. Conserved long noncoding RNAs transcriptionally regulated by Oct4 and Nanog modulate pluripotency in mouse embryonic stem cells.

    PubMed

    Sheik Mohamed, Jameelah; Gaughwin, Philip Michael; Lim, Bing; Robson, Paul; Lipovich, Leonard

    2010-02-01

    The genetic networks controlling stem cell identity are the focus of intense interest, due to their obvious therapeutic potential as well as exceptional relevance to models of early development. Genome-wide mapping of transcriptional networks in mouse embryonic stem cells (mESCs) reveals that many endogenous noncoding RNA molecules, including long noncoding RNAs (lncRNAs), may play a role in controlling the pluripotent state. We performed a genome-wide screen that combined full-length mESC transcriptome genomic mapping data with chromatin immunoprecipitation genomic location maps of the key mESC transcription factors Oct4 and Nanog. We henceforth identified four mESC-expressed, conserved lncRNA-encoding genes residing proximally to active genomic binding sites of Oct4 and Nanog. Accordingly, these four genes have potential roles in pluripotency. We show that two of these lncRNAs, AK028326 (Oct4-activated) and AK141205 (Nanog-repressed), are direct targets of Oct4 and Nanog. Most importantly, we demonstrate that these lncRNAs are not merely controlled by mESC transcription factors, but that they themselves regulate developmental state: knockdown and overexpression of these transcripts lead to robust changes in Oct4 and Nanog mRNA levels, in addition to alterations in cellular lineage-specific gene expression and in the pluripotency of mESCs. We further characterize AK028326 as a co-activator of Oct4 in a regulatory feedback loop. These results for the first time implicate lncRNAs in the modulation of mESC pluripotency and expand the established mESC regulatory network model to include functional lncRNAs directly controlled by key mESC transcription factors.

  14. Differential L1 regulation in pluripotent stem cells of humans and apes.

    PubMed

    Marchetto, Maria C N; Narvaiza, Iñigo; Denli, Ahmet M; Benner, Christopher; Lazzarini, Thomas A; Nathanson, Jason L; Paquola, Apuã C M; Desai, Keval N; Herai, Roberto H; Weitzman, Matthew D; Yeo, Gene W; Muotri, Alysson R; Gage, Fred H

    2013-11-28

    Identifying cellular and molecular differences between human and non-human primates (NHPs) is essential to the basic understanding of the evolution and diversity of our own species. Until now, preserved tissues have been the main source for most comparative studies between humans, chimpanzees (Pan troglodytes) and bonobos (Pan paniscus). However, these tissue samples do not fairly represent the distinctive traits of live cell behaviour and are not amenable to genetic manipulation. We propose that induced pluripotent stem (iPS) cells could be a unique biological resource to determine relevant phenotypical differences between human and NHPs, and that those differences could have potential adaptation and speciation value. Here we describe the generation and initial characterization of iPS cells from chimpanzees and bonobos as new tools to explore factors that may have contributed to great ape evolution. Comparative gene expression analysis of human and NHP iPS cells revealed differences in the regulation of long interspersed element-1 (L1, also known as LINE-1) transposons. A force of change in mammalian evolution, L1 elements are retrotransposons that have remained active during primate evolution. Decreased levels of L1-restricting factors APOBEC3B (also known as A3B) and PIWIL2 (ref. 7) in NHP iPS cells correlated with increased L1 mobility and endogenous L1 messenger RNA levels. Moreover, results from the manipulation of A3B and PIWIL2 levels in iPS cells supported a causal inverse relationship between levels of these proteins and L1 retrotransposition. Finally, we found increased copy numbers of species-specific L1 elements in the genome of chimpanzees compared to humans, supporting the idea that increased L1 mobility in NHPs is not limited to iPS cells in culture and may have also occurred in the germ line or embryonic cells developmentally upstream to germline specification during primate evolution. We propose that differences in L1 mobility may have

  15. Role of nitric oxide in the maintenance of pluripotency and regulation of the hypoxia response in stem cells

    PubMed Central

    Beltran-Povea, Amparo; Caballano-Infantes, Estefania; Salguero-Aranda, Carmen; Martín, Franz; Soria, Bernat; Bedoya, Francisco J; Tejedo, Juan R; Cahuana, Gladys M

    2015-01-01

    Stem cell pluripotency and differentiation are global processes regulated by several pathways that have been studied intensively over recent years. Nitric oxide (NO) is an important molecule that affects gene expression at the level of transcription and translation and regulates cell survival and proliferation in diverse cell types. In embryonic stem cells NO has a dual role, controlling differentiation and survival, but the molecular mechanisms by which it modulates these functions are not completely defined. NO is a physiological regulator of cell respiration through the inhibition of cytochrome c oxidase. Many researchers have been examining the role that NO plays in other aspects of metabolism such as the cellular bioenergetics state, the hypoxia response and the relationship of these areas to stem cell stemness. PMID:25914767

  16. Zfp281 Coordinates Opposing Functions of Tet1 and Tet2 in Pluripotent States.

    PubMed

    Fidalgo, Miguel; Huang, Xin; Guallar, Diana; Sanchez-Priego, Carlos; Valdes, Victor Julian; Saunders, Arven; Ding, Junjun; Wu, Wen-Shu; Clavel, Carlos; Wang, Jianlong

    2016-09-01

    Pluripotency is increasingly recognized as a spectrum of cell states defined by their growth conditions. Although naive and primed pluripotency states have been characterized molecularly, our understanding of events regulating state acquisition is wanting. Here, we performed comparative RNA sequencing of mouse embryonic stem cells (ESCs) and defined a pluripotent cell fate (PCF) gene signature associated with acquisition of naive and primed pluripotency. We identify Zfp281 as a key transcriptional regulator for primed pluripotency that also functions as a barrier toward achieving naive pluripotency in both mouse and human ESCs. Mechanistically, Zfp281 interacts with Tet1, but not Tet2, and its direct transcriptional target, miR-302/367, to negatively regulate Tet2 expression to establish and maintain primed pluripotency. Conversely, ectopic Tet2 alone, but not Tet1, efficiently reprograms primed cells toward naive pluripotency. Our study reveals a molecular circuitry in which opposing functions of Tet1 and Tet2 control acquisition of alternative pluripotent states. PMID:27345836

  17. Collagen/β1 integrin interaction is required for embryoid body formation during cardiogenesis from murine induced pluripotent stem cells

    PubMed Central

    2013-01-01

    Background The interactions between stem cells and extracellular matrix (ECM) mediated by integrins play important roles in the processes that determine stem cell fate. However, the role of ECM/integrin interaction in the formation of embryoid bodies (EBs) during cardiogenesis from murine induced pluripotent stem cells (miPSCs) remains unclear. Results In the present study, collagen type I and β1 integrin were expressed and upregulated synergistically during the formation of miPSC-derived EBs, with a peak expression at day 3 of differentiation. The blockage of collagen/β1 integrin interaction by β1 integrin blocking antibody resulted in the production of defective EBs that were characterized by decreased size and the absence of a shell-like layer composed of primitive endoderm cells. The quantification of spontaneous beating activity, cardiac-specific gene expression and cardiac troponin T (cTnT) immunostaining showed that the cardiac differentiation of these defective miPSC-derived EBs was lower than that of control EBs. Conclusions These findings indicate that collagen/β1 integrin interaction is required for the growth and cardiac differentiation of miPSC-derived EBs and will be helpful in future engineering of the matrix microenvironment within EBs to efficiently direct the cardiac fate of pluripotent stem cells to promote cardiovascular regeneration. PMID:23350814

  18. The nucleoporin Nup153 regulates embryonic stem cell pluripotency through gene silencing

    PubMed Central

    Jacinto, Filipe V.; Benner, Chris; Hetzer, Martin W.

    2015-01-01

    Nucleoporins (Nups) are a family of proteins best known as the constituent building blocks of nuclear pore complexes (NPCs), membrane-embedded channels that mediate nuclear transport across the nuclear envelope. Recent evidence suggests that several Nups have additional roles in controlling the activation and silencing of developmental genes; however, the mechanistic details of these functions remain poorly understood. Here, we show that depletion of Nup153 in mouse embryonic stem cells (mESCs) causes the derepression of developmental genes and induction of early differentiation. This loss of stem cell identity is not associated with defects in the nuclear import of key pluripotency factors. Rather, Nup153 binds around the transcriptional start site (TSS) of developmental genes and mediates the recruitment of the polycomb-repressive complex 1 (PRC1) to a subset of its target loci. Our results demonstrate a chromatin-associated role of Nup153 in maintaining stem cell pluripotency by functioning in mammalian epigenetic gene silencing. PMID:26080816

  19. Embryonic stem cell-specific microRNAs contribute to pluripotency by inhibiting regulators of multiple differentiation pathways

    PubMed Central

    Gruber, Andreas J.; Grandy, William A.; Balwierz, Piotr J.; Dimitrova, Yoana A.; Pachkov, Mikhail; Ciaudo, Constance; van Nimwegen, Erik; Zavolan, Mihaela

    2014-01-01

    The findings that microRNAs (miRNAs) are essential for early development in many species and that embryonic miRNAs can reprogram somatic cells into induced pluripotent stem cells suggest that these miRNAs act directly on transcriptional and chromatin regulators of pluripotency. To elucidate the transcription regulatory networks immediately downstream of embryonic miRNAs, we extended the motif activity response analysis approach that infers the regulatory impact of both transcription factors (TFs) and miRNAs from genome-wide expression states. Applying this approach to multiple experimental data sets generated from mouse embryonic stem cells (ESCs) that did or did not express miRNAs of the ESC-specific miR-290-295 cluster, we identified multiple TFs that are direct miRNA targets, some of which are known to be active during cell differentiation. Our results provide new insights into the transcription regulatory network downstream of ESC-specific miRNAs, indicating that these miRNAs act on cell cycle and chromatin regulators at several levels and downregulate TFs that are involved in the innate immune response. PMID:25030899

  20. N-Myc regulates expression of pluripotency genes in neuroblastoma including lif, klf2, klf4, and lin28b.

    PubMed

    Cotterman, Rebecca; Knoepfler, Paul S

    2009-01-01

    myc genes are best known for causing tumors when overexpressed, but recent studies suggest endogenous myc regulates pluripotency and self-renewal of stem cells. For example, N-myc is associated with a number of tumors including neuroblastoma, but also plays a central role in the function of normal neural stem and precursor cells (NSC). Both c- and N-myc also enhance the production of induced pluripotent stem cells (iPSC) and are linked to neural tumor stem cells. The mechanisms by which myc regulates normal and neoplastic stem-related functions remain largely open questions. Here from a global, unbiased search for N-Myc bound genes using ChIP-chip assays in neuroblastoma, we found lif as a putative N-Myc bound gene with a number of strong N-Myc binding peaks in the promoter region enriched for E-boxes. Amongst putative N-Myc target genes in expression microarray studies in neuroblastoma we also found lif and three additional important embryonic stem cell (ESC)-related factors that are linked to production of iPSC: klf2, klf4, and lin28b. To examine the regulation of these genes by N-Myc, we measured their expression using neuroblastoma cells that contain a Tet-regulatable N-myc transgene (TET21N) as well as NSC with a nestin-cre driven N-myc knockout. N-myc levels closely correlated with the expression of all of these genes in neuroblastoma and all but lif in NSC. Direct ChIP assays also indicate that N-Myc directly binds the lif promoter. N-Myc regulates trimethylation of lysine 4 of histone H3 in the promoter of lif and possibly in the promoters of several other stem-related genes. Together these findings indicate that N-Myc regulates overlapping stem-related gene expression programs in neuroblastoma and NSC, supporting a novel model by which amplification of the N-myc gene may drive formation of neuroblastoma. They also suggest mechanisms by which Myc proteins more generally contribute to maintenance of pluripotency and self-renewal of ESC as well as to i

  1. TRPV3 Channel Negatively Regulates Cell Cycle Progression and Safeguards the Pluripotency of Embryonic Stem Cells.

    PubMed

    Lo, Iek Chi; Chan, Hing Chung; Qi, Zenghua; Ng, Kwun Lam; So, Chun; Tsang, Suk Ying

    2016-02-01

    Embryonic stem cells (ESCs) have tremendous potential for research and future therapeutic purposes. However, the calcium handling mechanism in ESCs is not fully elucidated. Aims of this study are (1) to investigate if transient receptor potential vanilloid-3 (TRPV3) channels are present in mouse ESCs (mESCs) and their subcellular localization; (2) to investigate the role of TRPV3 in maintaining the characteristics of mESCs. Western blot and immunocytochemistry showed that TRPV3 was present at the endoplasmic reticulum (ER) of mESCs. Calcium imaging showed that, in the absence of extracellular calcium, TRPV3 activators camphor and 6-tert-butyl-m-cresol increased the cytosolic calcium. However, depleting the ER store in advance of activator addition abolished the calcium increase, suggesting that TRPV3 released calcium from the ER. To dissect the functional role of TRPV3, TRPV3 was activated and mESC proliferation was measured by trypan blue exclusion and MTT assays. The results showed that TRPV3 activation led to a decrease in mESC proliferation. Cell cycle analysis revealed that TRPV3 activation increased the percentage of cells in G2 /M phase; consistently, Western blot also revealed a concomitant increase in the expression of inactive form of cyclin-dependent kinase 1, suggesting that TRPV3 activation arrested mESCs at G2 /M phase. TRPV3 activation did not alter the expression of pluripotency markers Oct-4, Klf4 and c-Myc, suggesting that the pluripotency was preserved. Our study is the first study to show the presence of TRPV3 at ER. Our study also reveals the novel role of TRPV3 in controlling the cell cycle and preserving the pluripotency of ESCs.

  2. miR-302 regulates pluripotency, teratoma formation and differentiation in stem cells via an AKT1/OCT4-dependent manner

    PubMed Central

    Li, H-L; Wei, J-F; Fan, L-Y; Wang, S-H; Zhu, L; Li, T-P; Lin, G; Sun, Y; Sun, Z-J; Ding, J; Liang, X-L; Li, J; Han, Q; Zhao, R-C-H

    2016-01-01

    Pluripotency makes human pluripotent stem cells (hPSCs) promising for regenerative medicine, but the teratoma formation has been considered to be a major obstacle for their clinical applications. Here, we determined that the downregulation of miR-302 suppresses the teratoma formation, hampers the self-renewal and pluripotency, and promotes hPSC differentiation. The underlying mechanism is that the high endogenous expression of miR-302 suppresses the AKT1 expression by directly targeting its 3'UTR and subsequently maintains the pluripotent factor OCT4 at high level. Our findings reveal that miR-302 regulates OCT4 by suppressing AKT1, which provides hPSCs two characteristics related to their potential for clinical applications: the benefit of pluripotency and the hindrance of teratoma formation. More importantly, we demonstrate that miR-302 upregulation cannot lead OCT4 negative human adult mesenchymal stem cells (hMSCs) to acquire the teratoma formation in vivo. Whether miR-302 upregulation can drive hMSCs to acquire a higher differentiation potential is worthy of deep investigation. PMID:26821070

  3. TGFβ signaling regulates the choice between pluripotent and neural fates during reprogramming of human urine derived cells

    PubMed Central

    Wang, Lihui; Li, Xirui; Huang, Wenhao; Zhou, Tiancheng; Wang, Haitao; Lin, Aiping; Hutchins, Andrew Paul; Su, Zhenghui; Chen, Qianyu; Pei, Duanqing; Pan, Guangjin

    2016-01-01

    Human urine cells (HUCs) can be reprogrammed into neural progenitor cells (NPCs) or induced pluripotent stem cells (iPSCs) with defined factors and a small molecule cocktail, but the underlying fate choice remains unresolved. Here, through sequential removal of individual compound from small molecule cocktail, we showed that A8301, a TGFβ signaling inhibitor, is sufficient to switch the cell fate from iPSCs into NPCs in OSKM-mediated HUCs reprogramming. However, TGFβ exposure at early stage inhibits HUCs reprogramming by promoting EMT. Base on these data, we developed an optimized approach for generation of NPCs or iPSCs from HUCs with significantly improved efficiency by regulating TGFβ activity at different reprogramming stages. This approach provides a simplified and improved way for HUCs reprogramming, thus would be valuable for banking human iPSCs or NPCs from people with different genetic background. PMID:26935433

  4. Regulation of stem cell pluripotency and differentiation involves a mutual regulatory circuit of the NANOG, OCT4, and SOX2 pluripotency transcription factors with polycomb repressive complexes and stem cell microRNAs.

    PubMed

    Kashyap, Vasundhra; Rezende, Naira C; Scotland, Kymora B; Shaffer, Sebastian M; Persson, Jenny Liao; Gudas, Lorraine J; Mongan, Nigel P

    2009-09-01

    Coordinated transcription factor networks have emerged as the master regulatory mechanisms of stem cell pluripotency and differentiation. Many stem cell-specific transcription factors, including the pluripotency transcription factors, OCT4, NANOG, and SOX2 function in combinatorial complexes to regulate the expression of loci, which are involved in embryonic stem (ES) cell pluripotency and cellular differentiation. This review will address how these pathways form a reciprocal regulatory circuit whereby the equilibrium between stem cell self-renewal, proliferation, and differentiation is in perpetual balance. We will discuss how distinct epigenetic repressive pathways involving polycomb complexes, DNA methylation, and microRNAs cooperate to reduce transcriptional noise and to prevent stochastic and aberrant induction of differentiation. We will provide a brief overview of how these networks cooperate to modulate differentiation along hematopoietic and neuronal lineages. Finally, we will describe how aberrant functioning of components of the stem cell regulatory network may contribute to malignant transformation of adult stem cells and the establishment of a "cancer stem cell" phenotype and thereby underlie multiple types of human malignancies.

  5. MiRNA-mediated regulation of the SWI/SNF chromatin remodeling complex controls pluripotency and endodermal differentiation in human ES cells

    PubMed Central

    Wade, Staton L.; Langer, Lee F.; Ward, James M.; Archer, Trevor K.

    2015-01-01

    MicroRNAs and chromatin remodeling complexes represent powerful epigenetic mechanisms that regulate the pluripotent state. miR-302 is a strong inducer of pluripotency, which is characterized by a distinct chromatin architecture. This suggests that miR-302 regulates global chromatin structure; however, a direct relationship between miR-302 and chromatin remodelers has not been established. Here, we provide data to show that miR-302 regulates Brg1 chromatin remodeling complex composition in human embryonic stem (hESs) cells through direct repression of the BAF53a and BAF170 subunits. With the subsequent overexpression of BAF170 in hESCs, we show that miR-302’s inhibition of BAF170 protein levels can affect the expression of genes involved in cell proliferation. Furthermore, miR-302-mediated repression of BAF170 regulates pluripotency by positively influencing mesendodermal differentiation. Overexpression of BAF170 in hESCs led to biased differentiation toward the ectoderm lineage during EB formation and severely hindered directed definitive endoderm differentiation. Taken together, these data uncover a direct regulatory relationship between miR-302 and the Brg1 chromatin remodeling complex that controls gene expression and cell fate decisions in hESCs and suggests that similar mechanisms are at play during early human development. PMID:26119756

  6. A hybrid microfluidic system for regulation of neural differentiation in induced pluripotent stem cells.

    PubMed

    Hesari, Zahra; Soleimani, Massoud; Atyabi, Fatemeh; Sharifdini, Meysam; Nadri, Samad; Warkiani, Majid Ebrahimi; Zare, Mehrak; Dinarvand, Rassoul

    2016-06-01

    Controlling cellular orientation, proliferation, and differentiation is valuable in designing organ replacements and directing tissue regeneration. In the present study, we developed a hybrid microfluidic system to produce a dynamic microenvironment by placing aligned PDMS microgrooves on surface of biodegradable polymers as physical guidance cues for controlling the neural differentiation of human induced pluripotent stem cells (hiPSCs). The neuronal differentiation capacity of cultured hiPSCs in the microfluidic system and other control groups was investigated using quantitative real time PCR (qPCR) and immunocytochemistry. The functionally of differentiated hiPSCs inside hybrid system's scaffolds was also evaluated on the rat hemisected spinal cord in acute phase. Implanted cell's fate was examined using tissue freeze section and the functional recovery was evaluated according to the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale. Our results confirmed the differentiation of hiPSCs to neuronal cells on the microfluidic device where the expression of neuronal-specific genes was significantly higher compared to those cultured on the other systems such as plain tissue culture dishes and scaffolds without fluidic channels. Although survival and integration of implanted hiPSCs did not lead to a significant functional recovery, we believe that combination of fluidic channels with nanofiber scaffolds provides a great microenvironment for neural tissue engineering, and can be used as a powerful tool for in situ monitoring of differentiation potential of various kinds of stem cells. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1534-1543, 2016. PMID:26914600

  7. Cell Pluripotency Levels Associated with Imprinted Genes in Human

    PubMed Central

    Yuan, Liyun; Tang, Xiaoyan; Zhang, Binyan; Ding, Guohui

    2015-01-01

    Pluripotent stem cells are exhibited similarly in the morphology, gene expression, growth properties, and epigenetic modification with embryonic stem cells (ESCs). However, it is still controversial that the pluripotency of induced pluripotent stem cell (iPSC) is much inferior to ESC, and the differentiation capacity of iPSC and ESC can also be separated by transcriptome and epigenetics. miRNAs, which act in posttranscriptional regulation of gene expression and are involved in many basic cellular processes, may reveal the answer. In this paper, we focused on identifying the hidden relationship between miRNAs and imprinted genes in cell pluripotency. Total miRNA expression patterns in iPSC and ES cells were comprehensively analysed and linked with human imprinted genes, which show a global picture of their potential function in pluripotent level. A new CPA4-KLF14 region which locates in chromosomal homologous segments (CHSs) within mammals and include both imprinted genes and significantly expressed miRNAs was first identified. Molecular network analysis showed genes interacted with imprinted genes closely and enriched in modules such as cancer, cell death and survival, and tumor morphology. This imprinted region may provide a new look for those who are interested in cell pluripotency of hiPSCs and hESCs. PMID:26504487

  8. Cell Pluripotency Levels Associated with Imprinted Genes in Human.

    PubMed

    Yuan, Liyun; Tang, Xiaoyan; Zhang, Binyan; Ding, Guohui

    2015-01-01

    Pluripotent stem cells are exhibited similarly in the morphology, gene expression, growth properties, and epigenetic modification with embryonic stem cells (ESCs). However, it is still controversial that the pluripotency of induced pluripotent stem cell (iPSC) is much inferior to ESC, and the differentiation capacity of iPSC and ESC can also be separated by transcriptome and epigenetics. miRNAs, which act in posttranscriptional regulation of gene expression and are involved in many basic cellular processes, may reveal the answer. In this paper, we focused on identifying the hidden relationship between miRNAs and imprinted genes in cell pluripotency. Total miRNA expression patterns in iPSC and ES cells were comprehensively analysed and linked with human imprinted genes, which show a global picture of their potential function in pluripotent level. A new CPA4-KLF14 region which locates in chromosomal homologous segments (CHSs) within mammals and include both imprinted genes and significantly expressed miRNAs was first identified. Molecular network analysis showed genes interacted with imprinted genes closely and enriched in modules such as cancer, cell death and survival, and tumor morphology. This imprinted region may provide a new look for those who are interested in cell pluripotency of hiPSCs and hESCs. PMID:26504487

  9. Modeling Dengue Virus-Hepatic Cell Interactions Using Human Pluripotent Stem Cell-Derived Hepatocyte-like Cells.

    PubMed

    Lang, Jianshe; Vera, Daniel; Cheng, Yichen; Tang, Hengli

    2016-09-13

    The development of dengue antivirals and vaccine has been hampered by the incomplete understanding of molecular mechanisms of dengue virus (DENV) infection and pathology, partly due to the limited suitable cell culture or animal models that can capture the comprehensive cellular changes induced by DENV. In this study, we differentiated human pluripotent stem cells (hPSCs) into hepatocytes, one of the target cells of DENV, to investigate various aspects of DENV-hepatocyte interaction. hPSC-derived hepatocyte-like cells (HLCs) supported persistent and productive DENV infection. The activation of interferon pathways by DENV protected bystander cells from infection and protected the infected cells from massive apoptosis. Furthermore, DENV infection activated the NF-κB pathway, which led to production of proinflammatory cytokines and downregulated many liver-specific genes such as albumin and coagulation factor V. Our study demonstrates the utility of hPSC-derived hepatocytes as an in vitro model for DENV infection and reveals important aspects of DENV-host interactions. PMID:27546535

  10. MeCP2 Regulates the Synaptic Expression of a Dysbindin-BLOC-1 Network Component in Mouse Brain and Human Induced Pluripotent Stem Cell-Derived Neurons

    PubMed Central

    Larimore, Jennifer; Ryder, Pearl V.; Kim, Kun-Yong; Ambrose, L. Alex; Chapleau, Christopher; Calfa, Gaston; Gross, Christina; Bassell, Gary J.; Pozzo-Miller, Lucas; Smith, Yoland; Talbot, Konrad; Park, In-Hyun; Faundez, Victor

    2013-01-01

    Clinical, epidemiological, and genetic evidence suggest overlapping pathogenic mechanisms between autism spectrum disorder (ASD) and schizophrenia. We tested this hypothesis by asking if mutations in the ASD gene MECP2 which cause Rett syndrome affect the expression of genes encoding the schizophrenia risk factor dysbindin, a subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), and associated interacting proteins. We measured mRNA and protein levels of key components of a dysbindin interaction network by, quantitative real time PCR and quantitative immunohistochemistry in hippocampal samples of wild-type and Mecp2 mutant mice. In addition, we confirmed results by performing immunohistochemistry of normal human hippocampus and quantitative qRT-PCR of human inducible pluripotent stem cells (iPSCs)-derived human neurons from Rett syndrome patients. We defined the distribution of the BLOC-1 subunit pallidin in human and mouse hippocampus and contrasted this distribution with that of symptomatic Mecp2 mutant mice. Neurons from mutant mice and Rett syndrome patients displayed selectively reduced levels of pallidin transcript. Pallidin immunoreactivity decreased in the hippocampus of symptomatic Mecp2 mutant mice, a feature most prominent at asymmetric synapses as determined by immunoelectron microcopy. Pallidin immunoreactivity decreased concomitantly with reduced BDNF content in the hippocampus of Mecp2 mice. Similarly, BDNF content was reduced in the hippocampus of BLOC-1 deficient mice suggesting that genetic defects in BLOC-1 are upstream of the BDNF phenotype in Mecp2 deficient mice. Our results demonstrate that the ASD-related gene Mecp2 regulates the expression of components belonging to the dysbindin interactome and these molecular differences may contribute to synaptic phenotypes that characterize Mecp2 deficiencies and ASD. PMID:23750231

  11. Super-Enhancers at the Nanog Locus Differentially Regulate Neighboring Pluripotency-Associated Genes.

    PubMed

    Blinka, Steven; Reimer, Michael H; Pulakanti, Kirthi; Rao, Sridhar

    2016-09-27

    Super-enhancers are tissue-specific cis-regulatory elements that drive expression of genes associated with cell identity and malignancy. A cardinal feature of super-enhancers is that they are transcribed to produce enhancer-derived RNAs (eRNAs). It remains unclear whether super-enhancers robustly activate genes in situ and whether their functions are attributable to eRNAs or the DNA element. CRISPR/Cas9 was used to systematically delete three discrete super-enhancers at the Nanog locus in embryonic stem cells, revealing functional differences in Nanog transcriptional regulation. One distal super-enhancer 45 kb upstream of Nanog (-45 enhancer) regulates both nearest neighbor genes, Nanog and Dppa3. Interestingly, eRNAs produced at the -45 enhancer specifically regulate Dppa3 expression by stabilizing looping of the -45 enhancer and Dppa3. Our work illustrates that genomic editing is required to determine enhancer function and points to a method to selectively target a subset of super-enhancer-regulated genes by depleting eRNAs. PMID:27681417

  12. Modeling host interactions with hepatitis B virus using primary and induced pluripotent stem cell-derived hepatocellular systems.

    PubMed

    Shlomai, Amir; Schwartz, Robert E; Ramanan, Vyas; Bhatta, Ankit; de Jong, Ype P; Bhatia, Sangeeta N; Rice, Charles M

    2014-08-19

    Hepatitis B virus (HBV) chronically infects 400 million people worldwide and is a leading driver of end-stage liver disease and liver cancer. Research into the biology and treatment of HBV requires an in vitro cell-culture system that supports the infection of human hepatocytes, and accurately recapitulates virus-host interactions. Here, we report that micropatterned cocultures of primary human hepatocytes with stromal cells (MPCCs) reliably support productive HBV infection, and infection can be enhanced by blocking elements of the hepatocyte innate immune response associated with the induction of IFN-stimulated genes. MPCCs maintain prolonged, productive infection and represent a facile platform for studying virus-host interactions and for developing antiviral interventions. Hepatocytes obtained from different human donors vary dramatically in their permissiveness to HBV infection, suggesting that factors--such as divergence in genetic susceptibility to infection--may influence infection in vitro. To establish a complementary, renewable system on an isogenic background in which candidate genetics can be interrogated, we show that inducible pluripotent stem cells differentiated into hepatocyte-like cells (iHeps) support HBV infection that can also be enhanced by blocking interferon-stimulated gene induction. Notably, the emergence of the capacity to support HBV transcriptional activity and initial permissiveness for infection are marked by distinct stages of iHep differentiation, suggesting that infection of iHeps can be used both to study HBV, and conversely to assess the degree of iHep differentiation. Our work demonstrates the utility of these infectious systems for studying HBV biology and the virus' interactions with host hepatocyte genetics and physiology. PMID:25092305

  13. Axolotl Nanog activity in mouse embryonic stem cells demonstrates that ground state pluripotency is conserved from urodele amphibians to mammals.

    PubMed

    Dixon, James E; Allegrucci, Cinzia; Redwood, Catherine; Kump, Kevin; Bian, Yuhong; Chatfield, Jodie; Chen, Yi-Hsien; Sottile, Virginie; Voss, S Randal; Alberio, Ramiro; Johnson, Andrew D

    2010-09-01

    Cells in the pluripotent ground state can give rise to somatic cells and germ cells, and the acquisition of pluripotency is dependent on the expression of Nanog. Pluripotency is conserved in the primitive ectoderm of embryos from mammals and urodele amphibians, and here we report the isolation of a Nanog ortholog from axolotls (axNanog). axNanog does not contain a tryptophan repeat domain and is expressed as a monomer in the axolotl animal cap. The monomeric form is sufficient to regulate pluripotency in mouse embryonic stem cells, but axNanog dimers are required to rescue LIF-independent self-renewal. Our results show that protein interactions mediated by Nanog dimerization promote proliferation. More importantly, they demonstrate that the mechanisms governing pluripotency are conserved from urodele amphibians to mammals.

  14. Molecular Analysis of Neutrophil Differentiation from Human Induced Pluripotent Stem Cells Delineates the Kinetics of Key Regulators of Hematopoiesis.

    PubMed

    Sweeney, Colin L; Teng, Ruifeng; Wang, Hongmei; Merling, Randall K; Lee, Janet; Choi, Uimook; Koontz, Sherry; Wright, Daniel G; Malech, Harry L

    2016-06-01

    In vitro generation of mature neutrophils from human induced pluripotent stem cells (iPSCs) requires hematopoietic progenitor development followed by myeloid differentiation. The purpose of our studies was to extensively characterize this process, focusing on the critical window of development between hemogenic endothelium, hematopoietic stem/progenitor cells (HSPCs), and myeloid commitment, to identify associated regulators and markers that might enable the stem cell field to improve the efficiency and efficacy of iPSC hematopoiesis. We utilized a four-stage differentiation protocol involving: embryoid body (EB) formation (stage-1); EB culture with hematopoietic cytokines (stage-2); HSPC expansion (stage-3); and neutrophil maturation (stage-4). CD34(+) CD45(-) putative hemogenic endothelial cells were observed in stage-3 cultures, and expressed VEGFR-2/Flk-1/KDR and VE-cadherin endothelial markers, GATA-2, AML1/RUNX1, and SCL/TAL1 transcription factors, and endothelial/HSPC-associated microRNAs miR-24, miR-125a-3p, miR-126/126*, and miR-155. Upon further culture, CD34(+) CD45(-) cells generated CD34(+) CD45(+) HSPCs that produced hematopoietic CFUs. Mid-stage-3 CD34(+) CD45(+) HSPCs exhibited increased expression of GATA-2, AML1/RUNX1, SCL/TAL1, C/EBPα, and PU.1 transcription factors, but exhibited decreased expression of HSPC-associated microRNAs, and failed to engraft in immune-deficient mice. Mid-stage-3 CD34(-) CD45(+) cells maintained PU.1 expression and exhibited increased expression of hematopoiesis-associated miR-142-3p/5p and a trend towards increased miR-223 expression, indicating myeloid commitment. By late Stage-4, increased CD15, CD16b, and C/EBPɛ expression were observed, with 25%-65% of cells exhibiting morphology and functions of mature neutrophils. These studies demonstrate that hematopoiesis and neutrophil differentiation from human iPSCs recapitulates many features of embryonic hematopoiesis and neutrophil production in marrow, but reveals

  15. The epigenome in pluripotency and differentiation.

    PubMed

    Thiagarajan, Rathi D; Morey, Robert; Laurent, Louise C

    2014-02-01

    The ability to culture pluripotent stem cells and direct their differentiation into specific cell types in vitro provides a valuable experimental system for modeling pluripotency, development and cellular differentiation. High-throughput profiling of the transcriptomes and epigenomes of pluripotent stem cells and their differentiated derivatives has led to identification of patterns characteristic of each cell type, discovery of new regulatory features in the epigenome and early insights into the complexity of dynamic interactions among regulatory elements. This work has also revealed potential limitations of the use of pluripotent stem cells as in vitro models of developmental events, due to epigenetic variability among different pluripotent stem cell lines and epigenetic instability during derivation and culture, particularly at imprinted and X-inactivated loci. This review focuses on the two most well-studied epigenetic mechanisms, DNA methylation and histone modifications, within the context of pluripotency and differentiation. PMID:24579950

  16. Maintenance of Self-Renewal and Pluripotency in J1 Mouse Embryonic Stem Cells through Regulating Transcription Factor and MicroRNA Expression Induced by PD0325901

    PubMed Central

    Ai, Zhiying; Shao, Jingjing; Shi, Xinglong; Yu, Mengying; Wu, Yongyan; Du, Juan; Zhang, Yong; Guo, Zekun

    2016-01-01

    Embryonic stem cells (ESCs) have the ability to grow indefinitely and retain their pluripotency in culture, and this self-renewal capacity is governed by several crucial molecular pathways controlled by specific regulatory genes and epigenetic modifications. It is reported that multiple epigenetic regulators, such as miRNA and pluripotency factors, can be tightly integrated into molecular pathways and cooperate to maintain self-renewal of ESCs. However, mouse ESCs in serum-containing medium seem to be heterogeneous due to the self-activating differentiation signal of MEK/ERK. Thus, to seek for the crucial miRNA and key regulatory genes that establish ESC properties in MEK/ERK pathway, we performed microarray analysis and small RNA deep-sequencing of J1 mESCs treated with or without PD0325901 (PD), a well-known inhibitor of MEK/ERK signal pathway, followed by verification of western blot analysis and quantitative real-time PCR verification; we found that PD regulated the transcript expressions related to self-renewal and differentiation and antagonized the action of retinoic acid- (RA-) induced differentiation. Moreover, PD can significantly modulate the expressions of multiple miRNAs that have crucial functions in ESC development. Overall, our results demonstrate that PD could enhance ESC self-renewal capacity both by key regulatory genes and ES cell-specific miRNA, which in turn influences ESC self-renewal and cellular differentiation. PMID:26770202

  17. mTOR-regulated senescence and autophagy during reprogramming of somatic cells to pluripotency: a roadmap from energy metabolism to stem cell renewal and aging.

    PubMed

    Menendez, Javier A; Vellon, Luciano; Oliveras-Ferraros, Cristina; Cufí, Sílvia; Vazquez-Martin, Alejandro

    2011-11-01

    mitochondrial function and in the accelerated onset of the glycolytic metabolism that is required to fuel reprogramming. By critically exploring how mTOR-regulated senescence, bioenergetic infrastructure and autophagy can actively drive the reprogramming of somatic cells to pluripotency, we define a metabolic roadmap that may be helpful for designing pharmacological and behavioral interventions to prevent or retard the dysfunction/exhaustion of aging stem cell populations. PMID:22052357

  18. Genome-Wide Studies Reveal that H3K4me3 Modification in Bivalent Genes Is Dynamically Regulated during the Pluripotent Cell Cycle and Stabilized upon Differentiation

    PubMed Central

    Grandy, Rodrigo A.; Whitfield, Troy W.; Wu, Hai; Fitzgerald, Mark P.; VanOudenhove, Jennifer J.; Zaidi, Sayyed K.; Montecino, Martin A.; Lian, Jane B.; van Wijnen, André J.; Stein, Janet L.

    2015-01-01

    Stem cell phenotypes are reflected by posttranslational histone modifications, and this chromatin-related memory must be mitotically inherited to maintain cell identity through proliferative expansion. In human embryonic stem cells (hESCs), bivalent genes with both activating (H3K4me3) and repressive (H3K27me3) histone modifications are essential to sustain pluripotency. Yet, the molecular mechanisms by which this epigenetic landscape is transferred to progeny cells remain to be established. By mapping genomic enrichment of H3K4me3/H3K27me3 in pure populations of hESCs in G2, mitotic, and G1 phases of the cell cycle, we found striking variations in the levels of H3K4me3 through the G2-M-G1 transition. Analysis of a representative set of bivalent genes revealed that chromatin modifiers involved in H3K4 methylation/demethylation are recruited to bivalent gene promoters in a cell cycle-dependent fashion. Interestingly, bivalent genes enriched with H3K4me3 exclusively during mitosis undergo the strongest upregulation after induction of differentiation. Furthermore, the histone modification signature of genes that remain bivalent in differentiated cells resolves into a cell cycle-independent pattern after lineage commitment. These results establish a new dimension of chromatin regulation important in the maintenance of pluripotency. PMID:26644406

  19. Genome-Wide Studies Reveal that H3K4me3 Modification in Bivalent Genes Is Dynamically Regulated during the Pluripotent Cell Cycle and Stabilized upon Differentiation.

    PubMed

    Grandy, Rodrigo A; Whitfield, Troy W; Wu, Hai; Fitzgerald, Mark P; VanOudenhove, Jennifer J; Zaidi, Sayyed K; Montecino, Martin A; Lian, Jane B; van Wijnen, André J; Stein, Janet L; Stein, Gary S

    2016-02-01

    Stem cell phenotypes are reflected by posttranslational histone modifications, and this chromatin-related memory must be mitotically inherited to maintain cell identity through proliferative expansion. In human embryonic stem cells (hESCs), bivalent genes with both activating (H3K4me3) and repressive (H3K27me3) histone modifications are essential to sustain pluripotency. Yet, the molecular mechanisms by which this epigenetic landscape is transferred to progeny cells remain to be established. By mapping genomic enrichment of H3K4me3/H3K27me3 in pure populations of hESCs in G2, mitotic, and G1 phases of the cell cycle, we found striking variations in the levels of H3K4me3 through the G2-M-G1 transition. Analysis of a representative set of bivalent genes revealed that chromatin modifiers involved in H3K4 methylation/demethylation are recruited to bivalent gene promoters in a cell cycle-dependent fashion. Interestingly, bivalent genes enriched with H3K4me3 exclusively during mitosis undergo the strongest upregulation after induction of differentiation. Furthermore, the histone modification signature of genes that remain bivalent in differentiated cells resolves into a cell cycle-independent pattern after lineage commitment. These results establish a new dimension of chromatin regulation important in the maintenance of pluripotency. PMID:26644406

  20. Mediator Subunit Med28 Is Essential for Mouse Peri-Implantation Development and Pluripotency

    PubMed Central

    Li, Lin; Beauchamp, Roberta L.; Chang, Yuh-Shin; Gusella, James F.; Hochedlinger, Konrad; Ramesh, Vijaya

    2015-01-01

    The multi-subunit mammalian Mediator complex acts as an integrator of transcriptional regulation by RNA Polymerase II, and has emerged as a master coordinator of development and cell fate determination. We previously identified the Mediator subunit, MED28, as a cytosolic binding partner of merlin, the Neurofibromatosis 2 (NF2) tumor suppressor, and thus MED28 is distinct in having a cytosolic role as an NF2 interacting protein as well as a nuclear role as a Mediator complex subunit. Although limited in vitro studies have been performed on MED28, its in vivo function remains unknown. Employing a knockout mouse model, we describe for the first time the requirement for Med28 in the developing mouse embryo. Med28-deficiency causes peri-implantation lethality resulting from the loss of pluripotency of the inner cell mass accompanied by reduced expression of key pluripotency transcription factors Oct4 and Nanog. Further, overexpression of Med28 in mouse embryonic fibroblasts enhances the efficiency of their reprogramming to pluripotency. Cre-mediated inactivation of Med28 in induced pluripotent stem cells shows that Med28 is required for their survival. Intriguingly, heterozygous loss of Med28 results in differentiation of induced pluripotent stem cells into extraembryonic trophectoderm and primitive endoderm lineages. Our findings document the essential role of Med28 in the developing embryo as well as in acquisition and maintenance of pluripotency during reprogramming. PMID:26445504

  1. Nanog interact with CDK6 to regulates astrocyte cells proliferation following spinal cord injury.

    PubMed

    Gu, Jun; Ni, Yingjie; Xu, Lin; Xu, Hongliang; Cai, Zhengdong

    2016-01-22

    Previous research had reported transcription factors Nanog expressed in pluripotent embryonic stem cells (ESCS) that played an important role in regulating the cell proliferation. Nanog levels are frequently elevated in ESCS, but the role in the spinal cord was not clear. To examine the biological relevance of Nanog, we studied its properties in spinal cord injury model. The expression of Nanog and PCNA was gradually increased and reached a peak at 3 day by western blot analysis. The expression of Nanog was further analyzed by immunohistochemistry. Double immunofluorescent staining uncovered that Nanog can co-labeled with PCNA and GFAP in the spinal cord tissue. In vitro, Nanog can promote the proliferation of astrocyte cell by Fluorescence Activating Cell Sorter (FACS) and CCK8. Meanwhile, the cell-cycle protein CDK6 could interact with Nanog in the spinal cord tissue. Taken together, these data suggested that both Nanog may play important roles in spinal cord pathophysiology via interact with CDK6.

  2. Regulation of pluripotency of inner cell mass and growth and differentiation of trophectoderm of the bovine embryo by colony stimulating factor 2.

    PubMed

    Dobbs, Kyle B; Khan, Firdous A; Sakatani, Miki; Moss, James I; Ozawa, Manabu; Ealy, Alan D; Hansen, Peter J

    2013-12-01

    Colony-stimulating factor 2 (CSF2) enhances competence of the bovine embryo to establish and maintain pregnancy after the embryo is transferred into a recipient. Mechanisms involved could include regulation of lineage commitment, growth, or differentiation of the inner cell mass (ICM) and trophectoderm (TE). Experiments were conducted to evaluate regulation by CSF2 of pluripotency of the ICM and differentiation and growth of the TE. Embryos were cultured with 10 ng/ml recombinant bovine CSF2 or a vehicle control from Days 5 to 7 or 6 to 8 postinsemination. CSF2 increased the number of putative zygotes that developed to blastocysts when the percent of embryos becoming blastocysts in the control group was low but decreased blastocyst yield when blastocyst development in controls was high. ICM isolated from blastocysts by lysing the trophectoderm using antibody and complement via immunosurgery were more likely to survive passage when cultured on mitomycin C-treated fetal fibroblasts if derived from blastocysts treated with CSF2 than if from control blastocysts. There was little effect of CSF2 on characteristics of TE outgrowths from blastocysts. The exception was a decrease in outgrowth size for embryos treated with CSF2 from Days 5 to 7 and an increase in expression of CDX2 when treatment was from Days 6 to 8. Expression of the receptor subunit gene CSF2RA increased from the zygote stage to the 9-16 cell stage before decreasing to the blastocyst stage. In contrast, CSF2RB was undetectable at all stages. In conclusion, CSF2 improves competence of the ICM to survive in a pluripotent state and alters TE outgrowths. Actions of CSF2 occur through a signaling pathway that is likely to be independent of CSF2RB.

  3. LncRNA Dum interacts with Dnmts to regulate Dppa2 expression during myogenic differentiation and muscle regeneration.

    PubMed

    Wang, Lijun; Zhao, Yu; Bao, Xichen; Zhu, Xihua; Kwok, Yvonne Ka-Yin; Sun, Kun; Chen, Xiaona; Huang, Yongheng; Jauch, Ralf; Esteban, Miguel A; Sun, Hao; Wang, Huating

    2015-03-01

    Emerging studies document the roles of long non-coding RNAs (LncRNAs) in regulating gene expression at chromatin level but relatively less is known how they regulate DNA methylation. Here we identify an lncRNA, Dum (developmental pluripotency-associated 2 (Dppa2) Upstream binding Muscle lncRNA) in skeletal myoblast cells. The expression of Dum is dynamically regulated during myogenesis in vitro and in vivo. It is also transcriptionally induced by MyoD binding upon myoblast differentiation. Functional analyses show that it promotes myoblast differentiation and damage-induced muscle regeneration. Mechanistically, Dum was found to silence its neighboring gene, Dppa2, in cis through recruiting Dnmt1, Dnmt3a and Dnmt3b. Furthermore, intrachromosomal looping between Dum locus and Dppa2 promoter is necessary for Dum/Dppa2 interaction. Collectively, we have identified a novel lncRNA that interacts with Dnmts to regulate myogenesis.

  4. LncRNA Dum interacts with Dnmts to regulate Dppa2 expression during myogenic differentiation and muscle regeneration

    PubMed Central

    Wang, Lijun; Zhao, Yu; Bao, Xichen; Zhu, Xihua; Kwok, Yvonne Ka-yin; Sun, Kun; Chen, Xiaona; Huang, Yongheng; Jauch, Ralf; Esteban, Miguel A; Sun, Hao; Wang, Huating

    2015-01-01

    Emerging studies document the roles of long non-coding RNAs (LncRNAs) in regulating gene expression at chromatin level but relatively less is known how they regulate DNA methylation. Here we identify an lncRNA, Dum (developmental pluripotency-associated 2 (Dppa2) Upstream binding Muscle lncRNA) in skeletal myoblast cells. The expression of Dum is dynamically regulated during myogenesis in vitro and in vivo. It is also transcriptionally induced by MyoD binding upon myoblast differentiation. Functional analyses show that it promotes myoblast differentiation and damage-induced muscle regeneration. Mechanistically, Dum was found to silence its neighboring gene, Dppa2, in cis through recruiting Dnmt1, Dnmt3a and Dnmt3b. Furthermore, intrachromosomal looping between Dum locus and Dppa2 promoter is necessary for Dum/Dppa2 interaction. Collectively, we have identified a novel lncRNA that interacts with Dnmts to regulate myogenesis. PMID:25686699

  5. LncRNA Dum interacts with Dnmts to regulate Dppa2 expression during myogenic differentiation and muscle regeneration.

    PubMed

    Wang, Lijun; Zhao, Yu; Bao, Xichen; Zhu, Xihua; Kwok, Yvonne Ka-Yin; Sun, Kun; Chen, Xiaona; Huang, Yongheng; Jauch, Ralf; Esteban, Miguel A; Sun, Hao; Wang, Huating

    2015-03-01

    Emerging studies document the roles of long non-coding RNAs (LncRNAs) in regulating gene expression at chromatin level but relatively less is known how they regulate DNA methylation. Here we identify an lncRNA, Dum (developmental pluripotency-associated 2 (Dppa2) Upstream binding Muscle lncRNA) in skeletal myoblast cells. The expression of Dum is dynamically regulated during myogenesis in vitro and in vivo. It is also transcriptionally induced by MyoD binding upon myoblast differentiation. Functional analyses show that it promotes myoblast differentiation and damage-induced muscle regeneration. Mechanistically, Dum was found to silence its neighboring gene, Dppa2, in cis through recruiting Dnmt1, Dnmt3a and Dnmt3b. Furthermore, intrachromosomal looping between Dum locus and Dppa2 promoter is necessary for Dum/Dppa2 interaction. Collectively, we have identified a novel lncRNA that interacts with Dnmts to regulate myogenesis. PMID:25686699

  6. Tissue Interactions Regulating Tooth Development and Renewal.

    PubMed

    Balic, Anamaria; Thesleff, Irma

    2015-01-01

    Reciprocal interactions between epithelial and mesenchymal tissues play a fundamental role in the morphogenesis of teeth and regulate all aspects of tooth development. Extensive studies on mouse tooth development over the past 25 years have uncovered the molecular details of the signaling networks mediating these interactions (reviewed by Jussila & Thesleff, 2012; Lan, Jia, & Jiang, 2014). Five conserved signaling pathways, namely, the Wnt, BMP, FGF, Shh, and Eda, are involved in the mediation of the successive reciprocal epithelial-mesenchymal cross talk which follows the general principle of morphogenetic interactions (Davidson, 1993). The pathways regulate the expression of transcription factors which confer the identity of dental epithelium and mesenchyme. The signals and transcription factors are integrated in complex signaling networks whose fine-tuning allows the generation of the variation in tooth morphologies. In this review, we describe the principles and molecular mechanisms of the epithelial-mesenchymal interactions regulating successive stages of tooth formation: (i) the initiation of tooth development, with special reference to the shift of tooth-forming potential from epithelium to mesenchyme; (ii) the morphogenesis of the tooth crown, focusing on the roles of epithelial signaling centers; (iii) the differentiation of odontoblasts and ameloblasts, which produce dentin and enamel, respectively; and (iv) the maintenance of dental stem cells, which support the continuous growth of teeth. PMID:26589925

  7. Tissue Interactions Regulating Tooth Development and Renewal.

    PubMed

    Balic, Anamaria; Thesleff, Irma

    2015-01-01

    Reciprocal interactions between epithelial and mesenchymal tissues play a fundamental role in the morphogenesis of teeth and regulate all aspects of tooth development. Extensive studies on mouse tooth development over the past 25 years have uncovered the molecular details of the signaling networks mediating these interactions (reviewed by Jussila & Thesleff, 2012; Lan, Jia, & Jiang, 2014). Five conserved signaling pathways, namely, the Wnt, BMP, FGF, Shh, and Eda, are involved in the mediation of the successive reciprocal epithelial-mesenchymal cross talk which follows the general principle of morphogenetic interactions (Davidson, 1993). The pathways regulate the expression of transcription factors which confer the identity of dental epithelium and mesenchyme. The signals and transcription factors are integrated in complex signaling networks whose fine-tuning allows the generation of the variation in tooth morphologies. In this review, we describe the principles and molecular mechanisms of the epithelial-mesenchymal interactions regulating successive stages of tooth formation: (i) the initiation of tooth development, with special reference to the shift of tooth-forming potential from epithelium to mesenchyme; (ii) the morphogenesis of the tooth crown, focusing on the roles of epithelial signaling centers; (iii) the differentiation of odontoblasts and ameloblasts, which produce dentin and enamel, respectively; and (iv) the maintenance of dental stem cells, which support the continuous growth of teeth.

  8. When X-inactivation meets pluripotency: an intimate rendezvous.

    PubMed

    Navarro, Pablo; Avner, Philip

    2009-06-01

    The integration of X-inactivation with development is a crucial aspect of this classical paradigm of epigenetic regulation. During early female mouse development, X-inactivation reprogramming occurs in pluripotent cells of the inner cell mass of the blastocyst and in pluripotent primordial germ cells. Here we discuss the developmental strategies which ensure the coupling of the regulation of X-inactivation to the acquisition of pluripotency through the regulation of the master of X-inactivation, the non-coding Xist gene, by the key factors which support pluripotency Nanog, Oct4 and Sox2.

  9. Path from schizophrenia genomics to biology: gene regulation and perturbation in neurons derived from induced pluripotent stem cells and genome editing.

    PubMed

    Duan, Jubao

    2015-02-01

    Schizophrenia (SZ) is a devastating mental disorder afflicting 1% of the population. Recent genome-wide association studies (GWASs) of SZ have identified >100 risk loci. However, the causal variants/genes and the causal mechanisms remain largely unknown, which hinders the translation of GWAS findings into disease biology and drug targets. Most risk variants are noncoding, thus likely regulate gene expression. A major mechanism of transcriptional regulation is chromatin remodeling, and open chromatin is a versatile predictor of regulatory sequences. MicroRNA-mediated post-transcriptional regulation plays an important role in SZ pathogenesis. Neurons differentiated from patient-specific induced pluripotent stem cells (iPSCs) provide an experimental model to characterize the genetic perturbation of regulatory variants that are often specific to cell type and/or developmental stage. The emerging genome-editing technology enables the creation of isogenic iPSCs and neurons to efficiently characterize the effects of SZ-associated regulatory variants on SZ-relevant molecular and cellular phenotypes involving dopaminergic, glutamatergic, and GABAergic neurotransmissions. SZ GWAS findings equipped with the emerging functional genomics approaches provide an unprecedented opportunity for understanding new disease biology and identifying novel drug targets.

  10. Pluripotent states of human embryonic stem cells.

    PubMed

    Chen, Yifei; Lai, Dongmei

    2015-02-01

    Since human embryonic stem cells (hESCs) were first isolated and successfully cultured in vitro, the pluripotent potential of hESCs has been underestimated. The pluripotency of mouse embryonic stem cells (mESCs) can be categorized as naïve and primed, depending on their corresponding in vivo developing phases. mESC morphology differs at distinct pluripotent states, which differ in signaling dependence, gene expression, epigenetic features, and developmental potential. hESCs resemble mouse stem cells at primed pluripotency, and consequently are believed to correspond to a later developmental stage in vivo than mESCs. Nevertheless, recent studies indicate that a naïve state of pluripotency may exist in hESCs, and the pluripotency of hESCs also can be enhanced by genetic modification or optimized culture systems. These findings provide novel insight into the properties and differentiation potential of hESCs. Here, we review the recent advances in characterization of ESC states and investigate the mechanisms regulating hESC pluripotency. PMID:25393391

  11. Pluripotency-associated miR-290/302 family of microRNAs promote the dismantling of naive pluripotency

    PubMed Central

    Gu, Kai-Li; Zhang, Qiang; Yan, Ying; Li, Ting-Ting; Duan, Fei-Fei; Hao, Jing; Wang, Xi-Wen; Shi, Ming; Wu, Da-Ren; Guo, Wen-Ting; Wang, Yangming

    2016-01-01

    The molecular mechanism controlling the dismantling of naive pluripotency is poorly understood. Here we show that microRNAs (miRNAs) have important roles during naive to primed pluripotency transition. Dgcr8−/− embryonic stem cells (ESCs) failed to completely silence the naive pluripotency program, as well as to establish the primed pluripotency program during differentiation. miRNA profiling revealed that expression levels of a large number of miRNAs changed dynamically and rapidly during naive to primed pluripotency transition. Furthermore, a miRNA screen identified numerous miRNAs promoting naive to primed pluripotency transition. Unexpectedly, multiple miRNAs from miR-290 and miR-302 clusters, previously shown as pluripotency-promoting miRNAs, demonstrated the strongest effects in silencing naive pluripotency. Knockout of both miR-290 and miR-302 clusters but not either alone blocked the silencing of naive pluripotency program. Mechanistically, the miR-290/302 family of miRNAs may facilitate the exit of naive pluripotency in part by promoting the activity of MEK pathway and through directly repressing Akt1. Our study reveals miRNAs as an important class of regulators potentiating ESCs to transition from naive to primed pluripotency, and uncovers context-dependent functions of the miR-290/302 family of miRNAs at different developmental stages. PMID:26742694

  12. Growth factor transgenes interactively regulate articular chondrocytes.

    PubMed

    Shi, Shuiliang; Mercer, Scott; Eckert, George J; Trippel, Stephen B

    2013-04-01

    Adult articular chondrocytes lack an effective repair response to correct damage from injury or osteoarthritis. Polypeptide growth factors that stimulate articular chondrocyte proliferation and cartilage matrix synthesis may augment this response. Gene transfer is a promising approach to delivering such factors. Multiple growth factor genes regulate these cell functions, but multiple growth factor gene transfer remains unexplored. We tested the hypothesis that multiple growth factor gene transfer selectively modulates articular chondrocyte proliferation and matrix synthesis. We tested the hypothesis by delivering combinations of the transgenes encoding insulin-like growth factor I (IGF-I), fibroblast growth factor-2 (FGF-2), transforming growth factor beta1 (TGF-β1), bone morphogenetic protein-2 (BMP-2), and bone morphogenetic protien-7 (BMP-7) to articular chondrocytes and measured changes in the production of DNA, glycosaminoglycan, and collagen. The transgenes differentially regulated all these chondrocyte activities. In concert, the transgenes interacted to generate widely divergent responses from the cells. These interactions ranged from inhibitory to synergistic. The transgene pair encoding IGF-I and FGF-2 maximized cell proliferation. The three-transgene group encoding IGF-I, BMP-2, and BMP-7 maximized matrix production and also optimized the balance between cell proliferation and matrix production. These data demonstrate an approach to articular chondrocyte regulation that may be tailored to stimulate specific cell functions, and suggest that certain growth factor gene combinations have potential value for cell-based articular cartilage repair.

  13. Long non-coding RNAs in pluripotent stem cell biology.

    PubMed

    Lammens, Tim; D'hont, Inge; D'Herde, Katharina; Benoit, Yves; Diez-Fraile, Araceli

    2013-12-01

    Pluripotent stem cells are defined by their unlimited self-renewal capacities and potential to differentiate into any cell lineage. Many crucial determinants for the induction and maintenance of this pluripotent state have been identified. Long non-coding RNAs have recently emerged as key regulators of pluripotent stem cells and have enhanced our understanding of their potential functions in tissue regeneration. This review provides an overview of recent important insights into the roles of long non-coding RNAs as regulators and markers of pluripotency.

  14. Fate by RNA methylation: m6A steers stem cell pluripotency.

    PubMed

    Zhao, Boxuan Simen; He, Chuan

    2015-01-01

    The N 6-methyladenosine (m6A) modification of mRNA has a crucial function in regulating pluripotency in murine stem cells: it facilitates resolution of naïve pluripotency towards differentiation. PMID:25723450

  15. Fyn Kinase regulates GluN2B subunit-dominant NMDA receptors in human induced pluripotent stem cell-derived neurons

    PubMed Central

    Zhang, Wen-Bo; Ross, P. Joel; Tu, YuShan; Wang, Yongqian; Beggs, Simon; Sengar, Ameet S.; Ellis, James; Salter, Michael W.

    2016-01-01

    NMDA receptor (NMDAR)-mediated fast excitatory neurotransmission is implicated in a broad range of physiological and pathological processes in the mammalian central nervous system. The function and regulation of NMDARs have been extensively studied in neurons from rodents and other non-human species, and in recombinant expression systems. Here, we investigated human NMDARs in situ by using neurons produced by directed differentiation of human induced pluripotent stem cells (iPSCs). The resultant cells showed electrophysiological characteristics demonstrating that they are bona fide neurons. In particular, human iPSC-derived neurons expressed functional ligand-gated ion channels, including NMDARs, AMPA receptors, GABAA receptors, as well as glycine receptors. Pharmacological and electrophysiological properties of NMDAR-mediated currents indicated that these were dominated by receptors containing GluN2B subunits. The NMDAR currents were suppressed by genistein, a broad-spectrum tyrosine kinase inhibitor. The NMDAR currents were also inhibited by a Fyn-interfering peptide, Fyn(39–57), but not a Src-interfering peptide, Src(40–58). Together, these findings are the first evidence that tyrosine phosphorylation regulates the function of NMDARs in human iPSC-derived neurons. Our findings provide a basis for utilizing human iPSC-derived neurons in screening for drugs targeting NMDARs in neurological disorders. PMID:27040756

  16. Fyn Kinase regulates GluN2B subunit-dominant NMDA receptors in human induced pluripotent stem cell-derived neurons.

    PubMed

    Zhang, Wen-Bo; Ross, P Joel; Tu, YuShan; Wang, Yongqian; Beggs, Simon; Sengar, Ameet S; Ellis, James; Salter, Michael W

    2016-04-04

    NMDA receptor (NMDAR)-mediated fast excitatory neurotransmission is implicated in a broad range of physiological and pathological processes in the mammalian central nervous system. The function and regulation of NMDARs have been extensively studied in neurons from rodents and other non-human species, and in recombinant expression systems. Here, we investigated human NMDARs in situ by using neurons produced by directed differentiation of human induced pluripotent stem cells (iPSCs). The resultant cells showed electrophysiological characteristics demonstrating that they are bona fide neurons. In particular, human iPSC-derived neurons expressed functional ligand-gated ion channels, including NMDARs, AMPA receptors, GABAA receptors, as well as glycine receptors. Pharmacological and electrophysiological properties of NMDAR-mediated currents indicated that these were dominated by receptors containing GluN2B subunits. The NMDAR currents were suppressed by genistein, a broad-spectrum tyrosine kinase inhibitor. The NMDAR currents were also inhibited by a Fyn-interfering peptide, Fyn(39-57), but not a Src-interfering peptide, Src(40-58). Together, these findings are the first evidence that tyrosine phosphorylation regulates the function of NMDARs in human iPSC-derived neurons. Our findings provide a basis for utilizing human iPSC-derived neurons in screening for drugs targeting NMDARs in neurological disorders.

  17. Snapshots of Pluripotency

    PubMed Central

    Tesar, Paul J.

    2016-01-01

    Summary Pluripotency is a unique developmental state that lays the foundation upon which the entire embryo is built. Pluripotent cells possess the unique capacity to generate, in an exquisitely defined sequence, all the distinct cell types comprising the fetal and adult organism. The discovery of pluripotent stem cells and now the ability to generate them from differentiated cells has had a profound impact on a vast array of scientific disciplines. In addition to their clinical potential as a source of therapeutic cell types, pluripotent stem cells provide scalable access to otherwise experimentally inaccessible development- and disease-associated biology. Here I provide my perspective on the continuum of pluripotency in the early mammalian embryo. I also discuss how novel genomic technologies are now enabling the capture of molecular “snapshots” of the several distinct pluripotent states that stem cells undergo during this pivotal developmental period. PMID:26833092

  18. Regulation of amino acid transporters in pluripotent cell populations in the embryo and in culture; novel roles for sodium-coupled neutral amino acid transporters.

    PubMed

    Tan, Boon Siang Nicholas; Rathjen, Peter D; Harvey, Alexandra J; Gardner, David K; Rathjen, Joy

    2016-08-01

    The developmental outcomes of preimplantation mammalian embryos are regulated directly by the surrounding microenvironment, and inappropriate concentrations of amino acids, or the loss of amino acid-sensing mechanisms, can be detrimental and impact further development. A specific role for l-proline in the differentiation of embryonic stem (ES) cells, a cell population derived from the blastocyst, has been shown in culture. l-proline acts as a signalling molecule, exerting its effects through cell uptake and subsequent metabolism. Uptake in ES cells occurs predominantly through the sodium-coupled neutral amino acid transporter 2, Slc38a2 (SNAT2). Dynamic expression of amino acid transporters has been shown in the early mammalian embryo, reflecting functional roles for amino acids in embryogenesis. The expression of SNAT2 and family member Slc38a1 (SNAT1) was determined in mouse embryos from the 2-cell stage through to the early post-implantation pre-gastrulation embryo. Key changes in expression were validated in cell culture models of development. Both transporters showed temporal dynamic expression patterns and changes in intracellular localisation as differentiation progressed. Changes in transporter expression likely reflect different amino acid requirements during development. Findings include the differential expression of SNAT1 in the inner and outer cells of the compacted morula and nuclear localisation of SNAT2 in the trophectoderm and placental lineages. Furthermore, SNAT2 expression was up-regulated in the epiblast prior to primitive ectoderm formation, an expression pattern consistent with a role for the transporter in later developmental decisions within the pluripotent lineage. We propose that the differential expression of SNAT2 in the epiblast provides evidence for an l-proline-mediated mechanism contributing to the regulation of embryonic development. PMID:27373508

  19. Endothelial lineage differentiation from induced pluripotent stem cells is regulated by microRNA-21 and transforming growth factor β2 (TGF-β2) pathways.

    PubMed

    Di Bernardini, Elisabetta; Campagnolo, Paola; Margariti, Andriana; Zampetaki, Anna; Karamariti, Eirini; Hu, Yanhua; Xu, Qingbo

    2014-02-01

    Finding a suitable cell source for endothelial cells (ECs) for cardiovascular regeneration is a challenging issue for regenerative medicine. In this paper, we describe a novel mechanism regulating induced pluripotent stem cells (iPSC) differentiation into ECs, with a particular focus on miRNAs and their targets. We first established a protocol using collagen IV and VEGF to drive the functional differentiation of iPSCs into ECs and compared the miRNA signature of differentiated and undifferentiated cells. Among the miRNAs overrepresented in differentiated cells, we focused on microRNA-21 (miR-21) and studied its role in iPSC differentiation. Overexpression of miR-21 in predifferentiated iPSCs induced EC marker up-regulation and in vitro and in vivo capillary formation; accordingly, inhibition of miR-21 produced the opposite effects. Importantly, miR-21 overexpression increased TGF-β2 mRNA and secreted protein level, consistent with the strong up-regulation of TGF-β2 during iPSC differentiation. Indeed, treatment of iPSCs with TGFβ-2 induced EC marker expression and in vitro tube formation. Inhibition of SMAD3, a downstream effector of TGFβ-2, strongly decreased VE-cadherin expression. Furthermore, TGFβ-2 neutralization and knockdown inhibited miR-21-induced EC marker expression. Finally, we confirmed the PTEN/Akt pathway as a direct target of miR-21, and we showed that PTEN knockdown is required for miR-21-mediated endothelial differentiation. In conclusion, we elucidated a novel signaling pathway that promotes the differentiation of iPSC into functional ECs suitable for regenerative medicine applications.

  20. Systems biology discoveries using non-human primate pluripotent stem and germ cells: novel gene and genomic imprinting interactions as well as unique expression patterns.

    PubMed

    Ben-Yehudah, Ahmi; Easley, Charles A; Hermann, Brian P; Castro, Carlos; Simerly, Calvin; Orwig, Kyle E; Mitalipov, Shoukhrat; Schatten, Gerald

    2010-08-05

    The study of pluripotent stem cells has generated much interest in both biology and medicine. Understanding the fundamentals of biological decisions, including what permits a cell to maintain pluripotency, that is, its ability to self-renew and thereby remain immortal, or to differentiate into multiple types of cells, is of profound importance. For clinical applications, pluripotent cells, including both embryonic stem cells and adult stem cells, have been proposed for cell replacement therapy for a number of human diseases and disorders, including Alzheimer's, Parkinson's, spinal cord injury and diabetes. One challenge in their usage for such therapies is understanding the mechanisms that allow the maintenance of pluripotency and controlling the specific differentiation into required functional target cells. Because of regulatory restrictions and biological feasibilities, there are many crucial investigations that are just impossible to perform using pluripotent stem cells (PSCs) from humans (for example, direct comparisons among panels of inbred embryonic stem cells from prime embryos obtained from pedigreed and fertile donors; genomic analysis of parent versus progeny PSCs and their identical differentiated tissues; intraspecific chimera analyses for pluripotency testing; and so on). However, PSCs from nonhuman primates are being investigated to bridge these knowledge gaps between discoveries in mice and vital information necessary for appropriate clinical evaluations. In this review, we consider the mRNAs and novel genes with unique expression and imprinting patterns that were discovered using systems biology approaches with primate pluripotent stem and germ cells.

  1. Switching between self-renewal and lineage commitment of human induced pluripotent stem cells via cell-substrate and cell-cell interactions on a dendrimer-immobilized surface.

    PubMed

    Kim, Mee-Hae; Kino-oka, Masahiro

    2014-07-01

    Understanding mechanisms that govern cell fate determination of human induced pluripotent stem cells (hiPSCs) could assist in maintenance of the undifferentiated state during cell expansion. We used polyamidoamine dendrimer surfaces with first-generation (G1), third-generation (G3) and fifth-generation (G5) of dendron structure in cultures of hiPSCs with SNL feeder cells. Cells on the G1 surface formed tightly packed colony with close cell-cell contacts during division and migration; those on the G3 surface exhibited loose or dispersed colony pattern by enhanced migration. On the G5 surface, formation of aggregated colony with ring-like structures occurred spontaneously. We found that the substrate-adsorbed fibronectin and feeder cell-secreted fibronectin appeared elevated levels with the varied generation numbers of dendrimer surfaces. This subsequently resulted in cell migration and in activation of paxillin of hiPSCs. Location-dependent expression of Rac1 induced rearrangement of E-cadherin-mediated cell-cell interactions on dendrimer surfaces, and was associated with alterations in the cell and colony morphology, and migratory behavior. Furthermore, caspase-3 occurred in apoptotic cells on dendrimer surfaces, concomitant with the loss of E-cadherin-mediated cell-cell interactions. Cells on the G1 surface were maintained in an undifferentiated state, while those on the G5 surface exhibited the early commitment to differentiation toward endodermal fates. We conclude that morphological changes associated with altered migration on the dendrimer surfaces were responsible for the coordinated regulation of balance between cell-cell and cell-substrate interactions, thereby switching their transition from self-renewal state to early endoderm differentiation in hiPSCs. PMID:24746960

  2. Phytohormone regulation of legume-rhizobia interactions.

    PubMed

    Ferguson, Brett J; Mathesius, Ulrike

    2014-07-01

    The symbiosis between legumes and nitrogen fixing bacteria called rhizobia leads to the formation of root nodules. Nodules are highly organized root organs that form in response to Nod factors produced by rhizobia, and they provide rhizobia with a specialized niche to optimize nutrient exchange and nitrogen fixation. Nodule development and invasion by rhizobia is locally controlled by feedback between rhizobia and the plant host. In addition, the total number of nodules on a root system is controlled by a systemic mechanism termed 'autoregulation of nodulation'. Both the local and the systemic control of nodulation are regulated by phytohormones. There are two mechanisms by which phytohormone signalling is altered during nodulation: through direct synthesis by rhizobia and through indirect manipulation of the phytohormone balance in the plant, triggered by bacterial Nod factors. Recent genetic and physiological evidence points to a crucial role of Nod factor-induced changes in the host phytohormone balance as a prerequisite for successful nodule formation. Phytohormones synthesized by rhizobia enhance symbiosis effectiveness but do not appear to be necessary for nodule formation. This review provides an overview of recent advances in our understanding of the roles and interactions of phytohormones and signalling peptides in the regulation of nodule infection, initiation, positioning, development, and autoregulation. Future challenges remain to unify hormone-related findings across different legumes and to test whether hormone perception, response, or transport differences among different legumes could explain the variety of nodules types and the predisposition for nodule formation in this plant family. In addition, the molecular studies carried out under controlled conditions will need to be extended into the field to test whether and how phytohormone contributions by host and rhizobial partners affect the long term fitness of the host and the survival and

  3. Interactions between JARID2 and noncoding RNAs regulate PRC2 recruitment to chromatin.

    PubMed

    Kaneko, Syuzo; Bonasio, Roberto; Saldaña-Meyer, Ricardo; Yoshida, Takahaki; Son, Jinsook; Nishino, Koichiro; Umezawa, Akihiro; Reinberg, Danny

    2014-01-23

    JARID2 is an accessory component of Polycomb repressive complex-2 (PRC2) required for the differentiation of embryonic stem cells (ESCs). A role for JARID2 in the recruitment of PRC2 to target genes silenced during differentiation has been put forward, but the molecular details remain unclear. We identified a 30-amino-acid region of JARID2 that mediates interactions with long noncoding RNAs (lncRNAs) and found that the presence of lncRNAs stimulated JARID2-EZH2 interactions in vitro and JARID2-mediated recruitment of PRC2 to chromatin in vivo. Native and crosslinked RNA immunoprecipitations of JARID2 revealed that Meg3 and other lncRNAs from the imprinted Dlk1-Dio3 locus, an important regulator of development, interacted with PRC2 via JARID2. Lack of MEG3 expression in human induced pluripotent cells altered the chromatin distribution of JARID2, PRC2, and H3K27me3. Our findings show that lncRNAs facilitate JARID2-PRC2 interactions on chromatin and suggest a mechanism by which lncRNAs contribute to PRC2 recruitment.

  4. Human induced pluripotent stem cell-derived hepatic cell lines as a new model for host interaction with hepatitis B virus

    PubMed Central

    Kaneko, Shun; Kakinuma, Sei; Asahina, Yasuhiro; Kamiya, Akihide; Miyoshi, Masato; Tsunoda, Tomoyuki; Nitta, Sayuri; Asano, Yu; Nagata, Hiroko; Otani, Satoshi; Kawai-Kitahata, Fukiko; Murakawa, Miyako; Itsui, Yasuhiro; Nakagawa, Mina; Azuma, Seishin; Nakauchi, Hiromitsu; Nishitsuji, Hironori; Ujino, Saneyuki; Shimotohno, Kunitada; Iwamoto, Masashi; Watashi, Koichi; Wakita, Takaji; Watanabe, Mamoru

    2016-01-01

    Hepatitis B virus (HBV) is not eradicated by current antiviral therapies due to persistence of HBV covalently closed circular DNA (cccDNA) in host cells, and thus development of novel culture models for productive HBV infection is urgently needed, which will allow the study of HBV cccDNA eradication. To meet this need, we developed culture models of HBV infection using human induced pluripotent stem cell-derived hepatocyte lineages, including immature proliferating hepatic progenitor-like cell lines (iPS-HPCs) and differentiated hepatocyte-like cells (iPS-Heps). These cells were susceptible to HBV infection, produced HBV particles, and maintained innate immune responses. The infection efficiency of HBV in iPS-HPCs predominantly depended on the expression levels of sodium taurocholate cotransporting polypeptide (NTCP), and was low relative to iPS-Heps: however, long-term culture of iPS-Heps was difficult. To provide a model for HBV persistence, iPS-HPCs overexpressing NTCP were established. The long-term persistence of HBV cccDNA was detected in iPS-HPCs overexpressing NTCP, and depended on the inhibition of the Janus-kinase signaling pathway. In conclusion, this study provides evidence that iPS-derived hepatic cell lines can be utilized for novel HBV culture models with genetic variation to investigate the interactions between HBV and host cells and the development of anti-HBV strategies. PMID:27386799

  5. Non-coding RNAs in pluripotency and neural differentiation of human pluripotent stem cells.

    PubMed

    Lukovic, Dunja; Moreno-Manzano, Victoria; Klabusay, Martin; Stojkovic, Miodrag; Bhattacharya, Shomi S; Erceg, Slaven

    2014-01-01

    Several studies have demonstrated the important role of non-coding RNAs as regulators of posttranscriptional processes, including stem cells self-renewal and neural differentiation. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (ihPSCs) show enormous potential in regenerative medicine due to their capacity to differentiate to virtually any type of cells of human body. Deciphering the role of non-coding RNAs in pluripotency, self-renewal and neural differentiation will reveal new molecular mechanisms involved in induction and maintenances of pluripotent state as well as triggering these cells toward clinically relevant cells for transplantation. In this brief review we will summarize recently published studies which reveal the role of non-coding RNAs in pluripotency and neural differentiation of hESCs and ihPSC.

  6. Molecular manipulation targeting regulation of dopaminergic differentiation and proliferation of neural stem cells or pluripotent stem cells.

    PubMed

    Ding, Yin-Xiu; Wei, Li-Chun; Wang, Ya-Zhou; Cao, Rong; Wang, Xi; Chen, Liang-Wei

    2011-06-01

    Parkinson's disease (PD) is a severe deliberating neurological disease caused by progressive degenerative death of dopaminergic neurons in the substantia nigra of midbrain. While cell replacement strategy by transplantation of neural stem cells and inducement of dopaminergic neurons is recommended for the treatment of PD, understanding the differentiation mechanism and controlled proliferation of grafted stem cells remain major concerns in their clinical application. Here we review recent studies on molecular signaling pathways in regulation of dopaminergic differentiation and proliferation of stem cells, particularly Wnt/beta-catenin signaling in stimulating formation of the dopaminergic phenotype, Notch signaling in inhibiting stem cell differentiation, and Sonic hedgehog functioning in neural stem cell proliferation and neuronal cell production. Activation of oncogenes involved in uncontrolled proliferation or tumorigenicity of stem cells is also discussed. It is proposed that a selective molecular manipulation targeting strategy will greatly benefit cell replacement therapy for PD by effectively promoting dopaminergic neuronal cell generation and reducing risk of tumorigenicity of in vivo stem cell applications.

  7. The Anti-inflammatory Protein TSG-6 Regulates Chemokine Function by Inhibiting Chemokine/Glycosaminoglycan Interactions*

    PubMed Central

    Dyer, Douglas P.; Salanga, Catherina L.; Johns, Scott C.; Valdambrini, Elena; Fuster, Mark M.; Milner, Caroline M.; Day, Anthony J.; Handel, Tracy M.

    2016-01-01

    TNF-stimulated gene-6 (TSG-6) is a multifunctional protein secreted in response to pro-inflammatory stimuli by a wide range of cells, including neutrophils, monocytes, and endothelial cells. It has been shown to mediate anti-inflammatory and protective effects when administered in disease models, in part, by reducing neutrophil infiltration. Human TSG-6 inhibits neutrophil migration by binding CXCL8 through its Link module (Link_TSG6) and interfering with the presentation of CXCL8 on cell-surface glycosaminoglycans (GAGs), an interaction that is vital for the function of many chemokines. TSG-6 was also found to interact with chemokines CXCL11 and CCL5, suggesting the possibility that it may function as a broad specificity chemokine-binding protein, functionally similar to those encoded by viruses. This study was therefore undertaken to explore the ability of TSG-6 to regulate the function of other chemokines. Herein, we demonstrate that Link_TSG6 binds chemokines from both the CXC and CC families, including CXCL4, CXCL12, CCL2, CCL5, CCL7, CCL19, CCL21, and CCL27. We also show that the Link_TSG6-binding sites on chemokines overlap with chemokine GAG-binding sites, and that the affinities of Link_TSG6 for these chemokines (KD values 1–85 nm) broadly correlate with chemokine-GAG affinities. Link_TSG6 also inhibits chemokine presentation on endothelial cells not only through a direct interaction with chemokines but also by binding and therefore masking the availability of GAGs. Along with previous work, these findings suggest that TSG-6 functions as a pluripotent regulator of chemokines by modulating chemokine/GAG interactions, which may be a major mechanism by which TSG-6 produces its anti-inflammatory effects in vivo. PMID:27044744

  8. Steps to Pluripotent Learning: Provocative Teaching

    ERIC Educational Resources Information Center

    Rangachari, P. K.

    2011-01-01

    Education involves interactions between students and teachers in a societal framework. Teachers can best serve their students and society by making students flexible enough to thrive under uncertain conditions. They should, in a sense, nourish, nurture, provoke, and stimulate pluripotent "educatoblasts."

  9. Neural circuits: Interacting interneurons regulate fear learning.

    PubMed

    Ozawa, Takaaki; Johansen, Joshua P

    2014-08-01

    A recent study has found that, during associative fear learning, different sensory stimuli activate subsets of inhibitory interneurons in distinct ways to dynamically regulate glutamatergic neural activity and behavioral memory formation. PMID:25093560

  10. Jun‐Mediated Changes in Cell Adhesion Contribute to Mouse Embryonic Stem Cell Exit from Ground State Pluripotency

    PubMed Central

    Veluscek, Giulia; Li, Yaoyong; Yang, Shen‐Hsi

    2016-01-01

    Abstract Embryonic stem cells (ESC) are able to give rise to any somatic cell type. A lot is known about how ESC pluripotency is maintained, but comparatively less is known about how differentiation is promoted. Cell fate decisions are regulated by interactions between signaling and transcriptional networks. Recent studies have shown that the overexpression or downregulation of the transcription factor Jun can affect the ESC fate. Here we have focussed on the role of the Jun in the exit of mouse ESCs from ground state pluripotency and the onset of early differentiation. Transcriptomic analysis of differentiating ESCs reveals that Jun is required to upregulate a programme of genes associated with cell adhesion as ESCs exit the pluripotent ground state. Several of these Jun‐regulated genes are shown to be required for efficient adhesion. Importantly this adhesion is required for the timely regulated exit of ESCs from ground state pluripotency and the onset of early differentiation events. Stem Cells 2016;34:1213–1224 PMID:26850660

  11. Intricacies of Pluripotency.

    PubMed

    Bhartiya, Deepa

    2015-01-01

    Pluripotent stem cells have the potential to differentiate into 200 odd cell types present in adult body. Pluripotent stem cells available for regenerative medicine include embryonic stem (ES) cells, induced pluripotent stem (iPS) cells and very small ES-like stem (VSELs) cells. Nuclear OCT-4 is one of the crucial factors that dictate pluripotent state. Compared to ES/iPS cells grown in Petri dish, VSELs exist in adult body organs and results are emerging to suggest that they may have better potential to regenerate adult organs. This is because of their distinct epigenetic status as they are closer to the primordial germ cells from the epiblast-stage embryo compared to inner cell mass from which ES cells are obtained in vitro. We need to make special efforts to study them as they are very small in size and tend to get lost during processing. VSELs exist in adult organs, get mobilized in response to stress, undergo asymmetric cell divisions to give rise to tissue specific progenitors which further differentiate into various cell types and are possibly better candidates for regenerative medicine because they have no associated risk of tumor formation or immunological rejection. They are possibly also the 'embryonic remnants' in adult organs responsible for initiating cancer. Thus, rather than not accepting VSELs because they neither form teratoma nor divide in vitro like ES cells, it is time that scientific community should think of revising the definition of the term 'pluripotency'. PMID:26195889

  12. State-federal interactions in nuclear regulation

    SciTech Connect

    Pasternak, A.D.; Budnitz, R.J.

    1987-12-01

    The Atomic Energy Act of 1954 established, and later Congressional amendments have confirmed, that except in areas which have been explicitly granted to the states, the federal government possesses preemptive authority to regulate radiation hazards associated with the development and use of atomic energy. Since the passage of the original Act, numerous decisions by the courts have reaffirmed the legitimacy of federal preemption, and have defined and redefined its scope. In this study, the aim is to explore the underlying issues involved in federal preemption of radiation-hazard regulation, and to recommend actions that the Department of Energy and other agencies and groups should consider undertaking in the near term to protect the preemption principle. Appropriate roles of the states are discussed, as well as recent state-level activities and their rationale, and several current arenas in which state-federal conflicts about regulation of hazards are being played out. The emphasis here is on four particular arenas that are now important arenas of conflict, but the issues discussed are far broader in scope. These four arenas are: state-level moratorium activity; emergency planning for reactors; conflicts arising from state financial regulation; and inroads in federal preemption through litigation under state law.

  13. The Affective Regulation of Social Interaction

    ERIC Educational Resources Information Center

    Clore, Gerald L.; Pappas, Jesse

    2007-01-01

    The recent publication of David Heise's "Expressive Order" (2007) provides an occasion for discussing some of the key ideas in Affect Control Theory. The theory proposes that a few dimensions of affective meaning provide a common basis for interrelating personal identities and social actions. It holds that during interpersonal interactions, social…

  14. Nanog, Oct4 and Tet1 interplay in establishing pluripotency.

    PubMed

    Olariu, Victor; Lövkvist, Cecilia; Sneppen, Kim

    2016-01-01

    A few central transcription factors inside mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are believed to control the cells' pluripotency. Characterizations of pluripotent state were put forward on both transcription factor and epigenetic levels. Whereas core players have been identified, it is desirable to map out gene regulatory networks which govern the reprogramming of somatic cells as well as the early developmental decisions. Here we propose a multiple level model where the regulatory network of Oct4, Nanog and Tet1 includes positive feedback loops involving DNA-demethylation around the promoters of Oct4 and Tet1. We put forward a mechanistic understanding of the regulatory dynamics which account for i) Oct4 overexpression is sufficient to induce pluripotency in somatic cell types expressing the other Yamanaka reprogramming factors endogenously; ii) Tet1 can replace Oct4 in reprogramming cocktail; iii) Nanog is not necessary for reprogramming however its over-expression leads to enhanced self-renewal; iv) DNA methylation is the key to the regulation of pluripotency genes; v) Lif withdrawal leads to loss of pluripotency. Overall, our paper proposes a novel framework combining transcription regulation with DNA methylation modifications which, takes into account the multi-layer nature of regulatory mechanisms governing pluripotency acquisition through reprogramming. PMID:27146218

  15. Nanog, Oct4 and Tet1 interplay in establishing pluripotency

    PubMed Central

    Olariu, Victor; Lövkvist, Cecilia; Sneppen, Kim

    2016-01-01

    A few central transcription factors inside mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are believed to control the cells’ pluripotency. Characterizations of pluripotent state were put forward on both transcription factor and epigenetic levels. Whereas core players have been identified, it is desirable to map out gene regulatory networks which govern the reprogramming of somatic cells as well as the early developmental decisions. Here we propose a multiple level model where the regulatory network of Oct4, Nanog and Tet1 includes positive feedback loops involving DNA-demethylation around the promoters of Oct4 and Tet1. We put forward a mechanistic understanding of the regulatory dynamics which account for i) Oct4 overexpression is sufficient to induce pluripotency in somatic cell types expressing the other Yamanaka reprogramming factors endogenously; ii) Tet1 can replace Oct4 in reprogramming cocktail; iii) Nanog is not necessary for reprogramming however its over-expression leads to enhanced self-renewal; iv) DNA methylation is the key to the regulation of pluripotency genes; v) Lif withdrawal leads to loss of pluripotency. Overall, our paper proposes a novel framework combining transcription regulation with DNA methylation modifications which, takes into account the multi-layer nature of regulatory mechanisms governing pluripotency acquisition through reprogramming. PMID:27146218

  16. NaNog: A pluripotency homeobox (master) molecule

    PubMed Central

    Allouba, Mona H.; ElGuindy, Ahmed M.; Krishnamoorthy, Navaneethakrishnan; Yacoub, Magdi H.; Aguib, Yasmine E.

    2015-01-01

    One of the most intriguing aspects of cell biology is the state of pluripotency, where the cell is capable of self-renewal for as many times as deemed “necessary”, then at a specified time can differentiate into any type of cell. This fundamental process is required during organogenesis in foetal life and importantly during tissue repair in health and disease. Pluripotency is very tightly regulated, as any dysregulation can result in congenital defects, inability to repair damage, or cancer. Fuelled by the relatively recent interest in stem cell biology and tissue regeneration, the molecules implicated in regulating pluripotency have been the subject of extensive research. One of the important molecules involved in pluripotency, is NaNog, the subject of this article. PMID:26566529

  17. Intricacies of Pluripotency

    PubMed Central

    Bhartiya, Deepa

    2015-01-01

    Pluripotent stem cells have the potential to differentiate into 200 odd cell types present in adult body. Pluripotent stem cells available for regenerative medicine include embryonic stem (ES) cells, induced pluripotent stem (iPS) cells and very small ES-like stem (VSELs) cells. Nuclear OCT-4 is one of the crucial factors that dictate pluripotent state. Compared to ES/iPS cells grown in Petri dish, VSELs exist in adult body organs and results are emerging to suggest that they may have better potential to regenerate adult organs. This is because of their distinct epigenetic status as they are closer to the primordial germ cells from the epiblast-stage embryo compared to inner cell mass from which ES cells are obtained in vitro. We need to make special efforts to study them as they are very small in size and tend to get lost during processing. VSELs exist in adult organs, get mobilized in response to stress, undergo asymmetric cell divisions to give rise to tissue specific progenitors which further differentiate into various cell types and are possibly better candidates for regenerative medicine because they have no associated risk of tumor formation or immunological rejection. They are possibly also the ‘embryonic remnants’ in adult organs responsible for initiating cancer. Thus, rather than not accepting VSELs because they neither form teratoma nor divide in vitro like ES cells, it is time that scientific community should think of revising the definition of the term ‘pluripotency’. PMID:26195889

  18. Induction of pluripotency.

    PubMed

    Heffernan, Corey; Liu, Jun; Sumer, Huseyin; Malaver-Ortega, Luis F; Verma, Rajneesh; Carvalho, Edmund; Verma, Paul J

    2013-01-01

    The molecular and phenotypic irreversibility of mammalian cell differentiation was a fundamental principle of developmental biology at least until the 1980s, despite numerous reports dating back to the 1950s of the induction of pluripotency in amphibian cells by nuclear transfer (NT). Landmark reports in the 1980s and 1990s in sheep progressively challenged this dogmatic assumption; firstly, embryonic development of reconstructed embryos comprising whole (donor) blastomeres fused to enucleated oocytes, and famously, the cloning of Dolly from a terminally differentiated cell. Thus, the intrinsic ability of oocyte-derived factors to reverse the differentiated phenotype was confirmed. The concomitant elucidation of methods for human embryonic stem cell isolation and cultivation presented opportunities for therapeutic cell replacement strategies, particularly through NT of patient nuclei to enucleated oocytes for subsequent isolation of patient-specific (autologous), pluripotent cells from the resulting blastocysts. Associated logistical limitations of working with human oocytes, in addition to ethical and moral objections prompted exploration of alternative approaches to generate autologous stem cells for therapy, utilizing the full repertoire of factors characteristic of pluripotency, primarily through cell fusion and use of pluripotent cell extracts. Stunningly, in 2006, Japanese scientists described somatic cell reprogramming through delivery of four key factors (identified through a deductive approach from 24 candidate genes). Although less efficient than previous approaches, much of current stem cell research adopts this focused approach to cell reprogramming and (autologous) cell therapy. This chapter is a quasi-historical commentary of the various aforementioned approaches for the induction of pluripotency in lineage-committed cells, and introduces transcriptional and epigenetic changes occurring during reprogramming.

  19. Induction of pluripotency.

    PubMed

    Heffernan, Corey; Liu, Jun; Sumer, Huseyin; Malaver-Ortega, Luis F; Verma, Rajneesh; Carvalho, Edmund; Verma, Paul J

    2013-01-01

    The molecular and phenotypic irreversibility of mammalian cell differentiation was a fundamental principle of developmental biology at least until the 1980s, despite numerous reports dating back to the 1950s of the induction of pluripotency in amphibian cells by nuclear transfer (NT). Landmark reports in the 1980s and 1990s in sheep progressively challenged this dogmatic assumption; firstly, embryonic development of reconstructed embryos comprising whole (donor) blastomeres fused to enucleated oocytes, and famously, the cloning of Dolly from a terminally differentiated cell. Thus, the intrinsic ability of oocyte-derived factors to reverse the differentiated phenotype was confirmed. The concomitant elucidation of methods for human embryonic stem cell isolation and cultivation presented opportunities for therapeutic cell replacement strategies, particularly through NT of patient nuclei to enucleated oocytes for subsequent isolation of patient-specific (autologous), pluripotent cells from the resulting blastocysts. Associated logistical limitations of working with human oocytes, in addition to ethical and moral objections prompted exploration of alternative approaches to generate autologous stem cells for therapy, utilizing the full repertoire of factors characteristic of pluripotency, primarily through cell fusion and use of pluripotent cell extracts. Stunningly, in 2006, Japanese scientists described somatic cell reprogramming through delivery of four key factors (identified through a deductive approach from 24 candidate genes). Although less efficient than previous approaches, much of current stem cell research adopts this focused approach to cell reprogramming and (autologous) cell therapy. This chapter is a quasi-historical commentary of the various aforementioned approaches for the induction of pluripotency in lineage-committed cells, and introduces transcriptional and epigenetic changes occurring during reprogramming. PMID:23696349

  20. Dynamic Status of REST in the Mouse ESC Pluripotency Network

    PubMed Central

    Singh, Sanjay K.; Veo, Bethany L.; Kagalwala, Mohamedi N.; Shi, Weiwei; Liang, Shoudan; Majumder, Sadhan

    2012-01-01

    Background REST is abundantly expressed in mouse embryonic stem cells (ESCs). Many genome-wide analyses have found REST to be an integral part of the ESC pluripotency network. However, experimental systems have produced contradictory findings: (1) REST is required for the maintenance of ESC pluripotency and loss of REST causes increased expression of differentiation markers, (2) REST is not required for the maintenance of ESC pluripotency and loss of REST does not change expression of differentiation markers, and (3) REST is not required for the maintenance of ESC pluripotency but loss of REST causes decreased expression of differentiation markers. These reports highlight gaps in our knowledge of the ESC network. Methods Employing biochemical and genome-wide analyses of various culture conditions and ESC lines, we have attempted to resolve some of the discrepancies in the literature. Results We show that Rest+/− and Rest−/− AB-1 mutant ESCs, which did not exhibit a role of REST in ESC pluripotency when cultured in the presence of feeder cells, did show impaired self-renewal when compared with the parental cells under feeder-free culture conditions, but only in early passage cells. In late passage cells, both Rest+/− and Rest−/− AB-1 ESCs restored pluripotency, suggesting a passage and culture condition-dependent response. Genome-wide analysis followed by biochemical validation supported this response and further indicated that the restoration of pluripotency was associated by increased expression of the ESC pluripotency factors. E14Tg2a.4 ESCs with REST-knockdown, which earlier showed a REST-dependent pluripotency when cultured under feeder-free conditions, as well as Rest−/− AB-1 ESCs, showed no REST-dependent pluripotency when cultured in the presence of either feeder cells or laminin, indicating that extracellular matrix components can rescue REST's role in ESC pluripotency. Conclusions REST regulates ESC pluripotency in culture condition- and

  1. Cops2 promotes pluripotency maintenance by Stabilizing Nanog Protein and Repressing Transcription

    PubMed Central

    Zhang, Weiyu; Ni, Peiling; Mou, Chunlin; Zhang, Yanqin; Guo, Hongchao; Zhao, Tong; Loh, Yuin-Han; Chen, Lingyi

    2016-01-01

    The COP9 signalosome has been implicated in pluripotency maintenance of human embryonic stem cells. Yet, the mechanism for the COP9 signalosome to regulate pluripotency remains elusive. Through knocking down individual COP9 subunits, we demonstrate that Cops2, but not the whole COP9 signalosome, is essential for pluripotency maintenance in mouse embryonic stem cells. Down-regulation of Cops2 leads to reduced expression of pluripotency genes, slower proliferation rate, G2/M cell cycle arrest, and compromised embryoid differentiation of embryonic stem cells. Cops2 also facilitates somatic cell reprogramming. We further show that Cops2 binds to Nanog protein and prevent the degradation of Nanog by proteasome. Moreover, Cops2 functions as transcriptional corepressor to facilitate pluripotency maintenance. Altogether, our data reveal the essential role and novel mechanisms of Cops2 in pluripotency maintenance. PMID:27226076

  2. Cops2 promotes pluripotency maintenance by Stabilizing Nanog Protein and Repressing Transcription.

    PubMed

    Zhang, Weiyu; Ni, Peiling; Mou, Chunlin; Zhang, Yanqin; Guo, Hongchao; Zhao, Tong; Loh, Yuin-Han; Chen, Lingyi

    2016-01-01

    The COP9 signalosome has been implicated in pluripotency maintenance of human embryonic stem cells. Yet, the mechanism for the COP9 signalosome to regulate pluripotency remains elusive. Through knocking down individual COP9 subunits, we demonstrate that Cops2, but not the whole COP9 signalosome, is essential for pluripotency maintenance in mouse embryonic stem cells. Down-regulation of Cops2 leads to reduced expression of pluripotency genes, slower proliferation rate, G2/M cell cycle arrest, and compromised embryoid differentiation of embryonic stem cells. Cops2 also facilitates somatic cell reprogramming. We further show that Cops2 binds to Nanog protein and prevent the degradation of Nanog by proteasome. Moreover, Cops2 functions as transcriptional corepressor to facilitate pluripotency maintenance. Altogether, our data reveal the essential role and novel mechanisms of Cops2 in pluripotency maintenance. PMID:27226076

  3. Nanoengineered Platforms to Guide Pluripotent Stem Cell Fate

    PubMed Central

    Rutledge, Katy; Jabbarzadeh, Ehsan

    2016-01-01

    Tissue engineering utilizes cells, signaling molecules, and scaffolds towards creating functional tissue to repair damaged organs. Pluripotent stem cells (PSCs) are a promising cell source due to their ability to self-renewal indefinitely and their potential to differentiate into almost any cell type. Great strides have been taken to parse the physiological mechanisms by which PSCs respond to their microenvironment and commit to a specific lineage. The combination of physical cues and chemical factors is thought to have the most profound influence on stem cell behavior, therefore a major focus of tissue engineering strategies is scaffold design to incorporate these signals. One overlooked component of the in vivo microenvironment researchers attempt to recapitulate with three dimensional (3D) substrates is the nanoarchitecture formed by the fibrillar network of extracellular matrix (ECM) proteins. These nanoscale features have the ability to impact cell adhesion, migration, proliferation, and lineage commitment. Significant advances have been made in deciphering how these nanoscale cues interact with stem cells to determine phenotype, but much is still unknown as to how the interplay between physical and chemical signals regulate in vitro and in vivo cellular fate. This review dives deeper to investigate nanoscale platforms for engineering tissue, as well use the use of these nanotechnologies to drive pluripotent stem cell lineage determination. PMID:26918198

  4. BMP-SMAD signaling: From pluripotent stem cells to cardiovascular commitment.

    PubMed

    Orlova, Valeria V; Chuva de Sousa Lopes, Susana; Valdimarsdottir, Gudrun

    2016-02-01

    Human pluripotent stem cells (hPSCs) can form all somatic cells of the body. They thus offer opportunities for understanding (i) the basic steps of early human development, (ii) the pathophysiology in human degenerative diseases and (iii) approaches to regenerative medicine and drug development. Methods for improving their differentiation to defined mesodermal derivatives in particular will benefit their use in all of these areas but most particularly applications that require cardiac and vascular tissue. However, the molecular mechanisms that regulate mesodermal development in humans are still poorly understood. Gene ablation studies in mice have shown that the signaling pathways activated by the transforming growth factor beta (TGFβ) superfamily, including the bone morphogenetic proteins (BMP), play crucial roles in mesoderm differentiation and patterning the early embryo. Understanding their interplay and interaction with other signaling pathways, how they activate and inhibit transcription factors and epigenetic regulators during self-renewal, maintenance and exit from pluripotency and differentiation could provide vital information for a range of applications. This includes disease modeling when the hPSCs are derived from patients or drug screens for diseases of mesodermal organs. Here, we review the role of the BMP-SMAD signaling pathway in pluripotent stem cells and during mesoderm differentiation with focus on the cells that make up the cardiovascular system.

  5. DNA and Chromatin Modification Networks Distinguish Stem Cell Pluripotent Ground States*

    PubMed Central

    Song, Jing; Saha, Sudipto; Gokulrangan, Giridharan; Tesar, Paul J.; Ewing, Rob M.

    2012-01-01

    Pluripotent stem cells are capable of differentiating into all cell types of the body and therefore hold tremendous promise for regenerative medicine. Despite their widespread use in laboratories across the world, a detailed understanding of the molecular mechanisms that regulate the pluripotent state is currently lacking. Mouse embryonic (mESC) and epiblast (mEpiSC) stem cells are two closely related classes of pluripotent stem cells, derived from distinct embryonic tissues. Although both mESC and mEpiSC are pluripotent, these cell types show important differences in their properties suggesting distinct pluripotent ground states. To understand the molecular basis of pluripotency, we analyzed the nuclear proteomes of mESCs and mEpiSCs to identify protein networks that regulate their respective pluripotent states. Our study used label-free LC-MS/MS to identify and quantify 1597 proteins in embryonic and epiblast stem cell nuclei. Immunoblotting of a selected protein subset was used to confirm that key components of chromatin regulatory networks are differentially expressed in mESCs and mEpiSCs. Specifically, we identify differential expression of DNA methylation, ATP-dependent chromatin remodeling and nucleosome remodeling networks in mESC and mEpiSC nuclei. This study is the first comparative study of protein networks in cells representing the two distinct, pluripotent states, and points to the importance of DNA and chromatin modification processes in regulating pluripotency. In addition, by integrating our data with existing pluripotency networks, we provide detailed maps of protein networks that regulate pluripotency that will further both the fundamental understanding of pluripotency as well as efforts to reliably control the differentiation of these cells into functional cell fates. PMID:22822199

  6. Long non-coding RNAs in stem cell pluripotency.

    PubMed

    Ng, Shi-Yan; Stanton, Lawrence W

    2013-01-01

    Pluripotency refers to the self-renewal of undifferentiated embryonic stem cells (ESCs), and is maintained by a tightly regulated gene regulatory network involving an intricate interplay between transcription factors and their genomic targets, as well as epigenetic processes that influence gene expression. Long non-coding RNAs (lncRNAs) are newly discovered members of gene regulatory networks that govern a variety of cell functions. Defined as RNA transcripts larger than 200 nucleotides, lncRNAs have little or no protein-coding capacity and have been shown to act via various mechanisms, and are important in a variety of biological functions. Recent reports have described the discovery of pluripotent lncRNAs involved in the maintenance and induction of stem cell pluripotency. Here, we discuss how lncRNAs may integrate into the pluripotency network, as well as prominent questions in this emerging field.

  7. Transcriptional and epigenetic mechanisms of cellular reprogramming to induced pluripotency.

    PubMed

    van den Hurk, Mark; Kenis, Gunter; Bardy, Cedric; van den Hove, Daniel L; Gage, Fred H; Steinbusch, Harry W; Rutten, Bart P

    2016-08-01

    Enforced ectopic expression of a cocktail of pluripotency-associated genes such as Oct4, Sox2, Klf4 and c-Myc can reprogram somatic cells into induced pluripotent stem cells (iPSCs). The remarkable proliferation ability of iPSCs and their aptitude to redifferentiate into any cell lineage makes these cells a promising tool for generating a variety of human tissue in vitro. Yet, pluripotency induction is an inefficient process, as cells undergoing reprogramming need to overcome developmentally imposed epigenetic barriers. Recent work has shed new light on the molecular mechanisms that drive the reprogramming of somatic cells to iPSCs. Here, we present current knowledge on the transcriptional and epigenetic regulation of pluripotency induction and discuss how variability in epigenetic states impacts iPSCs' inherent biological properties. PMID:27419933

  8. Regulator artifacts in uniform matter for chiral interactions

    NASA Astrophysics Data System (ADS)

    Dyhdalo, A.; Furnstahl, R. J.; Hebeler, K.; Tews, I.

    2016-09-01

    Regulator functions applied to two- and three-nucleon forces are a necessary ingredient in many-body calculations based on chiral effective field theory interactions. These interactions have been developed recently with a variety of different cutoff forms, including regulating both the momentum transfer (local) and the relative momentum (nonlocal). While in principle any regulator that suppresses high-momentum modes can be employed, in practice artifacts are inevitable in current power counting schemes. Artifacts from particular regulators may cause significant distortions of the physics or may affect many-body convergence rates, so understanding their nature is important. Here we characterize the differences between cutoff effects using uniform matter at Hartree-Fock and second order in the interaction as a test-bed. This provides a clean laboratory to isolate phase-space effects of various regulators on both two- and three-nucleon interactions. We test the normal-ordering approximation for three-nucleon forces in nuclear matter and find that the relative size of the residual 3 N contributions is sensitive to the employed regularization scheme.

  9. Proximity interactions among centrosome components identify regulators of centriole duplication.

    PubMed

    Firat-Karalar, Elif Nur; Rauniyar, Navin; Yates, John R; Stearns, Tim

    2014-03-17

    The centrosome consists of a pair of centrioles and surrounding pericentriolar material (PCM). Many vertebrate cells also have an array of granules, termed centriolar satellites, that localize around the centrosome and are associated with centrosome and cilium function. Centriole duplication occurs once per cell cycle and is effected by a set of proteins including PLK4, CEP192, CEP152, CEP63, and CPAP. Information on the relationships between these components is limited due to the difficulty in assaying interactions in the context of the centrosome. Here, we used proximity-dependent biotin identification (BioID) to identify proximity interactions among centriole duplication proteins. PLK4, CEP192, and CEP152 BioID identified known physically interacting proteins and a new interaction between CEP152 and CDK5RAP2 consistent with a function of CEP152 in PCM recruitment. BioID for CEP63 and its paralog CCDC67 revealed extensive proximity interactions with centriolar satellite proteins. Focusing on these satellite proteins identified two new regulators of centriole duplication, CCDC14 and KIAA0753. Both proteins colocalize with CEP63 to satellites, bind to CEP63, and identify other satellite proteins by BioID. KIAA0753 positively regulates centriole duplication and CEP63 centrosome localization, whereas CCDC14 negatively regulates both processes. These results suggest that centriolar satellites have a previously unappreciated function in regulating centriole duplication. PMID:24613305

  10. Proximity interactions among centrosome components identify regulators of centriole duplication.

    PubMed

    Firat-Karalar, Elif Nur; Rauniyar, Navin; Yates, John R; Stearns, Tim

    2014-03-17

    The centrosome consists of a pair of centrioles and surrounding pericentriolar material (PCM). Many vertebrate cells also have an array of granules, termed centriolar satellites, that localize around the centrosome and are associated with centrosome and cilium function. Centriole duplication occurs once per cell cycle and is effected by a set of proteins including PLK4, CEP192, CEP152, CEP63, and CPAP. Information on the relationships between these components is limited due to the difficulty in assaying interactions in the context of the centrosome. Here, we used proximity-dependent biotin identification (BioID) to identify proximity interactions among centriole duplication proteins. PLK4, CEP192, and CEP152 BioID identified known physically interacting proteins and a new interaction between CEP152 and CDK5RAP2 consistent with a function of CEP152 in PCM recruitment. BioID for CEP63 and its paralog CCDC67 revealed extensive proximity interactions with centriolar satellite proteins. Focusing on these satellite proteins identified two new regulators of centriole duplication, CCDC14 and KIAA0753. Both proteins colocalize with CEP63 to satellites, bind to CEP63, and identify other satellite proteins by BioID. KIAA0753 positively regulates centriole duplication and CEP63 centrosome localization, whereas CCDC14 negatively regulates both processes. These results suggest that centriolar satellites have a previously unappreciated function in regulating centriole duplication.

  11. Sphingosine Phosphate Lyase Regulates Murine Embryonic Stem Cell Proliferation and Pluripotency through an S1P2/STAT3 Signaling Pathway.

    PubMed

    Smith, Gaelen S; Kumar, Ashok; Saba, Julie D

    2013-06-24

    Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that activates a family of G protein coupled-receptors (GPCRs) implicated in mammalian development, angiogenesis, immunity and tissue regeneration. S1P functions as a trophic factor for many cell types, including embryonic stem cells (ESCs). Sphingosine phosphate lyase (SPL) is an intracellular enzyme that catalyzes the irreversible degradation of S1P. We found SPL to be highly expressed in murine ESCs (mESCs). To investigate the role of SPL in mESC biology, we silenced SPL in mESCs via stable transfection with a lentiviral SPL-specific short hairpin RNA (shRNA) construct. SPL-knockdown (SPL-KD) mESCs showed a 5-fold increase in cellular S1P levels, increased proliferation rates and high expression of cell surface pluripotency markers SSEA1 and OCT4 compared to vector control cells. Compared to control mESCs, SPL-KD cells showed robust activation of STAT3 and a 10-fold increase in S1P2 expression. Inhibition of S1P2 or STAT3 reversed the proliferation and pluripotency phenotypes of SPL-KD mESCs. Further, inhibition of S1P2 attenuated, in a dose-dependent fashion, the high levels of OCT4 and STAT3 activation observed in SPL-KD mESCs. Finally, we showed that SPL-KD cells are capable of generating embryoid bodies from which muscle stem cells, called satellite cells, can be isolated. These findings demonstrate an important role for SPL in ESC homeostasis and suggest that SPL inhibition could facilitate ex vivo ESC expansion for therapeutic purposes.

  12. Variations in Glycogen Synthesis in Human Pluripotent Stem Cells with Altered Pluripotent States.

    PubMed

    Chen, Richard J; Zhang, Guofeng; Garfield, Susan H; Shi, Yi-Jun; Chen, Kevin G; Robey, Pamela G; Leapman, Richard D

    2015-01-01

    Human pluripotent stem cells (hPSCs) represent very promising resources for cell-based regenerative medicine. It is essential to determine the biological implications of some fundamental physiological processes (such as glycogen metabolism) in these stem cells. In this report, we employ electron, immunofluorescence microscopy, and biochemical methods to study glycogen synthesis in hPSCs. Our results indicate that there is a high level of glycogen synthesis (0.28 to 0.62 μg/μg proteins) in undifferentiated human embryonic stem cells (hESCs) compared with the glycogen levels (0 to 0.25 μg/μg proteins) reported in human cancer cell lines. Moreover, we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway. Our observation of glycogen bodies and sustained expression of the pluripotent factor Oct-4 mediated by the potent GSK-3 inhibitor CHIR-99021 reveals an altered pluripotent state in hPSC culture. We further confirmed glycogen variations under different naïve pluripotent cell growth conditions based on the addition of the GSK-3 inhibitor BIO. Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states, similar to those naïve-like hPSCs, with increased glycogen synthesis. Furthermore, we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis. Thus, our novel findings regarding the dynamic changes in glycogen metabolism provide new markers to assess the energetic and various pluripotent states in hPSCs. The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions.

  13. Changes in microRNA expression during differentiation of embryonic and induced pluripotent stem cells to definitive endoderm.

    PubMed

    Francis, Natalie; Moore, Melanie; Asan, Simona G; Rutter, Guy A; Burns, Chris

    2015-01-01

    Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have the potential to treat type 1 diabetes through cell replacement therapy. However, the protocols used to generate insulin-expressing cells in vitro frequently result in cells which have an immature phenotype and are functionally restricted. MicroRNAs (miRNAs) are now known to be important in cell fate specification, and a unique miRNA signature characterises pancreatic development at the definitive endoderm stage. Several studies have described differences in miRNA expression between ESCs and iPSCs. Here we have used microarray analysis both to identify miRNAs up- or down-regulated upon endoderm formation, and also miRNAs differentially expressed between ESCs and iPSCs. Several miRNAs fulfilling both these criteria were identified, suggesting that differences in the expression of these miRNAs may affect the ability of pluripotent stem cells to differentiate into definitive endoderm. The expression of these miRNAs was validated by qRT-PCR, and the relationship between one of these miRNAs, miR-151a-5p, and its predicted target gene, SOX17, was investigated by luciferase assay, and suggested an interaction between miR-151a-5p and this key transcription factor. In conclusion, these findings demonstrate a unique miRNA expression pattern for definitive endoderm derived from both embryonic and induced pluripotent stem cells.

  14. Otx2-PNN Interaction to Regulate Cortical Plasticity.

    PubMed

    Bernard, Clémence; Prochiantz, Alain

    2016-01-01

    The ability of the environment to shape cortical function is at its highest during critical periods of postnatal development. In the visual cortex, critical period onset is triggered by the maturation of parvalbumin inhibitory interneurons, which gradually become surrounded by a specialized glycosaminoglycan-rich extracellular matrix: the perineuronal nets. Among the identified factors regulating cortical plasticity in the visual cortex, extracortical homeoprotein Otx2 is transferred specifically into parvalbumin interneurons and this transfer regulates both the onset and the closure of the critical period of plasticity for binocular vision. Here, we review the interaction between the complex sugars of the perineuronal nets and homeoprotein Otx2 and how this interaction regulates cortical plasticity during critical period and in adulthood.

  15. Otx2-PNN Interaction to Regulate Cortical Plasticity

    PubMed Central

    Bernard, Clémence; Prochiantz, Alain

    2016-01-01

    The ability of the environment to shape cortical function is at its highest during critical periods of postnatal development. In the visual cortex, critical period onset is triggered by the maturation of parvalbumin inhibitory interneurons, which gradually become surrounded by a specialized glycosaminoglycan-rich extracellular matrix: the perineuronal nets. Among the identified factors regulating cortical plasticity in the visual cortex, extracortical homeoprotein Otx2 is transferred specifically into parvalbumin interneurons and this transfer regulates both the onset and the closure of the critical period of plasticity for binocular vision. Here, we review the interaction between the complex sugars of the perineuronal nets and homeoprotein Otx2 and how this interaction regulates cortical plasticity during critical period and in adulthood. PMID:26881132

  16. Sensor–response regulator interactions in a cross-regulated signal transduction network

    PubMed Central

    Huynh, TuAnh Ngoc; Chen, Li-Ling

    2015-01-01

    Two-component signal transduction involves phosphoryl transfer between a histidine kinase sensor and a response regulator effector. The nitrate-responsive two-component signal transduction systems in Escherichia coli represent a paradigm for a cross-regulation network, in which the paralogous sensor–response regulator pairs, NarX–NarL and NarQ–NarP, exhibit both cognate (e.g. NarX–NarL) and non-cognate (e.g. NarQ–NarL) interactions to control output. Here, we describe results from bacterial adenylate cyclase two-hybrid (BACTH) analysis to examine sensor dimerization as well as interaction between sensor–response regulator cognate and non-cognate pairs. Although results from BACTH analysis indicated that the NarX and NarQ sensors interact with each other, results from intragenic complementation tests demonstrate that they do not form functional heterodimers. Additionally, intragenic complementation shows that both NarX and NarQ undergo intermolecular autophosphorylation, deviating from the previously reported correlation between DHp (dimerization and histidyl phosphotransfer) domain loop handedness and autophosphorylation mode. Results from BACTH analysis revealed robust interactions for the NarX–NarL, NarQ–NarL and NarQ–NarP pairs but a much weaker interaction for the NarX–NarP pair. This demonstrates that asymmetrical cross-regulation results from differential binding affinities between different sensor–regulator pairs. Finally, results indicate that the NarL effector (DNA-binding) domain inhibits NarX–NarL interaction. Missense substitutions at receiver domain residue Ser-80 enhanced NarX–NarL interaction, apparently by destabilizing the NarL receiver–effector domain interface. PMID:25873583

  17. Vasodilator interactions in skeletal muscle blood flow regulation

    PubMed Central

    Hellsten, Y; Nyberg, M; Jensen, L G; Mortensen, S P

    2012-01-01

    During exercise, oxygen delivery to skeletal muscle is elevated to meet the increased oxygen demand. The increase in blood flow to skeletal muscle is achieved by vasodilators formed locally in the muscle tissue, either on the intraluminal or on the extraluminal side of the blood vessels. A number of vasodilators have been shown to bring about this increase in blood flow and, importantly, interactions between these compounds seem to be essential for the precise regulation of blood flow. Two compounds stand out as central in these vasodilator interactions: nitric oxide (NO) and prostacyclin. These two vasodilators are both stimulated by several compounds, e.g. adenosine, ATP, acetylcholine and bradykinin, and are affected by mechanically induced signals, such as shear stress. NO and prostacyclin have also been shown to interact in a redundant manner where one system can take over when formation of the other is compromised. Although numerous studies have examined the role of single and multiple pharmacological inhibition of different vasodilator systems, and important vasodilators and interactions have been identified, a large part of the exercise hyperaemic response remains unexplained. It is plausible that this remaining hyperaemia may be explained by cAMP- and cGMP-independent smooth muscle relaxation, such as effects of endothelial derived hyperpolarization factors (EDHFs) or through metabolic modulation of sympathetic effects. The nature and role of EDHF as well as potential novel mechanisms in muscle blood flow regulation remain to be further explored to fully elucidate the regulation of exercise hyperaemia. PMID:22988140

  18. MicroRNA-122 Influences the Development of Sperm Abnormalities from Human Induced Pluripotent Stem Cells by Regulating TNP2 Expression

    PubMed Central

    Huang, Yongyi; Liu, Jianjun; Zhao, Yanhui; Jiang, Lizhen; Huang, Qin

    2013-01-01

    Sperm abnormalities are one of the main factors responsible for male infertility; however, their pathogenesis remains unclear. The role of microRNAs in the development of sperm abnormalities in infertile men has not yet been investigated. Here, we used human induced pluripotent stem cells to investigate the influence of miR-122 expression on the differentiation of these cells into spermatozoa-like cells in vitro. After induction, mutant miR-122-transfected cells formed spermatozoa-like cells. Flow cytometry of DNA content revealed a significant increase in the haploid cell population in spermatozoa-like cells derived from mutant miR-122-transfected cells as compared to those derived from miR-122-transfected cells. During induction, TNP2 and protamine mRNA and protein levels were significantly higher in mutant miR-122-transfected cells than in miR-122-transfected cells. High-throughput isobaric tags for relative and absolute quantification were used to identify and quantify the different protein expression levels in miR-122- and mutant miR-122-transfected cells. Among all the proteins analyzed, the expression of lipoproteins, for example, APOB and APOA1, showed the most significant difference between the two groups. This study illustrates that miR-122 expression is associated with abnormal sperm development. MiR-122 may influence spermatozoa-like cells by suppressing TNP2 expression and inhibiting the expression of proteins associated with sperm development. PMID:23327642

  19. Transcription is regulated by NusA:NusG interaction

    PubMed Central

    Strauß, Martin; Vitiello, Christal; Schweimer, Kristian; Gottesman, Max; Rösch, Paul; Knauer, Stefan H.

    2016-01-01

    NusA and NusG are major regulators of bacterial transcription elongation, which act either in concert or antagonistically. Both bind to RNA polymerase (RNAP), regulating pausing as well as intrinsic and Rho-dependent termination. Here, we demonstrate by nuclear magnetic resonance spectroscopy that the Escherichia coli NusG amino-terminal domain forms a complex with the acidic repeat domain 2 (AR2) of NusA. The interaction surface of either transcription factor overlaps with the respective binding site for RNAP. We show that NusA-AR2 is able to remove NusG from RNAP. Our in vivo and in vitro results suggest that interaction between NusA and NusG could play various regulatory roles during transcription, including recruitment of NusG to RNAP, resynchronization of transcription:translation coupling, and modulation of termination efficiency. PMID:27174929

  20. The Schreber case revisited: schizophrenia as a disorder of self-regulation and of interactional regulation.

    PubMed

    Grotstein, J S

    1985-01-01

    The Schreber case has been used by generations of psychoanalysts and psychiatrists to exemplify many features of the psychoanalytic conception of psychosis. It has generally been considered the origin of a great debate in psychoanalysis as to whether schizophrenia is a disorder of nature or of nurture. I seek in this contribution to proffer a newer theory of psychopathology, one which is based upon the conception of primary and secondary disorders of attachment (bonding) and which presents itself clinically as disorders of self-regulation and of interactional regulation. I attempt to explicate this theory in the Schreber case by demonstrating that his symptoms revealed: (a) failures of normal mental state regulations, (b) the emergence of symptoms which then secondarily and pathologically restore regulation in a pathological manner, and finally (c) his/her very symptoms seem to regulate a state in the family system and/or in the system of the culture at large. PMID:4049912

  1. The Schreber case revisited: schizophrenia as a disorder of self-regulation and of interactional regulation.

    PubMed

    Grotstein, J S

    1985-01-01

    The Schreber case has been used by generations of psychoanalysts and psychiatrists to exemplify many features of the psychoanalytic conception of psychosis. It has generally been considered the origin of a great debate in psychoanalysis as to whether schizophrenia is a disorder of nature or of nurture. I seek in this contribution to proffer a newer theory of psychopathology, one which is based upon the conception of primary and secondary disorders of attachment (bonding) and which presents itself clinically as disorders of self-regulation and of interactional regulation. I attempt to explicate this theory in the Schreber case by demonstrating that his symptoms revealed: (a) failures of normal mental state regulations, (b) the emergence of symptoms which then secondarily and pathologically restore regulation in a pathological manner, and finally (c) his/her very symptoms seem to regulate a state in the family system and/or in the system of the culture at large.

  2. Parent conflict predicts infants' vagal regulation in social interaction.

    PubMed

    Moore, Ginger A

    2010-01-01

    Parent conflict during infancy may affect rapidly developing physiological regulation. To examine the association between parent conflict and infants' vagal tone functioning, mothers (N = 48) reported levels of parent conflict and their 6-month-old male and female infants' respiratory sinus arrhythmia (RSA) was measured in the still-face paradigm. Higher parent conflict was related to lower RSA at baseline and each episode of the still-face paradigm. Infants in relatively higher conflict families showed attenuated RSA withdrawal in response to mothers' disengagement and attenuated RSA activation when interacting with mothers. Findings suggest atypical RSA regulation and reliance on self-regulation for infants in families with moderate levels of parent conflict. Implications for later development and future research are discussed.

  3. Harvester ants use interactions to regulate forager activation and availability

    PubMed Central

    Pinter-Wollman, Noa; Bala, Ashwin; Merrell, Andrew; Queirolo, Jovel; Stumpe, Martin C.; Holmes, Susan; Gordon, Deborah M.

    2013-01-01

    Social groups balance flexibility and robustness in their collective response to environmental changes using feedback between behavioural processes that operate at different timescales. Here we examine how behavioural processes operating at two timescales regulate the foraging activity of colonies of the harvester ant, Pogonomyrmex barbatus, allowing them to balance their response to food availability and predation. Previous work showed that the rate at which foragers return to the nest with food influences the rate at which foragers leave the nest. To investigate how interactions inside the nest link the rates of returning and outgoing foragers, we observed outgoing foragers inside the nest in field colonies using a novel observation method. We found that the interaction rate experienced by outgoing foragers inside the nest corresponded to forager return rate, and that the interactions of outgoing foragers were spatially clustered. Activation of a forager occurred on the timescale of seconds: a forager left the nest 3–8 s after a substantial increase in interactions with returning foragers. The availability of outgoing foragers to become activated was adjusted on the timescale of minutes: when forager return was interrupted for more than 4–5 min, available foragers waiting near the nest entrance went deeper into the nest. Thus, forager activation and forager availability both increased with the rate at which foragers returned to the nest. This process was checked by negative feedback between forager activation and forager availability. Regulation of foraging activation on the timescale of seconds provides flexibility in response to fluctuations in food abundance, whereas regulation of forager availability on the timescale of minutes provides robustness in response to sustained disturbance such as predation. PMID:24031094

  4. Harvester ants use interactions to regulate forager activation and availability.

    PubMed

    Pinter-Wollman, Noa; Bala, Ashwin; Merrell, Andrew; Queirolo, Jovel; Stumpe, Martin C; Holmes, Susan; Gordon, Deborah M

    2013-07-01

    Social groups balance flexibility and robustness in their collective response to environmental changes using feedback between behavioural processes that operate at different timescales. Here we examine how behavioural processes operating at two timescales regulate the foraging activity of colonies of the harvester ant, Pogonomyrmex barbatus, allowing them to balance their response to food availability and predation. Previous work showed that the rate at which foragers return to the nest with food influences the rate at which foragers leave the nest. To investigate how interactions inside the nest link the rates of returning and outgoing foragers, we observed outgoing foragers inside the nest in field colonies using a novel observation method. We found that the interaction rate experienced by outgoing foragers inside the nest corresponded to forager return rate, and that the interactions of outgoing foragers were spatially clustered. Activation of a forager occurred on the timescale of seconds: a forager left the nest 3-8 s after a substantial increase in interactions with returning foragers. The availability of outgoing foragers to become activated was adjusted on the timescale of minutes: when forager return was interrupted for more than 4-5 min, available foragers waiting near the nest entrance went deeper into the nest. Thus, forager activation and forager availability both increased with the rate at which foragers returned to the nest. This process was checked by negative feedback between forager activation and forager availability. Regulation of foraging activation on the timescale of seconds provides flexibility in response to fluctuations in food abundance, whereas regulation of forager availability on the timescale of minutes provides robustness in response to sustained disturbance such as predation.

  5. Regulation of cell proliferation of human induced pluripotent stem cell-derived mesenchymal stem cells via ether-à-go-go 1 (hEAG1) potassium channel.

    PubMed

    Zhang, Jiao; Chan, Yau-Chi; Ho, Jenny Chung-Yee; Siu, Chung-Wah; Lian, Qizhou; Tse, Hung-Fat

    2012-07-15

    The successful generation of a high yield of mesenchymal stem cells (MSCs) from human induced pluripotent stem cells (iPSCs) may represent an unlimited cell source with superior therapeutic benefits for tissue regeneration to bone marrow (BM)-derived MSCs. We investigated whether the differential expression of ion channels in iPSC-MSCs was responsible for their higher proliferation capacity than BM-MSCs. The expression of ion channels for K(+), Na(+), Ca(2+), and Cl(-) was examined by RT-PCR. The electrophysiological properties of iPSC-MSCs and BM-MSCs were then compared by patch-clamp experiments to verify their functional roles. Significant mRNA expression of ion channel genes including KCa1.1, KCa3.1, KCNH1, Kir2.1, SCN9A, CACNA1C, and Clcn3 was observed in both human iPSC-MSCs and BM-MSCs, whereas Kir2.2 and Kir2.3 were only detected in human iPSC-MSCs. Five types of currents [big-conductance Ca(2+)-activated K(+) current (BK(Ca)), delayed rectifier K(+) current (IK(DR)), inwardly rectifying K(+) current (I(Kir)), Ca(2+)-activated K(+) current (IK(Ca)), and chloride current (I(Cl))] were found in iPSC-MSCs (83%, 47%, 11%, 5%, and 4%, respectively) but only four of them (BK(Ca), IK(DR), I(Kir), and IK(Ca)) were identified in BM-MSCs (76%, 25%, 22%, and 11%, respectively). Cell proliferation was examined with MTT or bromodeoxyuridine assay, and doubling times were 2.66 and 3.72 days for iPSC-MSCs and BM-MSCs, respectively, showing a 1.4-fold discrepancy. Blockade of IK(DR) with short hairpin RNA or human ether-à-go-go 1 (hEAG1) channel blockers, 4-AP and astemizole, significantly reduced the rate of proliferation of human iPSC-MSCs. These treatments also decreased the rate of proliferation of human BM-MSCs albeit to a lesser extent. These findings demonstrate that the hEAG1 channel plays a crucial role in controlling the proliferation rate of human iPSC-MSCs and to a lesser extent in BM-MSCs. PMID:22357737

  6. CEP120 interacts with CPAP and positively regulates centriole elongation.

    PubMed

    Lin, Yi-Nan; Wu, Chien-Ting; Lin, Yu-Chih; Hsu, Wen-Bin; Tang, Chieh-Ju C; Chang, Ching-Wen; Tang, Tang K

    2013-07-22

    Centriole duplication begins with the formation of a single procentriole next to a preexisting centriole. CPAP (centrosomal protein 4.1-associated protein) was previously reported to participate in centriole elongation. Here, we show that CEP120 is a cell cycle-regulated protein that directly interacts with CPAP and is required for centriole duplication. CEP120 levels increased gradually from early S to G2/M and decreased significantly after mitosis. Forced overexpression of either CEP120 or CPAP not only induced the assembly of overly long centrioles but also produced atypical supernumerary centrioles that grew from these long centrioles. Depletion of CEP120 inhibited CPAP-induced centriole elongation and vice versa, implying that these proteins work together to regulate centriole elongation. Furthermore, CEP120 was found to contain an N-terminal microtubule-binding domain, a C-terminal dimerization domain, and a centriolar localization domain. Overexpression of a microtubule binding-defective CEP120-K76A mutant significantly suppressed the formation of elongated centrioles. Together, our results indicate that CEP120 is a CPAP-interacting protein that positively regulates centriole elongation.

  7. Microbe–Host Interactions are Positively and Negatively Regulated by Galectin–Glycan Interactions

    PubMed Central

    Baum, Linda G.; Garner, Omai B.; Schaefer, Katrin; Lee, Benhur

    2014-01-01

    Microbe–host interactions are complex processes that are directly and indirectly regulated by a variety of factors, including microbe presentation of specific molecular signatures on the microbial surface, as well as host cell presentation of receptors that recognize these pathogen signatures. Cell surface glycans are one important class of microbial signatures that are recognized by a variety of host cell lectins. Host cell lectins that recognize microbial glycans include members of the galectin family of lectins that recognize specific glycan ligands on viruses, bacteria, fungi, and parasites. In this review, we will discuss the ways that the interactions of microbial glycans with host cell galectins positively and negatively regulate pathogen attachment, invasion, and survival, as well as regulate host responses that mitigate microbial pathogenesis. PMID:24995007

  8. Induced Pluripotency for Translational Research

    PubMed Central

    Wu, Menghua; Chen, Guilai; Hu, Baoyang

    2013-01-01

    The advent of induced pluripotent stem cells (iPSCs) has revolutionized the concept of cellular reprogramming and potentially will solve the immunological compatibility issues that have so far hindered the application of human pluripotent stem cells in regenerative medicine. Recent findings showed that pluripotency is defined by a state of balanced lineage potency, which can be artificially instated through various procedures, including the conventional Yamanaka strategy. As a type of pluripotent stem cell, iPSCs are subject to the usual concerns over purity of differentiated derivatives and risks of tumor formation when used for cell-based therapy, though they provide certain advantages in translational research, especially in the areas of personalized medicine, disease modeling and drug screening. iPSC-based technology, human embryonic stem cells (hESCs) and direct lineage conversion each will play distinct roles in specific aspects of translational medicine, and continue yielding surprises for scientists and the public. PMID:24056061

  9. Long noncoding RNAs: new players in the molecular mechanism for maintenance and differentiation of pluripotent stem cells.

    PubMed

    Ghosal, Suman; Das, Shaoli; Chakrabarti, Jayprokas

    2013-08-15

    Maintenance of the pluripotent state or differentiation of the pluripotent state into any germ layer depends on the factors that orchestrate expression of thousands of genes through epigenetic, transcriptional, and post-transcriptional regulation. Long noncoding RNAs (lncRNAs) are implicated in the complex molecular circuitry in the developmental processes. The ENCODE project has opened up new avenues for studying these lncRNA transcripts with the availability of new datasets for lncRNA annotation and regulation. Expression studies identified hundreds of long noncoding RNAs differentially expressed in the pluripotent state, and many of these lncRNAs are found to control the pluripotency and stemness in embryonic and induced pluripotent stem cells or, in the reverse way, promote differentiation of pluripotent cells. They are generally transcriptionally activated or repressed by pluripotency-associated transcription factors and function as molecular mediators of gene expression that determine the pluripotent state of the cell. They can act as molecular scaffolds or guides for the chromatin-modifying complexes to direct them to bind into specific genomic loci to impart a repressive or activating effect on gene expression, or they can transcriptionally or post-transcriptionally regulate gene expression by diverse molecular mechanisms. This review focuses on recent findings on the regulatory role of lncRNAs in two main aspects of pluripotency, namely, self renewal and differentiation into any lineage, and elucidates the underlying molecular mechanisms that are being uncovered lately.

  10. Deterministic direct reprogramming of somatic cells to pluripotency.

    PubMed

    Rais, Yoach; Zviran, Asaf; Geula, Shay; Gafni, Ohad; Chomsky, Elad; Viukov, Sergey; Mansour, Abed AlFatah; Caspi, Inbal; Krupalnik, Vladislav; Zerbib, Mirie; Maza, Itay; Mor, Nofar; Baran, Dror; Weinberger, Leehee; Jaitin, Diego A; Lara-Astiaso, David; Blecher-Gonen, Ronnie; Shipony, Zohar; Mukamel, Zohar; Hagai, Tzachi; Gilad, Shlomit; Amann-Zalcenstein, Daniela; Tanay, Amos; Amit, Ido; Novershtern, Noa; Hanna, Jacob H

    2013-10-01

    Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.

  11. Structural Interaction and Functional Regulation of Polycystin-2 by Filamin

    PubMed Central

    Wang, Qian; Dai, Xiao-Qing; Li, Qiang; Wang, Zuocheng; Cantero, María del Rocío; Li, Shu; Shen, Ji; Tu, Jian-Cheng; Cantiello, Horacio; Chen, Xing-Zhen

    2012-01-01

    Filamins are important actin cross-linking proteins implicated in scaffolding, membrane stabilization and signal transduction, through interaction with ion channels, receptors and signaling proteins. Here we report the physical and functional interaction between filamins and polycystin-2, a TRP-type cation channel mutated in 10–15% patients with autosomal dominant polycystic kidney disease. Yeast two-hybrid and GST pull-down experiments demonstrated that the C-termini of filamin isoforms A, B and C directly bind to both the intracellular N- and C-termini of polycystin-2. Reciprocal co-immunoprecipitation experiments showed that endogenous polycystin-2 and filamins are in the same complexes in renal epithelial cells and human melanoma A7 cells. We then examined the effect of filamin on polycystin-2 channel function by electrophysiology studies with a lipid bilayer reconstitution system and found that filamin-A substantially inhibits polycystin-2 channel activity. Our study indicates that filamins are important regulators of polycystin-2 channel function, and further links actin cytoskeletal dynamics to the regulation of this channel protein. PMID:22802962

  12. Dynamic and social behaviors of human pluripotent stem cells

    PubMed Central

    Phadnis, Smruti M.; Loewke, Nathan O.; Dimov, Ivan K.; Pai, Sunil; Amwake, Christine E.; Solgaard, Olav; Baer, Thomas M.; Chen, Bertha; Pera, Renee A. Reijo

    2015-01-01

    Human pluripotent stem cells (hPSCs) can self-renew or differentiate to diverse cell types, thus providing a platform for basic and clinical applications. However, pluripotent stem cell populations are heterogeneous and functional properties at the single cell level are poorly documented leading to inefficiencies in differentiation and concerns regarding reproducibility and safety. Here, we use non-invasive time-lapse imaging to continuously examine hPSC maintenance and differentiation and to predict cell viability and fate. We document dynamic behaviors and social interactions that prospectively distinguish hPSC survival, self-renewal, and differentiation. Results highlight the molecular role of E-cadherin not only for cell-cell contact but also for clonal propagation of hPSCs. Results indicate that use of continuous time-lapse imaging can distinguish cellular heterogeneity with respect to pluripotency as well as a subset of karyotypic abnormalities whose dynamic properties were monitored. PMID:26381699

  13. Epistatic study reveals two genetic interactions in blood pressure regulation

    PubMed Central

    2013-01-01

    Background Although numerous candidate gene and genome-wide association studies have been performed on blood pressure, a small number of regulating genetic variants having a limited effect have been identified. This phenomenon can partially be explained by possible gene-gene/epistasis interactions that were little investigated so far. Methods We performed a pre-planned two-phase investigation: in phase 1, one hundred single nucleotide polymorphisms (SNPs) in 65 candidate genes were genotyped in 1,912 French unrelated adults in order to study their two-locus combined effects on blood pressure (BP) levels. In phase 2, the significant epistatic interactions observed in phase 1 were tested in an independent population gathering 1,755 unrelated European adults. Results Among the 9 genetic variants significantly associated with systolic and diastolic BP in phase 1, some may act through altering the corresponding protein levels: SNPs rs5742910 (Padjusted≤0.03) and rs6046 (Padjusted =0.044) in F7 and rs1800469 (Padjusted ≤0.036) in TGFB1; whereas some may be functional through altering the corresponding protein structure: rs1800590 (Padjusted =0.028, SE=0.088) in LPL and rs2228570 (Padjusted ≤9.48×10-4) in VDR. The two epistatic interactions found for systolic and diastolic BP in the discovery phase: VCAM1 (rs1041163) * APOB (rs1367117), and SCGB1A1 (rs3741240) * LPL (rs1800590), were tested in the replication population and we observed significant interactions on DBP. In silico analyses yielded putative functional properties of the SNPs involved in these epistatic interactions trough the alteration of corresponding protein structures. Conclusions These findings support the hypothesis that different pathways and then different genes may act synergistically in order to modify BP. This could highlight novel pathophysiologic mechanisms underlying hypertension. PMID:23298194

  14. Wise Regulates Bone Deposition through Genetic Interactions with Lrp5

    PubMed Central

    Ellies, Debra L.; Economou, Androulla; Viviano, Beth; Rey, Jean-Philippe; Paine-Saunders, Stephenie; Krumlauf, Robb; Saunders, Scott

    2014-01-01

    In this study using genetic approaches in mouse we demonstrate that the secreted protein Wise plays essential roles in regulating early bone formation through its ability to modulate Wnt signaling via interactions with the Lrp5 co-receptor. In Wise−/− mutant mice we find an increase in the rate of osteoblast proliferation and a transient increase in bone mineral density. This change in proliferation is dependent upon Lrp5, as Wise;Lrp5 double mutants have normal bone mass. This suggests that Wise serves as a negative modulator of Wnt signaling in active osteoblasts. Wise and the closely related protein Sclerostin (Sost) are expressed in osteoblast cells during temporally distinct early and late phases in a manner consistent with the temporal onset of their respective increased bone density phenotypes. These data suggest that Wise and Sost may have common roles in regulating bone development through their ability to control the balance of Wnt signaling. We find that Wise is also required to potentiate proliferation in chondrocytes, serving as a potential positive modulator of Wnt activity. Our analyses demonstrate that Wise plays a key role in processes that control the number of osteoblasts and chondrocytes during bone homeostasis and provide important insight into mechanisms regulating the Wnt pathway during skeletal development. PMID:24789067

  15. Regulatory non-coding RNAs in pluripotent stem cells.

    PubMed

    Rosa, Alessandro; Brivanlou, Ali H

    2013-01-01

    The most part of our genome encodes for RNA transcripts are never translated into proteins. These include families of RNA molecules with a regulatory function, which can be arbitrarily subdivided in short (less than 200 nucleotides) and long non-coding RNAs (ncRNAs). MicroRNAs, which act post-transcriptionally to repress the function of target mRNAs, belong to the first group. Included in the second group are multi-exonic and polyadenylated long ncRNAs (lncRNAs), localized either in the nucleus, where they can associate with chromatin remodeling complexes to regulate transcription, or in the cytoplasm, acting as post-transcriptional regulators. Pluripotent stem cells, such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), represent useful systems for modeling normal development and human diseases, as well as promising tools for regenerative medicine. To fully explore their potential, however, a deep understanding of the molecular basis of stemness is crucial. In recent years, increasing evidence of the importance of regulation by ncRNAs in pluripotent cells is accumulating. In this review, we will discuss recent findings pointing to multiple roles played by regulatory ncRNAs in ESC and iPSCs, where they act in concert with signaling pathways, transcriptional regulatory circuitries and epigenetic factors to modulate the balance between pluripotency and differentiation.

  16. Interaction between amiodarone and hepatitis-C virus nucleotide inhibitors in human induced pluripotent stem cell-derived cardiomyocytes and HEK-293 Cav1.2 over-expressing cells.

    PubMed

    Lagrutta, Armando; Zeng, Haoyu; Imredy, John; Balasubramanian, Bharathi; Dech, Spencer; Lis, Edward; Wang, Jixin; Zhai, Jin; DeGeorge, Joseph; Sannajust, Frederick

    2016-10-01

    Several clinical cases of severe bradyarrhythmias have been reported upon co-administration of the Hepatitis-C NS5B Nucleotide Polymerase Inhibitor (HCV-NI) direct-acting antiviral agent, sofosbuvir (SOF), and the Class-III anti-arrhythmic amiodarone (AMIO). We model the cardiac drug-drug interaction (DDI) between AMIO and SOF, and between AMIO and a closely-related SOF analog, MNI-1 (Merck Nucleotide Inhibitor #1), in functional assays of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), to provide mechanistic insights into recently reported clinical cases. AMIO co-applied with SOF or MNI-1 increased beating rate or field potential (FP) rate and decreased impedance (IMP) and Ca(2+) transient amplitudes in hiPSC-CM syncytia. This action resembled that of Ca(2+) channel blockers (CCBs) in the model, but CCBs did not substitute for AMIO in the DDI. AMIO analog dronedarone (DRON) did not substitute for, but competed with AMIO in the DDI. Ryanodine and thapsigargin, decreasing intracellular Ca(2+) stores, and SEA-0400, a Na(+)/Ca(2+) exchanger-1 (NCX1) inhibitor, partially antagonized or suppressed DDI effects. Other agents affecting FP rate only exerted additive or subtractive effects, commensurate with their individual effects. We also describe an interaction between AMIO and MNI-1 on Cav1.2 ion channels in an over-expressing HEK-293 cell line. MNI-1 enhanced Cav1.2 channel inhibition by AMIO, but did not affect inhibition of Cav1.2 by DRON, verapamil, nifedipine, or diltiazem. Our data in hiPSC-CMs indicate that HCV-NI agents such as SOF and MNI-1 interact with key intracellular Ca(2+)-handling mechanisms. Additional study in a Cav1.2 HEK-293 cell-line suggests that HCV-NIs potentiate the inhibitory action of AMIO on L-type Ca(2+) channels.

  17. Chaotic expression dynamics implies pluripotency: when theory and experiment meet

    PubMed Central

    Furusawa, Chikara; Kaneko, Kunihiko

    2009-01-01

    Background During normal development, cells undergo a unidirectional course of differentiation that progressively decreases the number of cell types they can potentially become. Pluripotent stem cells can differentiate into several types of cells, but terminally differentiated cells cannot differentiate any further. A fundamental problem in stem cell biology is the characterization of the difference in cellular states, e.g., gene expression profiles, between pluripotent stem cells and terminally differentiated cells. Presentation of the hypothesis To address the problem, we developed a dynamical systems model of cells with intracellular protein expression dynamics and interactions with each other. According to extensive simulations, cells with irregular (chaotic) oscillations in gene expression dynamics have the potential to differentiate into other cell types. During development, such complex oscillations are lost successively, leading to a loss of pluripotency. These simulation results, together with recent single-cell-level measurements in stem cells, led us to the following hypothesis regarding pluripotency: Chaotic oscillation in the expression of some genes leads to cell pluripotency and affords cellular state heterogeneity, which is supported by itinerancy over quasi-stable states. Differentiation stabilizes these states, leading to a loss of pluripotency. Testing the hypothesis To test the hypothesis, it is crucial to measure the time course of gene expression levels at the single-cell level by fluorescence microscopy and fluorescence-activated cell sorting (FACS) analysis. By analyzing the time series of single-cell-level expression data, one can distinguish whether the variation in protein expression level over time is due only to stochasticity in expression dynamics or originates from the chaotic dynamics inherent to cells, as our hypothesis predicts. By further analyzing the expression in differentiated cell types, one can examine whether the loss of

  18. Direct dynamin–actin interactions regulate the actin cytoskeleton

    PubMed Central

    Gu, Changkyu; Yaddanapudi, Suma; Weins, Astrid; Osborn, Teresia; Reiser, Jochen; Pollak, Martin; Hartwig, John; Sever, Sanja

    2010-01-01

    The large GTPase dynamin assembles into higher order structures that are thought to promote endocytosis. Dynamin also regulates the actin cytoskeleton through an unknown, GTPase-dependent mechanism. Here, we identify a highly conserved site in dynamin that binds directly to actin filaments and aligns them into bundles. Point mutations in the actin-binding domain cause aberrant membrane ruffling and defective actin stress fibre formation in cells. Short actin filaments promote dynamin assembly into higher order structures, which in turn efficiently release the actin-capping protein (CP) gelsolin from barbed actin ends in vitro, allowing for elongation of actin filaments. Together, our results support a model in which assembled dynamin, generated through interactions with short actin filaments, promotes actin polymerization via displacement of actin-CPs. PMID:20935625

  19. TPX2 regulates neuronal morphology through kinesin-5 interaction

    PubMed Central

    Kahn, Olga I.; Ha, Ngoc; Baird, Michelle A.; Davidson, Michael W.; Baas, Peter W.

    2015-01-01

    TPX2 (targeting protein for Xklp2) is a multifunctional mitotic spindle assembly factor that in mammalian cells localizes and regulates mitotic motor protein kinesin-5 (also called Eg5 or kif11). We previously showed that upon depletion or inhibition of kinesin-5 in cultured neurons, microtubule movements increase, resulting in faster growing axons and thinner dendrites. Here, we show that depletion of TPX2 from cultured neurons speeds their rate of process outgrowth, similarly to kinesin-5 inhibition. The phenotype is rescued by TPX2 re-expression, but not if TPX2’s kinesin-5-interacting domain is deleted. These results, together with studies showing a spike in TPX2 expression during dendritic differentiation, suggest that the levels and distribution of TPX2 are likely to be determinants of when and where kinesin-5 acts in neurons. PMID:26257190

  20. Diabetes regulates fructose absorption through thioredoxin-interacting protein

    PubMed Central

    Dotimas, James R; Lee, Austin W; Schmider, Angela B; Carroll, Shannon H; Shah, Anu; Bilen, Julide; Elliott, Kayla R; Myers, Ronald B; Soberman, Roy J; Yoshioka, Jun; Lee, Richard T

    2016-01-01

    Metabolic studies suggest that the absorptive capacity of the small intestine for fructose is limited, though the molecular mechanisms controlling this process remain unknown. Here we demonstrate that thioredoxin-interacting protein (Txnip), which regulates glucose homeostasis in mammals, binds to fructose transporters and promotes fructose absorption by the small intestine. Deletion of Txnip in mice reduced fructose transport into the peripheral bloodstream and liver, as well as the severity of adverse metabolic outcomes resulting from long-term fructose consumption. We also demonstrate that fructose consumption induces expression of Txnip in the small intestine. Diabetic mice had increased expression of Txnip in the small intestine as well as enhanced fructose uptake and transport into the hepatic portal circulation. The deletion of Txnip in mice abolished the diabetes-induced increase in fructose absorption. Our results indicate that Txnip is a critical regulator of fructose metabolism and suggest that a diabetic state can promote fructose uptake. DOI: http://dx.doi.org/10.7554/eLife.18313.001 PMID:27725089

  1. Inducing pluripotency in vitro: recent advances and highlights in induced pluripotent stem cells generation and pluripotency reprogramming.

    PubMed

    Rony, I K; Baten, A; Bloomfield, J A; Islam, M E; Billah, M M; Islam, K D

    2015-04-01

    Induced pluripotent stem cells (iPSCs) are considered patient-specific counterparts of embryonic stem cells as they originate from somatic cells after forced expression of pluripotency reprogramming factors Oct4, Sox2, Klf4 and c-Myc. iPSCs offer unprecedented opportunity for personalized cell therapies in regenerative medicine. In recent years, iPSC technology has undergone substantial improvement to overcome slow and inefficient reprogramming protocols, and to ensure clinical-grade iPSCs and their functional derivatives. Recent developments in iPSC technology include better reprogramming methods employing novel delivery systems such as non-integrating viral and non-viral vectors, and characterization of alternative reprogramming factors. Concurrently, small chemical molecules (inhibitors of specific signalling or epigenetic regulators) have become crucial to iPSC reprogramming; they have the ability to replace putative reprogramming factors and boost reprogramming processes. Moreover, common dietary supplements, such as vitamin C and antioxidants, when introduced into reprogramming media, have been found to improve genomic and epigenomic profiles of iPSCs. In this article, we review the most recent advances in the iPSC field and potent application of iPSCs, in terms of cell therapy and tissue engineering.

  2. Macro histone variants are critical for the differentiation of human pluripotent cells.

    PubMed

    Barrero, María J; Sese, Borja; Martí, Mercè; Izpisua Belmonte, Juan Carlos

    2013-05-31

    We have previously shown that macro histone variants (macroH2A) are expressed at low levels in stem cells and are up-regulated during differentiation. Here we show that the knockdown of macro histone variants impaired the in vitro and in vivo differentiation of human pluripotent cells, likely through defects in the silencing of pluripotency-related genes. ChIP experiments showed that during differentiation macro histone variants are recruited to the regulatory regions of pluripotency and developmental genes marked with H3K27me3 contributing to the silencing of these genes.

  3. Mitochondria in human pluripotent stem cell apoptosis.

    PubMed

    TeSlaa, Tara; Setoguchi, Kiyoko; Teitell, Michael A

    2016-04-01

    Human pluripotent stem cells (hPSCs) have great potential in regenerative medicine because they can differentiate into any cell type in the body. Genome integrity is vital for human development and for high fidelity passage of genetic information across generations through the germ line. To ensure genome stability, hPSCs maintain a lower rate of mutation than somatic cells and undergo rapid apoptosis in response to DNA damage and additional cell stresses. Furthermore, cellular metabolism and the cell cycle are also differentially regulated between cells in pluripotent and differentiated states and can aid in protecting hPSCs against DNA damage and damaged cell propagation. Despite these safeguards, clinical use of hPSC derivatives could be compromised by tumorigenic potential and possible malignant transformation from failed to differentiate cells. Since hPSCs and mature cells differentially respond to cell stress, it may be possible to specifically target undifferentiated cells for rapid apoptosis in mixed cell populations to enable safer use of hPSC-differentiated cells in patients.

  4. Mitochondria in human pluripotent stem cell apoptosis.

    PubMed

    TeSlaa, Tara; Setoguchi, Kiyoko; Teitell, Michael A

    2016-04-01

    Human pluripotent stem cells (hPSCs) have great potential in regenerative medicine because they can differentiate into any cell type in the body. Genome integrity is vital for human development and for high fidelity passage of genetic information across generations through the germ line. To ensure genome stability, hPSCs maintain a lower rate of mutation than somatic cells and undergo rapid apoptosis in response to DNA damage and additional cell stresses. Furthermore, cellular metabolism and the cell cycle are also differentially regulated between cells in pluripotent and differentiated states and can aid in protecting hPSCs against DNA damage and damaged cell propagation. Despite these safeguards, clinical use of hPSC derivatives could be compromised by tumorigenic potential and possible malignant transformation from failed to differentiate cells. Since hPSCs and mature cells differentially respond to cell stress, it may be possible to specifically target undifferentiated cells for rapid apoptosis in mixed cell populations to enable safer use of hPSC-differentiated cells in patients. PMID:26828436

  5. PREFACE: Physics approaches to protein interactions and gene regulation Physics approaches to protein interactions and gene regulation

    NASA Astrophysics Data System (ADS)

    Nussinov, Ruth; Panchenko, Anna R.; Przytycka, Teresa

    2011-06-01

    networks have been identified, including scale free distribution of the vertex degree, network motifs, and modularity, to name a few. These studies of network organization require the network to be as complete as possible, which given the limitations of experimental techniques is not currently the case. Therefore, experimental procedures for detecting biomolecular interactions should be complemented by computational approaches. The paper by Lees et al provides a review of computational methods, integrating multiple independent sources of data to infer physical and functional protein-protein interaction networks. One of the important aspects of protein interactions that should be accounted for in the prediction of protein interaction networks is that many proteins are composed of distinct domains. Protein domains may mediate protein interactions while proteins and their interaction networks may gain complexity through gene duplication and expansion of existing domain architectures via domain rearrangements. The latter mechanisms have been explored in detail in the paper by Cohen-Gihon et al. Protein-protein interactions are not the only component of the cell's interactome. Regulation of cell activity can be achieved at the level of transcription and involve a transcription factor—DNA binding which typically requires recognition of a specific DNA sequence motif. Chip-Chip and the more recent Chip-Seq technologies allow in vivo identification of DNA binding sites and, together with novel in vitro approaches, provide data necessary for deciphering the corresponding binding motifs. Such information, complemented by structures of protein-DNA complexes and knowledge of the differences in binding sites among homologs, opens the door to constructing predictive binding models. The paper by Persikov and Singh provides an example of such a model in the Cys2His2 zinc finger family. Recent studies have indicated that the presence of such binding motifs is, however, neither necessary

  6. Adhesive interactions regulate transcriptional diversity in malignant B cells.

    PubMed

    Nadav-Dagan, Liat; Shay, Tal; Dezorella, Nili; Naparstek, Elizabeth; Domany, Eytan; Katz, Ben-Zion; Geiger, Benjamin

    2010-04-01

    The genetic profiling of B-cell malignancies is rapidly expanding, providing important information on the tumorigenic potential, response to treatment, and clinical outcome of these diseases. However, the relative contributions of inherent gene expression versus microenvironmental effects are poorly understood. The regulation of gene expression programs by means of adhesive interactions was studied here in ARH-77 human malignant B-cell variants, derived from the same cell line by selective adhesion to a fibronectin matrix. The populations included cells that adhere to fibronectin and are highly tumorigenic (designated "type A" cells) and cells that fail to adhere to fibronectin and fail to develop tumors in vivo ("type F" cells). To identify genes directly affected by cell adhesion to fibronectin, type A cells deprived of an adhesive substrate (designated "AF cells") were also examined. Bioinformatic analyses revealed a remarkable correlation between cell adhesion and both B-cell differentiation state and the expression of multiple myeloma (MM)-associated genes. The highly adherent type A cells expressed higher levels of NFkappaB-regulated genes, many of them associated with MM. Moreover, we found that the transcription of several MM-related proto-oncogenes is stimulated by adhesion to fibronectin. In contrast, type F cells, which display poor adhesive and tumorigenic properties, expressed genes associated with higher levels of B-cell differentiation. Our findings indicate that B-cell differentiation, as manifested by gene expression profiles, is attenuated by cell adhesion to fibronectin, leading to upregulation of specific genes known to be associated with the pathogenesis of MM.

  7. lncRNA maturation to initiate heterochromatin formation in the nucleolus is required for exit from pluripotency in ESCs.

    PubMed

    Savić, Nataša; Bär, Dominik; Leone, Sergio; Frommel, Sandra C; Weber, Fabienne A; Vollenweider, Eva; Ferrari, Elena; Ziegler, Urs; Kaech, Andres; Shakhova, Olga; Cinelli, Paolo; Santoro, Raffaella

    2014-12-01

    The open chromatin of embryonic stem cells (ESCs) condenses into repressive heterochromatin as cells exit the pluripotent state. How the 3D genome organization is orchestrated and implicated in pluripotency and lineage specification is not understood. Here, we find that maturation of the long noncoding RNA (lncRNA) pRNA is required for establishment of heterochromatin at ribosomal RNA genes, the genetic component of nucleoli, and this process is inactivated in pluripotent ESCs. By using mature pRNA to tether heterochromatin at nucleoli of ESCs, we find that localized heterochromatin condensation of ribosomal RNA genes initiates establishment of highly condensed chromatin structures outside of the nucleolus. Moreover, we reveal that formation of such highly condensed, transcriptionally repressed heterochromatin promotes transcriptional activation of differentiation genes and loss of pluripotency. Our findings unravel the nucleolus as an active regulator of chromatin plasticity and pluripotency and challenge current views on heterochromatin regulation and function in ESCs.

  8. Totipotency, Pluripotency and Nuclear Reprogramming

    NASA Astrophysics Data System (ADS)

    Mitalipov, Shoukhrat; Wolf, Don

    Mammalian development commences with the totipotent zygote which is capable of developing into all the specialized cells that make up the adult animal. As development unfolds, cells of the early embryo proliferate and differentiate into the first two lineages, the pluripotent inner cell mass and the trophectoderm. Pluripotent cells can be isolated, adapted and propagated indefinitely in vitro in an undifferentiated state as embryonic stem cells (ESCs). ESCs retain their ability to differentiate into cells representing the three major germ layers: endoderm, mesoderm or ectoderm or any of the 200+ cell types present in the adult body. Since many human diseases result from defects in a single cell type, pluripotent human ESCs represent an unlimited source of any cell or tissue type for replacement therapy thus providing a possible cure for many devastating conditions. Pluripotent cells resembling ESCs can also be derived experimentally by the nuclear reprogramming of somatic cells. Reprogrammed somatic cells may have an even more important role in cell replacement therapies since the patient's own somatic cells can be used for reprogramming thereby eliminating immune based rejection of transplanted cells. In this review, we summarize two major approaches to reprogramming: (1) somatic cell nuclear transfer and (2) direct reprogramming using genetic manipulations.

  9. Novel repressor regulates insulin sensitivity through interaction with Foxo1

    PubMed Central

    Nakae, Jun; Cao, Yongheng; Hakuno, Fumihiko; Takemori, Hiroshi; Kawano, Yoshinaga; Sekioka, Risa; Abe, Takaya; Kiyonari, Hiroshi; Tanaka, Toshiya; Sakai, Juro; Takahashi, Shin-Ichiro; Itoh, Hiroshi

    2012-01-01

    Forkhead box-containing protein o (Foxo) 1 is a key transcription factor in insulin and glucose metabolism. We identified a Foxo1-CoRepressor (FCoR) protein in mouse adipose tissue that inhibits Foxo1's activity by enhancing acetylation via impairment of the interaction between Foxo1 and the deacetylase Sirt1 and via direct acetylation. FCoR is phosphorylated at Threonine 93 by catalytic subunit of protein kinase A and is translocated into nucleus, making it possible to bind to Foxo1 in both cytosol and nucleus. Knockdown of FCoR in 3T3-F442A cells enhanced expression of Foxo target and inhibited adipocyte differentiation. Overexpression of FCoR in white adipose tissue decreased expression of Foxo-target genes and adipocyte size and increased insulin sensitivity in Leprdb/db mice and in mice fed a high-fat diet. In contrast, Fcor knockout mice were lean, glucose intolerant, and had decreased insulin sensitivity that was accompanied by increased expression levels of Foxo-target genes and enlarged adipocytes. Taken together, these data suggest that FCoR is a novel repressor that regulates insulin sensitivity and energy metabolism in adipose tissue by acting to fine-tune Foxo1 activity. PMID:22510882

  10. Aurora B interaction of centrosomal Nlp regulates cytokinesis.

    PubMed

    Yan, Jie; Jin, Shunqian; Li, Jia; Zhan, Qimin

    2010-12-17

    Cytokinesis is a fundamental cellular process, which ensures equal abscission and fosters diploid progenies. Aberrant cytokinesis may result in genomic instability and cell transformation. However, the underlying regulatory machinery of cytokinesis is largely undefined. Here, we demonstrate that Nlp (Ninein-like protein), a recently identified BRCA1-associated centrosomal protein that is required for centrosomes maturation at interphase and spindle formation in mitosis, also contributes to the accomplishment of cytokinesis. Through immunofluorescent analysis, Nlp is found to localize at midbody during cytokinesis. Depletion of endogenous Nlp triggers aborted division and subsequently leads to multinucleated phenotypes. Nlp can be recruited by Aurora B to the midbody apparatus via their physical association at the late stage of mitosis. Disruption of their interaction induces aborted cytokinesis. Importantly, Nlp is characterized as a novel substrate of Aurora B and can be phosphorylated by Aurora B. The specific phosphorylation sites are mapped at Ser-185, Ser-448, and Ser-585. The phosphorylation at Ser-448 and Ser-585 is likely required for Nlp association with Aurora B and localization at midbody. Meanwhile, the phosphorylation at Ser-185 is vital to Nlp protein stability. Disruptions of these phosphorylation sites abolish cytokinesis and lead to chromosomal instability. Collectively, these observations demonstrate that regulation of Nlp by Aurora B is critical for the completion of cytokinesis, providing novel insights into understanding the machinery of cell cycle progression.

  11. A BRCA1-interacting lncRNA regulates homologous recombination.

    PubMed

    Sharma, Vivek; Khurana, Simran; Kubben, Nard; Abdelmohsen, Kotb; Oberdoerffer, Philipp; Gorospe, Myriam; Misteli, Tom

    2015-11-01

    Long non-coding RNAs (lncRNAs) are important players in diverse biological processes. Upon DNA damage, cells activate a complex signaling cascade referred to as the DNA damage response (DDR). Using a microarray screen, we identify here a novel lncRNA, DDSR1 (DNA damage-sensitive RNA1), which is induced upon DNA damage. DDSR1 induction is triggered in an ATM-NF-κB pathway-dependent manner by several DNA double-strand break (DSB) agents. Loss of DDSR1 impairs cell proliferation and DDR signaling and reduces DNA repair capacity by homologous recombination (HR). The HR defect in the absence of DDSR1 is marked by aberrant accumulation of BRCA1 and RAP80 at DSB sites. In line with a role in regulating HR, DDSR1 interacts with BRCA1 and hnRNPUL1, an RNA-binding protein involved in DNA end resection. Our results suggest a role for the lncRNA DDSR1 in modulating DNA repair by HR.

  12. Cellular regulation and molecular interactions of the ferritins.

    PubMed

    Hintze, K J; Theil, E C

    2006-03-01

    Controlling iron/oxygen chemistry in biology depends on multiple genes, regulatory messenger RNA (mRNA) structures, signaling pathways and protein catalysts. Ferritin, a protein nanocage around an iron/oxy mineral, centralizes the control. Complementary DNA (antioxidant responsive element/Maf recognition element) and mRNA (iron responsive element) responses regulate ferritin synthesis rates. Multiple iron-protein interactions control iron and oxygen substrate movement through the protein cage, from dynamic gated pores to catalytic sites related to di-iron oxygenase cofactor sites. Maxi-ferritins concentrate iron for the bio-synthesis of iron/heme proteins, trapping oxygen; bacterial mini-ferritins, DNA protection during starvation proteins, reverse the substrate roles, destroying oxidants, trapping iron and protecting DNA. Ferritin is nature's unique and conserved approach to controlled, safe use of iron and oxygen, with protein synthesis in animals adjusted by dual, genetic DNA and mRNA sequences that selectively respond to iron or oxidant signals and link ferritin to proteins of iron, oxygen and antioxidant metabolism.

  13. Regulating social interactions: Developing a functional theory of collaboration

    NASA Astrophysics Data System (ADS)

    Borge, Marcela

    A role-playing intervention was developed and implemented in a fifth grade classroom. The goal of the intervention was to address serious problems that researchers have connected to dysfunctional collaborative interactions. These problems include an inability to: engage in important aspects of argumentation and communication, monitor and regulate group processes, and ensure equity in participation. To this end, a comprehensive theory of collaboration was presented to students through the use of four sociocognitive roles: mediation manager, collaboration manager, communication manager, and productivity manager. Each role came with a written guide that included specific goals and strategies related to the role. Metacognitive activities, including planning and reflection, were also used during class sessions to support students' understanding and role-use. Each of the students in the class was assigned one of the roles to manage during a two part collaborative science project. Students took quizzes on the roles and provided verbal and written feedback about their role-use and metacognitive activities. Students from one of the video-recorded groups were also interviewed after the intervention. Analyses of data from video sessions, quizzes, and interviews supported three important findings: (1) students were able to learn goals, and strategies for all of the roles, even though they only managed a single role, (2) students demonstrated the ability to take the information they learned and put it into practice, and (3) when students employed the roles while their group was working, members of the group accepted the role-use. These findings related to the learning and utilization of the roles are important because they: (1) imply that the intervention was successful at developing students' knowledge of the theory of collaboration that the roles represented, (2) indicate that students used this knowledge to monitor and regulate behaviors in an authentic context, and (3

  14. 3D Chromosome Regulatory Landscape of Human Pluripotent Cells.

    PubMed

    Ji, Xiong; Dadon, Daniel B; Powell, Benjamin E; Fan, Zi Peng; Borges-Rivera, Diego; Shachar, Sigal; Weintraub, Abraham S; Hnisz, Denes; Pegoraro, Gianluca; Lee, Tong Ihn; Misteli, Tom; Jaenisch, Rudolf; Young, Richard A

    2016-02-01

    In this study, we describe the 3D chromosome regulatory landscape of human naive and primed embryonic stem cells. To devise this map, we identified transcriptional enhancers and insulators in these cells and placed them within the context of cohesin-associated CTCF-CTCF loops using cohesin ChIA-PET data. The CTCF-CTCF loops we identified form a chromosomal framework of insulated neighborhoods, which in turn form topologically associating domains (TADs) that are largely preserved during the transition between the naive and primed states. Regulatory changes in enhancer-promoter interactions occur within insulated neighborhoods during cell state transition. The CTCF anchor regions we identified are conserved across species, influence gene expression, and are a frequent site of mutations in cancer cells, underscoring their functional importance in cellular regulation. These 3D regulatory maps of human pluripotent cells therefore provide a foundation for future interrogation of the relationships between chromosome structure and gene control in development and disease. PMID:26686465

  15. Vascular Potential of Human Pluripotent Stem Cells

    PubMed Central

    Iacobas, Ionela; Vats, Archana; Hirschi, Karen K.

    2010-01-01

    Cardiovascular disease is the number one cause of death and disability in the US. Understanding the biological activity of stem and progenitor cells, and their ability to contribute to the repair, regeneration and remodeling of the heart and blood vessels affected by pathologic processes is an essential part of the paradigm in enabling us to achieve a reduction in related deaths. Both human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are promising sources of cells for clinical cardiovascular therapies. Additional in vitro studies are needed, however, to understand their relative phenotypes and molecular regulation toward cardiovascular cell fates. Further studies in translational animal models are also needed to gain insights into the potential and function of both human ES- and iPS-derived cardiovascular cells, and enable translation from experimental and pre-clinical studies to human trials. PMID:20453170

  16. Imaging Pluripotency: Time-Lapse Analysis of Mouse Embryonic Stem Cells.

    PubMed

    Pezzarossa, Anna; Guedes, Ana M V; Henrique, Domingos; Abranches, Elsa

    2016-01-01

    The current view of the pluripotent state is that of a transient, dynamic state, maintained by the balance between opposing cues. Understanding how this dynamic state is established in pluripotent cells and how it relates to gene expression is essential to obtain a more detailed description of the pluripotent state.In this chapter, we describe how to study the dynamic expression of a core pluripotency gene regulator-Nanog-by exploiting single-cell time-lapse imaging of a reporter mESC line grown in different cell culture media. We further describe an automated image analysis method and discuss how to extract information from the generated quantitative time-course data.

  17. SILAC proteomics of planarians identifies Ncoa5 as a conserved component of pluripotent stem cells.

    PubMed

    Böser, Alexander; Drexler, Hannes C A; Reuter, Hanna; Schmitz, Henning; Wu, Guangming; Schöler, Hans R; Gentile, Luca; Bartscherer, Kerstin

    2013-11-27

    Planarian regeneration depends on the presence of pluripotent stem cells in the adult. We developed an in vivo stable isotope labeling by amino acids in cell culture (SILAC) protocol in planarians to identify proteins that are enriched in planarian stem cells. Through a comparison of SILAC proteomes of normal and stem cell-depleted planarians and of a stem cell-enriched population of sorted cells, we identified hundreds of stem cell proteins. One of these is an ortholog of nuclear receptor coactivator-5 (Ncoa5/CIA), which is known to regulate estrogen-receptor-mediated transcription in human cells. We show that Ncoa5 is essential for the maintenance of the pluripotent stem cell population in planarians and that a putative mouse ortholog is expressed in pluripotent cells of the embryo. Our study thus identifies a conserved component of pluripotent stem cells, demonstrating that planarians, in particular, when combined with in vivo SILAC, are a powerful model in stem cell research.

  18. Expression of stem cell pluripotency factors during regeneration in the earthworm Eisenia foetida.

    PubMed

    Zheng, Pengfei; Shao, Qiang; Diao, Xiaoping; Li, Zandong; Han, Qian

    2016-01-01

    Stem cell pluripotency factors can induce somatic cells to form induced pluripotent stem cells, which are involved in cell reprogramming and dedifferentiation. The tissue regeneration in the earthworm Eisenia foetida may involve cell dedifferentiation. There is limited information about associations between pluripotency factors and the regeneration. In this report, cDNA sequences of pluripotency factors, oct4, nanog, sox2, c-myc and lin28 genes from the earthworm E. foetida were cloned, and quantitative PCR analysis was performed for their mRNA expressions in the head, clitellum and tail. The maximum up-regulation of oct4, nanog, sox2, c-myc and lin28 occurred at 12h, 4 days, 12h, 2 days, and 24h after amputation for 110, 178, 21, 251 and 325-fold, respectively, in comparison with the controls. The results suggest that the tissues are regenerated via cellular dedifferentiation and reprogramming. PMID:26299657

  19. OSKM Induce Extraembryonic Endoderm Stem Cells in Parallel to Induced Pluripotent Stem Cells

    PubMed Central

    Parenti, Anthony; Halbisen, Michael A.; Wang, Kai; Latham, Keith; Ralston, Amy

    2016-01-01

    Summary The reprogramming factors OCT4, SOX2, KLF4, and MYC (OSKM) can reactivate the pluripotency network in terminally differentiated cells, but also regulate expression of non-pluripotency genes in other contexts, such as the mouse primitive endoderm. The primitive endoderm is an extraembryonic lineage established in parallel to the pluripotent epiblast in the blastocyst, and is the progenitor pool for extraembryonic endoderm stem (XEN) cells. We show that OSKM induce expression of endodermal genes, leading to formation of induced XEN (iXEN) cells, which possess key properties of blastocyst-derived XEN cells, including morphology, transcription profile, self-renewal, and multipotency. Our data show that iXEN cells arise in parallel to induced pluripotent stem cells, indicating that OSKM drive cells to two distinct cell fates during reprogramming. PMID:26947975

  20. Zinc Chloride Transiently Maintains Mouse Embryonic Stem Cell Pluripotency by Activating Stat3 Signaling

    PubMed Central

    Hu, Jing; Yang, Zhiyong; Wang, Jinbo; Yu, Jia; Guo, Jing; Liu, Shiying; Qian, Chunmei; Song, Liwen; Wu, Yi; Cheng, Jiajing

    2016-01-01

    An improved understanding of the pluripotency maintenance of embryonic stem (ES) cells is important for investigations of early embryo development and for cell replacement therapy, but the mechanism behind pluripotency is still incompletely understood. Recent findings show that zinc, an essential trace element in humans, is critically involved in regulating various signaling pathways and genes expression. However, its role in ES cell fate determination remains to be further explored. Here we showed that 2μM zinc chloride (ZnCl2) transiently maintained mouse ES cell pluripotency in vitro. The cultured mouse ES cells remained undifferentiated under 2μM ZnCl2 treatment in leukemia inhibitory factor (LIF) withdrawal, retinoic acid (RA) or embryoid bodies (EBs) differentiation assays. In addition, ZnCl2 increased pluripotency genes expression and inhibited differentiation genes expression. Further mechanistic studies revealed that ZnCl2 transiently activated signal transducers and activators of transcription 3 (Stat3) signaling through promoting Stat3 phosphorylation. Inhibition of Stat3 signaling abrogated the effects of ZnCl2 on mouse ES cell pluripotency. Taken together, this study demonstrated a critical role of zinc in the pluripotency maintenance of mouse ES cells, as well as an important regulator of Stat3 signaling. PMID:26910359

  1. The transcriptional foundation of pluripotency.

    PubMed

    Chambers, Ian; Tomlinson, Simon R

    2009-07-01

    A fundamental goal in biology is to understand the molecular basis of cell identity. Pluripotent embryonic stem (ES) cell identity is governed by a set of transcription factors centred on the triumvirate of Oct4, Sox2 and Nanog. These proteins often bind to closely localised genomic sites. Recent studies have identified additional transcriptional modulators that bind to chromatin near sites occupied by Oct4, Sox2 and Nanog. This suggests that the combinatorial control of gene transcription might be fundamental to the ES cell state. Here we discuss how these observations advance our understanding of the transcription factor network that controls pluripotent identity and highlight unresolved issues that arise from these studies. PMID:19542351

  2. Self-renewal and pluripotency acquired through somatic reprogramming to human cancer stem cells.

    PubMed

    Nagata, Shogo; Hirano, Kunio; Kanemori, Michele; Sun, Liang-Tso; Tada, Takashi

    2012-01-01

    Human induced pluripotent stem cells (iPSCs) are reprogrammed by transient expression of transcription factors in somatic cells. Approximately 1% of somatic cells can be reprogrammed into iPSCs, while the remaining somatic cells are differentially reprogrammed. Here, we established induced pluripotent cancer stem-like cells (iCSCs) as self-renewing pluripotent cell clones. Stable iCSC lines were established from unstable induced epithelial stem cell (iESC) lines through re-plating followed by embryoid body formation and serial transplantation. iCSCs shared the expression of pluripotent marker genes with iPSCs, except for REX1 and LIN28, while exhibited the expression of somatic marker genes EMP1 and PPARγ. iESCs and iCSCs could generate teratomas with high efficiency by implantation into immunodeficient mice. The second iCSCs isolated from dissociated cells of teratoma from the first iCSCs were stably maintained, showing a gene expression profile similar to the first iCSCs. In the first and second iCSCs, transgene-derived Oct4, Sox2, Klf4, and c-Myc were expressed. Comparative global gene expression analyses demonstrated that the first iCSCs were similar to iESCs, and clearly different from human iPSCs and somatic cells. In iCSCs, gene expression kinetics of the core pluripotency factor and the Myc-related factor were pluripotent type, whereas the polycomb complex factor was somatic type. These findings indicate that pluripotent tumorigenicity can be conferred on somatic cells through up-regulation of the core pluripotency and Myc-related factors, prior to establishment of the iPSC molecular network by full reprogramming through down-regulation of the polycomb complex factor.

  3. Interactions of Metacognition with Motivation and Affect in Self-Regulated Learning: The MASRL Model

    ERIC Educational Resources Information Center

    Efklides, Anastasia

    2011-01-01

    Metacognition, motivation, and affect are components of self-regulated learning (SRL) that interact. The "metacognitive and affective model of self-regulated learning" (the MASRL model) distinguishes two levels of functioning in SRL, namely, the Person level and the Task x Person level. At the Person level interactions between trait-like…

  4. Induction of pluripotency by defined factors

    PubMed Central

    TANABE, Koji; TAKAHASHI, Kazutoshi; YAMANAKA, Shinya

    2014-01-01

    The “reversion of cell fate from differentiated states back into totipotent or pluripotent states” has been an interest of many scientists for a long time. With the help of knowledge accumulated by those scientists, we succeeded in converting somatic cells to a pluripotent cell lineage by the forced expression of defined factors. These established induced pluripotent stem (iPS) cells have similar features to embryonic stem (ES) cells, including pluripotency and immortality. The iPS cell technology provides unprecedented opportunities for regenerative medicine and drug discovery. PMID:24621955

  5. Peptide-decorated polyvinyl alcohol/hyaluronan nanofibers for human induced pluripotent stem cell culture.

    PubMed

    Deng, Yi; Zhang, Xiaohong; Zhao, Yinghui; Liang, Shangshang; Xu, Anxiu; Gao, Xiang; Deng, Feng; Fang, Jing; Wei, Shicheng

    2014-01-30

    Realization of the full potential of human induced pluripotent stem cells (hiPSCs) in clinical applications requires development of well-defined conditions for their growth and differentiation. A novel fully defined polyvinyl alcohol/hyaluronan (PVA/HA) polysaccharide nanofiber was developed for hiPSCs culture in commercially available xeno-free, chemically defined medium. Vitronectin peptide (VP) was immobilized to PVA/HA nanofibers through NHS/EDC chemistry. The hiPSCs successfully grew and proliferated on the VP-decorated PVA/HA nanofibers, similar to those on Matrigel™. Such well-defined, xeno-free and safe nanofiber substrate that supports culture of hiPSCs will not only help to accelerate the translational perspectives of hiPSCs, but also provide a platform to investigate the cell-nanofiber interaction mechanisms that regulate stem cell proliferation and differentiation.

  6. Methods to Manipulate and Monitor Wnt Signaling in Human Pluripotent Stem Cells.

    PubMed

    Huggins, Ian J; Brafman, David; Willert, Karl

    2016-01-01

    Human pluripotent stem cells (hPSCs) may revolutionize medical practice by providing: (a) a renewable source of cells for tissue replacement therapies, (b) a powerful system to model human diseases in a dish, and (c) a platform for examining efficacy and safety of novel drugs. Furthermore, these cells offer a unique opportunity to study early human development in vitro, in particular, the process by which a seemingly uniform cell population interacts to give rise to the three main embryonic lineages: ectoderm, endoderm. and mesoderm. This process of lineage allocation is regulated by a number of inductive signals that are mediated by growth factors, including FGF, TGFβ, and Wnt. In this book chapter, we introduce a set of tools, methods, and protocols to specifically manipulate the Wnt signaling pathway with the intention of altering the cell fate outcome of hPSCs. PMID:27590161

  7. Human pluripotent stem cell culture density modulates YAP signaling.

    PubMed

    Hsiao, Cheston; Lampe, Michael; Nillasithanukroh, Songkhun; Han, Wenqing; Lian, Xiaojun; Palecek, Sean P

    2016-05-01

    Human pluripotent stem cell (hPSC) density is an important factor in self-renewal and differentiation fates; however, the mechanisms through which hPSCs sense cell density and process this information in making cell fate decisions remain to be fully understood. One particular pathway that may prove important in density-dependent signaling in hPSCs is the Hippo pathway, which is regulated by cell-cell contact and mechanosensing through the cytoskeleton and has been linked to the maintenance of stem cell pluripotency. To probe regulation of Hippo pathway activity in hPSCs, we assessed whether Hippo pathway transcriptional activator YAP was differentially modulated by cell density. At higher cell densities, YAP phosphorylation and localization to the cytoplasm increased, which led to decreased YAP-mediated transcriptional activity. Furthermore, total YAP protein levels diminished at high cell density due to the phosphorylation-targeted degradation of YAP. Inducible shRNA knockdown of YAP reduced expression of YAP target genes and pluripotency genes. Finally, the density-dependent increase of neuroepithelial cell differentiation was mitigated by shRNA knockdown of YAP. Our results suggest a pivotal role of YAP in cell density-mediated fate decisions in hPSCs.

  8. Electromagnetic fields mediate efficient cell reprogramming into a pluripotent state.

    PubMed

    Baek, Soonbong; Quan, Xiaoyuan; Kim, Soochan; Lengner, Christopher; Park, Jung-Keug; Kim, Jongpil

    2014-10-28

    Life on Earth is constantly exposed to natural electromagnetic fields (EMFs), and it is generally accepted that EMFs may exert a variety of effects on biological systems. Particularly, extremely low-frequency electromagnetic fields (EL-EMFs) affect biological processes such as cell development and differentiation; however, the fundamental mechanisms by which EMFs influence these processes remain unclear. Here we show that EMF exposure induces epigenetic changes that promote efficient somatic cell reprogramming to pluripotency. These epigenetic changes resulted from EMF-induced activation of the histone lysine methyltransferase Mll2. Remarkably, an EMF-free system that eliminates Earth's naturally occurring magnetic field abrogates these epigenetic changes, resulting in a failure to undergo reprogramming. Therefore, our results reveal that EMF directly regulates dynamic epigenetic changes through Mll2, providing an efficient tool for epigenetic reprogramming including the acquisition of pluripotency. PMID:25248035

  9. Epigenetic Biomarker to Support Classification into Pluripotent and Non-Pluripotent Cells

    NASA Astrophysics Data System (ADS)

    Lenz, Michael; Goetzke, Roman; Schenk, Arne; Schubert, Claudia; Veeck, Jürgen; Hemeda, Hatim; Koschmieder, Steffen; Zenke, Martin; Schuppert, Andreas; Wagner, Wolfgang

    2015-03-01

    Quality control of human induced pluripotent stem cells (iPSCs) can be performed by several methods. These methods are usually relatively labor-intensive, difficult to standardize, or they do not facilitate reliable quantification. Here, we describe a biomarker to distinguish between pluripotent and non-pluripotent cells based on DNA methylation (DNAm) levels at only three specific CpG sites. Two of these CpG sites were selected by their discriminatory power in 258 DNAm profiles - they were either methylated in pluripotent or non-pluripotent cells. The difference between these two β-values provides an Epi-Pluri-Score that was validated on independent DNAm-datasets (264 pluripotent and 1,951 non-pluripotent samples) with 99.9% specificity and 98.9% sensitivity. This score was complemented by a third CpG within the gene POU5F1 (OCT4), which better demarcates early differentiation events. We established pyrosequencing assays for the three relevant CpG sites and thereby correctly classified DNA of 12 pluripotent cell lines and 31 non-pluripotent cell lines. Furthermore, DNAm changes at these three CpGs were tracked in the course of differentiation of iPSCs towards mesenchymal stromal cells. The Epi-Pluri-Score does not give information on lineage-specific differentiation potential, but it provides a simple, reliable, and robust biomarker to support high-throughput classification into either pluripotent or non-pluripotent cells.

  10. CD24 tracks divergent pluripotent states in mouse and human cells

    PubMed Central

    Shakiba, Nika; White, Carl A.; Lipsitz, Yonatan Y.; Yachie-Kinoshita, Ayako; Tonge, Peter D; Hussein, Samer M. I.; Puri, Mira C.; Elbaz, Judith; Morrissey-Scoot, James; Li, Mira; Munoz, Javier; Benevento, Marco; Rogers, Ian M.; Hanna, Jacob H.; Heck, Albert J. R.; Wollscheid, Bernd; Nagy, Andras; Zandstra, Peter W

    2015-01-01

    Reprogramming is a dynamic process that can result in multiple pluripotent cell types emerging from divergent paths. Cell surface protein expression is a particularly desirable tool to categorize reprogramming and pluripotency as it enables robust quantification and enrichment of live cells. Here we use cell surface proteomics to interrogate mouse cell reprogramming dynamics and discover CD24 as a marker that tracks the emergence of reprogramming-responsive cells, while enabling the analysis and enrichment of transgene-dependent (F-class) and -independent (traditional) induced pluripotent stem cells (iPSCs) at later stages. Furthermore, CD24 can be used to delineate epiblast stem cells (EpiSCs) from embryonic stem cells (ESCs) in mouse pluripotent culture. Importantly, regulated CD24 expression is conserved in human pluripotent stem cells (PSCs), tracking the conversion of human ESCs to more naive-like PSC states. Thus, CD24 is a conserved marker for tracking divergent states in both reprogramming and standard pluripotent culture. PMID:26076835

  11. Mga is essential for the survival of pluripotent cells during peri-implantation development.

    PubMed

    Washkowitz, Andrew J; Schall, Caroline; Zhang, Kun; Wurst, Wolfgang; Floss, Thomas; Mager, Jesse; Papaioannou, Virginia E

    2015-01-01

    The maintenance and control of pluripotency is of great interest in stem cell biology. The dual specificity T-box/basic-helix-loop-helix-zipper transcription factor Mga is expressed in the pluripotent cells of the inner cell mass (ICM) and epiblast of the peri-implantation mouse embryo, but its function has not been investigated previously. Here, we use a loss-of-function allele and RNA knockdown to demonstrate that Mga depletion leads to the death of proliferating pluripotent ICM cells in vivo and in vitro, and the death of embryonic stem cells (ESCs) in vitro. Additionally, quiescent pluripotent cells lacking Mga are lost during embryonic diapause. Expression of Odc1, the rate-limiting enzyme in the conversion of ornithine into putrescine in the synthesis of polyamines, is reduced in Mga mutant cells, and the survival of mutant ICM cells as well as ESCs is rescued in culture by the addition of exogenous putrescine. These results suggest a mechanism whereby Mga influences pluripotent cell survival through regulation of the polyamine pool in pluripotent cells of the embryo, whether they are in a proliferative or quiescent state.

  12. The developmental dismantling of pluripotency is reversed by ectopic Oct4 expression

    PubMed Central

    Osorno, Rodrigo; Tsakiridis, Anestis; Wong, Frederick; Cambray, Noemí; Economou, Constantinos; Wilkie, Ronald; Blin, Guillaume; Scotting, Paul J.; Chambers, Ian; Wilson, Valerie

    2012-01-01

    The transcription factors Nanog and Oct4 regulate pluripotency in the pre-implantation epiblast and in derivative embryonic stem cells. During post-implantation development, the precise timing and mechanism of the loss of pluripotency is unknown. Here, we show that in the mouse, pluripotency is extinguished at the onset of somitogenesis, coincident with reduced expression and chromatin accessibility of Oct4 and Nanog regulatory regions. Prior to somitogenesis expression of both Nanog and Oct4 is regionalized. We show that pluripotency tracks the in vivo level of Oct4 and not Nanog by assessing the ability to reactivate or maintain Nanog expression in cell culture. Enforced Oct4 expression in somitogenesis-stage tissue provokes rapid reopening of Oct4 and Nanog chromatin, Nanog re-expression and resuscitates moribund pluripotency. Our data suggest that decreasing Oct4 expression is converted to a sudden drop in competence to maintain pluripotency gene regulatory network activity that is subsequently stabilized by epigenetic locks. PMID:22669820

  13. Concise Review: Control of Cell Fate Through Cell Cycle and Pluripotency Networks.

    PubMed

    Boward, Ben; Wu, Tianming; Dalton, Stephen

    2016-06-01

    Pluripotent stem cells (PSCs) proliferate rapidly with a characteristic cell cycle structure consisting of short G1- and G2-gap phases. This applies broadly to PSCs of peri-implantation stage embryos, cultures of embryonic stem cells, induced pluripotent stem cells, and embryonal carcinoma cells. During the early stages of PSC differentiation however, cell division times increase as a consequence of cell cycle remodeling. Most notably, this is indicated by elongation of the G1-phase. Observations linking changes in the cell cycle with exit from pluripotency have raised questions about the role of cell cycle control in maintenance of the pluripotent state. Until recently however, this has been a difficult question to address because of limitations associated with experimental tools. Recent studies now show that pluripotency and cell cycle regulatory networks are intertwined and that cell cycle control mechanisms are an integral, mechanistic part of the PSC state. Studies in embryonal carcinoma, some 30 years ago, first suggested that pluripotent cells initiate differentiation when in the G1-phase. More recently, a molecular "priming" mechanism has been proposed to explain these observations in human embryonic stem cells. Complexity in this area has been increased by the realization that pluripotent cells exist in multiple developmental states and that in addition to each having their own characteristic gene expression and epigenetic signatures, they potentially have alternate modes of cell cycle regulation. This review will summarize current knowledge in these areas and will highlight important aspects of interconnections between the cell cycle, self-renewal, pluripotency, and cell fate decisions. Stem Cells 2016;34:1427-1436.

  14. Resetting Human Naïve Pluripotency

    PubMed Central

    Xiao, Jifang; Mai, Daniel H.; Xie, Liangqi

    2016-01-01

    The rodent naive pluripotent state is believed to represent the preimplantation inner cell mass state of the developing blastocyst and can derive self-renewing pluripotent embryonic stem cells (ESCs) in vitro. Nevertheless, human ESCs exhibit epigenetic, metabolic, and transcriptomic characteristics more akin to primed pluripotent stem cells (PSCs) derived from the postimplantation epiblast. Understanding the genetic and epigenetic mechanisms that constrain human ESCs in the primed state is crucial for the human naive pluripotent state resetting and numerous applications in regenerative medicine. In this review, we begin by defining the naive and primed states in the murine model and compare the epigenetic characteristics of those states to the human PSCs. We also examine the various reprogramming schemes to derive the human naive pluripotent state. Finally, we discuss future perspectives of studying and deriving the human naive PSCs in the context of cellular engineering and regenerative medicine. PMID:27512340

  15. Expression of stem cell pluripotency factors during regeneration in newts.

    PubMed

    Maki, Nobuyasu; Suetsugu-Maki, Rinako; Tarui, Hiroshi; Agata, Kiyokazu; Del Rio-Tsonis, Katia; Tsonis, Panagiotis A

    2009-06-01

    In this study, we present data indicating that mammalian stem cell pluripotency-inducing factors are expressed during lens and limb regeneration in newts. The apparent expression even in intact tissues and the ensued regulation during regeneration raises the possibility that these factors might regulate tissue-specific reprogramming and regeneration. Furthermore, these factors should enable us to understand the similarities and differences between animal regeneration in the newt and stem cell strategies in mammals. Developmental Dynamics 238:1613-1616, 2009. (c) 2009 Wiley-Liss, Inc.

  16. Novel regulation and functional interaction of polycistronic miRNAs.

    PubMed

    Truscott, Mary; Islam, Abul B M M K; Frolov, Maxim V

    2016-01-01

    The importance of microRNAs in gene expression and disease is well recognized. However, what is less appreciated is that almost half of miRNA genes are organized in polycistronic clusters and are therefore coexpressed. The mir-11∼998 cluster consists of two miRNAs, miR-11 and miR-998. Here, we describe a novel layer of regulation that links the processing and expression of miR-998 to the presence of the mir-11 gene. We show that the presence of miR-11 in the pri-miRNA is required for processing by Drosha, and deletion of mir-11 prevents the expression of miR-998. Replacing mir-11 with an unrelated miRNA rescued miR-998 expression in vivo and in vitro, as did expressing miR-998 from a shorter, more canonical miRNA scaffold. The embedded regulation of miR-998 is functionally important because unchecked miR-998 expression in the absence of miR-11 resulted in pleiotropic developmental defects. This novel regulation of expression of miRNAs within a cluster is not limited to the mir-11∼998 cluster and, thus, likely reflects the more general cis-regulation of expression of individual miRNAs. Collectively, our results uncover a novel layer of regulation within miRNA clusters that tempers the functions of the individual miRNAs. Unlinking their expression has the potential to change the expression of multiple miRNA targets and shift a biological response. PMID:26554028

  17. Inhibition of β-catenin–TCF1 interaction delays differentiation of mouse embryonic stem cells

    PubMed Central

    Chatterjee, Sujash S.; Saj, Abil; Gocha, Tenzin; Murphy, Matthew; Gonsalves, Foster C.; Zhang, Xiaoqian; Hayward, Penelope; Akgöl Oksuz, Betül; Shen, Steven S.; Madar, Aviv; Martinez Arias, Alfonso

    2015-01-01

    The ability of mouse embryonic stem cells (mESCs) to self-renew or differentiate into various cell lineages is regulated by signaling pathways and a core pluripotency transcriptional network (PTN) comprising Nanog, Oct4, and Sox2. The Wnt/β-catenin pathway promotes pluripotency by alleviating T cell factor TCF3-mediated repression of the PTN. However, it has remained unclear how β-catenin’s function as a transcriptional activator with TCF1 influences mESC fate. Here, we show that TCF1-mediated transcription is up-regulated in differentiating mESCs and that chemical inhibition of β-catenin/TCF1 interaction improves long-term self-renewal and enhances functional pluripotency. Genetic loss of TCF1 inhibited differentiation by delaying exit from pluripotency and conferred a transcriptional profile strikingly reminiscent of self-renewing mESCs with high Nanog expression. Together, our data suggest that β-catenin’s function in regulating mESCs is highly context specific and that its interaction with TCF1 promotes differentiation, further highlighting the need for understanding how its individual protein–protein interactions drive stem cell fate. PMID:26459597

  18. Multiple cell and population-level interactions with mouse embryonic stem cell heterogeneity.

    PubMed

    Cannon, Danielle; Corrigan, Adam M; Miermont, Agnes; McDonel, Patrick; Chubb, Jonathan R

    2015-08-15

    Much of development and disease concerns the generation of gene expression differences between related cells sharing similar niches. However, most analyses of gene expression only assess population and time-averaged levels of steady-state transcription. The mechanisms driving differentiation are buried within snapshots of the average cell, lacking dynamic information and the diverse regulatory history experienced by individual cells. Here, we use a quantitative imaging platform with large time series data sets to determine the regulation of developmental gene expression by cell cycle, lineage, motility and environment. We apply this technology to the regulation of the pluripotency gene Nanog in mouse embryonic stem cells. Our data reveal the diversity of cell and population-level interactions with Nanog dynamics and heterogeneity, and how this regulation responds to triggers of pluripotency. Cell cycles are highly heterogeneous and cycle time increases with Nanog reporter expression, with longer, more variable cycle times as cells approach ground-state pluripotency. Nanog reporter expression is highly stable over multiple cell generations, with fluctuations within cycles confined by an attractor state. Modelling reveals an environmental component to expression stability, in addition to any cell-autonomous behaviour, and we identify interactions of cell density with both cycle behaviour and Nanog. Rex1 expression dynamics showed shared and distinct regulatory effects. Overall, our observations of multiple partially overlapping dynamic heterogeneities imply complex cell and environmental regulation of pluripotent cell behaviour, and suggest simple deterministic views of stem cell states are inappropriate.

  19. Multiple cell and population-level interactions with mouse embryonic stem cell heterogeneity

    PubMed Central

    Cannon, Danielle; Corrigan, Adam M.; Miermont, Agnes; McDonel, Patrick; Chubb, Jonathan R.

    2015-01-01

    Much of development and disease concerns the generation of gene expression differences between related cells sharing similar niches. However, most analyses of gene expression only assess population and time-averaged levels of steady-state transcription. The mechanisms driving differentiation are buried within snapshots of the average cell, lacking dynamic information and the diverse regulatory history experienced by individual cells. Here, we use a quantitative imaging platform with large time series data sets to determine the regulation of developmental gene expression by cell cycle, lineage, motility and environment. We apply this technology to the regulation of the pluripotency gene Nanog in mouse embryonic stem cells. Our data reveal the diversity of cell and population-level interactions with Nanog dynamics and heterogeneity, and how this regulation responds to triggers of pluripotency. Cell cycles are highly heterogeneous and cycle time increases with Nanog reporter expression, with longer, more variable cycle times as cells approach ground-state pluripotency. Nanog reporter expression is highly stable over multiple cell generations, with fluctuations within cycles confined by an attractor state. Modelling reveals an environmental component to expression stability, in addition to any cell-autonomous behaviour, and we identify interactions of cell density with both cycle behaviour and Nanog. Rex1 expression dynamics showed shared and distinct regulatory effects. Overall, our observations of multiple partially overlapping dynamic heterogeneities imply complex cell and environmental regulation of pluripotent cell behaviour, and suggest simple deterministic views of stem cell states are inappropriate. PMID:26209649

  20. Multiple cell and population-level interactions with mouse embryonic stem cell heterogeneity.

    PubMed

    Cannon, Danielle; Corrigan, Adam M; Miermont, Agnes; McDonel, Patrick; Chubb, Jonathan R

    2015-08-15

    Much of development and disease concerns the generation of gene expression differences between related cells sharing similar niches. However, most analyses of gene expression only assess population and time-averaged levels of steady-state transcription. The mechanisms driving differentiation are buried within snapshots of the average cell, lacking dynamic information and the diverse regulatory history experienced by individual cells. Here, we use a quantitative imaging platform with large time series data sets to determine the regulation of developmental gene expression by cell cycle, lineage, motility and environment. We apply this technology to the regulation of the pluripotency gene Nanog in mouse embryonic stem cells. Our data reveal the diversity of cell and population-level interactions with Nanog dynamics and heterogeneity, and how this regulation responds to triggers of pluripotency. Cell cycles are highly heterogeneous and cycle time increases with Nanog reporter expression, with longer, more variable cycle times as cells approach ground-state pluripotency. Nanog reporter expression is highly stable over multiple cell generations, with fluctuations within cycles confined by an attractor state. Modelling reveals an environmental component to expression stability, in addition to any cell-autonomous behaviour, and we identify interactions of cell density with both cycle behaviour and Nanog. Rex1 expression dynamics showed shared and distinct regulatory effects. Overall, our observations of multiple partially overlapping dynamic heterogeneities imply complex cell and environmental regulation of pluripotent cell behaviour, and suggest simple deterministic views of stem cell states are inappropriate. PMID:26209649

  1. Learning Emotional Understanding and Emotion Regulation through Sibling Interaction

    ERIC Educational Resources Information Center

    Kramer, Laurie

    2014-01-01

    Research Findings: Young children's relationships with their sisters and brothers offer unique and important opportunities for learning about emotions and developing emotional understanding. Through a critical analysis, this article examines sibling interaction in 3 different but normative contexts (conflict/conflict management, play, and…

  2. Regulation of Bax/mitochondria interaction by AKT.

    PubMed

    Simonyan, Lilit; Renault, Thibaud T; Novais, Maria João da Costa; Sousa, Maria João; Côrte-Real, Manuela; Camougrand, Nadine; Gonzalez, Cécile; Manon, Stéphen

    2016-01-01

    Bax-dependent mitochondrial permeabilization during apoptosis is controlled by multiple factors, including the phosphorylation by the protein kinase AKT. We used the heterologous co-expression of human Bax and AKT1 in yeast to investigate how the kinase modulates the different steps underlying Bax activation. We found that AKT activated Bax and increased its cellular content. Both effects were dependent on Ser184, but a phosphorylation of this residue did not fully explain the effects of AKT. Additional experiments with mutants substituted on Ser184 suggested that the regulation of Bax dynamic equilibrium between the cytosol and mitochondria might be more tightly regulated by Bcl-xL when Bax is phosphorylated. PMID:26763134

  3. The implementation of novel collaborative structures for the identification and resolution of barriers to pluripotent stem cell translation.

    PubMed

    Brindley, David A; French, Anna; Suh, Jane; Roberts, MacKenna; Davies, Benjamin; Pinedo-Villanueva, Rafael; Wartolowska, Karolina; Rooke, Kelly; Kramm, Anneke; Judge, Andrew; Morrey, Mark; Chandra, Amit; Hurley, Hannah; Grover, Liam; Bingham, Ian; Siegel, Bernard; Rattley, Matt S; Buckler, R Lee; McKeon, David; Krumholz, Katie; Hook, Lilian; May, Michael; Rikabi, Sarah; Pigott, Rosie; Morys, Megan; Sabokbar, Afsie; Titus, Emily; Laabi, Yacine; Lemaitre, Gilles; Zahkia, Raymond; Sipp, Doug; Horne, Robert; Bravery, Christopher; Williams, David; Wall, Ivan; Snyder, Evan Y; Karp, Jeffrey M; Barker, Richard W; Bure, Kim; Carr, Andrew J; Reeve, Brock

    2013-12-01

    Increased global connectivity has catalyzed technological development in almost all industries, in part through the facilitation of novel collaborative structures. Notably, open innovation and crowd-sourcing-of expertise and/or funding-has tremendous potential to increase the efficiency with which biomedical ecosystems interact to deliver safe, efficacious and affordable therapies to patients. Consequently, such practices offer tremendous potential in advancing development of cellular therapies. In this vein, the CASMI Translational Stem Cell Consortium (CTSCC) was formed to unite global thought-leaders, producing academically rigorous and commercially practicable solutions to a range of challenges in pluripotent stem cell translation. Critically, the CTSCC research agenda is defined through continuous consultation with its international funding and research partners. Herein, initial findings for all research focus areas are presented to inform global product development strategies, and to stimulate continued industry interaction around biomanufacturing, strategic partnerships, standards, regulation and intellectual property and clinical adoption.

  4. The Implementation of Novel Collaborative Structures for the Identification and Resolution of Barriers to Pluripotent Stem Cell Translation

    PubMed Central

    Brindley, David A.; French, Anna; Suh, Jane; Roberts, MacKenna; Davies, Benjamin; Pinedo-Villanueva, Rafael; Wartolowska, Karolina; Rooke, Kelly; Kramm, Anneke; Judge, Andrew; Morrey, Mark; Chandra, Amit; Hurley, Hannah; Grover, Liam; Bingham, Ian; Siegel, Bernard; Rattley, Matt S.; Buckler, R. Lee; McKeon, David; Krumholz, Katie; Hook, Lilian; May, Michael; Rikabi, Sarah; Pigott, Rosie; Morys, Megan; Sabokbar, Afsie; Titus, Emily; Laabi, Yacine; Lemaitre, Gilles; Zahkia, Raymond; Sipp, Doug; Horne, Robert; Bravery, Christopher; Williams, David; Wall, Ivan; Snyder, Evan Y.; Karp, Jeffrey M.; Barker, Richard W.; Bure, Kim; Carr, Andrew J.; Reeve, Brock

    2013-01-01

    Abstract Increased global connectivity has catalyzed technological development in almost all industries, in part through the facilitation of novel collaborative structures. Notably, open innovation and crowd-sourcing—of expertise and/or funding—has tremendous potential to increase the efficiency with which biomedical ecosystems interact to deliver safe, efficacious and affordable therapies to patients. Consequently, such practices offer tremendous potential in advancing development of cellular therapies. In this vein, the CASMI Translational Stem Cell Consortium (CTSCC) was formed to unite global thought-leaders, producing academically rigorous and commercially practicable solutions to a range of challenges in pluripotent stem cell translation. Critically, the CTSCC research agenda is defined through continuous consultation with its international funding and research partners. Herein, initial findings for all research focus areas are presented to inform global product development strategies, and to stimulate continued industry interaction around biomanufacturing, strategic partnerships, standards, regulation and intellectual property and clinical adoption. PMID:24304079

  5. Stem cells. m6A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation.

    PubMed

    Geula, Shay; Moshitch-Moshkovitz, Sharon; Dominissini, Dan; Mansour, Abed AlFatah; Kol, Nitzan; Salmon-Divon, Mali; Hershkovitz, Vera; Peer, Eyal; Mor, Nofar; Manor, Yair S; Ben-Haim, Moshe Shay; Eyal, Eran; Yunger, Sharon; Pinto, Yishay; Jaitin, Diego Adhemar; Viukov, Sergey; Rais, Yoach; Krupalnik, Vladislav; Chomsky, Elad; Zerbib, Mirie; Maza, Itay; Rechavi, Yoav; Massarwa, Rada; Hanna, Suhair; Amit, Ido; Levanon, Erez Y; Amariglio, Ninette; Stern-Ginossar, Noam; Novershtern, Noa; Rechavi, Gideon; Hanna, Jacob H

    2015-02-27

    Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N(6)-methyladenosine (m(6)A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout preimplantation epiblasts and naïve embryonic stem cells are depleted for m(6)A in mRNAs, yet are viable. However, they fail to adequately terminate their naïve state and, subsequently, undergo aberrant and restricted lineage priming at the postimplantation stage, which leads to early embryonic lethality. m(6)A predominantly and directly reduces mRNA stability, including that of key naïve pluripotency-promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo and identifies regulatory modules that functionally influence naïve and primed pluripotency in an opposing manner.

  6. Stem cells. m6A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation.

    PubMed

    Geula, Shay; Moshitch-Moshkovitz, Sharon; Dominissini, Dan; Mansour, Abed AlFatah; Kol, Nitzan; Salmon-Divon, Mali; Hershkovitz, Vera; Peer, Eyal; Mor, Nofar; Manor, Yair S; Ben-Haim, Moshe Shay; Eyal, Eran; Yunger, Sharon; Pinto, Yishay; Jaitin, Diego Adhemar; Viukov, Sergey; Rais, Yoach; Krupalnik, Vladislav; Chomsky, Elad; Zerbib, Mirie; Maza, Itay; Rechavi, Yoav; Massarwa, Rada; Hanna, Suhair; Amit, Ido; Levanon, Erez Y; Amariglio, Ninette; Stern-Ginossar, Noam; Novershtern, Noa; Rechavi, Gideon; Hanna, Jacob H

    2015-02-27

    Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N(6)-methyladenosine (m(6)A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout preimplantation epiblasts and naïve embryonic stem cells are depleted for m(6)A in mRNAs, yet are viable. However, they fail to adequately terminate their naïve state and, subsequently, undergo aberrant and restricted lineage priming at the postimplantation stage, which leads to early embryonic lethality. m(6)A predominantly and directly reduces mRNA stability, including that of key naïve pluripotency-promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo and identifies regulatory modules that functionally influence naïve and primed pluripotency in an opposing manner. PMID:25569111

  7. Reversible mitochondrial DNA accumulation in nuclei of pluripotent stem cells.

    PubMed

    Schneider, Joel S; Cheng, Xin; Zhao, Qingshi; Underbayev, Chingiz; Gonzalez, J Patrick; Raveche, Elizabeth S; Fraidenraich, Diego; Ivessa, Andreas S

    2014-11-15

    According to the endosymbiotic hypothesis, the precursor of mitochondria invaded the precursor of eukaryotic cells, a process that began roughly 2 billion years ago. Since then, the majority of the genetic material translocated from the mitochondria to the nucleus, where now almost all mitochondrial proteins are expressed. Only a tiny amount of DNA remained in the mitochondria, known as mitochondrial DNA (mtDNA). In this study, we report that the transfer of mtDNA fragments to the nucleus of pluripotent stem cells is still ongoing. We show by in situ hybridization and agarose DNA two-dimensional gel technique that induced pluripotent stem (iPS) cells contain high levels of mtDNA in the nucleus. We found that a large proportion of the accumulated mtDNA sequences appear to be extrachromosomal. Accumulation of mtDNA in the nucleus is present not only in the iPS cells, but also in embryonic stem (ES) cells. However upon differentiation, the level of mtDNA in the nuclei of iPS and ES cells is substantially reduced. This reversible accumulation of mtDNA in the nucleus supports the notion that the nuclear copy number of mtDNA sequences may provide a novel mechanism by which chromosomal DNA is dynamically regulated in pluripotent stem cells.

  8. Advances in culture and manipulation of human pluripotent stem cells.

    PubMed

    Qian, X; Villa-Diaz, L G; Krebsbach, P H

    2013-11-01

    Recent advances in the understanding of pluripotent stem cell biology and emerging technologies to reprogram somatic cells to a stem cell-like state are helping bring stem cell therapies for a range of human disorders closer to clinical reality. Human pluripotent stem cells (hPSCs) have become a promising resource for regenerative medicine and research into early development because these cells are able to self-renew indefinitely and are capable of differentiation into specialized cell types of all 3 germ layers and trophoectoderm. Human PSCs include embryonic stem cells (hESCs) derived from the inner cell mass of blastocyst-stage embryos and induced pluripotent stem cells (hiPSCs) generated via the reprogramming of somatic cells by the overexpression of key transcription factors. The application of hiPSCs and the finding that somatic cells can be directly reprogrammed into different cell types will likely have a significant impact on regenerative medicine. However, a major limitation for successful therapeutic application of hPSCs and their derivatives is the potential xenogeneic contamination and instability of current culture conditions. This review summarizes recent advances in hPSC culture and methods to induce controlled lineage differentiation through regulation of cell-signaling pathways and manipulation of gene expression as well as new trends in direct reprogramming of somatic cells.

  9. A genetic interaction map of cell cycle regulators

    PubMed Central

    Billmann, Maximilian; Horn, Thomas; Fischer, Bernd; Sandmann, Thomas; Huber, Wolfgang; Boutros, Michael

    2016-01-01

    Cell-based RNA interference (RNAi) is a powerful approach to screen for modulators of many cellular processes. However, resulting candidate gene lists from cell-based assays comprise diverse effectors, both direct and indirect, and further dissecting their functions can be challenging. Here we screened a genome-wide RNAi library for modulators of mitosis and cytokinesis in Drosophila S2 cells. The screen identified many previously known genes as well as modulators that have previously not been connected to cell cycle control. We then characterized ∼300 candidate modifiers further by genetic interaction analysis using double RNAi and a multiparametric, imaging-based assay. We found that analyzing cell cycle–relevant phenotypes increased the sensitivity for associating novel gene function. Genetic interaction maps based on mitotic index and nuclear size grouped candidates into known regulatory complexes of mitosis or cytokinesis, respectively, and predicted previously uncharacterized components of known processes. For example, we confirmed a role for the Drosophila CCR4 mRNA processing complex component l(2)NC136 during the mitotic exit. Our results show that the combination of genome-scale RNAi screening and genetic interaction analysis using process-directed phenotypes provides a powerful two-step approach to assigning components to specific pathways and complexes. PMID:26912791

  10. Mitochondrial Protein Interaction Mapping Identifies Regulators of Respiratory Chain Function.

    PubMed

    Floyd, Brendan J; Wilkerson, Emily M; Veling, Mike T; Minogue, Catie E; Xia, Chuanwu; Beebe, Emily T; Wrobel, Russell L; Cho, Holly; Kremer, Laura S; Alston, Charlotte L; Gromek, Katarzyna A; Dolan, Brendan K; Ulbrich, Arne; Stefely, Jonathan A; Bohl, Sarah L; Werner, Kelly M; Jochem, Adam; Westphall, Michael S; Rensvold, Jarred W; Taylor, Robert W; Prokisch, Holger; Kim, Jung-Ja P; Coon, Joshua J; Pagliarini, David J

    2016-08-18

    Mitochondria are essential for numerous cellular processes, yet hundreds of their proteins lack robust functional annotation. To reveal functions for these proteins (termed MXPs), we assessed condition-specific protein-protein interactions for 50 select MXPs using affinity enrichment mass spectrometry. Our data connect MXPs to diverse mitochondrial processes, including multiple aspects of respiratory chain function. Building upon these observations, we validated C17orf89 as a complex I (CI) assembly factor. Disruption of C17orf89 markedly reduced CI activity, and its depletion is found in an unresolved case of CI deficiency. We likewise discovered that LYRM5 interacts with and deflavinates the electron-transferring flavoprotein that shuttles electrons to coenzyme Q (CoQ). Finally, we identified a dynamic human CoQ biosynthetic complex involving multiple MXPs whose topology we map using purified components. Collectively, our data lend mechanistic insight into respiratory chain-related activities and prioritize hundreds of additional interactions for further exploration of mitochondrial protein function. PMID:27499296

  11. On the regulator-insurer interaction in a structural model

    NASA Astrophysics Data System (ADS)

    Bernard, Carole; Chen, An

    2009-11-01

    In this paper, we provide a new insight to the previous work of Briys and de Varenne [E. Briys, F. de Varenne, Life insurance in a contingent claim framework: Pricing and regulatory implications, Geneva Papers on Risk and Insurance Theory 19 (1) (1994) 53-72], Grosen and Jørgensen [A. Grosen, P.L. Jørgensen, Life insurance liabilities at market value: An analysis of insolvency risk, bonus policy, and regulatory intervention rules in a barrier option framework, Journal of Risk and Insurance 69 (1) (2002) 63-91] and Chen and Suchanecki [A. Chen, M. Suchanecki, Default risk, bankruptcy procedures and the market value of life insurance liabilities, Insurance: Mathematics and Economics 40 (2007) 231-255]. We show that the particular risk management strategy followed by the insurance company can significantly change the risk exposure of the company, and that it should thus be taken into account by regulators. We first study how the regulator establishes regulation intervention levels in order to control for instance the default probability of the insurance company. This part of the analysis is based on a constant volatility. Given that the insurance company is informed of regulatory rules, we study how results can be significantly different when the insurance company follows a risk management strategy with non-constant volatilities. We thus highlight some limits of the prior literature and believe that the risk management strategy of the company should be taken into account in the estimation of the risk exposure as well as in that of the market value of liabilities.

  12. Cripto is essential to capture mouse epiblast stem cell and human embryonic stem cell pluripotency.

    PubMed

    Fiorenzano, Alessandro; Pascale, Emilia; D'Aniello, Cristina; Acampora, Dario; Bassalert, Cecilia; Russo, Francesco; Andolfi, Gennaro; Biffoni, Mauro; Francescangeli, Federica; Zeuner, Ann; Angelini, Claudia; Chazaud, Claire; Patriarca, Eduardo J; Fico, Annalisa; Minchiotti, Gabriella

    2016-01-01

    Known molecular determinants of developmental plasticity are mainly transcription factors, while the extrinsic regulation of this process has been largely unexplored. Here we identify Cripto as one of the earliest epiblast markers and a key extracellular determinant of the naive and primed pluripotent states. We demonstrate that Cripto sustains mouse embryonic stem cell (ESC) self-renewal by modulating Wnt/β-catenin, whereas it maintains mouse epiblast stem cell (EpiSC) and human ESC pluripotency through Nodal/Smad2. Moreover, we provide unprecedented evidence that Cripto controls the metabolic reprogramming in ESCs to EpiSC transition. Remarkably, Cripto deficiency attenuates ESC lineage restriction in vitro and in vivo, and permits ESC transdifferentiation into trophectoderm lineage, suggesting that Cripto has earlier functions than previously recognized. All together, our studies provide novel insights into the current model of mammalian pluripotency and contribute to the understanding of the extrinsic regulation of the first cell lineage decision in the embryo. PMID:27586544

  13. Cripto is essential to capture mouse epiblast stem cell and human embryonic stem cell pluripotency

    PubMed Central

    Fiorenzano, Alessandro; Pascale, Emilia; D'Aniello, Cristina; Acampora, Dario; Bassalert, Cecilia; Russo, Francesco; Andolfi, Gennaro; Biffoni, Mauro; Francescangeli, Federica; Zeuner, Ann; Angelini, Claudia; Chazaud, Claire; Patriarca, Eduardo J.; Fico, Annalisa; Minchiotti, Gabriella

    2016-01-01

    Known molecular determinants of developmental plasticity are mainly transcription factors, while the extrinsic regulation of this process has been largely unexplored. Here we identify Cripto as one of the earliest epiblast markers and a key extracellular determinant of the naive and primed pluripotent states. We demonstrate that Cripto sustains mouse embryonic stem cell (ESC) self-renewal by modulating Wnt/β-catenin, whereas it maintains mouse epiblast stem cell (EpiSC) and human ESC pluripotency through Nodal/Smad2. Moreover, we provide unprecedented evidence that Cripto controls the metabolic reprogramming in ESCs to EpiSC transition. Remarkably, Cripto deficiency attenuates ESC lineage restriction in vitro and in vivo, and permits ESC transdifferentiation into trophectoderm lineage, suggesting that Cripto has earlier functions than previously recognized. All together, our studies provide novel insights into the current model of mammalian pluripotency and contribute to the understanding of the extrinsic regulation of the first cell lineage decision in the embryo. PMID:27586544

  14. Replication timing: a fingerprint for cell identity and pluripotency.

    PubMed

    Ryba, Tyrone; Hiratani, Ichiro; Sasaki, Takayo; Battaglia, Dana; Kulik, Michael; Zhang, Jinfeng; Dalton, Stephen; Gilbert, David M

    2011-10-01

    Many types of epigenetic profiling have been used to classify stem cells, stages of cellular differentiation, and cancer subtypes. Existing methods focus on local chromatin features such as DNA methylation and histone modifications that require extensive analysis for genome-wide coverage. Replication timing has emerged as a highly stable cell type-specific epigenetic feature that is regulated at the megabase-level and is easily and comprehensively analyzed genome-wide. Here, we describe a cell classification method using 67 individual replication profiles from 34 mouse and human cell lines and stem cell-derived tissues, including new data for mesendoderm, definitive endoderm, mesoderm and smooth muscle. Using a Monte-Carlo approach for selecting features of replication profiles conserved in each cell type, we identify "replication timing fingerprints" unique to each cell type and apply a k nearest neighbor approach to predict known and unknown cell types. Our method correctly classifies 67/67 independent replication-timing profiles, including those derived from closely related intermediate stages. We also apply this method to derive fingerprints for pluripotency in human and mouse cells. Interestingly, the mouse pluripotency fingerprint overlaps almost completely with previously identified genomic segments that switch from early to late replication as pluripotency is lost. Thereafter, replication timing and transcription within these regions become difficult to reprogram back to pluripotency, suggesting these regions highlight an epigenetic barrier to reprogramming. In addition, the major histone cluster Hist1 consistently becomes later replicating in committed cell types, and several histone H1 genes in this cluster are downregulated during differentiation, suggesting a possible instrument for the chromatin compaction observed during differentiation. Finally, we demonstrate that unknown samples can be classified independently using site-specific PCR against

  15. Biological Effects of Culture Substrates on Human Pluripotent Stem Cells.

    PubMed

    Hayashi, Yohei; Furue, Miho Kusuda

    2016-01-01

    In recent years, as human pluripotent stem cells (hPSCs) have been commonly cultured in feeder-free conditions, a number of cell culture substrates have been applied or developed. However, the functional roles of these substrates in maintaining hPSC self-renewal remain unclear. Here in this review, we summarize the types of these substrates and their effect on maintaining hPSC self-renewal. Endogenous extracellular matrix (ECM) protein expression has been shown to be crucial in maintaining hPSC self-renewal. These ECM molecules interact with integrin cell-surface receptors and transmit their cellular signaling. We discuss the possible effect of integrin-mediated signaling pathways on maintaining hPSC self-renewal. Activation of integrin-linked kinase (ILK), which transmits ECM-integrin signaling to AKT (also known as protein kinase B), has been shown to be critical in maintaining hPSC self-renewal. Also, since naïve pluripotency has been widely recognized as an alternative pluripotent state of hPSCs, we discuss the possible effects of culture substrates and integrin signaling on naïve hPSCs based on the studies of mouse embryonic stem cells. Understanding the role of culture substrates in hPSC self-renewal and differentiation enables us to control hPSC behavior precisely and to establish scalable or microfabricated culture technologies for regenerative medicine and drug development. PMID:27656216

  16. Biological Effects of Culture Substrates on Human Pluripotent Stem Cells

    PubMed Central

    2016-01-01

    In recent years, as human pluripotent stem cells (hPSCs) have been commonly cultured in feeder-free conditions, a number of cell culture substrates have been applied or developed. However, the functional roles of these substrates in maintaining hPSC self-renewal remain unclear. Here in this review, we summarize the types of these substrates and their effect on maintaining hPSC self-renewal. Endogenous extracellular matrix (ECM) protein expression has been shown to be crucial in maintaining hPSC self-renewal. These ECM molecules interact with integrin cell-surface receptors and transmit their cellular signaling. We discuss the possible effect of integrin-mediated signaling pathways on maintaining hPSC self-renewal. Activation of integrin-linked kinase (ILK), which transmits ECM-integrin signaling to AKT (also known as protein kinase B), has been shown to be critical in maintaining hPSC self-renewal. Also, since naïve pluripotency has been widely recognized as an alternative pluripotent state of hPSCs, we discuss the possible effects of culture substrates and integrin signaling on naïve hPSCs based on the studies of mouse embryonic stem cells. Understanding the role of culture substrates in hPSC self-renewal and differentiation enables us to control hPSC behavior precisely and to establish scalable or microfabricated culture technologies for regenerative medicine and drug development. PMID:27656216

  17. The role of telomeres and telomerase reverse transcriptase isoforms in pluripotency induction and maintenance.

    PubMed

    Teichroeb, Jonathan H; Kim, Joohwan; Betts, Dean H

    2016-08-01

    Telomeres are linear guanine-rich DNA structures at the ends of chromosomes. The length of telomeric DNA is actively regulated by a number of mechanisms in highly proliferative cells such as germ cells, cancer cells, and pluripotent stem cells. Telomeric DNA is synthesized by way of the ribonucleoprotein called telomerase containing a reverse transcriptase (TERT) subunit and RNA component (TERC). TERT is highly conserved across species and ubiquitously present in their respective pluripotent cells. Recent studies have uncovered intricate associations between telomeres and the self-renewal and differentiation properties of pluripotent stem cells. Interestingly, the past decade's work indicates that the TERT subunit also has the capacity to modulate mitochondrial function, to remodel chromatin structure, and to participate in key signaling pathways such as the Wnt/β-catenin pathway. Many of these non-canonical functions do not require TERT's catalytic activity, which hints at possible functions for the extensive number of alternatively spliced TERT isoforms that are highly expressed in pluripotent stem cells. In this review, some of the established and potential routes of pluripotency induction and maintenance are highlighted from the perspectives of telomere maintenance, known TERT isoform functions and their complex regulation. PMID:26786236

  18. Pluripotency-associated genes in human nasopharyngeal carcinoma CNE-2 cells are reactivated by a unique epigenetic sub-microenvironment

    PubMed Central

    2010-01-01

    Background There is increasing evidence that cancers contain their own stem-like cells, and particular attention has been paid to one subset of cancer-stem cells termed side population (SP). Stem cells under normal physical conditions are tightly controlled by their microenvironment, however, the regulatory role of the microenvironment surrounding cancer stem cells is not well characterized yet. In this study we found that the phenotype of SP can be "generated" by macrophage-like cells under conditioned culture. Furthermore the gene regulation pathway involved in cellular reprogramming process was investigated. Methods The selection and identification of SP in 50 CNE-2 single cell clones were performed by flow cytometry. The transwell assay and immunofluorescence staining were used to measure migration and cancer stem cell characters of non-SP single clone cells cultured with conditioned medium respectively. The subtraction suppression hybridization (SSH) technique and northern blotting analysis was applied to explore the pluripotency-associated genes under a unique epigenetic sub-microenvironment. Results Among 50 clones, only one did not possess SP subpopulation while others did. The non-SP cells induced by macrophage-like cells showed more aggressive characters, which increased cell migration compared with the control cells and showed some fraction of SP phenotype. These cells expressed distinguished level of pluripotency-associated genes such as ADP-ribosylation factor-like 6 interacting protein (ARMER), poly (rC) binding protein 1 (PCBP1) and pyruvate dehydrogenase E1-β subunit (PDHB) when subjected to the environment. Conclusion To our knowledge, this is the first study to demonstrate that non-SP single-clone cells can be induced to generate a SP phenotype when they are cultured with conditioned medium of macrophage-like cells, which is associated with the reactivation of pluripotency-associated genes. PMID:20181293

  19. Regulation of mouse gamete interaction by a sperm tyrosine kinase.

    PubMed

    Leyton, L; LeGuen, P; Bunch, D; Saling, P M

    1992-12-15

    A 95-kDa mouse sperm protein has been previously identified as a putative receptor involved in the sperm-egg interactions that lead to fertilization. The ligand for this receptor is the zona pellucida glycoprotein ZP3. This constituent of the oocyte-specific extracellular matrix mediates not only sperm binding to the zona but also triggers acrosomal exocytosis. The latter, also termed the acrosome reaction, is a key regulatory event upon which fertilization is absolutely dependent. Previously, we showed that the 95-kDa protein that binds ZP3 is a substrate for tyrosine kinase, and its phosphotyrosine content increases after sperm-zona pellucida binding. Here, we show the presence of protein tyrosine kinase activity in sperm plasma membranes and in electroeluted 95-kDa protein. The tyrosine kinase activity of the isolated protein is stimulated by solubilized zona pellucida and inhibited by tyrphostin RG-50864, a membrane-permeable tyrosine kinase inhibitor. Furthermore, tyrphostin inhibits zona-triggered acrosomal exocytosis in a dose-dependent manner. These findings indicate that the 95-kDa protein participates in a critical regulatory event of gamete interaction; moreover, our experiments suggest that sperm protein tyrosine kinase may be an excellent target for the control of fertility.

  20. Impact of retrotransposons in pluripotent stem cells.

    PubMed

    Tanaka, Yoshiaki; Chung, Leeyup; Park, In-Hyun

    2012-12-01

    Retrotransposons, which constitute approximately 40% of the human genome, have the capacity to 'jump' across the genome. Their mobility contributes to oncogenesis, evolution, and genomic plasticity of the host genome. Induced pluripotent stem cells as well as embryonic stem cells are more susceptible than differentiated cells to genomic aberrations including insertion, deletion and duplication. Recent studies have revealed specific behaviors of retrotransposons in pluripotent cells. Here, we review recent progress in understanding retrotransposons and provide a perspective on the relationship between retrotransposons and genomic variation in pluripotent stem cells. PMID:23135636

  1. Relationship of Maternal Negative Moods to Child Emotion Regulation during Family Interaction

    PubMed Central

    Dagne, Getachew A.; Snyder, James

    2016-01-01

    The relationship of maternal hostile and depressive moods to children’s down-regulation of unprovoked anger and sadness/fear was assessed in a community sample of 267 five year old boys and girls. The speed of children’s down-regulation of unprovoked anger and sadness/fear was based on real-time observations during mother-child interaction. The association of down-regulation with maternal mood was estimated using Bayesian event history analysis. As mothers reported higher depressive mood, both boys and girls were faster to down regulate anger displays as those displays accumulated during mother child interaction. The speed of boys’ down regulation of anger and of sadness/fear was not associated with maternal hostile mood. As mothers reported more hostile mood, girls were faster to down regulate displays of sadness/fear, but the speed of this down regulation slowed as those displays accumulated during ongoing mother-child interaction. These associations of child down regulation and maternal mood were observed after controlling for child adjustment. The data suggest frequent exposure to different negative maternal moods affect children’s expression and regulation of emotions in relatively specific ways, conditional on the type of maternal mood, the type of child emotion, and child gender. PMID:21262049

  2. Stress Regulation in Adolescents: Physiological Reactivity during the Adult Attachment Interview and Conflict Interaction

    ERIC Educational Resources Information Center

    Beijersbergen, Marielle D.; Bakermans-Kranenburg, Marian J.; van IJzendoorn, Marinus H.; Juffer, Femmie

    2008-01-01

    The current study examined whether adolescents' attachment representations were associated with differences in emotion regulation during the Adult Attachment Interview (AAI; C. George, N. Kaplan, & M. Main, 1996) and during a mother-adolescent conflict interaction task (Family Interaction Task [FIT]; J. P. Allen et al., 2003). Participants were…

  3. Dorsomedial hypothalamic GABA regulates anxiety in the social interaction test.

    PubMed

    Shekhar, A; Katner, J S

    1995-02-01

    Blockade of GABAA function in the region of the dorsomedial hypothalamus (DMH) of rats is known to elicit a constellation of physiologic responses including increases in heart rate (HR), mean arterial blood pressure (BP), respiratory rate, and plasma catecholamine levels, as well as behavioral responses such as increases in locomotor activity and anxiogenic-like effects as measured in a conflict test and the elevated plus-maze test. The aim of the present study was to test the effects of microinjecting GABAA antagonists bicuculline methiodide (BMI) and picrotoxin, as well as the GABAA agonist muscimol, into the DMH of rats placed in the social interaction (SI) test. Muscimol decreased HR and BP but increased SI, whereas the GABA antagonists increased HR and BP but decreased SI time. Blocking the HR changes elicited by GABAergic drugs injected into the DMH with systemic injections of atenolol and atropine methylbromide did not block their effects on SI.

  4. Pleiohomeotic Interacts with the Core Transcription Elongation Factor Spt5 to Regulate Gene Expression in Drosophila

    PubMed Central

    Jennings, Barbara H.

    2013-01-01

    The early elongation checkpoint regulated by Positive Transcription Elongation Factor b (P-TEFb) is a critical control point for the expression of many genes. Spt5 interacts directly with RNA polymerase II and has an essential role in establishing this checkpoint, and also for further transcript elongation. Here we demonstrate that Drosophila Spt5 interacts both physically and genetically with the Polycomb Group (PcG) protein Pleiohomeotic (Pho), and the majority of Pho binding sites overlap with Spt5 binding sites across the genome in S2 cells. Our results indicate that Pho can interact with Spt5 to regulate transcription elongation in a gene specific manner. PMID:23894613

  5. Task relevance regulates the interaction between reward expectation and emotion.

    PubMed

    Wei, Ping; Kang, Guanlan

    2014-06-01

    In the present study, we investigated the impact of reward expectation on the processing of emotional facial expression using a cue-target paradigm. A cue indicating the reward condition of each trial (incentive vs. non-incentive) was followed by the presentation of a picture of an emotional face, the target. Participants were asked to discriminate the emotional expression of the target face in Experiment 1, to discriminate the gender of the target face in Experiment 2, and to judge a number superimposed on the center of the target face as even or odd in Experiment 3, rendering the emotional expression of the target face as task relevant in Experiment 1 but task irrelevant in Experiments 2 and 3. Faster reaction times (RTs) were observed in the monetary incentive condition than in the non-incentive condition, demonstrating the effect of reward on facilitating task concentration. Moreover, the reward effect (i.e., RTs in non-incentive conditions versus incentive conditions) was larger for emotional faces than for neutral faces when emotional expression was task relevant but not when it was task irrelevant. The findings suggest that top-down incentive motivation biased attentional processing toward task-relevant stimuli, and that task relevance played an important role in regulating the influence of reward expectation on the processing of emotional stimuli. PMID:24553754

  6. PTRcombiner: mining combinatorial regulation of gene expression from post-transcriptional interaction maps

    PubMed Central

    2014-01-01

    Background The progress in mapping RNA-protein and RNA-RNA interactions at the transcriptome-wide level paves the way to decipher possible combinatorial patterns embedded in post-transcriptional regulation of gene expression. Results Here we propose an innovative computational tool to extract clusters of mRNA trans-acting co-regulators (RNA binding proteins and non-coding RNAs) from pairwise interaction annotations. In addition the tool allows to analyze the binding site similarity of co-regulators belonging to the same cluster, given their positional binding information. The tool has been tested on experimental collections of human and yeast interactions, identifying modules that coordinate functionally related messages. Conclusions This tool is an original attempt to uncover combinatorial patterns using all the post-transcriptional interaction data available so far. PTRcombiner is available at http://disi.unitn.it/~passerini/software/PTRcombiner/. PMID:24758252

  7. Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors

    PubMed Central

    El-Sayed, Ahmed Kamel; Zhang, Zhentao; Zhang, Lei; Liu, Zhiyong; Abbott, Louise C.; Zhang, Yani; Li, Bichun

    2014-01-01

    Reprogramming of somatic cells has great potential to provide therapeutic treatments for a number of diseases as well as provide insight into mechanisms underlying early embryonic development. Improvement of induced Pluripotent Stem Cells (iPSCs) generation through mRNA-based methods is currently an area of intense research. This approach provides a number of advantages over previously used methods such as DNA integration and insertional mutagenesis. Using transfection of specifically synthesized mRNAs of various pluripotency factors, we generated iPSCs from mouse embryonic fibroblast (MEF) cells. The genetic, epigenetic and functional properties of the iPSCs were evaluated at different times during the reprogramming process. We successfully introduced synthesized mRNAs, which localized correctly inside the cells and exhibited efficient and stable translation into proteins. Our work demonstrated a robust up-regulation and a gradual promoter de-methylation of the pluripotency markers, including non-transfected factors such as Nanog, SSEA-1 (stage-specific embryonic antigen 1) and Rex-1 (ZFP-42, zinc finger protein 42). Using embryonic stem cells (ESCs) conditions to culture the iPS cells resulted in formation of ES-like colonies after approximately 12 days with only five daily repeated transfections. The colonies were positive for alkaline phosphatase and pluripotency-specific markers associated with ESCs. This study revealed the ability of pluripotency induction and generation of mouse mRNA induced pluripotent stem cells (mRNA iPSCs) using transfection of specifically synthesized mRNAs of various pluripotency factors into mouse embryonic fibroblast (MEF) cells. These generated iPSCs exhibited molecular and functional properties similar to ESCs, which indicate that this method is an efficient and viable alternative to ESCs and can be used for further biological, developmental and therapeutic investigations. PMID:25437916

  8. Threonine 393 of beta-catenin regulates interaction with Axin.

    PubMed

    Wu, Hao; Symes, Karen; Seldin, David C; Dominguez, Isabel

    2009-09-01

    CK2 is a regulatory kinase implicated in embryonic development and in cancer. Among the CK2 substrates is beta-catenin, a protein with dual function in Wnt signaling and cell adhesion. Previously, we reported that CK2 activity is required for beta-catenin stability and we identified threonine (T) 393 as a major CK2 phosphorylation site in beta-catenin. However, it is not known whether phosphorylation at T393 increases beta-catenin stability and if so, what is the mechanism. In this study we investigate the molecular mechanism of beta-catenin stabilization through phosphorylation at T393. We found that pseudophosphorylation of beta-catenin at T393 resulted in a stable activated form of beta-catenin with decreased affinity for Axin in vitro. This phosphomimetic mutant also displayed decreased regulation by Axin in vivo in a bioassay in Xenopus laevis embryos. In contrast, the binding of T393 pseudophosphorylated beta-catenin to E-cadherin was unaffected. Further analysis showed that pseudophosphorylation at T393 did not prevent beta-catenin phosphorylation by GSK3beta. Interestingly, we found that in the presence of pseudophophorylated beta-catenin and another activated form of beta-catenin, the recruitment of GSK3beta to Axin is enhanced. These findings indicate that phosphorylation of T393 by CK2 may affect the stability of beta-catenin through decreased binding to Axin. In addition, the increased recruitment of GSK3beta to the destruction complex in the presence of activated beta-catenin mutants could be a feedback mechanism to suppress overactive Wnt signaling.

  9. Proteome Analysis of Ground State Pluripotency

    PubMed Central

    Taleahmad, Sara; Mirzaei, Mehdi; Parker, Lindsay M.; Hassani, Seyedeh-Nafiseh; Mollamohammadi, Sepideh; Sharifi-Zarchi, Ali; Haynes, Paul A.; Baharvand, Hossein; Salekdeh, Ghasem Hosseini

    2015-01-01

    The differentiation potential of pluripotent embryonic stem cells (ESCs) can be manipulated via serum and medium conditions for direct cellular development or to maintain a naïve ground state. The self-renewal state of ESCs can thus be induced by adding inhibitors of mitogen activated protein kinase (MAPK) and glycogen synthase kinase-3 (Gsk3), known as 2 inhibitors (2i) treatment. We have used a shotgun proteomics approach to investigate differences in protein expressions between 2i- and serum-grown mESCs. The results indicated that 164 proteins were significantly upregulated and 107 proteins downregulated in 2i-grown cells compared to serum. Protein pathways in 2i-grown cells with the highest enrichment were associated with glycolysis and gluconeogenesis. Protein pathways related to organ development were downregulated in 2i-grown cells. In serum-grown ESCs, protein pathways involved in integrin and focal adhesion, and signaling proteins involved in the actin cytoskeleton regulation were enriched. We observed a number of nuclear proteins which were mostly involved in self-renewal maintenance and were expressed at higher levels in 2i compared to serum - Dnmt1, Map2k1, Parp1, Xpo4, Eif3g, Smarca4/Brg1 and Smarcc1/Baf155. Collectively, the results provided an insight into the key protein pathways used by ESCs in the ground state or metastable conditions through 2i or serum culture medium, respectively. PMID:26671762

  10. Hydrodynamic modulation of pluripotent stem cells

    PubMed Central

    2012-01-01

    Controlled expansion and differentiation of pluripotent stem cells (PSCs) using reproducible, high-throughput methods could accelerate stem cell research for clinical therapies. Hydrodynamic culture systems for PSCs are increasingly being used for high-throughput studies and scale-up purposes; however, hydrodynamic cultures expose PSCs to complex physical and chemical environments that include spatially and temporally modulated fluid shear stresses and heterogeneous mass transport. Furthermore, the effects of fluid flow on PSCs cannot easily be attributed to any single environmental parameter since the cellular processes regulating self-renewal and differentiation are interconnected and the complex physical and chemical parameters associated with fluid flow are thus difficult to independently isolate. Regardless of the challenges posed by characterizing fluid dynamic properties, hydrodynamic culture systems offer several advantages over traditional static culture, including increased mass transfer and reduced cell handling. This article discusses the challenges and opportunities of hydrodynamic culture environments for the expansion and differentiation of PSCs in microfluidic systems and larger-volume suspension bioreactors. Ultimately, an improved understanding of the effects of hydrodynamics on the self-renewal and differentiation of PSCs could yield improved bioprocessing technologies to attain scalable PSC culture strategies that will probably be requisite for the development of therapeutic and diagnostic applications. PMID:23168068

  11. Pluripotent Stem Cells from Domesticated Mammals.

    PubMed

    Ezashi, Toshihiko; Yuan, Ye; Roberts, R Michael

    2016-01-01

    This review deals with the latest advances in the study of embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) from domesticated species, with a focus on pigs, cattle, sheep, goats, horses, cats, and dogs. Whereas the derivation of fully pluripotent ESC from these species has proved slow, reprogramming of somatic cells to iPSC has been more straightforward. However, most of these iPSC depend on the continued expression of the introduced transgenes, a major drawback to their utility. The persistent failure in generating ESC and the dependency of iPSC on ectopic genes probably stem from an inability to maintain the stability of the endogenous gene networks necessary to maintain pluripotency. Based on work in humans and rodents, achievement of full pluripotency will likely require fine adjustments in the growth factors and signaling inhibitors provided to the cells. Finally, we discuss the future utility of these cells for biomedical and agricultural purposes. PMID:26566158

  12. Regulatory issues for personalized pluripotent cells.

    PubMed

    Condic, Maureen L; Rao, Mahendra

    2008-11-01

    The development of personalized pluripotent stem cells for research and for possible therapies holds out great hope for patients. However, such cells will face significant technical and regulatory challenges before they can be used as therapeutic reagents. Here we consider two possible sources of personalized pluripotent stem cells: embryonic stem cells derived from nuclear transfer (NT-ESCs) and induced pluripotent stem cells (iPSCs) derived from direct reprogramming of adult somatic cells. Both sources of personalized pluripotent stem cells face unique regulatory hurdles that are in some ways significantly higher than those facing stem cells derived from embryos produced by fertilization (ESCs). However, the outstanding long-term potential of iPSCs and their relative freedom from the ethical concerns raised by both ESCs and NT-ESCs makes direct reprogramming an exceptionally promising approach to advancing research and providing therapies in the field of regenerative medicine.

  13. Dichloroacetate, the Pyruvate Dehydrogenase Complex and the Modulation of mESC Pluripotency

    PubMed Central

    Rodrigues, Ana Sofia; Correia, Marcelo; Gomes, Andreia; Pereira, Sandro L.; Perestrelo, Tânia; Sousa, Maria Inês; Ramalho-Santos, João

    2015-01-01

    Introduction The pyruvate dehydrogenase (PDH) complex is localized in the mitochondrial matrix catalyzing the irreversible decarboxylation of pyruvate to acetyl-CoA and NADH. For proper complex regulation the E1-α subunit functions as an on/off switch regulated by phosphorylation/dephosphorylation. In different cell types one of the four-pyruvate dehydrogenase kinase isoforms (PDHK1-4) can phosphorylate this subunit leading to PDH inactivation. Our previous results with human Embryonic Stem Cells (hESC), suggested that PDHK could be a key regulator in the metabolic profile of pluripotent cells, as it is upregulated in pluripotent stem cells. Therefore, we wondered if metabolic modulation, via inexpensive pharmacological inhibition of PDHK, could impact metabolism and pluripotency. Methods/Results In order to assess the importance of the PDH cycle in mouse Embryonic Stem Cells (mESC), we incubated cells with the PDHK inhibitor dichloroacetate (DCA) and observed that in its presence ESC started to differentiate. Changes in mitochondrial function and proliferation potential were also found and protein levels for PDH (both phosphorylated and non-phosphorylated) and PDHK1 were monitored. Interestingly, we were also able to describe a possible pathway that involves Hif-1α and p53 during DCA-induced loss of pluripotency. Results with ESCs treated with DCA were comparable to those obtained for cells grown without Leukemia Inhibitor Factor (LIF), used in this case as a positive control for differentiation. Conclusions DCA negatively affects ESC pluripotency by changing cell metabolism and elements related to the PDH cycle, suggesting that PDHK could function as a possible metabolic gatekeeper in ESC, and may be a good target to modulate metabolism and differentiation. Although further molecular biology-based experiments are required, our data suggests that inactive PDH favors pluripotency and that ESC have similar strategies as cancer cells to maintain a glycolytic

  14. PRMT5 is required for human embryonic stem cell proliferation but not pluripotency.

    PubMed

    Gkountela, Sofia; Li, Ziwei; Chin, Chee Jia; Lee, Serena A; Clark, Amander T

    2014-04-01

    Human pluripotent stem cells (PSCs) are critical in vitro tools for understanding mechanisms that regulate lineage differentiation in the human embryo as well as a potentially unlimited supply of stem cells for regenerative medicine. Pluripotent human and mouse embryonic stem cells (ESCs) derived from the inner cell mass of blastocysts share a similar transcription factor network to maintain pluripotency and self-renewal, yet there are considerable molecular differences reflecting the diverse environments in which mouse and human ESCs are derived. In the current study we evaluated the role of Protein arginine methyltransferase 5 (PRMT5) in human ESC (hESC) self-renewal and pluripotency given its critical role in safeguarding mouse ESC pluripotency. Unlike the mouse, we discovered that PRMT5 has no role in hESC pluripotency. Using microarray analysis we discovered that a significant depletion in PRMT5 RNA and protein from hESCs changed the expression of only 78 genes, with the majority being repressed. Functionally, we discovered that depletion of PRMT5 had no effect on expression of OCT4, NANOG or SOX2, and did not prevent teratoma formation. Instead, we show that PRMT5 functions in hESCs to regulate proliferation in the self-renewing state by regulating the fraction of cells in Gap 1 (G1) of the cell cycle and increasing expression of the G1 cell cycle inhibitor P57. Taken together our data unveils a distinct role for PRMT5 in hESCs and identifies P57 as new target. PMID:24477620

  15. Interactive Regulation by the Bacillus subtilis Global Regulators CodY and ScoC

    PubMed Central

    BELITSKY, BORIS R.; BARBIERI, GIULIA; ALBERTINI, ALESSANDRA M.; FERRARI, EUGENIO; STRAUCH, MARK A.; SONENSHEIN, ABRAHAM L.

    2015-01-01

    SUMMARY CodY and ScoC are Bacillus subtilis transcriptional regulators that control the expression of dozens of genes and operons. Using scoC-lacZ fusions and DNA-binding experiments, we show here that scoC is directly repressed by CodY. This effect creates multiple forms of cascade regulation. For instance, expression of the dtpT gene, which is directly and negatively controlled by ScoC and encodes a putative oligopeptide permease, was activated indirectly by CodY due to CodY-mediated repression of scoC. The opp operon, which encodes an oligopeptide permease that is essential for sporulation and genetic competence development, proved to be a direct target of repression by both ScoC and CodY, but was not significantly affected in codY or scoC single mutants. The combined actions of CodY and ScoC maintain opp repression when either one of the regulators loses activity, but limit the level of repression to that provided by one of the regulators acting alone. Under conditions of nitrogen limitation, repression by ScoC of dtpT and opp was partly prevented by TnrA. Thus, the functioning of ScoC is determined by other transcription factors via modulation of its expression or DNA binding. PMID:25966844

  16. Nuclear transcriptome profiling of induced pluripotent stem cells and embryonic stem cells identify non-coding loci resistant to reprogramming.

    PubMed

    Fort, Alexandre; Yamada, Daisuke; Hashimoto, Kosuke; Koseki, Haruhiko; Carninci, Piero

    2015-01-01

    Identification of functionally relevant differences between induced pluripotent stem cells (iPSC) and reference embryonic stem cells (ESC) remains a central question for therapeutic applications. Differences in gene expression between iPSC and ESC have been examined by microarray and more recently with RNA-SEQ technologies. We here report an in depth analyses of nuclear and cytoplasmic transcriptomes, using the CAGE (cap analysis of gene expression) technology, for 5 iPSC clones derived from mouse lymphocytes B and 3 ESC lines. This approach reveals nuclear transcriptomes significantly more complex in ESC than in iPSC. Hundreds of yet not annotated putative non-coding RNAs and enhancer-associated transcripts specifically transcribed in ESC have been detected and supported with epigenetic and chromatin-chromatin interactions data. We identified super-enhancers transcriptionally active specifically in ESC and associated with genes implicated in the maintenance of pluripotency. Similarly, we detected non-coding transcripts of yet unknown function being regulated by ESC specific super-enhancers. Taken together, these results demonstrate that current protocols of iPSC reprogramming do not trigger activation of numerous cis-regulatory regions. It thus reinforces the need for already suggested deeper monitoring of the non-coding transcriptome when characterizing iPSC clones. Such differences in regulatory transcript expression may indeed impact their potential for clinical applications. PMID:25664506

  17. Maintaining embryonic stem cell pluripotency with Wnt signaling.

    PubMed

    Sokol, Sergei Y

    2011-10-01

    Wnt signaling pathways control lineage specification in vertebrate embryos and regulate pluripotency in embryonic stem (ES) cells, but how the balance between progenitor self-renewal and differentiation is achieved during axis specification and tissue patterning remains highly controversial. The context- and stage-specific effects of the different Wnt pathways produce complex and sometimes opposite outcomes that help to generate embryonic cell diversity. Although the results of recent studies of the Wnt/β-catenin pathway in ES cells appear to be surprising and controversial, they converge on the same conserved mechanism that leads to the inactivation of TCF3-mediated repression. PMID:21903672

  18. Temporospatial cell interactions regulating mandibular and maxillary arch patterning.

    PubMed

    Ferguson, C A; Tucker, A S; Sharpe, P T

    2000-01-01

    The cellular origin of the instructive information for hard tissue patterning of the jaws has been the subject of a long-standing controversy. Are the cranial neural crest cells prepatterned or does the epithelium pattern a developmentally uncommitted population of ectomesenchymal cells? In order to understand more about how orofacial patterning is controlled we have investigated the temporal signalling interactions and responses between epithelium and mesenchymal cells in the mandibular and maxillary primordia. We show that within the mandibular arch, homeobox genes that are expressed in different proximodistal spatial domains corresponding to presumptive molar and incisor ectomesenchymal cells are induced by signals from the oral epithelium. In mouse, prior to E10, all ectomesenchyme cells in the mandibular arch are equally responsive to epithelial signals such as Fgf8, indicating that there is no pre-specification of these cells into different populations and suggesting that patterning of the hard tissues of the mandible is instructed by the epithelium. By E10.5, ectomesenchymal cell gene expression domains are still dependent on epithelial signals but have become fixed and ectopic expression cannot be induced. At E11 expression becomes independent of epithelial signals such that removal of the epithelium does not affect spatial ectomesenchymal expression. Significantly, however, the response of ectomesenchyme cells to epithelial regulatory signals was found to be different in the mandibular and maxillary primordium. Thus, whereas both mandibular and maxillary arch epithelia could induce Dlx2 and Dlx5 expression in the mandible and Dlx2 expression in the maxilla, neither could induce Dlx5 expression in the maxilla. Reciprocal cell transplantations between mandibular and maxillary arch ectomesenchymal cells revealed intrinsic differences between these populations of cranial neural crest-derived cells. Research in odontogenesis has shown that the oral epithelium

  19. The Path from Pluripotency to Skeletal Muscle: Developmental Myogenesis Guides the Way

    PubMed Central

    Hicks, Michael; Pyle, April

    2016-01-01

    Following cues from mouse embryogenesis, Chal et al. (2015) identified key regulators of skeletal myogenesis from mouse and human pluripotent stem cells. Emerging myogenic progenitors were specified to form multinucleated fibers that enabled development of quiescent, satellite cell-like progenitors and a model for Duchenne Muscular Dystrophy. PMID:26340524

  20. Moral Identity and Adolescent Prosocial and Antisocial Behaviors: Interactions with Moral Disengagement and Self-regulation.

    PubMed

    Hardy, Sam A; Bean, Dallas S; Olsen, Joseph A

    2015-08-01

    Moral identity has been positively linked to prosocial behaviors and negatively linked to antisocial behaviors; but, the processes by which it is linked to such outcomes are unclear. The purpose of the present study was to examine moral identity not only as a predictor, but also as a moderator of relationships between other predictors (moral disengagement and self-regulation) and youth outcomes (prosocial and antisocial behaviors). The sample consisted of 384 adolescents (42 % female), ages 15-18 recruited from across the US using an online survey panel. Latent variables were created for moral identity, moral disengagement, and self-regulation. Structural equation models assessed these latent variables, and interactions of moral identity with moral disengagement and self-regulation, as predictors of prosocial (charity and civic engagement) and antisocial (aggression and rule breaking) behaviors. None of the interactions were significant predicting prosocial behaviors. For antisocial behaviors, the interaction between moral identity and moral disengagement predicted aggression, while the interaction between moral identity and self-regulation was significant in predicting aggression and rule breaking. Specifically, at higher levels of moral identity, the positive link between moral disengagement and aggression was weaker, and the negative link between self-regulation and both antisocial behaviors was weaker. Thus, moral identity may buffer against the maladaptive effects of high moral disengagement and low self-regulation. PMID:25146465

  1. Moral Identity and Adolescent Prosocial and Antisocial Behaviors: Interactions with Moral Disengagement and Self-regulation.

    PubMed

    Hardy, Sam A; Bean, Dallas S; Olsen, Joseph A

    2015-08-01

    Moral identity has been positively linked to prosocial behaviors and negatively linked to antisocial behaviors; but, the processes by which it is linked to such outcomes are unclear. The purpose of the present study was to examine moral identity not only as a predictor, but also as a moderator of relationships between other predictors (moral disengagement and self-regulation) and youth outcomes (prosocial and antisocial behaviors). The sample consisted of 384 adolescents (42 % female), ages 15-18 recruited from across the US using an online survey panel. Latent variables were created for moral identity, moral disengagement, and self-regulation. Structural equation models assessed these latent variables, and interactions of moral identity with moral disengagement and self-regulation, as predictors of prosocial (charity and civic engagement) and antisocial (aggression and rule breaking) behaviors. None of the interactions were significant predicting prosocial behaviors. For antisocial behaviors, the interaction between moral identity and moral disengagement predicted aggression, while the interaction between moral identity and self-regulation was significant in predicting aggression and rule breaking. Specifically, at higher levels of moral identity, the positive link between moral disengagement and aggression was weaker, and the negative link between self-regulation and both antisocial behaviors was weaker. Thus, moral identity may buffer against the maladaptive effects of high moral disengagement and low self-regulation.

  2. On human pluripotent stem cell control: The rise of 3D bioengineering and mechanobiology

    PubMed Central

    Shao, Yue; Sang, Jianming; Fu, Jianping

    2015-01-01

    Human pluripotent stem cells (hPSCs) provide promising resources for regenerating tissues and organs and modeling development and diseases in vitro. To fulfill their promise, the fate, function, and organization of hPSCs need to be precisely regulated in a three-dimensional (3D) environment to mimic cellular structures and functions of native tissues and organs. In the past decade, innovations in 3D culture systems with functional biomaterials have enabled efficient and versatile control of hPSC fate at the cellular level. However, we are just at the beginning of bringing hPSC-based regeneration and development and disease modeling to the tissue and organ levels. In this review, we summarize existing bioengineered culture platforms for controlling hPSC fate and function by regulating inductive mechanical and biochemical cues coexisting in the synthetic cell microenvironment. We highlight recent excitements in developing 3D hPSC-based in vitro tissue and organ models with in vivo-like cellular structures, interactions, and functions. We further discuss an emerging multifaceted mechanotransductive signaling network – with transcriptional coactivators YAP and TAZ at the center stage – that regulate fates and behaviors of mammalian cells, including hPSCs. Future development of 3D biomaterial systems should incorporate dynamically modulated mechanical and chemical properties targeting specific intracellular signaling events leading to desirable hPSC fate patterning and functional tissue formation in 3D. PMID:25818411

  3. Pluripotency factors and Polycomb Group proteins repress aryl hydrocarbon receptor expression in murine embryonic stem cells.

    PubMed

    Ko, Chia-I; Wang, Qin; Fan, Yunxia; Xia, Ying; Puga, Alvaro

    2014-01-01

    The aryl hydrocarbon receptor (AHR) is a transcription factor and environmental sensor that regulates expression of genes involved in drug-metabolism and cell cycle regulation. Chromatin immunoprecipitation analyses, Ahr ablation in mice and studies with orthologous genes in invertebrates suggest that AHR may also play a significant role in embryonic development. To address this hypothesis, we studied the regulation of Ahr expression in mouse embryonic stem cells and their differentiated progeny. In ES cells, interactions between OCT3/4, NANOG, SOX2 and Polycomb Group proteins at the Ahr promoter repress AHR expression, which can also be repressed by ectopic expression of reprogramming factors in hepatoma cells. In ES cells, unproductive RNA polymerase II binds at the Ahr transcription start site and drives the synthesis of short abortive transcripts. Activation of Ahr expression during differentiation follows from reversal of repressive marks in Ahr promoter chromatin, release of pluripotency factors and PcG proteins, binding of Sp factors, establishment of histone marks of open chromatin, and engagement of active RNAPII to drive full-length RNA transcript elongation. Our results suggest that reversible Ahr repression in ES cells holds the gene poised for expression and allows for a quick switch to activation during embryonic development.

  4. On human pluripotent stem cell control: The rise of 3D bioengineering and mechanobiology.

    PubMed

    Shao, Yue; Sang, Jianming; Fu, Jianping

    2015-06-01

    Human pluripotent stem cells (hPSCs) provide promising resources for regenerating tissues and organs and modeling development and diseases in vitro. To fulfill their promise, the fate, function, and organization of hPSCs need to be precisely regulated in a three-dimensional (3D) environment to mimic cellular structures and functions of native tissues and organs. In the past decade, innovations in 3D culture systems with functional biomaterials have enabled efficient and versatile control of hPSC fate at the cellular level. However, we are just at the beginning of bringing hPSC-based regeneration and development and disease modeling to the tissue and organ levels. In this review, we summarize existing bioengineered culture platforms for controlling hPSC fate and function by regulating inductive mechanical and biochemical cues coexisting in the synthetic cell microenvironment. We highlight recent excitements in developing 3D hPSC-based in vitro tissue and organ models with in vivo-like cellular structures, interactions, and functions. We further discuss an emerging multifaceted mechanotransductive signaling network--with transcriptional coactivators YAP and TAZ at the center stage--that regulate fates and behaviors of mammalian cells, including hPSCs. Future development of 3D biomaterial systems should incorporate dynamically modulated mechanical and chemical properties targeting specific intracellular signaling events leading to desirable hPSC fate patterning and functional tissue formation in 3D. PMID:25818411

  5. On human pluripotent stem cell control: The rise of 3D bioengineering and mechanobiology

    PubMed Central

    Shao, Yue; Sang, Jianming; Fu, Jianping

    2015-01-01

    Human pluripotent stem cells (hPSCs) provide promising resources for regenerating tissues and organs and modeling development and diseases in vitro. To fulfill their promise, the fate, function, and organization of hPSCs need to be precisely regulated in a three-dimensional (3D) environment to mimic cellular structures and functions of native tissues and organs. In the past decade, innovations in 3D culture systems with functional biomaterials have enabled efficient and versatile control of hPSC fate at the cellular level. However, we are just at the beginning of bringing hPSC-based regeneration and development and disease modeling to the tissue and organ levels. In this review, we summarize existing bioengineered culture platforms for controlling hPSC fate and function by regulating inductive mechanical and biochemical cues coexisting in the synthetic cell microenvironment. We highlight recent excitements in developing 3D hPSC-based in vitro tissue and organ models with in vivo-like cellular structures, interactions, and functions. We further discuss an emerging multifaceted mechanotransductive signaling network – with transcriptional coactivators YAP and TAZ at the center stage – that regulate fates and behaviors of mammalian cells, including hPSCs. Future development of 3D biomaterial systems should incorporate dynamically modulated mechanical and chemical properties targeting specific intracellular signaling events leading to desirable hPSC fate patterning and functional tissue formation in 3D. PMID:25818411

  6. Histamine regulation of microglia: Gene-environment interaction in the regulation of central nervous system inflammation.

    PubMed

    Frick, Luciana; Rapanelli, Maximiliano; Abbasi, Eeman; Ohtsu, Hiroshi; Pittenger, Christopher

    2016-10-01

    Microglia mediate neuroinflammation and regulate brain development and homeostasis. Microglial abnormalities are implicated in a range of neuropsychiatric pathology, including Tourette syndrome (TS) and autism. Histamine (HA) is both a neurotransmitter and an immune modulator. HA deficiency has been implicated as a rare cause of TS and may contribute to other neuropsychiatric conditions. In vitro studies suggest that HA can regulate microglia, but this has never been explored in vivo. We used immunohistochemistry to examine the effects of HA deficiency in histidine decarboxylase (Hdc) knockout mice and of HA receptor stimulation in wild-type animals. We find HA to regulate microglia in vivo, via the H4 receptor. Chronic HA deficiency in Hdc knockout mice reduces ramifications of microglia in the striatum and (at trend level) in the hypothalamus, but not elsewhere in the brain. Depletion of histaminergic neurons in the hypothalamus has a similar effect. Microglia expressing IGF-1 are particularly reduced, However, the microglial response to challenge with lipopolysacchariade (LPS) is potentiated in Hdc knockout mice. Genetic abnormalities in histaminergic signaling may produce a vulnerability to inflammatory challenge, setting the state for pathogenically dysregulated neuroimmune responses.

  7. Identification of a small molecule that turns ON the pluripotency gene circuitry in human fibroblasts.

    PubMed

    Pandian, Ganesh N; Sato, Shinsuke; Anandhakumar, Chandran; Taniguchi, Junichi; Takashima, Kazuhiro; Syed, Junetha; Han, Le; Saha, Abhijit; Bando, Toshikazu; Nagase, Hiroki; Sugiyama, Hiroshi

    2014-12-19

    A nontransgenic approach to reprogram mouse somatic cells into induced pluripotent stem cells using only small molecules got achieved to propose a potential clinical-friendly cellular reprogramming strategy. Consequently, the screening and identification of small molecules capable of inducing pluripotency genes in human cells are increasingly a focus of research. Because cellular reprogramming is multifactorial in nature, there is a need for versatile small molecules capable of modulating the complicated gene networks associated with pluripotency. We have developed a targeting small molecule called SAHA-PIP comprising the histone deacetylase inhibitor SAHA and the sequence-specific DNA binding pyrrole-imidazole polyamides for modulating distinct gene networks. Here, we report the identification of a SAHA-PIP termed Ì that could trigger genome-wide epigenetic reprogramming and turn ON the typically conserved core pluripotency gene network. Through independent lines of evidence, we report for the first time a synthetic small molecule inducer that target and activate the OCT-3/4 regulated pluripotency genes in human dermal fibroblasts.

  8. Identification of a small molecule that turns ON the pluripotency gene circuitry in human fibroblasts.

    PubMed

    Pandian, Ganesh N; Sato, Shinsuke; Anandhakumar, Chandran; Taniguchi, Junichi; Takashima, Kazuhiro; Syed, Junetha; Han, Le; Saha, Abhijit; Bando, Toshikazu; Nagase, Hiroki; Sugiyama, Hiroshi

    2014-12-19

    A nontransgenic approach to reprogram mouse somatic cells into induced pluripotent stem cells using only small molecules got achieved to propose a potential clinical-friendly cellular reprogramming strategy. Consequently, the screening and identification of small molecules capable of inducing pluripotency genes in human cells are increasingly a focus of research. Because cellular reprogramming is multifactorial in nature, there is a need for versatile small molecules capable of modulating the complicated gene networks associated with pluripotency. We have developed a targeting small molecule called SAHA-PIP comprising the histone deacetylase inhibitor SAHA and the sequence-specific DNA binding pyrrole-imidazole polyamides for modulating distinct gene networks. Here, we report the identification of a SAHA-PIP termed Ì that could trigger genome-wide epigenetic reprogramming and turn ON the typically conserved core pluripotency gene network. Through independent lines of evidence, we report for the first time a synthetic small molecule inducer that target and activate the OCT-3/4 regulated pluripotency genes in human dermal fibroblasts. PMID:25366962

  9. Role of the small subunit processome in the maintenance of pluripotent stem cells.

    PubMed

    You, Kwon Tae; Park, Joha; Kim, V Narry

    2015-10-01

    RNA-binding proteins (RBPs) play integral roles in gene regulation, yet only a small fraction of RBPs has been studied in the context of stem cells. Here we applied an RNAi screen for RBPs in mouse embryonic stem cells (ESCs) and identified 16 RBPs involved in pluripotency maintenance. Interestingly, six identified RBPs, including Krr1 and Ddx47, are part of a complex called small subunit processome (SSUP) that mediates 18S rRNA biogenesis. The SSUP components are preferentially expressed in stem cells and enhance the global translational rate, which is critical to sustain the protein levels of labile pluripotency factors such as Nanog and Esrrb. Furthermore, the SSUP proteins are required for efficient reprogramming of induced pluripotent stem cells. Our study uncovers the role of the SSUP and the importance of translational control in stem cell fate decision.

  10. Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology

    PubMed Central

    Sudhir, Putty-Reddy; Chen, Chung-Hsuan

    2016-01-01

    A protein complex consists of two or more proteins that are linked together through protein–protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions. The delineation of protein complexes is important to expand our knowledge on proteins functional roles in physiological and pathological conditions. The genetic yeast-2-hybrid method has been extensively used to characterize protein-protein interactions. Alternatively, a biochemical-based affinity purification coupled with mass spectrometry (AP-MS) approach has been widely used to characterize the protein complexes. In the AP-MS method, a protein complex of a target protein of interest is purified using a specific antibody or an affinity tag (e.g., DYKDDDDK peptide (FLAG) and polyhistidine (His)) and is subsequently analyzed by means of MS. Tandem affinity purification, a two-step purification system, coupled with MS has been widely used mainly to reduce the contaminants. We review here a general principle for AP-MS-based characterization of protein complexes and we explore several protein complexes identified in pluripotent stem cell biology and cancer biology as examples. PMID:27011181

  11. Tex10 Coordinates Epigenetic Control of Super-Enhancer Activity in Pluripotency and Reprogramming

    PubMed Central

    Ding, Junjun; Huang, Xin; Shao, Ningyi; Zhou, Hongwei; Lee, Dung-Fang; Faiola, Francesco; Fidalgo, Miguel; Guallar, Diana; Saunders, Arven; Shliaha, Pavel V.; Wang, Hailong; Waghray, Avinash; Papatsenko, Dmitri; Sánchez-Priego, Carlos; Li, Dan; Yuan, Ye; Lemischka, Ihor R.; Shen, Li; Kelley, Kevin; Deng, Haiteng; Shen, Xiaohua; Wang, Jianlong

    2015-01-01

    SUMMARY Super-enhancers (SEs) are large clusters of transcriptional enhancers that are co-occupied by multiple lineage specific transcription factors driving expression of genes that define cell identity. In embryonic stem cells (ESCs), SEs are highly enriched for the core pluripotency factors Oct4, Sox2, and Nanog. In this study, we sought to dissect the molecular control mechanism of SE activity in pluripotency and reprogramming. Starting from a protein interaction network surrounding Sox2, we identified Tex10 as a key pluripotency factor that plays a functionally significant role in ESC self-renewal, early embryo development, and reprogramming. Tex10 is enriched at SEs in a Sox2-dependent manner and coordinates histone acetylation and DNA demethylation at SEs. Tex10 activity is also important for pluripotency and reprogramming in human cells. Our study therefore highlights Tex10 as a core component of the pluripotency network and sheds light on its role in epigenetic control of SE activity for cell fate determination. PMID:25936917

  12. Regulation of claudin/zonula occludens-1 complexes by hetero-claudin interactions

    PubMed Central

    Schlingmann, Barbara; Overgaard, Christian E.; Molina, Samuel A.; Lynn, K. Sabrina; Mitchell, Leslie A.; Dorsainvil White, StevenClaude; Mattheyses, Alexa L.; Guidot, David M.; Capaldo, Christopher T.; Koval, Michael

    2016-01-01

    Claudins are tetraspan transmembrane tight-junction proteins that regulate epithelial barriers. In the distal airspaces of the lung, alveolar epithelial tight junctions are crucial to regulate airspace fluid. Chronic alcohol abuse weakens alveolar tight junctions, priming the lung for acute respiratory distress syndrome, a frequently lethal condition caused by airspace flooding. Here we demonstrate that in response to alcohol, increased claudin-5 paradoxically accompanies an increase in paracellular leak and rearrangement of alveolar tight junctions. Claudin-5 is necessary and sufficient to diminish alveolar epithelial barrier function by impairing the ability of claudin-18 to interact with a scaffold protein, zonula occludens 1 (ZO-1), demonstrating that one claudin affects the ability of another claudin to interact with the tight-junction scaffold. Critically, a claudin-5 peptide mimetic reverses the deleterious effects of alcohol on alveolar barrier function. Thus, claudin controlled claudin-scaffold protein interactions are a novel target to regulate tight-junction permeability. PMID:27452368

  13. Regulation of claudin/zonula occludens-1 complexes by hetero-claudin interactions.

    PubMed

    Schlingmann, Barbara; Overgaard, Christian E; Molina, Samuel A; Lynn, K Sabrina; Mitchell, Leslie A; Dorsainvil White, StevenClaude; Mattheyses, Alexa L; Guidot, David M; Capaldo, Christopher T; Koval, Michael

    2016-01-01

    Claudins are tetraspan transmembrane tight-junction proteins that regulate epithelial barriers. In the distal airspaces of the lung, alveolar epithelial tight junctions are crucial to regulate airspace fluid. Chronic alcohol abuse weakens alveolar tight junctions, priming the lung for acute respiratory distress syndrome, a frequently lethal condition caused by airspace flooding. Here we demonstrate that in response to alcohol, increased claudin-5 paradoxically accompanies an increase in paracellular leak and rearrangement of alveolar tight junctions. Claudin-5 is necessary and sufficient to diminish alveolar epithelial barrier function by impairing the ability of claudin-18 to interact with a scaffold protein, zonula occludens 1 (ZO-1), demonstrating that one claudin affects the ability of another claudin to interact with the tight-junction scaffold. Critically, a claudin-5 peptide mimetic reverses the deleterious effects of alcohol on alveolar barrier function. Thus, claudin controlled claudin-scaffold protein interactions are a novel target to regulate tight-junction permeability. PMID:27452368

  14. HPS4/SABRE regulates plant responses to phosphate starvation through antagonistic interaction with ethylene signalling

    PubMed Central

    Yu, Hailan; Luo, Nan; Sun, Lichao; Liu, Dong

    2012-01-01

    The phytohormone ethylene plays important roles in regulating plant responses to phosphate (Pi) starvation. To date, however, no molecular components have been identified that interact with ethylene signalling in regulating such responses. In this work, an Arabidopsis mutant, hps4, was characterized that exhibits enhanced responses to Pi starvation, including increased inhibition of primary root growth, enhanced expression of Pi starvation-induced genes, and overproduction of root-associated acid phosphatases. Molecular cloning indicated that hps4 is a new allele of SABRE, which was previously identified as an important regulator of cell expansion in Arabidopsis. HPS4/SABRE antagonistically interacts with ethylene signalling to regulate plant responses to Pi starvation. Furthermore, it is shown that Pi-starved hps4 mutants accumulate more auxin in their root tips than the wild type, which may explain the increased inhibition of their primary root growth when grown under Pi deficiency. PMID:22615140

  15. Making the Auroras glow: regulation of Aurora A and B kinase function by interacting proteins.

    PubMed

    Carmena, Mar; Ruchaud, Sandrine; Earnshaw, William C

    2009-12-01

    The conserved Aurora family of protein kinases have emerged as crucial regulators of mitosis and cytokinesis. Despite their high degree of homology, Aurora A and B have very distinctive localisations and functions: Aurora A associates with the spindle poles to regulate entry into mitosis, centrosome maturation and spindle assembly; Aurora B is a member of the Chromosomal Passenger Complex (CPC) that transfers from the inner centromere in early mitosis to the spindle midzone, equatorial cortex and midbody in late mitosis and cytokinesis. Aurora B functions include regulation of chromosome-microtubule interactions, cohesion, spindle stability and cytokinesis. This review will focus on how interacting proteins make this functional diversity possible by targeting the kinases to different subcellular locations and regulating their activity. PMID:19836940

  16. Making the Auroras glow: regulation of Aurora A and B kinase function by interacting proteins.

    PubMed

    Carmena, Mar; Ruchaud, Sandrine; Earnshaw, William C

    2009-12-01

    The conserved Aurora family of protein kinases have emerged as crucial regulators of mitosis and cytokinesis. Despite their high degree of homology, Aurora A and B have very distinctive localisations and functions: Aurora A associates with the spindle poles to regulate entry into mitosis, centrosome maturation and spindle assembly; Aurora B is a member of the Chromosomal Passenger Complex (CPC) that transfers from the inner centromere in early mitosis to the spindle midzone, equatorial cortex and midbody in late mitosis and cytokinesis. Aurora B functions include regulation of chromosome-microtubule interactions, cohesion, spindle stability and cytokinesis. This review will focus on how interacting proteins make this functional diversity possible by targeting the kinases to different subcellular locations and regulating their activity.

  17. The psychiatric disease risk factors DISC1 and TNIK interact to regulate synapse composition and function

    PubMed Central

    Wang, Q; Charych, EI; Pulito, VL; Lee, JB; Graziane, NM; Crozier, RA; Revilla-Sanchez, R; Kelly, MP; Dunlop, AJ; Murdoch, H; Taylor, N; Xie, Y; Pausch, M; Hayashi-Takagi, A; Ishizuka, K; Seshadri, S; Bates, B; Kariya, K; Sawa, A; Weinberg, RJ; Moss, SJ; Houslay, MD; Yan, Z; Brandon, NJ

    2011-01-01

    Disrupted in schizophrenia 1 (DISC1), a genetic risk factor for multiple serious psychiatric diseases including schizophrenia, bipolar disorder and autism, is a key regulator of multiple neuronal functions linked to both normal development and disease processes. As these diseases are thought to share a common deficit in synaptic function and architecture, we have analyzed the role of DISC1 using an approach that focuses on understanding the protein– protein interactions of DISC1 specifically at synapses. We identify the Traf2 and Nck-interacting kinase (TNIK), an emerging risk factor itself for disease, as a key synaptic partner for DISC1, and provide evidence that the DISC1–TNIK interaction regulates synaptic composition and activity by stabilizing the levels of key postsynaptic density proteins. Understanding the novel DISC1–TNIK interaction is likely to provide insights into the etiology and underlying synaptic deficits found in major psychiatric diseases. PMID:20838393

  18. Analysis of CD40-CD40 ligand interactions in the regulation of human B cell function.

    PubMed

    Lipsky, P E; Attrep, J F; Grammer, A C; McIlraith, M J; Nishioka, Y

    1997-04-01

    CD40-CD40 ligand interactions play an essential role in T cell/B cell collaboration. The data presented in this review have served to widen the scope of CD40-CD40 ligand interactions to include initial activation, proliferation, differentiation, and isotype switching of B cells, as well as subsequent downregulation of B cell function. Moreover, CD40 ligand expression by activated B cells is likely to play an essential role in facilitating ongoing responses of stimulated B cells maturing in germinal centers. Finally, CD40 expression by activated T cells may also play an important role in regulating the function of helper T cells within germinal centers. In summary, emerging data have expanded the role of CD40-CD40 ligand interaction during T cell/B cell collaboration and have emphasized its potential to regulate many of the functions of both partners in this essential interaction involved in antibody production.

  19. HIF-1α regulates the interaction of chronic lymphocytic leukemia cells with the tumor microenvironment

    PubMed Central

    Valsecchi, Roberta; Coltella, Nadia; Belloni, Daniela; Ponente, Manfredi; ten Hacken, Elisa; Scielzo, Cristina; Scarfò, Lydia; Bertilaccio, Maria Teresa Sabrina; Brambilla, Paola; Lenti, Elisa; Martinelli Boneschi, Filippo; Brendolan, Andrea; Ferrero, Elisabetta; Ferrarini, Marina; Ghia, Paolo; Tonon, Giovanni; Ponzoni, Maurilio; Caligaris-Cappio, Federico

    2016-01-01

    Hypoxia-inducible transcription factors (HIFs) regulate a wide array of adaptive responses to hypoxia and are often activated in solid tumors and hematologic malignancies due to intratumoral hypoxia and emerging new layers of regulation. We found that in chronic lymphocytic leukemia (CLL), HIF-1α is a novel regulator of the interaction of CLL cells with protective leukemia microenvironments and, in turn, is regulated by this interaction in a positive feedback loop that promotes leukemia survival and propagation. Through unbiased microarray analysis, we found that in CLL cells, HIF-1α regulates the expression of important chemokine receptors and cell adhesion molecules that control the interaction of leukemic cells with bone marrow and spleen microenvironments. Inactivation of HIF-1α impairs chemotaxis and cell adhesion to stroma, reduces bone marrow and spleen colonization in xenograft and allograft CLL mouse models, and prolongs survival in mice. Of interest, we found that in CLL cells, HIF-1α is transcriptionally regulated after coculture with stromal cells. Furthermore, HIF-1α messenger RNA levels vary significantly within CLL patients and correlate with the expression of HIF-1α target genes, including CXCR4, thus further emphasizing the relevance of HIF-1α expression to CLL pathogenesis. PMID:26825709

  20. Identification of two distinct transactivation domains in the pluripotency sustaining factor nanog.

    PubMed

    Pan, Guang Jin; Pei, Duan Qing

    2003-12-01

    Nanog is a newly identified homeodomain gene that functions to sustain the pluripotency of embryonic stem cells. However, the molecular mechanism through which nanog regulates stem cell pluripotency remains unknown. Mouse nanog encodes a polypeptide of 305 residues with a divergent homeodomain similar to those in the NK-2 family. The rest of nanog contains no apparent homology to any known proteins characterized so far. It is hypothesized that nanog encodes a transcription factor that regulates stem cell pluripotency by switching on or off target genes. To test this hypothesis, we constructed fusion proteins between nanog and DNA binding domains of the yeast transcription factor Gal4 and tested the transactivation potentials of these constructs. Our data demonstrate that both regions N- and C- terminal to the homeodomain have transcription activities. Despite the fact that it contains no apparent transactivation motifs, the C-terminal domain is about 7 times as active as the N-terminal one. This unique arrangement of dual transactivators may confer nanog the flexibility and specificity to regulate downstream genes critical for both pluripotency and differentiation of stem cells.

  1. Network motifs in integrated cellular networks of transcription-regulation and protein-protein interaction

    NASA Astrophysics Data System (ADS)

    Yeger-Lotem, Esti; Sattath, Shmuel; Kashtan, Nadav; Itzkovitz, Shalev; Milo, Ron; Pinter, Ron Y.; Alon, Uri; Margalit, Hanah

    2004-04-01

    Genes and proteins generate molecular circuitry that enables the cell to process information and respond to stimuli. A major challenge is to identify characteristic patterns in this network of interactions that may shed light on basic cellular mechanisms. Previous studies have analyzed aspects of this network, concentrating on either transcription-regulation or protein-protein interactions. Here we search for composite network motifs: characteristic network patterns consisting of both transcription-regulation and protein-protein interactions that recur significantly more often than in random networks. To this end we developed algorithms for detecting motifs in networks with two or more types of interactions and applied them to an integrated data set of protein-protein interactions and transcription regulation in Saccharomyces cerevisiae. We found a two-protein mixed-feedback loop motif, five types of three-protein motifs exhibiting coregulation and complex formation, and many motifs involving four proteins. Virtually all four-protein motifs consisted of combinations of smaller motifs. This study presents a basic framework for detecting the building blocks of networks with multiple types of interactions.

  2. Interaction of a plant pseudo-response regulator with a calmodulin-like protein

    SciTech Connect

    Perochon, Alexandre; Dieterle, Stefan; Pouzet, Cecile; Aldon, Didier; Galaud, Jean-Philippe

    2010-08-06

    Research highlights: {yields} The pseudo-response regulator PRR2 specifically binds CML9, a calmodulin-like protein {yields} The interaction is confirmed in plant cell nuclei {yields} The interaction requires an intact PRR2 protein. -- Abstract: Calmodulin (CaM) plays a crucial role in the regulation of diverse cellular processes by modulating the activities of numerous target proteins. Plants possess an extended CaM family including numerous CaM-like proteins (CMLs), most of which appear to be unique to plants. We previously demonstrated a role for CML9 in abiotic stress tolerance and seed germination in Arabidopsis thaliana. We report here the isolation of PRR2, a pseudo-response regulator as a CML9 interacting protein by screening an expression library prepared from Arabidopsis seedlings with CML9 as bait in a yeast two-hybrid system. PRR2 is similar to the response regulators of the two-component system, but lacks the invariant residue required for phosphorylation by which response regulators switch their output response, suggesting the existence of alternative regulatory mechanisms. PRR2 was found to bind CML9 and closely related CMLs but not a canonical CaM. Mapping analyses indicate that an almost complete form of PRR2 is required for interaction with CML9, suggesting a recognition mode different from the classical CaM-target peptide complex. PRR2 contains several features that are typical of transcription factors, including a GARP DNA recognition domain, a Pro-rich region and a Golden C-terminal box. PRR2 and CML9 as fusion proteins with fluorescent tags co-localized in the nucleus of plant cells, and their interaction in the nuclear compartment was validated in planta by using a fluorophore-tagged protein interaction assay. These findings suggest that binding of PRR2 to CML9 may be an important mechanism to modulate the physiological role of this transcription factor in plants.

  3. Mechanical stimulation of cyclic tensile strain induces reduction of pluripotent related gene expressions via activation of Rho/ROCK and subsequent decreasing of AKT phosphorylation in human induced pluripotent stem cells

    SciTech Connect

    Teramura, Takeshi; Takehara, Toshiyuki; Onodera, Yuta; Nakagawa, Koichi; Hamanishi, Chiaki; Fukuda, Kanji

    2012-01-13

    Highlights: Black-Right-Pointing-Pointer Mechanical stimulation is an important factor for regulation of stem cell fate. Black-Right-Pointing-Pointer Cyclic stretch to human induced pluripotent stem cells activated small GTPase Rho. Black-Right-Pointing-Pointer Rho-kinase activation attenuated pluripotency via inhibition of AKT activation. Black-Right-Pointing-Pointer This reaction could be reproduced only by transfection of dominant active Rho. Black-Right-Pointing-Pointer Rho/ROCK are important molecules in mechanotransduction and control of stemness. -- Abstract: Mechanical stimulation has been shown to regulate the proliferation and differentiation of stem cells. However, the effects of the mechanical stress on the stemness or related molecular mechanisms have not been well determined. Pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are used as good materials for cell transplantation therapy and research of mammalian development, since they can self-renew infinitely and differentiate into various cell lineages. Here we demonstrated that the mechanical stimulation to human iPS cells altered alignment of actin fibers and expressions of the pluripotent related genes Nanog, POU5f1 and Sox2. In the mechanically stimulated iPS cells, small GTPase Rho was activated and interestingly, AKT phosphorylation was decreased. Inhibition of Rho-associated kinase ROCK recovered the AKT phosphorylation and the gene expressions. These results clearly suggested that the Rho/ROCK is a potent primary effector of mechanical stress in the pluripotent stem cells and it participates to pluripotency-related signaling cascades as an upper stream regulator.

  4. NAC1, a cocaine-regulated POZ/BTB protein interacts with CoREST.

    PubMed

    Korutla, Laxminarayana; Degnan, Ryan; Wang, Peijie; Mackler, Scott A

    2007-05-01

    In this report, CoREST was identified as a protein that interacts with NAC1. NAC1 is a cocaine-regulated Pox virus and Zinc finger/Bric-a-brac Tramtrack Broad complex (POZ/BTB) repressor protein, which mediates interactions among several other transcriptional regulators. In the present study, an interaction between NAC1 and CoREST was detected in neuro-2A cells and HEK293T cells. We found that the POZ/BTB domain is necessary and sufficient for interaction with CoREST. Surprisingly, only one of five mutations in the POZ/BTB domain that disrupts homodimer assembly interfered with NAC1 and CoREST interactions. These results indicate that POZ/BTB homodimer formation is not required for NAC1-CoREST interaction. CoREST demonstrated protein-protein interactions with both isoforms of NAC1, sNAC1, and lNAC1. Coimmunoprecipitation studies show that NAC1 and CoREST are physically bound together. To further support the results, a direct interaction was demonstrated in glutathione-S-transferase pull down assays. siRNA directed against NAC1 mRNA significantly reduced NAC1 protein expression and resulted in reversal of CoREST-mediated repression in cells. This interaction between NAC1 and CoREST was not found for other POZ/BTB proteins tested. Endogenous interaction was demonstrated in lysates from rat brain samples. This is the first report to demonstrate that a POZ/BTB protein interacts with CoREST. Taken together, the results indicate that CoREST may be part of the NAC1 repressor mechanism.

  5. Regulation of plant growth and development by the GROWTH-REGULATING FACTOR and GRF-INTERACTING FACTOR duo.

    PubMed

    Hoe Kim, Jeong; Tsukaya, Hirokazu

    2015-10-01

    Transcription factors are key regulators of gene expression and play pivotal roles in all aspects of living organisms. Therefore, identification and functional characterization of transcription factors is a prerequisite step toward understanding life. This article reviews molecular and biological functions of the two transcription regulator families, GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF), which have only recently been recognized. A myriad of experimental evidence clearly illustrates that GRF and GIF are bona fide partner proteins and form a plant-specific transcriptional complex. One of the most conspicuous outcomes from this research field is that the GRF-GIF duo endows the primordial cells of vegetative and reproductive organs with a meristematic specification state, guaranteeing the supply of cells for organogenesis and successful reproduction. It has recently been shown that GIF1 proteins, also known as ANGUSTIFOLIA3, recruit chromatin remodelling complexes to target genes, and that AtGRF expression is directly activated by the floral identity factors, APETALA1 and SEPALLATA3, providing an important insight into understanding of the action of GRF-GIF. Moreover, GRF genes are extensively subjected to post-transcriptional control by microRNA396, revealing the presence of a complex regulatory circuit in regulation of plant growth and development by the GRF-GIF duo.

  6. PAR3-aPKC regulates Tiam1 by modulating suppressive internal interactions

    PubMed Central

    Matsuzawa, Kenji; Akita, Hiroki; Watanabe, Takashi; Kakeno, Mai; Matsui, Toshinori; Wang, Shujie; Kaibuchi, Kozo

    2016-01-01

    Tiam1 is one of the most extensively analyzed activators of the small GTPase Rac. However, fundamental aspects of its regulation are poorly understood. Here we demonstrate that Tiam1 is functionally suppressed by internal interactions and that the PAR complex participates in its full activation. The N-terminal region of Tiam1 binds to the protein-binding and catalytic domains to inhibit its localization and activation. Atypical PKCs phosphorylate Tiam1 to relieve its intramolecular interactions, and the subsequent stabilization of its interaction with PAR3 allows it to exert localized activity. By analyzing Tiam1 regulation by PAR3-aPKC within the context of PDGF signaling, we also show that PAR3 directly binds PDGF receptor β. Thus we provide the first evidence for the negative regulation of Tiam1 by internal interactions, elucidate the nature of Tiam1 regulation by the PAR complex, and reveal a novel role for the PAR complex in PDGF signaling. PMID:26941335

  7. Customizable Computer-Based Interaction Analysis for Coaching and Self-Regulation in Synchronous CSCL Systems

    ERIC Educational Resources Information Center

    Lonchamp, Jacques

    2010-01-01

    Computer-based interaction analysis (IA) is an automatic process that aims at understanding a computer-mediated activity. In a CSCL system, computer-based IA can provide information directly to learners for self-assessment and regulation and to tutors for coaching support. This article proposes a customizable computer-based IA approach for a…

  8. Interaction Effects of Hypervideo Navigation Variables in College Students' Self-Regulated Learning

    ERIC Educational Resources Information Center

    Azmy, Nabil

    2013-01-01

    The purpose of this study is to investigate the question of whether the interaction effects of hypervideo navigation variables (navigation control and navigation links) would affect college students' self-regulated learning just after their learning from instructional hypervideo programs. Navigation control (free navigation or free navigation with…

  9. Family Interactions, Exposure to Violence, and Emotion Regulation: Perceptions of Children and Early Adolescents at Risk

    ERIC Educational Resources Information Center

    Houltberg, Benjamin J.; Henry, Carolyn S.; Morris, Amanda Sheffield

    2012-01-01

    This study examined the protective nature of youth reports of family interactions in relation to perceived exposure to violence and anger regulation in 84 children and early adolescents (mean age of 10.5; 7-15 years old) primarily from ethnic minority groups and living in high-risk communities in a large southwestern city. Path analysis and…

  10. Students' Self-Regulation for Interaction with Others in Online Learning Environments

    ERIC Educational Resources Information Center

    Cho, Moon-Heum; Kim, B. Joon

    2013-01-01

    The purpose of this study was to explore variables explaining students' self-regulation (SR) for interaction with others, specifically peers and instructors, in online learning environments. A total of 407 students participated in the study. With hierarchical regression model (HRM), several variables were regressed on students' SR for interaction…

  11. Interactive regulation of affect in postpartum depressed mothers and their infants: an overview.

    PubMed

    Reck, Corinna; Hunt, Aoife; Fuchs, Thomas; Weiss, Robert; Noon, Andrea; Moehler, Eva; Downing, George; Tronick, Edward Z; Mundt, Christoph

    2004-01-01

    Specific patterns of interaction emerging in the first months of life are related to processes regulating mutual affects in the mother-child dyad. Particularly important for the dyad are the matching and interactive repair processes. The interaction between postpartum depressed mothers and their children is characterized by a lack of responsiveness, by passivity or intrusiveness, withdrawal and avoidance, as well as a low level of positive expression of affect. Thus, an impaired capability to regulate the child's affect has been demonstrated in depressed mothers. Maternal aggression, neglect toward infants, infanticidal thoughts, as well as infanticidal behavior are mainly linked to severe postpartum depression, especially with psychotic symptoms. The findings on mother-child interaction reported in this paper are based on mothers with mild to moderate depressive disorders without psychotic symptoms. Considering the stability of interaction patterns in the course of depressive illness as well as the long-term consequences of these interactions, it seems surprising that there are still few systematic studies of depressed mothers interacting with their infants. In connection with an overview on these issues, treatment models for parent-infant psychotherapy are discussed. PMID:15539778

  12. The heterotrimeric kinesin-2 complex interacts with and regulates GLI protein function

    PubMed Central

    Carpenter, Brandon S.; Barry, Renee L.; Verhey, Kristen J.; Allen, Benjamin L.

    2015-01-01

    ABSTRACT GLI transport to the primary cilium and nucleus is required for proper Hedgehog (HH) signaling; however, the mechanisms that mediate these trafficking events are poorly understood. Kinesin-2 motor proteins regulate ciliary transport of cargo, yet their role in GLI protein function remains unexplored. To examine a role for the heterotrimeric KIF3A–KIF3B–KAP3 kinesin-2 motor complex in regulating GLI activity, we performed a series of structure-function analyses using biochemical, cell signaling and in vivo approaches that define novel specific interactions between GLI proteins and two components of this complex, KAP3 and KIF3A. We find that all three mammalian GLI proteins interact with KAP3 and we map specific interaction sites in both proteins. Furthermore, we find that GLI proteins interact selectively with KIF3A, but not KIF3B, and that GLI interacts synergistically with KAP3 and KIF3A. Using a combination of cell signaling assays and chicken in ovo electroporation, we demonstrate that KAP3 interactions restrict GLI activator function but not GLI repressor function. These data suggest that GLI interactions with KIF3A–KIF3B–KAP3 complexes are essential for proper GLI transcriptional activity. PMID:25588831

  13. Phosphorylation regulates the Star-PAP-PIPKIα interaction and directs specificity toward mRNA targets.

    PubMed

    Mohan, Nimmy; Sudheesh, A P; Francis, Nimmy; Anderson, Richard; Laishram, Rakesh S

    2015-08-18

    Star-PAP is a nuclear non-canonical poly(A) polymerase (PAP) that shows specificity toward mRNA targets. Star-PAP activity is stimulated by lipid messenger phosphatidyl inositol 4,5 bisphoshate (PI4,5P2) and is regulated by the associated Type I phosphatidylinositol-4-phosphate 5-kinase that synthesizes PI4,5P2 as well as protein kinases. These associated kinases act as coactivators of Star-PAP that regulates its activity and specificity toward mRNAs, yet the mechanism of control of these interactions are not defined. We identified a phosphorylated residue (serine 6, S6) on Star-PAP in the zinc finger region, the domain required for PIPKIα interaction. We show that S6 is phosphorylated by CKIα within the nucleus which is required for Star-PAP nuclear retention and interaction with PIPKIα. Unlike the CKIα mediated phosphorylation at the catalytic domain, Star-PAP S6 phosphorylation is insensitive to oxidative stress suggesting a signal mediated regulation of CKIα activity. S6 phosphorylation together with coactivator PIPKIα controlled select subset of Star-PAP target messages by regulating Star-PAP-mRNA association. Our results establish a novel role for phosphorylation in determining Star-PAP target mRNA specificity and regulation of 3'-end processing.

  14. Phosphorylation regulates the Star-PAP-PIPKIα interaction and directs specificity toward mRNA targets

    PubMed Central

    Mohan, Nimmy; AP, Sudheesh; Francis, Nimmy; Anderson, Richard; Laishram, Rakesh S.

    2015-01-01

    Star-PAP is a nuclear non-canonical poly(A) polymerase (PAP) that shows specificity toward mRNA targets. Star-PAP activity is stimulated by lipid messenger phosphatidyl inositol 4,5 bisphoshate (PI4,5P2) and is regulated by the associated Type I phosphatidylinositol-4-phosphate 5-kinase that synthesizes PI4,5P2 as well as protein kinases. These associated kinases act as coactivators of Star-PAP that regulates its activity and specificity toward mRNAs, yet the mechanism of control of these interactions are not defined. We identified a phosphorylated residue (serine 6, S6) on Star-PAP in the zinc finger region, the domain required for PIPKIα interaction. We show that S6 is phosphorylated by CKIα within the nucleus which is required for Star-PAP nuclear retention and interaction with PIPKIα. Unlike the CKIα mediated phosphorylation at the catalytic domain, Star-PAP S6 phosphorylation is insensitive to oxidative stress suggesting a signal mediated regulation of CKIα activity. S6 phosphorylation together with coactivator PIPKIα controlled select subset of Star-PAP target messages by regulating Star-PAP-mRNA association. Our results establish a novel role for phosphorylation in determining Star-PAP target mRNA specificity and regulation of 3′-end processing. PMID:26138484

  15. Integrated Genomic Analysis of Diverse Induced Pluripotent Stem Cells from the Progenitor Cell Biology Consortium.

    PubMed

    Salomonis, Nathan; Dexheimer, Phillip J; Omberg, Larsson; Schroll, Robin; Bush, Stacy; Huo, Jeffrey; Schriml, Lynn; Ho Sui, Shannan; Keddache, Mehdi; Mayhew, Christopher; Shanmukhappa, Shiva Kumar; Wells, James; Daily, Kenneth; Hubler, Shane; Wang, Yuliang; Zambidis, Elias; Margolin, Adam; Hide, Winston; Hatzopoulos, Antonis K; Malik, Punam; Cancelas, Jose A; Aronow, Bruce J; Lutzko, Carolyn

    2016-07-12

    The rigorous characterization of distinct induced pluripotent stem cells (iPSC) derived from multiple reprogramming technologies, somatic sources, and donors is required to understand potential sources of variability and downstream potential. To achieve this goal, the Progenitor Cell Biology Consortium performed comprehensive experimental and genomic analyses of 58 iPSC from ten laboratories generated using a variety of reprogramming genes, vectors, and cells. Associated global molecular characterization studies identified functionally informative correlations in gene expression, DNA methylation, and/or copy-number variation among key developmental and oncogenic regulators as a result of donor, sex, line stability, reprogramming technology, and cell of origin. Furthermore, X-chromosome inactivation in PSC produced highly correlated differences in teratoma-lineage staining and regulator expression upon differentiation. All experimental results, and raw, processed, and metadata from these analyses, including powerful tools, are interactively accessible from a new online portal at https://www.synapse.org to serve as a reusable resource for the stem cell community.

  16. The Interactive Roles of Parenting, Emotion Regulation and Executive Functioning in Moral Reasoning during Middle Childhood

    PubMed Central

    Hinnant, J. Benjamin; Nelson, Jackie A.; O’Brien, Marion; Keane, Susan P.; Calkins, Susan D.

    2013-01-01

    We examined mother-child cooperative behavior, children’s emotion regulation and executive function, as well as combinations of these factors, as predictors of moral reasoning in 89 10-year-old children. Dyadic cooperation was coded from videotaped observations of laboratory puzzle and speech tasks. Emotion regulation was derived from maternal report, and executive functioning was assessed with the Tower of London task. Moral reasoning was coded during mother-child conversations about morally ambiguous, peer-conflict situations. Two significant interactions indicated that children from more cooperative dyads who also had higher executive function skills had higher moral reasoning scores than other children, and children lower in both emotion regulation and executive function had lower moral reasoning scores than other children. The results contribute to the literature on the multiple and interactive levels of influence on moral reasoning in childhood. PMID:23650955

  17. The interactive roles of parenting, emotion regulation and executive functioning in moral reasoning during middle childhood.

    PubMed

    Hinnant, J Benjamin; Nelson, Jackie A; O'Brien, Marion; Keane, Susan P; Calkins, Susan D

    2013-01-01

    We examined mother-child co-operative behaviour, children's emotion regulation and executive function, as well as combinations of these factors, as predictors of moral reasoning in 89 10-year-old children. Dyadic co-operation was coded from videotaped observations of laboratory puzzle and speech tasks. Emotion regulation was derived from maternal report, and executive functioning was assessed with the Tower of London task. Moral reasoning was coded during mother-child conversations about morally ambiguous, peer-conflict situations. Two significant interactions indicated that children from more co-operative dyads who also had higher executive function skills had higher moral reasoning scores than other children, and children lower in both emotion regulation and executive function had lower moral reasoning scores than other children. The results contribute to the literature on the multiple and interactive levels of influence on moral reasoning in childhood. PMID:23650955

  18. New balance in pluripotency: reprogramming with lineage specifiers.

    PubMed

    Ben-David, Uri; Nissenbaum, Jonathan; Benvenisty, Nissim

    2013-05-23

    Induction of pluripotency in somatic cells has been achieved by myriad combinations of transcription factors that belong to the core pluripotency circuitry. In this issue, Shu et al. report reprogramming with lineage specifiers, lending support to the view of the pluripotent state as a fine balance between competing differentiation forces.

  19. PIP2 regulates psychostimulant behaviors through its interaction with a membrane protein

    PubMed Central

    Khelashvili, George; Saunders, Christine; Erreger, Kevin; Javitch, Jonathan A.; Sitte, Harald H.; Weinstein, Harel; Matthies, Heinrich J.G.; Galli, Aurelio

    2014-01-01

    Phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the function of ion channels and transporters. Here, we demonstrate that PIP2 directly binds the human dopamine (DA) transporter (hDAT), a key regulator of DA homeostasis and a target of the psychostimulant amphetamine (AMPH). This binding occurs through electrostatic interactions with positively charged hDAT N-terminal residues and is shown to facilitate AMPH-induced, DAT-mediated DA efflux and the psychomotor properties of AMPH. Substitution of these residues with uncharged amino acids reduces hDAT-PIP2 interactions and AMPH-induced DA efflux, without altering the hDAT physiological function of DA uptake. We evaluated, for the first time, the significance of this interaction in vivo using locomotion as a behavioral assay in Drosophila melanogaster. Expression of mutated hDAT with reduced PIP2 interaction in Drosophila DA neurons impairs AMPH-induced locomotion without altering basal locomotion. We present the first demonstration of how PIP2 interactions with a membrane protein can regulate the behaviors of complex organisms. PMID:24880859

  20. SUMOylation regulates polo-like kinase 1-interacting checkpoint helicase (PICH) during mitosis.

    PubMed

    Sridharan, Vinidhra; Park, Hyewon; Ryu, Hyunju; Azuma, Yoshiaki

    2015-02-01

    Mitotic SUMOylation has an essential role in faithful chromosome segregation in eukaryotes, although its molecular consequences are not yet fully understood. In Xenopus egg extract assays, we showed that poly(ADP-ribose) polymerase 1 (PARP1) is modified by SUMO2/3 at mitotic centromeres and that its enzymatic activity could be regulated by SUMOylation. To determine the molecular consequence of mitotic SUMOylation, we analyzed SUMOylated PARP1-specific binding proteins. We identified Polo-like kinase 1-interacting checkpoint helicase (PICH) as an interaction partner of SUMOylated PARP1 in Xenopus egg extract. Interestingly, PICH also bound to SUMOylated topoisomerase IIα (TopoIIα), a major centromeric small ubiquitin-like modifier (SUMO) substrate. Purified recombinant human PICH interacted with SUMOylated substrates, indicating that PICH directly interacts with SUMO, and this interaction is conserved among species. Further analysis of mitotic chromosomes revealed that PICH localized to the centromere independent of mitotic SUMOylation. Additionally, we found that PICH is modified by SUMO2/3 on mitotic chromosomes and in vitro. PICH SUMOylation is highly dependent on protein inhibitor of activated STAT, PIASy, consistent with other mitotic chromosomal SUMO substrates. Finally, the SUMOylation of PICH significantly reduced its DNA binding capability, indicating that SUMOylation might regulate its DNA-dependent ATPase activity. Collectively, our findings suggest a novel SUMO-mediated regulation of the function of PICH at mitotic centromeres. PMID:25564610

  1. Focal adhesion kinase regulates expression of thioredoxin-interacting protein (TXNIP) in cancer cells.

    PubMed

    Ho, Baotran; Huang, Grace; Golubovskaya, Vita M

    2014-01-01

    Focal Adhesion Kinase (FAK) plays an important role in cancer cell survival. Previous microarray gene profiling study detected inverse regulation between expression of thioredoxin-interacting protein (TXNIP) and FAK, where down-regulation of FAK by siRNA in MCF-7 cells caused up-regulation of TXNIP mRNA level, and in contrast up-regulation of doxycyclin- induced FAK caused repression of TXNIP. In the present report, we show that overexpression of FAK in MCF-7 cells repressed TXNIP promoter activity. Treatment of MCF-7 cells with 1alpha, 25-dihydroxyvitamin D3 (1,25D) down-regulated endogenous FAK and up-regulated TXNIP protein level, and treatment with 5-FU decreased FAK protein expression and up-regulated TXNIP protein expression in 293 cells. Moreover, silencing of FAK with siRNA increased TXNIP protein expression, while overexpression of FAK inhibited TXNIP protein expression in 293 cells. In addition, treatment of DBTRG glioblastoma cells with FAK inhibitor Y15 increased TXNIP mRNA, decreased cancer cell viability and increased apoptosis. These results for the first time demonstrate FAK-regulated TXNIP expression which is important for apoptotic, survival and oxidative stress signaling pathways in cancer cells. PMID:23387972

  2. Performance goals in conflictual social interactions: towards the distinction between two modes of relational conflict regulation.

    PubMed

    Sommet, Nicolas; Darnon, Céline; Mugny, Gabriel; Quiamzade, Alain; Pulfrey, Caroline; Dompnier, Benoît; Butera, Fabrizio

    2014-03-01

    Socio-cognitive conflict has been defined as a situation of confrontation with a disagreeing other. Previous research suggests that individuals can regulate conflict in a relational way, namely by focusing on social comparison between relative levels of competences. Relational conflict regulation has been described as yielding particularly negative effects on social interactions and learning, but has been understudied. The present research addresses the question of the origin of relational conflict regulation by introducing a fundamental distinction between two types of regulation, one based on the affirmation of one's own point of view and the invalidation of the other's (i.e., 'competitive' regulation), the other corresponding to the protection of self-competence via compliance (i.e., 'protective' regulation). Three studies show that these modes of relational conflict regulation result from the endorsement of distinct performance goals, respectively, performance-approach goals (trying to outperform others) and performance-avoidance goals (avoiding performing more poorly than others). Theoretical implications for the literature on both conflict regulation and achievement goals are discussed.

  3. Nonviral methods for inducing pluripotency to cells.

    PubMed

    O'Doherty, Ryan; Greiser, Udo; Wang, Wenxin

    2013-01-01

    The concept of inducing pluripotency to adult somatic cells by introducing reprogramming factors to them is one that has recently emerged, gained widespread acclaim and garnered much attention among the scientific community. The idea that cells can be reprogrammed, and are not unidirectionally defined opens many avenues for study. With their clear potential for use in the clinic, these reprogrammed cells stand to have a huge impact in regenerative medicine. This realization did not occur overnight but is, however, the product of many decades worth of advancements in researching this area. It was a combination of such research that led to the development of induced pluripotent stem cells as we know it today. This review delivers a brief insight in to the roots of iPS research and focuses on succinctly describing current nonviral methods of inducing pluripotency using plasmid vectors, small molecules and chemicals, and RNAs. PMID:23841088

  4. Gene expression of pluripotency determinants is conserved between mammalian and planarian stem cells.

    PubMed

    Onal, Pinar; Grün, Dominic; Adamidi, Catherine; Rybak, Agnieszka; Solana, Jordi; Mastrobuoni, Guido; Wang, Yongbo; Rahn, Hans-Peter; Chen, Wei; Kempa, Stefan; Ziebold, Ulrike; Rajewsky, Nikolaus

    2012-06-13

    Freshwater planaria possess extreme regeneration capabilities mediated by abundant, pluripotent stem cells (neoblasts) in adult animals. Although planaria emerged as an attractive in vivo model system for stem cell biology, gene expression in neoblasts has not been profiled comprehensively and it is unknown how molecular mechanisms for pluripotency in neoblasts relate to those in mammalian embryonic stem cells (ESCs). We purified neoblasts and quantified mRNA and protein expression by sequencing and shotgun proteomics. We identified ∼4000 genes specifically expressed in neoblasts, including all ∼30 known neoblast markers. Genes important for pluripotency in ESCs, including regulators as well as targets of OCT4, were well conserved and upregulated in neoblasts. We found conserved expression of epigenetic regulators and demonstrated their requirement for planarian regeneration by knockdown experiments. Post-transcriptional regulatory genes characteristic for germ cells were also enriched in neoblasts, suggesting the existence of a common ancestral state of germ cells and ESCs. We conclude that molecular determinants of pluripotency are conserved throughout evolution and that planaria are an informative model system for human stem cell biology.

  5. Gene expression of pluripotency determinants is conserved between mammalian and planarian stem cells

    PubMed Central

    Önal, Pinar; Grün, Dominic; Adamidi, Catherine; Rybak, Agnieszka; Solana, Jordi; Mastrobuoni, Guido; Wang, Yongbo; Rahn, Hans-Peter; Chen, Wei; Kempa, Stefan; Ziebold, Ulrike; Rajewsky, Nikolaus

    2012-01-01

    Freshwater planaria possess extreme regeneration capabilities mediated by abundant, pluripotent stem cells (neoblasts) in adult animals. Although planaria emerged as an attractive in vivo model system for stem cell biology, gene expression in neoblasts has not been profiled comprehensively and it is unknown how molecular mechanisms for pluripotency in neoblasts relate to those in mammalian embryonic stem cells (ESCs). We purified neoblasts and quantified mRNA and protein expression by sequencing and shotgun proteomics. We identified ∼4000 genes specifically expressed in neoblasts, including all ∼30 known neoblast markers. Genes important for pluripotency in ESCs, including regulators as well as targets of OCT4, were well conserved and upregulated in neoblasts. We found conserved expression of epigenetic regulators and demonstrated their requirement for planarian regeneration by knockdown experiments. Post-transcriptional regulatory genes characteristic for germ cells were also enriched in neoblasts, suggesting the existence of a common ancestral state of germ cells and ESCs. We conclude that molecular determinants of pluripotency are conserved throughout evolution and that planaria are an informative model system for human stem cell biology. PMID:22543868

  6. Analysis of Mitochondrial DNA in Induced Pluripotent and Embryonic Stem Cells.

    PubMed

    Lee, William; Kelly, Richard D W; Yeung, Ka Yu; Cagnone, Gael; McKenzie, Matthew; St John, Justin C

    2015-01-01

    The mitochondrial genome has a major role to play in establishing and maintaining pluripotency. Furthermore, mitochondrial DNA (mtDNA) copy is strictly regulated during differentiation. Undifferentiated, pluripotent cells possess fewer than 300 copies of mtDNA, which establishes the mtDNA set point and promotes cell proliferation and, as a result, these cells rely on glycolysis with some support from oxidative phosphorylation (OXPHOS) for the generation of ATP. The mtDNA set point provides the starting point from which cells increase their mtDNA copy number as they differentiate into mature functional cells. Dependent on cell types, mtDNA copy number ranges from ~10 copies in sperm to several thousand in cardiomyocytes. Consequently, differentiating cell types can acquire the appropriate numbers of mtDNA copy to meet their specific requirements for ATP generated through OXPHOS. However, as reprogrammed somatic cells do not always achieve this, it is essential to analyze them for their OXPHOS potential and ability to regulate mtDNA copy number. Here, we describe how to assess mtDNA copy number in pluripotent and differentiating cells using real-time PCR protocols; assess expression of the mtDNA specific replication factors through real-time RT-PCR; identify mtDNA variants in embryonic and induced pluripotent stem cells; determine DNA methylation patterns of the mtDNA-specific replication factors; and assess mitochondrial OXPHOS capacity. PMID:26621601

  7. Gata6 potently initiates reprograming of pluripotent and differentiated cells to extraembryonic endoderm stem cells

    PubMed Central

    Wamaitha, Sissy E.; del Valle, Ignacio; Cho, Lily T.Y.; Wei, Yingying; Fogarty, Norah M.E.; Blakeley, Paul; Sherwood, Richard I.; Ji, Hongkai; Niakan, Kathy K.

    2015-01-01

    Transcription factor-mediated reprograming is a powerful method to study cell fate changes. In this study, we demonstrate that the transcription factor Gata6 can initiate reprograming of multiple cell types to induced extraembryonic endoderm stem (iXEN) cells. Intriguingly, Gata6 is sufficient to drive iXEN cells from mouse pluripotent cells and differentiated neural cells. Furthermore, GATA6 induction in human embryonic stem (hES) cells also down-regulates pluripotency gene expression and up-regulates extraembryonic endoderm (ExEn) genes, revealing a conserved function in mediating this cell fate switch. Profiling transcriptional changes following Gata6 induction in mES cells reveals step-wise pluripotency factor disengagement, with initial repression of Nanog and Esrrb, then Sox2, and finally Oct4, alongside step-wise activation of ExEn genes. Chromatin immunoprecipitation and subsequent high-throughput sequencing analysis shows Gata6 enrichment near pluripotency and endoderm genes, suggesting that Gata6 functions as both a direct repressor and activator. Together, this demonstrates that Gata6 is a versatile and potent reprograming factor that can act alone to drive a cell fate switch from diverse cell types. PMID:26109048

  8. Characterization of human pluripotent stem cells.

    PubMed

    Gokhale, Paul J; Andrews, Peter W

    2013-12-18

    Human pluripotent stem cells (PSCs), whether embryonic stem cells or induced PSCs, offer enormous opportunities for regenerative medicine and other biomedical applications once we have developed the ability to harness their capacity for extensive differentiation. Central to this is our ability to identify and characterize such PSCs, but this is fraught with potential difficulties that arise from a tension between functional definitions of pluripotency and the more convenient use of 'markers', a problem exacerbated by ethical issues, our lack of knowledge of early human embryonic development, and differences from the mouse paradigm.

  9. Lipid Raft-Mediated Regulation of Hyaluronan–CD44 Interactions in Inflammation and Cancer

    PubMed Central

    Murai, Toshiyuki

    2015-01-01

    Hyaluronan is a major component of the extracellular matrix and plays pivotal roles in inflammation and cancer. Hyaluronan oligomers are frequently found in these pathological conditions, in which they exert their effects via association with the transmembrane receptor CD44. Lipid rafts are cholesterol- and glycosphingolipid-enriched membrane microdomains that may regulate membrane receptors while serving as platforms for transmembrane signaling at the cell surface. This article focuses on the recent discovery that lipid rafts regulate the interaction between CD44 and hyaluronan, which depends largely on hyaluronan’s size. Lipid rafts regulate CD44’s ability to bind hyaluronan in T cells, control the rolling adhesion of lymphocytes on vascular endothelial cells, and regulate hyaluronan- and CD44-mediated cancer cell migration. The implications of these findings for preventing inflammatory disorders and cancer are also discussed. PMID:26347743

  10. Auxetic nuclei in embryonic stem cells exiting pluripotency

    NASA Astrophysics Data System (ADS)

    Pagliara, Stefano; Franze, Kristian; McClain, Crystal R.; Wylde, George W.; Fisher, Cynthia L.; Franklin, Robin J. M.; Kabla, Alexandre J.; Keyser, Ulrich F.; Chalut, Kevin J.

    2014-06-01

    Embryonic stem cells (ESCs) self-renew in a state of naïve pluripotency in which they are competent to generate all somatic cells. It has been hypothesized that, before irreversibly committing, ESCs pass through at least one metastable transition state. This transition would represent a gateway for differentiation and reprogramming of somatic cells. Here, we show that during the transition, the nuclei of ESCs are auxetic: they exhibit a cross-sectional expansion when stretched and a cross-sectional contraction when compressed, and their stiffness increases under compression. We also show that the auxetic phenotype of transition ESC nuclei is driven at least in part by global chromatin decondensation. Through the regulation of molecular turnover in the differentiating nucleus by external forces, auxeticity could be a key element in mechanotransduction. Our findings highlight the importance of nuclear structure in the regulation of differentiation and reprogramming.

  11. VE-cadherin interacts with cell polarity protein Pals1 to regulate vascular lumen formation.

    PubMed

    Brinkmann, Benjamin F; Steinbacher, Tim; Hartmann, Christian; Kummer, Daniel; Pajonczyk, Denise; Mirzapourshafiyi, Fatemeh; Nakayama, Masanori; Weide, Thomas; Gerke, Volker; Ebnet, Klaus

    2016-09-15

    Blood vessel tubulogenesis requires the formation of stable cell-to-cell contacts and the establishment of apicobasal polarity of vascular endothelial cells. Cell polarity is regulated by highly conserved cell polarity protein complexes such as the Par3-aPKC-Par6 complex and the CRB3-Pals1-PATJ complex, which are expressed by many different cell types and regulate various aspects of cell polarity. Here we describe a functional interaction of VE-cadherin with the cell polarity protein Pals1. Pals1 directly interacts with VE-cadherin through a membrane-proximal motif in the cytoplasmic domain of VE-cadherin. VE-cadherin clusters Pals1 at cell-cell junctions. Mutating the Pals1-binding motif in VE-cadherin abrogates the ability of VE-cadherin to regulate apicobasal polarity and vascular lumen formation. In a similar way, deletion of the Par3-binding motif at the C-terminus of VE-cadherin impairs apicobasal polarity and vascular lumen formation. Our findings indicate that the biological activity of VE-cadherin in regulating endothelial polarity and vascular lumen formation is mediated through its interaction with the two cell polarity proteins Pals1 and Par3.

  12. Role of TARP interaction in S-SCAM-mediated regulation of AMPA receptors.

    PubMed

    Danielson, Eric; Metallo, Jacob; Lee, Sang H

    2012-01-01

    Scaffolding proteins are involved in the incorporation, anchoring, maintenance, and removal of AMPA receptors (AMPARs) at synapses, either through a direct interaction with AMPARs or via indirect association through auxiliary subunits of transmembrane AMPAR regulatory proteins (TARPs). Synaptic scaffolding molecule (S-SCAM) is a newly characterized member of the scaffolding proteins critical for the regulation and maintenance of AMPAR levels at synapses, and directly binds to TARPs through a PDZ interaction. However, the functional significance of S-SCAM-TARP interaction in the regulation of AMPARs has not been tested. Here we show that overexpression of the C-terminal peptide of TARP-γ2 fused to EGFP abolished the S-SCAM-mediated enhancement of surface GluA2 expression. Conversely, the deletion of the PDZ-5 domain of S-SCAM that binds TARPs greatly attenuated the S-SCAM-induced increase of surface GluA2 expression. In contrast, the deletion of the guanylate kinase domain of S-SCAM did not show a significant effect on the regulation of AMPARs. Together, these results suggest that S-SCAM is regulating AMPARs through TARPs.

  13. Homeodomain-Interacting Protein Kinase (HPK-1) regulates stress responses and ageing in C. elegans

    PubMed Central

    Berber, Slavica; Wood, Mallory; Llamosas, Estelle; Thaivalappil, Priya; Lee, Karen; Liao, Bing Mana; Chew, Yee Lian; Rhodes, Aaron; Yucel, Duygu; Crossley, Merlin; Nicholas, Hannah R

    2016-01-01

    Proteins of the Homeodomain-Interacting Protein Kinase (HIPK) family regulate an array of processes in mammalian systems, such as the DNA damage response, cellular proliferation and apoptosis. The nematode Caenorhabditis elegans has a single HIPK homologue called HPK-1. Previous studies have implicated HPK-1 in longevity control and suggested that this protein may be regulated in a stress-dependent manner. Here we set out to expand these observations by investigating the role of HPK-1 in longevity and in the response to heat and oxidative stress. We find that levels of HPK-1 are regulated by heat stress, and that HPK-1 contributes to survival following heat or oxidative stress. Additionally, we show that HPK-1 is required for normal longevity, with loss of HPK-1 function leading to a faster decline of physiological processes that reflect premature ageing. Through microarray analysis, we have found that HPK-1-regulated genes include those encoding proteins that serve important functions in stress responses such as Phase I and Phase II detoxification enzymes. Consistent with a role in longevity assurance, HPK-1 also regulates the expression of age-regulated genes. Lastly, we show that HPK-1 functions in the same pathway as DAF-16 to regulate longevity and reveal a new role for HPK-1 in development. PMID:26791749

  14. Classroom Interactions, Dyadic Teacher-Child Relationships, and Self-Regulation in Socially Disadvantaged Young Children.

    PubMed

    Cadima, Joana; Verschueren, Karine; Leal, Teresa; Guedes, Carolina

    2016-01-01

    This study examined the quality of the classroom climate and dyadic teacher-child relationships as predictors of self-regulation in a sample of socially disadvantaged preschool children (N = 206; 52 % boys). Children's self-regulation was observed in preschool at the beginning and at the end of the school year. At the middle of the preschool year, classroom observations of interactions were conducted by trained observers and teachers rated the quality of dyadic teacher-child relationships. Results from multilevel analyses revealed that teacher-child closeness predicted improvements in observed self-regulation skills. Children showed larger gains in self-regulation when they experienced closer teacher-child relationships. Moreover, a moderating effect between classroom instructional quality and observed self-regulation was found such that children with low initial self-regulation skills benefit the most from classrooms with higher classroom quality. Findings have implications for understanding the role of classroom social processes on the development of self-regulation.

  15. Trauma exposure interacts with impulsivity in predicting emotion regulation and depressive mood

    PubMed Central

    Ceschi, Grazia; Billieux, Joël; Hearn, Melissa; Fürst, Guillaume; Van der Linden, Martial

    2014-01-01

    Background Traumatic exposure may modulate the expression of impulsive behavioral dispositions and change the implementation of emotion regulation strategies associated with depressive mood. Past studies resulted in only limited comprehension of these relationships, especially because they failed to consider impulsivity as a multifactorial construct. Objective Based on Whiteside and Lynam's multidimensional model that identifies four distinct dispositional facets of impulsive-like behaviors, namely urgency, (lack of) premeditation, (lack of) perseverance, and sensation seeking (UPPS), the current study used a sample of community volunteers to investigate whether an interaction exists between impulsivity facets and lifetime trauma exposure in predicting cognitive emotion regulation and depressive mood. Methods Ninety-three adults completed questionnaires measuring lifetime trauma exposure, impulsivity, cognitive emotion regulation, and depressive mood. Results Results showed that trauma-exposed participants with a strong disposition toward urgency (predisposition to act rashly in intense emotional contexts) tended to use fewer appropriate cognitive emotion regulation strategies than other individuals. Unexpectedly, participants lacking in perseverance (predisposition to have difficulties concentrating on demanding tasks) used more appropriate emotion regulation strategies if they had experienced traumatic events during their life than if they had not. Emotion regulation mediated the path between these two impulsivity facets and depressive mood. Conclusions Together, these findings suggest that impulsivity has a differential impact on emotion regulation and depressive mood depending on lifetime exposure to environmental factors, especially traumatic events. PMID:25317255

  16. 14-3-3 proteins regulate Tctp–Rheb interaction for organ growth in Drosophila

    PubMed Central

    Le, Thao Phuong; Vuong, Linh Thuong; Kim, Ah-Ram; Hsu, Ya-Chieh; Choi, Kwang-Wook

    2016-01-01

    14-3-3 family proteins regulate multiple signalling pathways. Understanding biological functions of 14-3-3 proteins has been limited by the functional redundancy of conserved isotypes. Here we provide evidence that 14-3-3 proteins regulate two interacting components of Tor signalling in Drosophila, translationally controlled tumour protein (Tctp) and Rheb GTPase. Single knockdown of 14-3-3ɛ or 14-3-3ζ isoform does not show obvious defects in organ development but causes synergistic genetic interaction with Tctp and Rheb to impair tissue growth. 14-3-3 proteins physically interact with Tctp and Rheb. Knockdown of both 14-3-3 isoforms abolishes the binding between Tctp and Rheb, disrupting organ development. Depletion of 14-3-3s also reduces the level of phosphorylated S6 kinase, phosphorylated Thor/4E-BP and cyclin E (CycE). Growth defects from knockdown of 14-3-3 and Tctp are suppressed by CycE overexpression. This study suggests a novel mechanism of Tor regulation mediated by 14-3-3 interaction with Tctp and Rheb. PMID:27151460

  17. Pluripotent Stem Cells: Current Understanding and Future Directions

    PubMed Central

    Romito, Antonio

    2016-01-01

    Pluripotent stem cells have the ability to undergo self-renewal and to give rise to all cells of the tissues of the body. However, this definition has been recently complicated by the existence of distinct cellular states that display these features. Here, we provide a detailed overview of the family of pluripotent cell lines derived from early mouse and human embryos and compare them with induced pluripotent stem cells. Shared and distinct features of these cells are reported as additional hallmark of pluripotency, offering a comprehensive scenario of pluripotent stem cells. PMID:26798367

  18. Pluripotent Stem Cells: Current Understanding and Future Directions.

    PubMed

    Romito, Antonio; Cobellis, Gilda

    2016-01-01

    Pluripotent stem cells have the ability to undergo self-renewal and to give rise to all cells of the tissues of the body. However, this definition has been recently complicated by the existence of distinct cellular states that display these features. Here, we provide a detailed overview of the family of pluripotent cell lines derived from early mouse and human embryos and compare them with induced pluripotent stem cells. Shared and distinct features of these cells are reported as additional hallmark of pluripotency, offering a comprehensive scenario of pluripotent stem cells.

  19. Chromatin organization in pluripotent cells: emerging approaches to study and disrupt function

    PubMed Central

    Lopes Novo, Clara

    2016-01-01

    Translating the vast amounts of genomic and epigenomic information accumulated on the linear genome into three-dimensional models of nuclear organization is a current major challenge. In response to this challenge, recent technological innovations based on chromosome conformation capture methods in combination with increasingly powerful functional approaches have revealed exciting insights into key aspects of genome regulation. These findings have led to an emerging model where the genome is folded and compartmentalized into highly conserved topological domains that are further divided into functional subdomains containing physical loops that bring cis-regulatory elements to close proximity. Targeted functional experiments, largely based on designable DNA-binding proteins, have begun to define the major architectural proteins required to establish and maintain appropriate genome regulation. Here, we focus on the accessible and well-characterized system of pluripotent cells to review the functional role of chromatin organization in regulating pluripotency, differentiation and reprogramming. PMID:26206085

  20. Wheat CBL-interacting protein kinase 25 negatively regulates salt tolerance in transgenic wheat

    PubMed Central

    Jin, Xia; Sun, Tao; Wang, Xiatian; Su, Peipei; Ma, Jingfei; He, Guangyuan; Yang, Guangxiao

    2016-01-01

    CBL-interacting protein kinases are involved in plant responses to abiotic stresses, including salt stress. However, the negative regulating mechanism of this gene family in response to salinity is less reported. In this study, we evaluated the role of TaCIPK25 in regulating salt response in wheat. Under conditions of high salinity, TaCIPK25 expression was markedly down-regulated in roots. Overexpression of TaCIPK25 resulted in hypersensitivity to Na+ and superfluous accumulation of Na+ in transgenic wheat lines. TaCIPK25 expression did not decline in transgenic wheat and remained at an even higher level than that in wild-type wheat controls under high-salinity treatment. Furthermore, transmembrane Na+/H+ exchange was impaired in the root cells of transgenic wheat. These results suggested that TaCIPK25 negatively regulated salt response in wheat. Additionally, yeast-one-hybrid, β-glucuronidase activity and DNA-protein-interaction-enzyme-linked-immunosorbent assays showed that the transcription factor TaWRKY9 bound W-box in the TaCIPK25 promoter region. Quantitative real-time polymerase chain reaction assays showed concomitantly inverted expression patterns of TaCIPK25 and TaWRKY9 in wheat roots under salt treatment, ABA application and inhibition of endogenous ABA condition. Overall, based on our results, in a salt stress condition, the negative salt response in wheat involved TaCIPK25 with the expression regulated by TaWRKY9. PMID:27358166

  1. Wheat CBL-interacting protein kinase 25 negatively regulates salt tolerance in transgenic wheat.

    PubMed

    Jin, Xia; Sun, Tao; Wang, Xiatian; Su, Peipei; Ma, Jingfei; He, Guangyuan; Yang, Guangxiao

    2016-01-01

    CBL-interacting protein kinases are involved in plant responses to abiotic stresses, including salt stress. However, the negative regulating mechanism of this gene family in response to salinity is less reported. In this study, we evaluated the role of TaCIPK25 in regulating salt response in wheat. Under conditions of high salinity, TaCIPK25 expression was markedly down-regulated in roots. Overexpression of TaCIPK25 resulted in hypersensitivity to Na(+) and superfluous accumulation of Na(+) in transgenic wheat lines. TaCIPK25 expression did not decline in transgenic wheat and remained at an even higher level than that in wild-type wheat controls under high-salinity treatment. Furthermore, transmembrane Na(+)/H(+) exchange was impaired in the root cells of transgenic wheat. These results suggested that TaCIPK25 negatively regulated salt response in wheat. Additionally, yeast-one-hybrid, β-glucuronidase activity and DNA-protein-interaction-enzyme-linked-immunosorbent assays showed that the transcription factor TaWRKY9 bound W-box in the TaCIPK25 promoter region. Quantitative real-time polymerase chain reaction assays showed concomitantly inverted expression patterns of TaCIPK25 and TaWRKY9 in wheat roots under salt treatment, ABA application and inhibition of endogenous ABA condition. Overall, based on our results, in a salt stress condition, the negative salt response in wheat involved TaCIPK25 with the expression regulated by TaWRKY9.

  2. KCC2 regulates actin dynamics in dendritic spines via interaction with β-PIX.

    PubMed

    Llano, Olaya; Smirnov, Sergey; Soni, Shetal; Golubtsov, Andrey; Guillemin, Isabelle; Hotulainen, Pirta; Medina, Igor; Nothwang, Hans Gerd; Rivera, Claudio; Ludwig, Anastasia

    2015-06-01

    Chloride extrusion in mature neurons is largely mediated by the neuron-specific potassium-chloride cotransporter KCC2. In addition, independently of its chloride transport function, KCC2 regulates the development and morphology of dendritic spines through structural interactions with the actin cytoskeleton. The mechanism of this effect remains largely unknown. In this paper, we show a novel pathway for KCC2-mediated regulation of the actin cytoskeleton in neurons. We found that KCC2, through interaction with the b isoform of Rac/Cdc42 guanine nucleotide exchange factor β-PIX, regulates the activity of Rac1 GTPase and the phosphorylation of one of the major actin-regulating proteins, cofilin-1. KCC2-deficient neurons had abnormally high levels of phosphorylated cofilin-1. Consistently, dendritic spines of these neurons exhibited a large pool of stable actin, resulting in reduced spine motility and diminished density of functional synapses. In conclusion, we describe a novel signaling pathway that couples KCC2 to the cytoskeleton and regulates the formation of glutamatergic synapses. PMID:26056138

  3. KCC2 regulates actin dynamics in dendritic spines via interaction with β-PIX

    PubMed Central

    Llano, Olaya; Smirnov, Sergey; Soni, Shetal; Golubtsov, Andrey; Guillemin, Isabelle; Hotulainen, Pirta; Medina, Igor; Nothwang, Hans Gerd

    2015-01-01

    Chloride extrusion in mature neurons is largely mediated by the neuron-specific potassium-chloride cotransporter KCC2. In addition, independently of its chloride transport function, KCC2 regulates the development and morphology of dendritic spines through structural interactions with the actin cytoskeleton. The mechanism of this effect remains largely unknown. In this paper, we show a novel pathway for KCC2-mediated regulation of the actin cytoskeleton in neurons. We found that KCC2, through interaction with the b isoform of Rac/Cdc42 guanine nucleotide exchange factor β-PIX, regulates the activity of Rac1 GTPase and the phosphorylation of one of the major actin-regulating proteins, cofilin-1. KCC2-deficient neurons had abnormally high levels of phosphorylated cofilin-1. Consistently, dendritic spines of these neurons exhibited a large pool of stable actin, resulting in reduced spine motility and diminished density of functional synapses. In conclusion, we describe a novel signaling pathway that couples KCC2 to the cytoskeleton and regulates the formation of glutamatergic synapses. PMID:26056138

  4. Wheat CBL-interacting protein kinase 25 negatively regulates salt tolerance in transgenic wheat.

    PubMed

    Jin, Xia; Sun, Tao; Wang, Xiatian; Su, Peipei; Ma, Jingfei; He, Guangyuan; Yang, Guangxiao

    2016-01-01

    CBL-interacting protein kinases are involved in plant responses to abiotic stresses, including salt stress. However, the negative regulating mechanism of this gene family in response to salinity is less reported. In this study, we evaluated the role of TaCIPK25 in regulating salt response in wheat. Under conditions of high salinity, TaCIPK25 expression was markedly down-regulated in roots. Overexpression of TaCIPK25 resulted in hypersensitivity to Na(+) and superfluous accumulation of Na(+) in transgenic wheat lines. TaCIPK25 expression did not decline in transgenic wheat and remained at an even higher level than that in wild-type wheat controls under high-salinity treatment. Furthermore, transmembrane Na(+)/H(+) exchange was impaired in the root cells of transgenic wheat. These results suggested that TaCIPK25 negatively regulated salt response in wheat. Additionally, yeast-one-hybrid, β-glucuronidase activity and DNA-protein-interaction-enzyme-linked-immunosorbent assays showed that the transcription factor TaWRKY9 bound W-box in the TaCIPK25 promoter region. Quantitative real-time polymerase chain reaction assays showed concomitantly inverted expression patterns of TaCIPK25 and TaWRKY9 in wheat roots under salt treatment, ABA application and inhibition of endogenous ABA condition. Overall, based on our results, in a salt stress condition, the negative salt response in wheat involved TaCIPK25 with the expression regulated by TaWRKY9. PMID:27358166

  5. The pluripotency factor-bound intron 1 of Xist is dispensable for X chromosome inactivation and reactivation in vitro and in vivo.

    PubMed

    Minkovsky, Alissa; Barakat, Tahsin Stefan; Sellami, Nadia; Chin, Mark Henry; Gunhanlar, Nilhan; Gribnau, Joost; Plath, Kathrin

    2013-03-28

    X chromosome inactivation (XCI) is a dynamically regulated developmental process with inactivation and reactivation accompanying the loss and gain of pluripotency, respectively. A functional relationship between pluripotency and lack of XCI has been suggested, whereby pluripotency transcription factors repress the master regulator of XCI, the noncoding transcript Xist, by binding to its first intron (intron 1). To test this model, we have generated intron 1 mutant embryonic stem cells (ESCs) and two independent mouse models. We found that Xist's repression in ESCs, its transcriptional upregulation upon differentiation, and its silencing upon reprogramming to pluripotency are not dependent on intron 1. Although we observed subtle effects of intron 1 deletion on the randomness of XCI and in the absence of the antisense transcript Tsix in differentiating ESCs, these have little relevance in vivo because mutant mice do not deviate from Mendelian ratios of allele transmission. Altogether, our findings demonstrate that intron 1 is dispensable for the developmental dynamics of Xist expression.

  6. Advances in genetic modification of pluripotent stem cells.

    PubMed

    Fontes, Andrew; Lakshmipathy, Uma

    2013-11-15

    Genetically engineered stem cells aid in dissecting basic cell function and are valuable tools for drug discovery, in vivo cell tracking, and gene therapy. Gene transfer into pluripotent stem cells has been a challenge due to their intrinsic feature of growing in clusters and hence not amenable to common gene delivery methods. Several advances have been made in the rapid assembly of DNA elements, optimization of culture conditions, and DNA delivery methods. This has lead to the development of viral and non-viral methods for transient or stable modification of cells, albeit with varying efficiencies. Most methods require selection and clonal expansion that demand prolonged culture and are not suited for cells with limited proliferative potential. Choosing the right platform based on preferred length, strength, and context of transgene expression is a critical step. Random integration of the transgene into the genome can be complicated due to silencing or altered regulation of expression due to genomic effects. An alternative to this are site-specific methods that target transgenes followed by screening to identify the genomic loci that support long-term expression with stem cell proliferation and differentiation. A highly precise and accurate editing of the genome driven by homology can be achieved using traditional methods as well as the newer technologies such as zinc finger nuclease, TAL effector nucleases and CRISPR. In this review, we summarize the different genetic engineering methods that have been successfully used to create modified embryonic and induced pluripotent stem cells.

  7. Myc Depletion Induces a Pluripotent Dormant State Mimicking Diapause

    PubMed Central

    Scognamiglio, Roberta; Cabezas-Wallscheid, Nina; Thier, Marc Christian; Altamura, Sandro; Reyes, Alejandro; Prendergast, Áine M.; Baumgärtner, Daniel; Carnevalli, Larissa S.; Atzberger, Ann; Haas, Simon; von Paleske, Lisa; Boroviak, Thorsten; Wörsdörfer, Philipp; Essers, Marieke A.G.; Kloz, Ulrich; Eisenman, Robert N.; Edenhofer, Frank; Bertone, Paul; Huber, Wolfgang; van der Hoeven, Franciscus; Smith, Austin; Trumpp, Andreas

    2016-01-01

    Summary Mouse embryonic stem cells (ESCs) are maintained in a naive ground state of pluripotency in the presence of MEK and GSK3 inhibitors. Here, we show that ground-state ESCs express low Myc levels. Deletion of both c-myc and N-myc (dKO) or pharmacological inhibition of Myc activity strongly decreases transcription, splicing, and protein synthesis, leading to proliferation arrest. This process is reversible and occurs without affecting pluripotency, suggesting that Myc-depleted stem cells enter a state of dormancy similar to embryonic diapause. Indeed, c-Myc is depleted in diapaused blastocysts, and the differential expression signatures of dKO ESCs and diapaused epiblasts are remarkably similar. Following Myc inhibition, pre-implantation blastocysts enter biosynthetic dormancy but can progress through their normal developmental program after transfer into pseudo-pregnant recipients. Our study shows that Myc controls the biosynthetic machinery of stem cells without affecting their potency, thus regulating their entry and exit from the dormant state. PMID:26871632

  8. Modeling neurodevelopmental disorders using human pluripotent stem cells.

    PubMed

    Telias, Michael; Ben-Yosef, Dalit

    2014-08-01

    Neurodevelopmental disorders (NDs) are impairments that affect the development and growth of the brain and the central nervous system during embryonic and early postnatal life. Genetically manipulated animals have contributed greatly to the advancement of ND research, but many of them differ considerably from the human phenotype. Cellular in vitro models are also valuable, but the availability of human neuronal cells is limited and their lifespan in culture is short. Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, comprise a powerful tool for studying developmentally regulated diseases, including NDs. We reviewed all recent studies in which hPSCs were used as in vitro models for diseases and syndromes characterized by impairment of neurogenesis or synaptogenesis leading to intellectual disability and delayed neurodevelopment. We analyzed their methodology and results, focusing on the data obtained following in vitro neural differentiation and gene expression and profiling of the derived neurons. Electrophysiological recording of action potentials, synaptic currents and response to neurotransmitters is pivotal for validation of the neuronal fate as well as for assessing phenotypic dysfunctions linked to the disease in question. We therefore focused on the studies which included electrophysiological recordings on the in vitro-derived neurons. Finally, we addressed specific issues that are critical for the advancement of this area of research, specifically in providing a reliable human pre-clinical research model and drug screening platform. PMID:24728983

  9. Myc Depletion Induces a Pluripotent Dormant State Mimicking Diapause.

    PubMed

    Scognamiglio, Roberta; Cabezas-Wallscheid, Nina; Thier, Marc Christian; Altamura, Sandro; Reyes, Alejandro; Prendergast, Áine M; Baumgärtner, Daniel; Carnevalli, Larissa S; Atzberger, Ann; Haas, Simon; von Paleske, Lisa; Boroviak, Thorsten; Wörsdörfer, Philipp; Essers, Marieke A G; Kloz, Ulrich; Eisenman, Robert N; Edenhofer, Frank; Bertone, Paul; Huber, Wolfgang; van der Hoeven, Franciscus; Smith, Austin; Trumpp, Andreas

    2016-02-11

    Mouse embryonic stem cells (ESCs) are maintained in a naive ground state of pluripotency in the presence of MEK and GSK3 inhibitors. Here, we show that ground-state ESCs express low Myc levels. Deletion of both c-myc and N-myc (dKO) or pharmacological inhibition of Myc activity strongly decreases transcription, splicing, and protein synthesis, leading to proliferation arrest. This process is reversible and occurs without affecting pluripotency, suggesting that Myc-depleted stem cells enter a state of dormancy similar to embryonic diapause. Indeed, c-Myc is depleted in diapaused blastocysts, and the differential expression signatures of dKO ESCs and diapaused epiblasts are remarkably similar. Following Myc inhibition, pre-implantation blastocysts enter biosynthetic dormancy but can progress through their normal developmental program after transfer into pseudo-pregnant recipients. Our study shows that Myc controls the biosynthetic machinery of stem cells without affecting their potency, thus regulating their entry and exit from the dormant state. PMID:26871632

  10. Characterizing the radioresponse of pluripotent and multipotent human stem cells.

    PubMed

    Lan, Mary L; Acharya, Munjal M; Tran, Katherine K; Bahari-Kashani, Jessica; Patel, Neal H; Strnadel, Jan; Giedzinski, Erich; Limoli, Charles L

    2012-01-01

    The potential capability of stem cells to restore functionality to diseased or aged tissues has prompted a surge of research, but much work remains to elucidate the response of these cells to genotoxic agents. To more fully understand the impact of irradiation on different stem cell types, the present study has analyzed the radioresponse of human pluripotent and multipotent stem cells. Human embryonic stem (ES) cells, human induced pluripotent (iPS) cells, and iPS-derived human neural stem cells (iPS-hNSCs) cells were irradiated and analyzed for cell survival parameters, differentiation, DNA damage and repair and oxidative stress at various times after exposure. While irradiation led to dose-dependent reductions in survival, the fraction of surviving cells exhibited dose-dependent increases in metabolic activity. Irradiation did not preclude germ layer commitment of ES cells, but did promote neuronal differentiation. ES cells subjected to irradiation exhibited early apoptosis and inhibition of cell cycle progression, but otherwise showed normal repair of DNA double-strand breaks. Cells surviving irradiation also showed acute and persistent increases in reactive oxygen and nitrogen species that were significant at nearly all post-irradiation times analyzed. We suggest that stem cells alter their redox homeostasis to adapt to adverse conditions and that radiation-induced oxidative stress plays a role in regulating the function and fate of stem cells within tissues compromised by radiation injury.

  11. Interaction with the Yes-associated protein (YAP) allows TEAD1 to positively regulate NAIP expression.

    PubMed

    Landin Malt, André; Georges, Adrien; Silber, Joël; Zider, Alain; Flagiello, Domenico

    2013-10-01

    Although the expression of the neuronal apoptosis inhibitory protein (NAIP) gene is considered involved in apoptosis suppression as well as in inflammatory response, the molecular basis of the NAIP gene expression is poorly understood. Here we show that the TEA domain protein 1 (TEAD1) is able to positively activate the transcription of NAIP. We further demonstrate that this regulation is mediated by the presence of the endogenous Yes associated protein (YAP) cofactor, and requires the interaction with YAP. We finally identified an intronic region of the NAIP gene responding to TEAD1/YAP activity, suggesting that regulation of NAIP by TEAD1/YAP is at the transcriptional level. PMID:23994529

  12. Interaction with the Yes-associated protein (YAP) allows TEAD1 to positively regulate NAIP expression.

    PubMed

    Landin Malt, André; Georges, Adrien; Silber, Joël; Zider, Alain; Flagiello, Domenico

    2013-10-01

    Although the expression of the neuronal apoptosis inhibitory protein (NAIP) gene is considered involved in apoptosis suppression as well as in inflammatory response, the molecular basis of the NAIP gene expression is poorly understood. Here we show that the TEA domain protein 1 (TEAD1) is able to positively activate the transcription of NAIP. We further demonstrate that this regulation is mediated by the presence of the endogenous Yes associated protein (YAP) cofactor, and requires the interaction with YAP. We finally identified an intronic region of the NAIP gene responding to TEAD1/YAP activity, suggesting that regulation of NAIP by TEAD1/YAP is at the transcriptional level.

  13. Phospho-regulated ACAP4-Ezrin interaction is essential for histamine-stimulated parietal cell secretion.

    PubMed

    Ding, Xia; Deng, Hui; Wang, Dongmei; Zhou, Jiajia; Huang, Yuejia; Zhao, Xuannv; Yu, Xue; Wang, Ming; Wang, Fengsong; Ward, Tarsha; Aikhionbare, Felix; Yao, Xuebiao

    2010-06-11

    The ezrin-radixin-moesin proteins provide a regulated linkage between membrane proteins and the cortical cytoskeleton and also participate in signal transduction pathways. Ezrin is localized to the apical membrane of parietal cells and couples the protein kinase A activation cascade to the regulated HCl secretion. Our recent proteomic study revealed a protein complex of ezrin-ACAP4-ARF6 essential for volatile membrane remodeling (Fang, Z., Miao, Y., Ding, X., Deng, H., Liu, S., Wang, F., Zhou, R., Watson, C., Fu, C., Hu, Q., Lillard, J. W., Jr., Powell, M., Chen, Y., Forte, J. G., and Yao, X. (2006) Mol. Cell Proteomics 5, 1437-1449). However, knowledge of whether ACAP4 physically interacts with ezrin and how their interaction is integrated into membrane-cytoskeletal remodeling has remained elusive. Here we provide the first evidence that ezrin interacts with ACAP4 in a protein kinase A-mediated phosphorylation-dependent manner through the N-terminal 400 amino acids of ACAP4. ACAP4 locates in the cytoplasmic membrane in resting parietal cells but translocates to the apical plasma membrane upon histamine stimulation. ACAP4 was precipitated with ezrin from secreting but not resting parietal cell lysates, suggesting a phospho-regulated interaction. Indeed, this interaction is abolished by phosphatase treatment and validated by an in vitro reconstitution assay using phospho-mimicking ezrin(S66D). Importantly, ezrin specifies the apical distribution of ACAP4 in secreting parietal cells because either suppression of ezrin or overexpression of non-phosphorylatable ezrin prevents the apical localization of ACAP4. In addition, overexpressing GTPase-activating protein-deficient ACAP4 results in an inhibition of apical membrane-cytoskeletal remodeling and gastric acid secretion. Taken together, these results define a novel molecular mechanism linking ACAP4-ezrin interaction to polarized epithelial secretion.

  14. Methylobacterium-plant interaction genes regulated by plant exudate and quorum sensing molecules.

    PubMed

    Dourado, Manuella Nóbrega; Bogas, Andrea Cristina; Pomini, Armando M; Andreote, Fernando Dini; Quecine, Maria Carolina; Marsaioli, Anita J; Araújo, Welington Luiz

    2013-12-01

    Bacteria from the genus Methylobacterium interact symbiotically (endophytically and epiphytically) with different plant species. These interactions can promote plant growth or induce systemic resistance, increasing plant fitness. The plant colonization is guided by molecular communication between bacteria-bacteria and bacteria-plants, where the bacteria recognize specific exuded compounds by other bacteria (e.g. homoserine molecules) and/or by the plant roots (e.g. flavonoids, ethanol and methanol), respectively. In this context, the aim of this study was to evaluate the effect of quorum sensing molecules (N-acyl-homoserine lactones) and plant exudates (including ethanol) in the expression of a series of bacterial genes involved in Methylobacterium-plant interaction. The selected genes are related to bacterial metabolism (mxaF), adaptation to stressful environment (crtI, phoU and sss), to interactions with plant metabolism compounds (acdS) and pathogenicity (patatin and phoU). Under in vitro conditions, our results showed the differential expression of some important genes related to metabolism, stress and pathogenesis, thereby AHL molecules up-regulate all tested genes, except phoU, while plant exudates induce only mxaF gene expression. In the presence of plant exudates there is a lower bacterial density (due the endophytic and epiphytic colonization), which produce less AHL, leading to down regulation of genes when compared to the control. Therefore, bacterial density, more than plant exudate, influences the expression of genes related to plant-bacteria interaction.

  15. Methylobacterium-plant interaction genes regulated by plant exudate and quorum sensing molecules

    PubMed Central

    Dourado, Manuella Nóbrega; Bogas, Andrea Cristina; Pomini, Armando M.; Andreote, Fernando Dini; Quecine, Maria Carolina; Marsaioli, Anita J.; Araújo, Welington Luiz

    2013-01-01

    Bacteria from the genus Methylobacterium interact symbiotically (endophytically and epiphytically) with different plant species. These interactions can promote plant growth or induce systemic resistance, increasing plant fitness. The plant colonization is guided by molecular communication between bacteria-bacteria and bacteria-plants, where the bacteria recognize specific exuded compounds by other bacteria (e.g. homoserine molecules) and/or by the plant roots (e.g. flavonoids, ethanol and methanol), respectively. In this context, the aim of this study was to evaluate the effect of quorum sensing molecules (N-acyl-homoserine lactones) and plant exudates (including ethanol) in the expression of a series of bacterial genes involved in Methylobacterium-plant interaction. The selected genes are related to bacterial metabolism (mxaF), adaptation to stressful environment (crtI, phoU and sss), to interactions with plant metabolism compounds (acdS) and pathogenicity (patatin and phoU). Under in vitro conditions, our results showed the differential expression of some important genes related to metabolism, stress and pathogenesis, thereby AHL molecules up-regulate all tested genes, except phoU, while plant exudates induce only mxaF gene expression. In the presence of plant exudates there is a lower bacterial density (due the endophytic and epiphytic colonization), which produce less AHL, leading to down regulation of genes when compared to the control. Therefore, bacterial density, more than plant exudate, influences the expression of genes related to plant-bacteria interaction. PMID:24688531

  16. CCL2 enhances pluripotency of human induced pluripotent stem cells by activating hypoxia related genes.

    PubMed

    Hasegawa, Yuki; Tang, Dave; Takahashi, Naoko; Hayashizaki, Yoshihide; Forrest, Alistair R R; Suzuki, Harukazu

    2014-01-01

    Standard culture of human induced pluripotent stem cells (hiPSCs) requires basic Fibroblast Growth Factor (bFGF) to maintain the pluripotent state, whereas hiPSC more closely resemble epiblast stem cells than true naïve state ES which requires LIF to maintain pluripotency. Here we show that chemokine (C-C motif) ligand 2 (CCL2) enhances the expression of pluripotent marker genes through the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) protein. Moreover, comparison of transcriptomes between hiPSCs cultured with CCL2 versus with bFGF, we found that CCL2 activates hypoxia related genes, suggesting that CCL2 enhanced pluripotency by inducing a hypoxic-like response.Further, we show that hiPSCs cultured with CCL2 can differentiate at a higher efficiency than culturing withjust bFGF and we show CCL2 can be used in feeder-free conditions [corrected]. Taken together, our finding indicates the novel functions of CCL2 in enhancing its pluripotency in hiPSCs. PMID:24957798

  17. Development of the Human Interaction Dimension of the Self-Regulated Learning Questionnaire in Asynchronous Online Learning Environments

    ERIC Educational Resources Information Center

    Cho, Moon-Heum; Jonassen, David

    2009-01-01

    Two studies focusing on the development and validation of the Online Self-Regulated Learning Inventory (OSRLI) were conducted. The OSRLI is a self-report instrument assessing the human interaction dimension of online self-regulated learning. It consists of an affect/motivation scale and an interaction strategies scale. In Study 1, exploratory…

  18. Nephron reconstitution from pluripotent stem cells.

    PubMed

    Taguchi, Atsuhiro; Nishinakamura, Ryuichi

    2015-05-01

    It has been a challenge in developmental biology and regenerative medicine to generate nephron progenitors that reconstitute the three-dimensional (3D) nephron structure in vitro. Many studies have tried to induce nephron progenitors from pluripotent stem cells by mimicking the developmental processes in vivo. However, the current developmental model does not precisely address the spatiotemporal origin of nephron progenitors, hampering our understanding of cell fate decisions in the kidney. Here, we present a revised model of early-stage kidney specification, suggesting distinct origins of the two major kidney components: the ureteric bud and metanephric mesenchyme. This model enables the induction of metanephric nephron progenitors from both mouse and human pluripotent stem cells. The induced cells self-organize in the presence of Wnt signaling and reconstitute 3D nephron structures including both nephric tubules with a clear lumina and glomeruli with podocytes. The engrafted kidney tissue develops vascularized glomeruli and nephric tubules, but it does not produce urine, suggesting the requirement for further maturation. Nevertheless, the generation of nephron components from human-induced pluripotent stem cells will be useful for future application in regenerative therapy and modeling of congenital kidney diseases in vitro. This review discusses the possibility of de novo organogenesis of a functional kidney from pluripotent stem cells and the future direction toward clinical applications.

  19. DELLA proteins physically interact with CONSTANS to regulate flowering under long days in Arabidopsis.

    PubMed

    Xu, Feng; Li, Ting; Xu, Peng-Bo; Li, Ling; Du, Sha-Sha; Lian, Hong-Li; Yang, Hong-Quan

    2016-02-01

    Proper timing of flowering is essential for reproduction of plants. Although it is well known that both light and gibberellin (GA) signaling play critical roles in promoting flowering in Arabidopsis thaliana, whether and how they are integrated to regulate flowering remain largely unknown. Here, we show through biochemical studies that DELLA proteins physically interact with CONSTANS (CO). Furthermore, the interaction of CO with NF-YB2 is inhibited by the DELLA protein, RGA. Our findings suggest that regulation of flowering by GA signaling in leaves under long days is mediated, at least in part, through repression of DELLA proteins on CO, providing a molecular link between DELLA proteins, key components in GA signaling pathway, and CO, a critical flowering activator in photoperiod signaling pathway.

  20. An experimental study of emotion regulation during relationship conflict interactions: the moderating role of attachment orientations.

    PubMed

    Ben-Naim, Shiri; Hirschberger, Gilad; Ein-Dor, Tsachi; Mikulincer, Mario

    2013-06-01

    Romantic couples (N = 127) engaged in a relationship conflict interaction during which their autonomic physiology, emotional experience, and emotional behavior were recorded. Couples were assigned randomly to one of two interventions, or to a control condition: In the affective suppression condition, one partner was instructed to refrain from expressing emotions. In the positive mindset condition, one partner was instructed to think about the positive aspects of the relationship. Results revealed that emotion regulation interventions influenced the physiology, emotional behavior, and emotional experience of both the manipulated person and his or her partner, who was oblivious to regulation manipulations. Specifically, suppression increased, and positive mindset decreased cardiovascular arousal and negative affect. These effects were generally exacerbated among those high on attachment anxiety and attenuated among those high on attachment avoidance. The results of this research corroborate and extend the Temporal Interpersonal Emotion Systems model (Butler, 2011) in the context of relationship conflict interactions.

  1. Thyroid hormone and retinoic acid interact to regulate zebrafish craniofacial neural crest development.

    PubMed

    Bohnsack, Brenda L; Kahana, Alon

    2013-01-15

    Craniofacial and ocular morphogenesis require proper regulation of cranial neural crest migration, proliferation, survival and differentiation. Although alterations in maternal thyroid hormone (TH) are associated with congenital craniofacial anomalies, the role of TH on the neural crest has not been previously described. Using zebrafish, we demonstrate that pharmacologic and genetic alterations in TH signaling disrupt cranial neural crest migration, proliferation, and survival, leading to craniofacial, extraocular muscle, and ocular developmental abnormalities. In the rostral cranial neural crest that gives rise to the periocular mesenchyme and the frontonasal process, retinoic acid (RA) rescued migratory defects induced by decreased TH signaling. In the caudal cranial neural crest, TH and RA had reciprocal effects on anterior and posterior pharyngeal arch development. The interactions between TH and RA signaling were partially mediated by the retinoid X receptor. We conclude that TH regulates both rostral and caudal cranial neural crest. Further, coordinated interactions of TH and RA are required for proper craniofacial and ocular development.

  2. Thyroid Hormone and Retinoic Acid Interact to Regulate Zebrafish Craniofacial Neural Crest Development

    PubMed Central

    Bohnsack, Brenda L.; Kahana, Alon

    2012-01-01

    Craniofacial and ocular morphogenesis requires proper regulation of cranial neural crest migration, proliferation, survival and differentiation. Although alterations in maternal thyroid hormone (TH) are associated with congenital craniofacial anomalies, the role of TH on the neural crest has not been previously described. Using zebrafish, we demonstrate that pharmacologic and genetic alterations in TH signaling disrupt cranial neural crest migration, proliferation, and survival, leading to craniofacial, extraocular muscle, and ocular developmental abnormalities. In the rostral cranial neural crest that gives rise to the periocular mesenchyme and the frontonasal process, retinoic acid (RA) rescued migratory defects induced by decreased TH signaling. In the caudal cranial neural crest, TH and RA had reciprocal effects on anterior and posterior pharyngeal arch development. The interactions between TH and RA signaling were partially mediated by the retinoid X receptor. We conclude that TH regulates both rostral and caudal cranial neural crest. Further, coordinated interactions of TH and RA are required for proper craniofacial and ocular development. PMID:23165295

  3. Pluripotent Conversion of Muscle Stem Cells Without Reprogramming Factors or Small Molecules.

    PubMed

    Bose, Bipasha; Shenoy P, Sudheer

    2016-02-01

    Muscle derived stem cells (MDSCs) are multipotent stem cells that can differentiate into several lineages including skeletal muscle precursor cells. Here, we show that MDSCs from myostatin null mice (Mstn (-/-) ) can be readily induced into pluripotent stem cells without using reprogramming factors. Microarray studies revealed a strong upregulation of markers like Leukemia Inhibitory factor (LIF) and Leukemia Inhibitory factor receptor (LIFR) in Mstn (-/-) MDSCs as compared to wild type MDSCs (WT-MDSCs). Furthermore when cultured in mouse embryonic stem cell media with LIF for 95 days, Mstn (-/-) MDSCs formed embryonic stem cell (ES) like colonies. We termed such ES like cells as the culture-induced pluripotent stem cells (CiPSC). CiPSCs from Mstn (-/-) MDSCs were phenotypically similar to ESCs, expressed high levels of Oct4, Nanog, Sox2 and SSEA-1, maintained a normal karyotype. Furthermore, CiPSCs formed embryoid bodies and teratomas when injected into immunocompromised mice. In addition, CiPSCs differentiated into somatic cells of all three lineages. We further show that culturing in ES cell media, resulted in hypermethylation and downregulation of BMP2 in Mstn(-/-) MDSCs. Western blot further confirmed a down regulation of BMP2 signaling in Mstn (-/-) MDSCs in supportive of pluripotent reprogramming. Given that down regulation of BMP2 has been shown to induce pluripotency in cells, we propose that lack of myostatin epigenetically reprograms the MDSCs to become pluripotent stem cells. Thus, here we report the successful establishment of ES-like cells from adult stem cells of the non-germline origin under culture-induced conditions without introducing reprogramming genes.

  4. Morphology and its underlying genetic regulation impact the interaction between Cryptococcus neoformans and its hosts.

    PubMed

    Lin, Jianfeng; Idnurm, Alexander; Lin, Xiaorong

    2015-06-01

    Cryptococcus neoformans is a fungus that causes the majority of fatal cryptococcal meningitis cases worldwide. This pathogen is capable of assuming different morphotypes: yeast, pseudohypha, and hypha. The yeast form is the most common cell type observed clinically. The hyphal and pseudohyphal forms are rarely observed in the clinical setting and are considered attenuated in virulence. However, as a ubiquitous environmental pathogen, Cryptococcus interacts with various organisms, and it is known to be parasitic to different hosts. Capitalizing on recent discoveries, morphogenesis regulators were manipulated to examine the impact of cell shape on the cryptococcal interaction with three different host systems: the soil amoeba Acanthamoeba castellanii (a protist), the greater wax moth Galleria mellonella (an insect), and the murine macrophage cell line J774A.1 (mammalian cells). The regulation of Ace2 and morphogenesis (RAM) pathway is a highly conserved pathway among eukaryotes that regulates cytokinesis. Disruption of any of five RAM components in Cryptococcus renders cells constitutively in the pseudohyphal form. The transcription factor Znf2 is the master activator of the yeast to hyphal transition. Deletion of ZNF2 locks cells in the yeast form, while overexpression of this regulator drives hyphal growth. Genetic epistasis analyses indicate that the RAM and the Znf2 pathways regulate distinct aspects of cryptococcal morphogenesis and independently of each other. These investigations using the Cryptococcus RAM and ZNF2 mutants indicate that cell shape, cell size, and likely cell surface properties weigh differently on the outcome of cryptococcal interactions with different hosts. Thus, certain traits evolved in Cryptococcus that are beneficial within one host might be detrimental when a different host is encountered.

  5. Prolonged Proteasome Inhibition Cyclically Upregulates Oct3/4 and Nanog Gene Expression, but Reduces Induced Pluripotent Stem Cell Colony Formation

    PubMed Central

    Floyd, Elizabeth Z.; Staszkiewicz, Jaroslaw; Power, Rachel A.; Kilroy, Gail; Kirk-Ballard, Heather; Barnes, Christian W.; Strickler, Karen L.; Rim, Jong S.; Harkins, Lettie L.; Gao, Ru; Kim, Jeong

    2015-01-01

    Abstract There is ample evidence that the ubiquitin–proteasome system is an important regulator of transcription and its activity is necessary for maintaining pluripotency and promoting cellular reprogramming. Moreover, proteasome activity contributes to maintaining the open chromatin structure found in pluripotent stem cells, acting as a transcriptional inhibitor at specific gene loci generally associated with differentiation. The current study was designed to understand further the role of proteasome inhibition in reprogramming and its ability to modulate endogenous expression of pluripotency-related genes and induced pluripotent stem cells (iPSCs) colony formation. Herein, we demonstrate that acute combinatorial treatment with the proteasome inhibitors MG101 or MG132 and the histone deacetylase (HDAC) inhibitor valproic acid (VPA) increases gene expression of the pluripotency marker Oct3/4, and that MG101 alone is as effective as VPA in the induction of Oct3/4 mRNA expression in fibroblasts. Prolonged proteasome inhibition cyclically upregulates gene expression of Oct3/4 and Nanog, but reduces colony formation in the presence of the iPSC induction cocktail. In conclusion, our results demonstrate that the 26S proteasome is an essential modulator in the reprogramming process. Its inhibition enhances expression of pluripotency-related genes; however, efficient colony formation requires proteasome activity. Therefore, discovery of small molecules that increase proteasome activity might lead to more efficient cell reprogramming and generation of pluripotent cells. PMID:25826722

  6. Vinculin modulation of paxillin–FAK interactions regulates ERK to control survival and motility

    PubMed Central

    Subauste, M. Cecilia; Pertz, Olivier; Adamson, Eileen D.; Turner, Christopher E.; Junger, Sachiko; Hahn, Klaus M.

    2004-01-01

    Cells lacking vinculin are highly metastatic and motile. The reasons for this finding have remained unclear. Both enhanced survival and motility are critical to metastasis. Here, we show that vinculin null (vin−/−) cells and cells expressing a vinculin Y822F mutant have increased survival due to up-regulated activity of extracellular signal–regulated kinase (ERK). This increase is shown to result from vinculin's modulation of paxillin–FAK interactions. A vinculin fragment (amino acids 811–1066) containing the paxillin binding site restored apoptosis and suppressed ERK activity in vin−/− cells. Both vinY822F and vin−/− cells exhibit increased interaction between paxillin and focal adhesion kinase (FAK) and increased paxillin and FAK phosphorylation. Transfection with paxillin Y31FY118F dominant-negative mutant in these cells inhibits ERK activation and restores apoptosis. The enhanced motility of vin−/− and vinY822F cells is also shown to be due to a similar mechanism. Thus, vinculin regulates survival and motility via ERK by controlling the accessibility of paxillin for FAK interaction. PMID:15138291

  7. ZmMADS47 Regulates Zein Gene Transcription through Interaction with Opaque2.

    PubMed

    Qiao, Zhenyi; Qi, Weiwei; Wang, Qian; Feng, Ya'nan; Yang, Qing; Zhang, Nan; Wang, Shanshan; Tang, Yuanping; Song, Rentao

    2016-04-01

    Zeins, the predominent storage proteins in maize endosperm, are encoded by multiple genes and gene families. However, only a few transcriptional factors for zein gene regulation have been functionally characterized. In this study, a MADS-box protein, namely ZmMADS47, was identified as an Opaque2 (O2) interacting protein via yeast two-hybrid screening. The N-terminal portion of ZmMADS47 contains a nuclear localization signal (NLS), and its C-terminal portion contains a transcriptional activation domain (AD). Interestingly, the transcriptional activation activity is blocked in its full length form, suggesting conformational regulation of the AD. Molecular and RNA-seq analyses of ZmMADS47 RNAi lines revealed down regulation of α-zein and 50-kD γ-zein genes. ZmMADS47 binds the CATGT motif in promoters of these zein genes, but ZmMADS47 alone is not able to transactivate the promoters. However, when both O2 and ZmMADS47 are present, the transactivation of these promoters was greatly enhanced. This enhancement was dependent on the AD function of ZmMADS47 and the interaction between ZmMADS47 and O2, but it was independent from the AD function of O2. Therefore, it appears interaction with O2 activates ZmMADS47 on zein gene promoters. PMID:27077660

  8. ZmMADS47 Regulates Zein Gene Transcription through Interaction with Opaque2

    PubMed Central

    Qiao, Zhenyi; Qi, Weiwei; Wang, Qian; Feng, Ya’nan; Yang, Qing; Zhang, Nan; Wang, Shanshan; Tang, Yuanping; Song, Rentao

    2016-01-01

    Zeins, the predominent storage proteins in maize endosperm, are encoded by multiple genes and gene families. However, only a few transcriptional factors for zein gene regulation have been functionally characterized. In this study, a MADS-box protein, namely ZmMADS47, was identified as an Opaque2 (O2) interacting protein via yeast two-hybrid screening. The N-terminal portion of ZmMADS47 contains a nuclear localization signal (NLS), and its C-terminal portion contains a transcriptional activation domain (AD). Interestingly, the transcriptional activation activity is blocked in its full length form, suggesting conformational regulation of the AD. Molecular and RNA-seq analyses of ZmMADS47 RNAi lines revealed down regulation of α-zein and 50-kD γ-zein genes. ZmMADS47 binds the CATGT motif in promoters of these zein genes, but ZmMADS47 alone is not able to transactivate the promoters. However, when both O2 and ZmMADS47 are present, the transactivation of these promoters was greatly enhanced. This enhancement was dependent on the AD function of ZmMADS47 and the interaction between ZmMADS47 and O2, but it was independent from the AD function of O2. Therefore, it appears interaction with O2 activates ZmMADS47 on zein gene promoters. PMID:27077660

  9. Quantitative characterization of the interactions among c-myc transcriptional regulators FUSE, FBP, and FIR.

    PubMed

    Hsiao, Hsin-Hao; Nath, Abhinav; Lin, Chi-Yen; Folta-Stogniew, Ewa J; Rhoades, Elizabeth; Braddock, Demetrios T

    2010-06-01

    Human c-myc is critical for cell homeostasis and growth but is a potent oncogenic factor if improperly regulated. The c-myc far-upstream element (FUSE) melts into single-stranded DNA upon active transcription, and the noncoding strand FUSE recruits an activator [the FUSE-binding protein (FBP)] and a repressor [the FBP-interacting repressor (FIR)] to fine-tune c-myc transcription in a real-time manner. Despite detailed biological experiments describing this unique mode of transcriptional regulation, quantitative measurements of the physical constants regulating the protein-DNA interactions remain lacking. Here, we first demonstrate that the two FUSE strands adopt different conformations upon melting, with the noncoding strand DNA in an extended, linear form. FBP binds to the linear noncoding FUSE with a dissociation constant in the nanomolar range. FIR binds to FUSE more weakly, having its modest dissociation constants in the low micromolar range. FIR is monomeric under near-physiological conditions but upon binding of FUSE dimerizes into a 2:1 FIR(2)-FUSE complex mediated by the RRMs. In the tripartite interaction, our analysis suggests a stepwise addition of FIR onto an activating FBP-FUSE complex to form a quaternary FIR(2)-FBP-FUSE inhibitory complex. Our quantitative characterization enhances understanding of DNA strand preference and the mechanism of the stepwise complex formation in the FUSE-FBP-FIR regulatory system.

  10. The PSI-U1 snRNP interaction regulates male mating behavior in Drosophila.

    PubMed

    Wang, Qingqing; Taliaferro, J Matthew; Klibaite, Ugne; Hilgers, Valérie; Shaevitz, Joshua W; Rio, Donald C

    2016-05-10

    Alternative pre-mRNA splicing (AS) is a critical regulatory mechanism that operates extensively in the nervous system to produce diverse protein isoforms. Fruitless AS isoforms have been shown to influence male courtship behavior, but the underlying mechanisms are unknown. Using genome-wide approaches and quantitative behavioral assays, we show that the P-element somatic inhibitor (PSI) and its interaction with the U1 small nuclear ribonucleoprotein complex (snRNP) control male courtship behavior. PSI mutants lacking the U1 snRNP-interacting domain (PSIΔAB mutant) exhibit extended but futile mating attempts. The PSIΔAB mutant results in significant changes in the AS patterns of ∼1,200 genes in the Drosophila brain, many of which have been implicated in the regulation of male courtship behavior. PSI directly regulates the AS of at least one-third of these transcripts, suggesting that PSI-U1 snRNP interactions coordinate the behavioral network underlying courtship behavior. Importantly, one of these direct targets is fruitless, the master regulator of courtship. Thus, PSI imposes a specific mode of regulatory control within the neuronal circuit controlling courtship, even though it is broadly expressed in the fly nervous system. This study reinforces the importance of AS in the control of gene activity in neurons and integrated neuronal circuits, and provides a surprising link between a pleiotropic pre-mRNA splicing pathway and the precise control of successful male mating behavior.

  11. Aurora A orchestrates entosis by regulating a dynamic MCAK–TIP150 interaction

    PubMed Central

    Xia, Peng; Zhou, Jinhua; Song, Xiaoyu; Wu, Bing; Liu, Xing; Li, Di; Zhang, Shuyuan; Wang, Zhikai; Yu, Huijuan; Ward, Tarsha; Zhang, Jiancun; Li, Yinmei; Wang, Xiaoning; Chen, Yong; Guo, Zhen; Yao, Xuebiao

    2014-01-01

    Entosis, a cell-in-cell process, has been implicated in the formation of aneuploidy associated with an aberrant cell division control. Microtubule plus-end-tracking protein TIP150 facilitates the loading of MCAK onto the microtubule plus ends and orchestrates microtubule plus-end dynamics during cell division. Here we show that TIP150 cooperates with MCAK to govern entosis via a regulatory circuitry that involves Aurora A-mediated phosphorylation of MCAK. Our biochemical analyses show that MCAK forms an intra-molecular association, which is essential for TIP150 binding. Interestingly, Aurora A-mediated phosphorylation of MCAK modulates its intra-molecular association, which perturbs the MCAK–TIP150 interaction in vitro and inhibits entosis in vivo. To probe if MCAK–TIP150 interaction regulates microtubule plasticity to affect the mechanical properties of cells during entosis, we used an optical trap to measure the mechanical rigidity of live MCF7 cells. We find that the MCAK cooperates with TIP150 to promote microtubule dynamics and modulate the mechanical rigidity of the cells during entosis. Our results show that a dynamic interaction of MCAK–TIP150 orchestrated by Aurora A-mediated phosphorylation governs entosis via regulating microtubule plus-end dynamics and cell rigidity. These data reveal a previously unknown mechanism of Aurora A regulation in the control of microtubule plasticity during cell-in-cell processes. PMID:24847103

  12. Epigenetic regulation of traf2- and Nck-interacting kinase (TNIK) in polycystic ovary syndrome

    PubMed Central

    Li, Da; Jiao, Jiao; Zhou, Yi-Ming; Wang, Xiu-Xia

    2015-01-01

    Emerging evidence has led to considerable interest in the role of Traf2- and Nck-interacting kinase (TNIK) in polycystic ovary syndrome (PCOS) development. However, the epigenetic mechanism regulating TNIK transcription remains largely unknown. Here, we show that (i) TNIK mRNA expression is significantly increased in PCOS ovarian tissues, compared to normal ovarian tissues; (ii) PCOS ovarian tissues exhibit a hypermethylation pattern at the cg10180092 site, (iii) and cg10180092 is the critical site for the transcriptional regulation of TNIK. Mechanistically, hypermethylated cg10180092 site-mediated loss of holocarboxylase synthetase (HLCS)-related H3K9me enrichment activated TNIK transcription in PCOS ovarian tissues. Notably, a substantial body of evidence indicates that DNA hypermethylation is an alternative mechanism for gene inactivation, and a new role for DNA hypermethylationmediated TNIK activating was observed in this study. This may improve our understanding of divergent transcriptional regulation in the initiation and progression of TNIK-related PCOS. PMID:26279758

  13. Regulated phosphorylation and dephosphorylation of tau protein: effects on microtubule interaction, intracellular trafficking and neurodegeneration.

    PubMed Central

    Billingsley, M L; Kincaid, R L

    1997-01-01

    This review attempts to summarize what is known about tau phosphorylation in the context of both normal cellular function and dysfunction. However, conceptions of tau function continue to evolve, and it is likely that the regulation of tau distribution and metabolism is complex. The roles of microtubule-associated kinases and phosphatases have yet to be fully described, but may afford insight into how tau phosphorylation at the distal end of the axon regulates cytoskeletal-membrane interactions. Finally, lipid and glycosaminoglycan modification of tau structure affords yet more complexity for regulation and aggregation. Continued work will help to determine what is causal and what is coincidental in Alzheimer's disease, and may lead to identification of therapeutic targets for halting the progression of paired helical filament formation. PMID:9169588

  14. Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis.

    PubMed

    Liang, Xiubin; Da Paula, Ana Carina; Bozóky, Zoltán; Zhang, Hui; Bertrand, Carol A; Peters, Kathryn W; Forman-Kay, Julie D; Frizzell, Raymond A

    2012-03-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP/protein kinase A (PKA)-regulated chloride channel whose phosphorylation controls anion secretion across epithelial cell apical membranes. We examined the hypothesis that cAMP/PKA stimulation regulates CFTR biogenesis posttranslationally, based on predicted 14-3-3 binding motifs within CFTR and forskolin-induced CFTR expression. The 14-3-3β, γ, and ε isoforms were expressed in airway cells and interacted with CFTR in coimmunoprecipitation assays. Forskolin stimulation (15 min) increased 14-3-3β and ε binding to immature and mature CFTR (bands B and C), and 14-3-3 overexpression increased CFTR bands B and C and cell surface band C. In pulse-chase experiments, 14-3-3β increased the synthesis of immature CFTR, reduced its degradation rate, and increased conversion of immature to mature CFTR. Conversely, 14-3-3β knockdown decreased CFTR B and C bands (70 and 55%) and elicited parallel reductions in cell surface CFTR and forskolin-stimulated anion efflux. In vitro, 14-3-3β interacted with the CFTR regulatory region, and by nuclear magnetic resonance analysis, this interaction occurred at known PKA phosphorylated sites. In coimmunoprecipitation assays, forskolin stimulated the CFTR/14-3-3β interaction while reducing CFTR's interaction with coat protein complex 1 (COP1). Thus 14-3-3 binding to phosphorylated CFTR augments its biogenesis by reducing retrograde retrieval of CFTR to the endoplasmic reticulum. This mechanism permits cAMP/PKA stimulation to make more CFTR available for anion secretion.

  15. Regulation of β2-Adrenergic Receptor Maturation and Anterograde Trafficking by an Interaction with Rab Geranylgeranyltransferase

    PubMed Central

    Lachance, Véronik; Cartier, Andréane; Génier, Samuel; Munger, Sandra; Germain, Pascale; Labrecque, Pascale; Parent, Jean-Luc

    2011-01-01

    Previous reports by us and others demonstrated that G protein-coupled receptors interact functionally with Rab GTPases. Here, we show that the β2-adrenergic receptor (β2AR) interacts with the Rab geranylgeranyltransferase α-subunit (RGGTA). Confocal microscopy showed that β2AR co-localizes with RGGTA in intracellular compartments and at the plasma membrane. Site-directed mutagenesis revealed that RGGTA binds to the L339L340 motif in the β2AR C terminus known to be involved in the transport of the receptor from the endoplasmic reticulum to the cell surface. Modulation of the cellular levels of RGGTA protein by overexpression or siRNA-mediated knockdown of the endogenous protein demonstrated that RGGTA has a positive role in the maturation and anterograde trafficking of the β2AR, which requires the interaction of RGGTA with the β2AR L339L340 motif. Furthermore, the β2AR modulates the geranylgeranylation of Rab6a, Rab8a, and Rab11a, but not of other Rab proteins tested in this study. Regulation of Rab geranylgeranylation by the β2AR was dependent on the RGGTA-interacting L339L340 motif. Interestingly, a RGGTA-Y107F mutant was unable to regulate Rab geranylgeranylation but still promoted β2AR maturation, suggesting that RGGTA may have functions independent of Rab geranylgeranylation. We demonstrate for the first time an interaction between a transmembrane receptor and RGGTA which regulates the maturation and anterograde transport of the receptor, as well as geranylgeranylation of Rab GTPases. PMID:21990357

  16. Dax1 and Nanog act in parallel to stabilize mouse embryonic stem cells and induced pluripotency.

    PubMed

    Zhang, Junlei; Liu, Gaoke; Ruan, Yan; Wang, Jiali; Zhao, Ke; Wan, Ying; Liu, Bing; Zheng, Hongting; Peng, Tao; Wu, Wei; He, Ping; Hu, Fu-Quan; Jian, Rui

    2014-01-01

    Nanog expression is heterogeneous and dynamic in embryonic stem cells (ESCs). However, the mechanism for stabilizing pluripotency during the transitions between Nanog(high) and Nanog(low) states is not well understood. Here we report that Dax1 acts in parallel with Nanog to regulate mouse ESC (mESCs) identity. Dax1 stable knockdown mESCs are predisposed towards differentiation but do not lose pluripotency, whereas Dax1 overexpression supports LIF-independent self-renewal. Although partially complementary, Dax1 and Nanog function independently and cannot replace one another. They are both required for full reprogramming to induce pluripotency. Importantly, Dax1 is indispensable for self-renewal of Nanog(low) mESCs. Moreover, we report that Dax1 prevents extra-embryonic endoderm (ExEn) commitment by directly repressing Gata6 transcription. Dax1 may also mediate inhibition of trophectoderm differentiation independent or as a downstream effector of Oct4. These findings establish a basal role of Dax1 in maintaining pluripotency during the state transition of mESCs and somatic cell reprogramming. PMID:25284313

  17. Mediator MED23 regulates basal transcription in vivo via an interaction with P-TEFb.

    PubMed

    Wang, Wei; Yao, Xiao; Huang, Yan; Hu, Xiangming; Liu, Runzhong; Hou, Dongming; Chen, Ruichuan; Wang, Gang

    2013-01-01

    The Mediator is a multi-subunit complex that transduces regulatory information from transcription regulators to the RNA polymerase II apparatus. Growing evidence suggests that Mediator plays roles in multiple stages of eukaryotic transcription, including elongation. However, the detailed mechanism by which Mediator regulates elongation remains elusive. In this study, we demonstrate that Mediator MED23 subunit controls a basal level of transcription by recruiting elongation factor P-TEFb, via an interaction with its CDK9 subunit. The mRNA level of Egr1, a MED23-controlled model gene, is reduced 4-5 fold in Med23 (-/-) ES cells under an unstimulated condition, but Med23-deficiency does not alter the occupancies of RNAP II, GTFs, Mediator complex, or activator ELK1 at the Egr1 promoter. Instead, Med23 depletion results in a significant decrease in P-TEFb and RNAP II (Ser2P) binding at the coding region, but no changes for several other elongation regulators, such as DSIF and NELF. ChIP-seq revealed that Med23-deficiency partially reduced the P-TEFb occupancy at a set of MED23-regulated gene promoters. Further, we demonstrate that MED23 interacts with CDK9 in vivo and in vitro. Collectively, these results provide the mechanistic insight into how Mediator promotes RNAP II into transcription elongation.

  18. RILP interacts with HOPS complex via VPS41 subunit to regulate endocytic trafficking.

    PubMed

    Lin, Xiaosi; Yang, Ting; Wang, Shicong; Wang, Zhen; Yun, Ye; Sun, Lixiang; Zhou, Yunhe; Xu, Xiaohui; Akazawa, Chihiro; Hong, Wanjin; Wang, Tuanlao

    2014-01-01

    The HOPS complex serves as a tethering complex with GEF activity for Ypt7p in yeast to regulate late endosomal membrane maturation. While the role of HOPS complex is well established in yeast cells, its functional and mechanistic aspects in mammalian cells are less well defined. In this study, we report that RILP, a downstream effector of Rab7, interacts with HOPS complex and recruits HOPS subunits to the late endosomal compartment. Structurally, the amino-terminal portion of RILP interacts with HOPS complex. Unexpectedly, this interaction is independent of Rab7. VPS41 subunit of HOPS complex was defined to be the major partner for interacting with RILP. The carboxyl-terminal region of VPS41 was mapped to be responsible for the interaction. Functionally, either depletion of VPS41 by shRNA or overexpression of VPS41 C-terminal half retarded EGF-induced degradation of EGFR. These results suggest that interaction of RILP with HOPS complex via VPS41 plays a role in endocytic trafficking of EGFR. PMID:25445562

  19. Adhesive activity of Lu glycoproteins is regulated by interaction with spectrin

    SciTech Connect

    An, Xiuli; Gauthier, Emilie; Zhang, Xihui; Guo, Xinhua; Anstee, David; Mohandas, Narla; Anne Chasis, Joel

    2008-03-18

    The Lutheran (Lu) and Lu(v13) blood group glycoproteins function as receptors for extracellular matrix laminins. Lu and Lu(v13) are linked to the erythrocyte cytoskeleton through a direct interaction with spectrin. However, neither the molecular basis of the interaction nor its functional consequences have previously been delineated. In the present study, we defined the binding motifs of Lu and Lu(v13) on spectrin and identified a functional role for this interaction. We found that the cytoplasmic domains of both Lu and Lu(v13) bound to repeat 4 of the spectrin chain. The interaction of full-length spectrin dimer to Lu and Lu(v13) was inhibited by repeat 4 of {alpha}-spectrin. Further, resealing of this repeat peptide into erythrocytes led to weakened Lu-cytoskeleton interaction as demonstrated by increased detergent extractability of Lu. Importantly, disruption of the Lu-spectrin linkage was accompanied by enhanced cell adhesion to laminin. We conclude that the interaction of the Lu cytoplasmic tail with the cytoskeleton regulates its adhesive receptor function.

  20. Yeast growth plasticity is regulated by environment-specific multi-QTL interactions.

    PubMed

    Bhatia, Aatish; Yadav, Anupama; Zhu, Chenchen; Gagneur, Julien; Radhakrishnan, Aparna; Steinmetz, Lars M; Bhanot, Gyan; Sinha, Himanshu

    2014-01-28

    For a unicellular, nonmotile organism like Saccharomyces cerevisiae, carbon sources act as nutrients and as signaling molecules; consequently, these sources affect various fitness parameters, including growth. It is therefore advantageous for yeast strains to adapt their growth to carbon source variation. The ability of a given genotype to manifest different phenotypes in varying environments is known as phenotypic plasticity. To identify quantitative trait loci (QTL) that drive plasticity in growth, two growth parameters (growth rate and biomass) were measured for a set of meiotic recombinants of two genetically divergent yeast strains grown in different carbon sources. To identify QTL contributing to plasticity across pairs of environments, gene-environment interaction mapping was performed, which identified several QTL that have a differential effect across environments, some of which act antagonistically across pairs of environments. Multi-QTL analysis identified loci interacting with previously known growth affecting QTL as well as novel two-QTL interactions that affect growth. A QTL that had no significant independent effect was found to alter growth rate and biomass for several carbon sources through two-QTL interactions. Our study demonstrates that environment-specific epistatic interactions contribute to the growth plasticity in yeast. We propose that a targeted scan for epistatic interactions, such as the one described here, can help unravel mechanisms regulating phenotypic plasticity.

  1. DAZL limits pluripotency, differentiation, and apoptosis in developing primordial germ cells.

    PubMed

    Chen, Hsu-Hsin; Welling, Maaike; Bloch, Donald B; Muñoz, Javier; Mientjes, Edwin; Chen, Xinjie; Tramp, Cody; Wu, Jie; Yabuuchi, Akiko; Chou, Yu-Fen; Buecker, Christa; Krainer, Adrian; Willemsen, Rob; Heck, Albert J; Geijsen, Niels

    2014-11-11

    The scarcity of primordial germ cells (PGCs) in the developing mammalian embryo hampers robust biochemical analysis of the processes that underlie early germ cell formation. Here, we demonstrate that DAZL, a germ cell-specific RNA binding protein, is a robust PGC marker during in vitro germ cell development. Using Dazl-GFP reporter ESCs, we demonstrate that DAZL plays a central role in a large mRNA/protein interactive network that blocks the translation of core pluripotency factors, including Sox2 and Sall4, as well as of Suz12, a polycomb family member required for differentiation of pluripotent cells. Thus, DAZL limits both pluripotency and somatic differentiation in nascent PGCs. In addition, we observed that DAZL associates with mRNAs of key Caspases and similarly inhibits their translation. This elegant fail-safe mechanism ensures that, whereas loss of DAZL results in prolonged expression of pluripotency factors, teratoma formation is avoided due to the concomitant activation of the apoptotic cascade. PMID:25418731

  2. Cohesin regulates MHC class II genes through interactions with MHC class II insulators.

    PubMed

    Majumder, Parimal; Boss, Jeremy M

    2011-10-15

    Cohesin is a multiprotein, ringed complex that is most well-known for its role in stabilizing the association of sister chromatids between S phase and M. More recently, cohesin was found to be associated with transcriptional insulators, elements that are associated with the organization of chromatin into regulatory domains. The human MHC class II (MHC-II) locus contains 10 intergenic elements, termed MHC-II insulators, which bind the transcriptional insulator protein CCCTC-binding factor. MHC-II insulators interact with each other, forming a base architecture of discrete loops and potential regulatory domains. When MHC-II genes are expressed, their proximal promoter regulatory regions reorganize to the foci established by the interacting MHC-II insulators. MHC-II insulators also bind cohesin, but the functional role of cohesin in regulating this system is not known. In this article, we show that the binding of cohesin to MHC-II insulators occurred irrespective of MHC-II expression but was required for optimal expression of the HLA-DR and HLA-DQ genes. In a DNA-dependent manner, cohesin subunits interacted with CCCTC-binding factor and the MHC-II-specific transcription factors regulatory factor X and CIITA. Intriguingly, cohesin subunits were important for DNA looping interactions between the HLA-DRA promoter region and a 5' MHC-II insulator but were not required for interactions between the MHC-II insulators themselves. This latter observation introduces cohesin as a regulator of MHC-II expression by initiating or stabilizing MHC-II promoter regulatory element interactions with the MHC-II insulator elements, events that are required for maximal MHC-II transcription.

  3. Maternal Positive and Negative Interaction Behaviors and Early Adolescents' Depressive Symptoms: Adolescent Emotion Regulation as a Mediator

    ERIC Educational Resources Information Center

    Yap, Marie B. H.; Schwartz, Orli S.; Byrne, Michelle L.; Simmons, Julian G.; Allen, Nicholas B.

    2010-01-01

    This study examined the relation between mothers' positive and negative interaction behaviors during mother-child interactions and the emotion regulation (ER) and depressive symptoms of their adolescent offspring. Event-planning (EPI) and problem-solving interactions (PSI) were observed in 163 mother-adolescent dyads, and adolescents also provided…

  4. Generating human intestinal tissues from pluripotent stem cells to study development and disease.

    PubMed

    Sinagoga, Katie L; Wells, James M

    2015-05-01

    As one of the largest and most functionally complex organs of the human body, the intestines are primarily responsible for the breakdown and uptake of macromolecules from the lumen and the subsequent excretion of waste from the body. However, the intestine is also an endocrine organ, regulating digestion, metabolism, and feeding behavior. Intricate neuronal, lymphatic, immune, and vascular systems are integrated into the intestine and are required for its digestive and endocrine functions. In addition, the gut houses an extensive population of microbes that play roles in digestion, global metabolism, barrier function, and host-parasite interactions. With such an extensive array of cell types working and performing in one essential organ, derivation of functional intestinal tissues from human pluripotent stem cells (PSCs) represents a significant challenge. Here we will discuss the intricate developmental processes and cell types that are required for assembly of this highly complex organ and how embryonic processes, particularly morphogenesis, have been harnessed to direct differentiation of PSCs into 3-dimensional human intestinal organoids (HIOs) in vitro. We will further describe current uses of HIOs in development and disease research and how additional tissue complexity might be engineered into HIOs for better functionality and disease modeling. PMID:25792515

  5. Generating human intestinal tissues from pluripotent stem cells to study development and disease

    PubMed Central

    Sinagoga, Katie L; Wells, James M

    2015-01-01

    As one of the largest and most functionally complex organs of the human body, the intestines are primarily responsible for the breakdown and uptake of macromolecules from the lumen and the subsequent excretion of waste from the body. However, the intestine is also an endocrine organ, regulating digestion, metabolism, and feeding behavior. Intricate neuronal, lymphatic, immune, and vascular systems are integrated into the intestine and are required for its digestive and endocrine functions. In addition, the gut houses an extensive population of microbes that play roles in digestion, global metabolism, barrier function, and host–parasite interactions. With such an extensive array of cell types working and performing in one essential organ, derivation of functional intestinal tissues from human pluripotent stem cells (PSCs) represents a significant challenge. Here we will discuss the intricate developmental processes and cell types that are required for assembly of this highly complex organ and how embryonic processes, particularly morphogenesis, have been harnessed to direct differentiation of PSCs into 3-dimensional human intestinal organoids (HIOs) in vitro. We will further describe current uses of HIOs in development and disease research and how additional tissue complexity might be engineered into HIOs for better functionality and disease modeling. PMID:25792515

  6. Glycomics of human embryonic stem cells and human induced pluripotent stem cells.

    PubMed

    Furukawa, Jun-Ichi; Okada, Kazue; Shinohara, Yasuro

    2016-10-01

    Most cells are coated by a dense glycocalyx composed of glycoconjugates such as glycosphingolipids, glycoproteins, and proteoglycans. The overall glycomic profile is believed to be crucial for the diverse roles of glycans, which are mediated by specific interactions that regulate cell-cell adhesion, the immune response, microbial pathogenesis, and other cellular events. Many cell surface markers were discovered and identified as glycoconjugates such as stage-specific embryonic antigen, Tra-1-60/81 and various other cell surface molecules (e.g., cluster of differentiation). Recent progress in the development of analytical methodologies and strategies has begun to clarify the cellular glycomics of various cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). The glycomic profiles of these cells are highly cell type-specific and reflect cellular alterations, such as development, differentiation and cancerous change. In this mini review, we briefly summarize the glycosylation spectra specific to hESCs and hiPSCs, which cover glycans of all major glycoconjugates (i.e., glycosphingolipids, N- and O-glycans of glycoproteins, and glycosaminoglycans) and free oligosaccharides.

  7. Lin28 and let-7: ancient milestones on the road from pluripotency to neurogenesis.

    PubMed

    Rehfeld, Frederick; Rohde, Anna Maria; Nguyen, Duong Thi Thuy; Wulczyn, F Gregory

    2015-01-01

    Beginning with their discovery in the context of stem cell fate choice in Caenorhabditis elegans, the microRNA (miRNA) let-7 and the RNA-binding protein Lin28 have been recognized as a regulatory pair with far-reaching impact on stem cell behavior in a wide range of organisms and tissues, including the mammalian brain. In this review, we describe molecular interactions between Lin28 and let-7 and the biological role that each plays in implementing stem cell programs that either maintain stem cell self-renewal and plasticity or drive lineage commitment and differentiation. For Lin28, considerable progress has been made in defining let-7-dependent and let-7-independent functions in the maintenance of pluripotency, somatic cell reprogramming, tissue regeneration, and neural stem cell plasticity. For the pro-differentiation activity of let-7, we focus on emerging roles in mammalian neurogenesis and neuronal function. Specific targets and pathways for let-7 have been identified in embryonic and adult neurogenesis, including corticogenesis, retinal specification, and adult neurogenic niches. Special emphasis is given to examples of feedback and feedforward regulation, in particular within the miRNA biogenesis pathway.

  8. The Arabidopsis LYST INTERACTING PROTEIN 5 Acts in Regulating Abscisic Acid Signaling and Drought Response.

    PubMed

    Xia, Zongliang; Huo, Yongjin; Wei, Yangyang; Chen, Qiansi; Xu, Ziwei; Zhang, Wei

    2016-01-01

    Multivesicular bodies (MVBs) are unique endosomes containing vesicles in the lumens and play essential roles in many eukaryotic cellular processes. The Arabidopsis LYST INTERACTING PROTEIN 5 (LIP5), a positive regulator of MVB biogenesis, has critical roles in biotic and abiotic stress responses. However, whether the abscisic acid (ABA) signaling is involved in LIP5-mediated stress response is largely unknown. Here, we report that LIP5 functions in regulating ABA signaling and drought response in Arabidopsis. Analyses of a LIP5 promoter-β-glucuronidase (GUS) construct revealed substantial GUS activity in whole seedlings. The expression of LIP5 was induced by ABA and drought, and overexpression of LIP5 led to ABA hypersensitivity, enhanced stomatal closure, reduced water loss, and, therefore, increased drought tolerance. On the contrary, LIP5 knockdown mutants showed ABA-insensitive phenotypes and reduced drought tolerance; suggesting that LIP5 acts in regulating ABA response. Further analysis using a fluorescent dye revealed that ABA and water stress induced cell endocytosis or vesicle trafficking in a largely LIP5-dependent manner. Furthermore, expression of several drought- or ABA-inducible marker genes was significantly down-regulated in the lip5 mutant seedlings. Collectively, our data suggest that LIP5 positively regulates drought tolerance through ABA-mediated cell signaling. PMID:27313589

  9. The Arabidopsis LYST INTERACTING PROTEIN 5 Acts in Regulating Abscisic Acid Signaling and Drought Response

    PubMed Central

    Xia, Zongliang; Huo, Yongjin; Wei, Yangyang; Chen, Qiansi; Xu, Ziwei; Zhang, Wei

    2016-01-01

    Multivesicular bodies (MVBs) are unique endosomes containing vesicles in the lumens and play essential roles in many eukaryotic cellular processes. The Arabidopsis LYST INTERACTING PROTEIN 5 (LIP5), a positive regulator of MVB biogenesis, has critical roles in biotic and abiotic stress responses. However, whether the abscisic acid (ABA) signaling is involved in LIP5-mediated stress response is largely unknown. Here, we report that LIP5 functions in regulating ABA signaling and drought response in Arabidopsis. Analyses of a LIP5 promoter-β-glucuronidase (GUS) construct revealed substantial GUS activity in whole seedlings. The expression of LIP5 was induced by ABA and drought, and overexpression of LIP5 led to ABA hypersensitivity, enhanced stomatal closure, reduced water loss, and, therefore, increased drought tolerance. On the contrary, LIP5 knockdown mutants showed ABA-insensitive phenotypes and reduced drought tolerance; suggesting that LIP5 acts in regulating ABA response. Further analysis using a fluorescent dye revealed that ABA and water stress induced cell endocytosis or vesicle trafficking in a largely LIP5-dependent manner. Furthermore, expression of several drought- or ABA-inducible marker genes was significantly down-regulated in the lip5 mutant seedlings. Collectively, our data suggest that LIP5 positively regulates drought tolerance through ABA-mediated cell signaling. PMID:27313589

  10. BRCA1 interacts with Nrf2 to regulate antioxidant signaling and cell survival

    PubMed Central

    Gorrini, Chiara; Baniasadi, Pegah S.; Harris, Isaac S.; Silvester, Jennifer; Inoue, Satoshi; Snow, Bryan; Joshi, Purna A.; Wakeham, Andrew; Molyneux, Sam D.; Martin, Bernard; Bouwman, Peter; Cescon, David W.; Elia, Andrew J.; Winterton-Perks, Zoe; Cruickshank, Jennifer; Brenner, Dirk; Tseng, Alan; Musgrave, Melinda; Berman, Hal K.; Khokha, Rama; Jonkers, Jos

    2013-01-01

    Oxidative stress plays an important role in cancer development and treatment. Recent data implicate the tumor suppressor BRCA1 in regulating oxidative stress, but the molecular mechanism and the impact in BRCA1-associated tumorigenesis remain unclear. Here, we show that BRCA1 regulates Nrf2-dependent antioxidant signaling by physically interacting with Nrf2 and promoting its stability and activation. BRCA1-deficient mouse primary mammary epithelial cells show low expression of Nrf2-regulated antioxidant enzymes and accumulate reactive oxygen species (ROS) that impair survival in vivo. Increased Nrf2 activation rescues survival and ROS levels in BRCA1-null cells. Interestingly, 53BP1 inactivation, which has been shown to alleviate several defects associated with BRCA1 loss, rescues survival of BRCA1-null cells without restoring ROS levels. We demonstrate that estrogen treatment partially restores Nrf2 levels in the absence of BRCA1. Our data suggest that Nrf2-regulated antioxidant response plays a crucial role in controlling survival downstream of BRCA1 loss. The ability of estrogen to induce Nrf2 posits an involvement of an estrogen-Nrf2 connection in BRCA1 tumor suppression. Lastly, BRCA1-mutated tumors retain a defective antioxidant response that increases the sensitivity to oxidative stress. In conclusion, the role of BRCA1 in regulating Nrf2 activity suggests important implications for both the etiology and treatment of BRCA1-related cancers. PMID:23857982

  11. Quantitative Analysis of Human Pluripotency and Neural Specification by In-Depth (Phospho)Proteomic Profiling.

    PubMed

    Singec, Ilyas; Crain, Andrew M; Hou, Junjie; Tobe, Brian T D; Talantova, Maria; Winquist, Alicia A; Doctor, Kutbuddin S; Choy, Jennifer; Huang, Xiayu; La Monaca, Esther; Horn, David M; Wolf, Dieter A; Lipton, Stuart A; Gutierrez, Gustavo J; Brill, Laurence M; Snyder, Evan Y

    2016-09-13

    Controlled differentiation of human embryonic stem cells (hESCs) can be utilized for precise analysis of cell type identities during early development. We established a highly efficient neural induction strategy and an improved analytical platform, and determined proteomic and phosphoproteomic profiles of hESCs and their specified multipotent neural stem cell derivatives (hNSCs). This quantitative dataset (nearly 13,000 proteins and 60,000 phosphorylation sites) provides unique molecular insights into pluripotency and neural lineage entry. Systems-level comparative analysis of proteins (e.g., transcription factors, epigenetic regulators, kinase families), phosphorylation sites, and numerous biological pathways allowed the identification of distinct signatures in pluripotent and multipotent cells. Furthermore, as predicted by the dataset, we functionally validated an autocrine/paracrine mechanism by demonstrating that the secreted protein midkine is a regulator of neural specification. This resource is freely available to the scientific community, including a searchable website, PluriProt. PMID:27569059

  12. TIP30 interacts with an estrogen receptor alpha-interacting coactivator CIA and regulates c-myc transcription.

    PubMed

    Jiang, Chao; Ito, Mitsuhiro; Piening, Valerie; Bruck, Kristy; Roeder, Robert G; Xiao, Hua

    2004-06-25

    Deregulation of c-myc expression is implicated in the pathogenesis of many neoplasias. Estrogen receptor alpha (ERalpha) can increase the rate of c-myc transcription through the recruitment of a variety of cofactors to the promoter, yet the precise roles of these cofactors in transcription and tumorigenesis are largely unknown. We show here that a putative tumor suppressor TIP30, also called CC3 or Htatip2, interacts with an ERalpha-interacting coactivator CIA. Using chromatin immunoprecipitation assays, we demonstrate that TIP30 and CIA are distinct cofactors that are dynamically associated with the promoter and downstream regions of the c-myc gene in response to estrogen. Both TIP30 and CIA are recruited to the c-myc gene promoter by liganded ERalpha in the second transcription cycle. TIP30 overexpression represses ERalpha-mediated c-myc transcription, whereas TIP30 deficiency enhances c-myc transcription in both the absence and presence of estrogen. Ectopic CIA cooperates with TIP30 to repress ERalpha-mediated c-myc transcription. Moreover, virgin TIP30 knockout mice exhibit increased c-myc expression in mammary glands. Together, these results reveal an important role for TIP30 in the regulation of ERalpha-mediated c-myc transcription and suggest a mechanism for tumorigenesis promoted by TIP30 deficiency.

  13. Generation of serotonin neurons from human pluripotent stem cells.

    PubMed

    Lu, Jianfeng; Zhong, Xuefei; Liu, Huisheng; Hao, Ling; Huang, Cindy Tzu-Ling; Sherafat, Mohammad Amin; Jones, Jeffrey; Ayala, Melvin; Li, Lingjun; Zhang, Su-Chun

    2016-01-01

    Serotonin neurons located in the raphe nucleus of the hindbrain have crucial roles in regulating brain functions and have been implicated in various psychiatric disorders. Yet functional human serotonin neurons are not available for in vitro studies. Through manipulation of the WNT pathway, we demonstrate efficient differentiation of human pluripotent stem cells (hPSCs) to cells resembling central serotonin neurons, primarily those located in the rhombomeric segments 2-3 of the rostral raphe, which participate in high-order brain functions. The serotonin neurons express a series of molecules essential for serotonergic development, including tryptophan hydroxylase 2, exhibit typical electrophysiological properties and release serotonin in an activity-dependent manner. When treated with the FDA-approved drugs tramadol and escitalopram oxalate, they release or uptake serotonin in a dose- and time-dependent manner, suggesting the utility of these cells for the evaluation of drug candidates.

  14. Generation of serotonin neurons from human pluripotent stem cells

    PubMed Central

    Lu, Jianfeng; Zhong, Xuefei; Liu, Huisheng; Hao, Ling; Huang, Cindy Tzu-Ling; Sherafat, Mohammad Amin; Jones, Jeffrey; Ayala, Melvin; Li, Lingjun; Zhang, Su-Chun

    2016-01-01

    Serotonin neurons located in the raphe nucleus of the hindbrain have crucial roles in regulating brain functions and have been implicated in various psychiatric disorders. Yet functional human serotonin neurons are not available for in vitro studies. Through manipulation of the WNT pathway, we demonstrate efficient differentiation of human pluripotent stem cells (hPSCs) to cells resembling central serotonin neurons, primarily those located in the rhombomeric segments 2–3 of the rostral raphe, which participate in high-order brain functions. The serotonin neurons express a series of molecules essential for serotonergic development, including tryptophan hydroxylase 2, exhibit typical electrophysiological properties and release serotonin in an activity-dependent manner. When treated with the FDA-approved drugs tramadol and escitalopram oxalate, they release or uptake serotonin in a dose- and time-dependent manner, suggesting the utility of these cells for the evaluation of drug candidates. PMID:26655496

  15. ERECTA and BAK1 Receptor Like Kinases Interact to Regulate Immune Responses in Arabidopsis.

    PubMed

    Jordá, Lucía; Sopeña-Torres, Sara; Escudero, Viviana; Nuñez-Corcuera, Beatriz; Delgado-Cerezo, Magdalena; Torii, Keiko U; Molina, Antonio

    2016-01-01

    ERECTA (ER) receptor-like kinase (RLK) regulates Arabidopsis thaliana organ growth, and inflorescence and stomatal development by interacting with the ERECTA-family genes (ERf) paralogs, ER-like 1 (ERL1) and ERL2, and the receptor-like protein (RLP) TOO MANY MOUTHS (TMM). ER also controls immune responses and resistance to pathogens such as the bacterium Pseudomonas syringae pv. tomato DC3000 (Pto) and the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM). We found that er null-mutant plants overexpressing an ER dominant-negative version lacking the cytoplasmic kinase domain (ERΔK) showed an enhanced susceptibility to PcBMM, suggesting that ERΔK associates and forms inactive complexes with additional RLKs/RLPs required for PcBMM resistance. Genetic analyses demonstrated that ER acts in a combinatorial specific manner with ERL1, ERL2, and TMM to control PcBMM resistance. Moreover, BAK1 (BRASSINOSTEROID INSENSITIVE 1-associated kinase 1) RLK, which together with ERf/TMM regulates stomatal patterning and resistance to Pto, was also found to have an unequal contribution with ER in regulating immune responses and resistance to PcBMM. Co-immunoprecipitation experiments in Nicotiana benthamiana further demonstrated BAK1-ER protein interaction. The secreted epidermal pattern factor peptides (EPF1 and EPF2), which are perceived by ERf members to specify stomatal patterning, do not seem to regulate ER-mediated immunity to PcBMM, since their inducible overexpression in A. thaliana did not impact on PcBMM resistance. Our results indicate that the multiproteic receptorsome formed by ERf, TMM and BAK1 modulates A. thaliana resistance to PcBMM, and suggest that the cues underlying ERf/TMM/BAK1-mediated immune responses are distinct from those regulating stomatal pattering. PMID:27446127

  16. ERECTA and BAK1 Receptor Like Kinases Interact to Regulate Immune Responses in Arabidopsis

    PubMed Central

    Jordá, Lucía; Sopeña-Torres, Sara; Escudero, Viviana; Nuñez-Corcuera, Beatriz; Delgado-Cerezo, Magdalena; Torii, Keiko U.; Molina, Antonio

    2016-01-01

    ERECTA (ER) receptor-like kinase (RLK) regulates Arabidopsis thaliana organ growth, and inflorescence and stomatal development by interacting with the ERECTA-family genes (ERf) paralogs, ER-like 1 (ERL1) and ERL2, and the receptor-like protein (RLP) TOO MANY MOUTHS (TMM). ER also controls immune responses and resistance to pathogens such as the bacterium Pseudomonas syringae pv. tomato DC3000 (Pto) and the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM). We found that er null-mutant plants overexpressing an ER dominant-negative version lacking the cytoplasmic kinase domain (ERΔK) showed an enhanced susceptibility to PcBMM, suggesting that ERΔK associates and forms inactive complexes with additional RLKs/RLPs required for PcBMM resistance. Genetic analyses demonstrated that ER acts in a combinatorial specific manner with ERL1, ERL2, and TMM to control PcBMM resistance. Moreover, BAK1 (BRASSINOSTEROID INSENSITIVE 1-associated kinase 1) RLK, which together with ERf/TMM regulates stomatal patterning and resistance to Pto, was also found to have an unequal contribution with ER in regulating immune responses and resistance to PcBMM. Co-immunoprecipitation experiments in Nicotiana benthamiana further demonstrated BAK1-ER protein interaction. The secreted epidermal pattern factor peptides (EPF1 and EPF2), which are perceived by ERf members to specify stomatal patterning, do not seem to regulate ER-mediated immunity to PcBMM, since their inducible overexpression in A. thaliana did not impact on PcBMM resistance. Our results indicate that the multiproteic receptorsome formed by ERf, TMM and BAK1 modulates A. thaliana resistance to PcBMM, and suggest that the cues underlying ERf/TMM/BAK1-mediated immune responses are distinct from those regulating stomatal pattering. PMID:27446127

  17. ERECTA and BAK1 Receptor Like Kinases Interact to Regulate Immune Responses in Arabidopsis.

    PubMed

    Jordá, Lucía; Sopeña-Torres, Sara; Escudero, Viviana; Nuñez-Corcuera, Beatriz; Delgado-Cerezo, Magdalena; Torii, Keiko U; Molina, Antonio

    2016-01-01

    ERECTA (ER) receptor-like kinase (RLK) regulates Arabidopsis thaliana organ growth, and inflorescence and stomatal development by interacting with the ERECTA-family genes (ERf) paralogs, ER-like 1 (ERL1) and ERL2, and the receptor-like protein (RLP) TOO MANY MOUTHS (TMM). ER also controls immune responses and resistance to pathogens such as the bacterium Pseudomonas syringae pv. tomato DC3000 (Pto) and the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM). We found that er null-mutant plants overexpressing an ER dominant-negative version lacking the cytoplasmic kinase domain (ERΔK) showed an enhanced susceptibility to PcBMM, suggesting that ERΔK associates and forms inactive complexes with additional RLKs/RLPs required for PcBMM resistance. Genetic analyses demonstrated that ER acts in a combinatorial specific manner with ERL1, ERL2, and TMM to control PcBMM resistance. Moreover, BAK1 (BRASSINOSTEROID INSENSITIVE 1-associated kinase 1) RLK, which together with ERf/TMM regulates stomatal patterning and resistance to Pto, was also found to have an unequal contribution with ER in regulating immune responses and resistance to PcBMM. Co-immunoprecipitation experiments in Nicotiana benthamiana further demonstrated BAK1-ER protein interaction. The secreted epidermal pattern factor peptides (EPF1 and EPF2), which are perceived by ERf members to specify stomatal patterning, do not seem to regulate ER-mediated immunity to PcBMM, since their inducible overexpression in A. thaliana did not impact on PcBMM resistance. Our results indicate that the multiproteic receptorsome formed by ERf, TMM and BAK1 modulates A. thaliana resistance to PcBMM, and suggest that the cues underlying ERf/TMM/BAK1-mediated immune responses are distinct from those regulating stomatal pattering.

  18. Induced pluripotency of human prostatic epithelial cells.

    PubMed

    Zhao, Hongjuan; Sun, Ning; Young, Sarah R; Nolley, Rosalie; Santos, Jennifer; Wu, Joseph C; Peehl, Donna M

    2013-01-01

    Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen (PSA), a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified epigenetic changes in key pathways and genes involved in prostatic differentiation as E-PZ-iPS-like cells converted to differentiated AR- and PSA-expressing cells. Our results suggest that

  19. Hsp70-Bag3 interactions regulate cancer-related signaling networks.

    PubMed

    Colvin, Teresa A; Gabai, Vladimir L; Gong, Jianlin; Calderwood, Stuart K; Li, Hu; Gummuluru, Suryaram; Matchuk, Olga N; Smirnova, Svetlana G; Orlova, Nina V; Zamulaeva, Irina A; Garcia-Marcos, Mikel; Li, Xiaokai; Young, Z T; Rauch, Jennifer N; Gestwicki, Jason E; Takayama, Shinichi; Sherman, Michael Y

    2014-09-01

    Bag3, a nucleotide exchange factor of the heat shock protein Hsp70, has been implicated in cell signaling. Here, we report that Bag3 interacts with the SH3 domain of Src, thereby mediating the effects of Hsp70 on Src signaling. Using several complementary approaches, we established that the Hsp70-Bag3 module is a broad-acting regulator of cancer cell signaling by modulating the activity of the transcription factors NF-κB, FoxM1, Hif1α, the translation regulator HuR, and the cell-cycle regulators p21 and survivin. We also identified a small-molecule inhibitor, YM-1, that disrupts the Hsp70-Bag3 interaction. YM-1 mirrored the effects of Hsp70 depletion on these signaling pathways, and in vivo administration of this drug was sufficient to suppress tumor growth in mice. Overall, our results defined Bag3 as a critical factor in Hsp70-modulated signaling and offered a preclinical proof-of-concept that the Hsp70-Bag3 complex may offer an appealing anticancer target.

  20. The Interactive Effects of Emotion Regulation and Alcohol Intoxication on Lab-Based Intimate Partner Aggression

    PubMed Central

    Watkins, Laura E.; DiLillo, David; Maldonado, Rosalita C.

    2015-01-01

    This study draws on Finkel and Eckhardt’s (2013) I3 framework to examine the interactive effects of two emotion regulation strategies, anger rumination (an impellance factor) and reappraisal (an inhibition factor), and alcohol intoxication (a disinhibition factor), on intimate partner aggression (IPA) perpetration as measured with an analogue aggression task. Participants were 69 couples recruited from a large Midwestern university (total N = 138). Participants’ trait rumination and reappraisal were measured by self-report. Participants were randomized individually to an alcohol or placebo condition, then recalled an anger event while employing one of three randomly assigned emotion regulation conditions (rumination, reappraisal, or uninstructed). Following this, participants completed an analogue aggression task involving ostensibly assigning white noise blasts to their partner. Participants in the alcohol condition displayed greater IPA than participants in the placebo condition for provoked IPA, but not unprovoked IPA. Results also revealed interactions such that for those in the alcohol and rumination group, higher trait reappraisal was related to lower unprovoked IPA. For provoked IPA, higher trait rumination was related to greater IPA among those in the alcohol and rumination condition and those in the placebo and uninstructed condition. In general, results were consistent with I3 theory, suggesting that alcohol disinhibits, rumination impels, and trait reappraisal inhibits IPA. The theoretical and clinical implications of these findings are discussed in the context of current knowledge about the influence of alcohol intoxication and emotion regulation strategies on IPA perpetration. PMID:25844831

  1. Nucleophosmin and Nucleolin Regulate K-Ras Plasma Membrane Interactions and MAPK Signal Transduction*

    PubMed Central

    Inder, Kerry L.; Lau, Chiyan; Loo, Dorothy; Chaudhary, Natasha; Goodall, Andrew; Martin, Sally; Jones, Alun; van der Hoeven, Dharini; Parton, Robert G.; Hill, Michelle M.; Hancock, John F.

    2009-01-01

    The spatial organization of Ras proteins into nanoclusters on the inner leaflet of the plasma membrane is essential for high fidelity signaling through the MAPK pathway. Here we identify two selective regulators of K-Ras nanoclustering from a proteomic screen for K-Ras interacting proteins. Nucleophosmin (NPM) and nucleolin are predominantly localized to the nucleolus but also have extranuclear functions. We show that a subset of NPM and nucleolin localizes to the inner leaflet of plasma membrane and forms specific complexes with K-Ras but not other Ras isoforms. Active GTP-loaded and inactive GDP-loaded K-Ras both interact with NPM, although NPM-K-Ras binding is increased by growth factor receptor activation. NPM and nucleolin both stabilize K-Ras levels on the plasma membrane, but NPM concurrently increases the clustered fraction of GTP-K-Ras. The increase in nanoclustered GTP-K-Ras in turn enhances signal gain in the MAPK pathway. In summary these results reveal novel extranucleolar functions for NPM and nucleolin as regulators of K-Ras nanocluster formation and activation of the MAPK pathway. The study also identifies a new class of K-Ras nanocluster regulator that operates independently of the structural scaffold galectin-3. PMID:19661056

  2. The interactive effects of emotion regulation and alcohol intoxication on lab-based intimate partner aggression.

    PubMed

    Watkins, Laura E; DiLillo, David; Maldonado, Rosalita C

    2015-09-01

    This study draws on Finkel and Eckhardt's (2013) I³ framework to examine the interactive effects of 2 emotion regulation strategies-anger rumination (an impellance factor) and reappraisal (an inhibition factor), and alcohol intoxication (a disinhibition factor)-on intimate partner aggression (IPA) perpetration as measured with an analogue aggression task. Participants were 69 couples recruited from a large Midwestern university (total N = 138). Participants' trait rumination and reappraisal were measured by self-report. Participants were randomized individually to an alcohol or placebo condition, then recalled an anger event while using 1 of 3 randomly assigned emotion regulation conditions (rumination, reappraisal, or uninstructed). Following this, participants completed an analogue aggression task involving ostensibly assigning white noise blasts to their partner. Participants in the alcohol condition displayed greater IPA than participants in the placebo condition for provoked IPA, but not unprovoked IPA. Results also revealed interactions such that for those in the alcohol and rumination group, higher trait reappraisal was related to lower unprovoked IPA. For provoked IPA, higher trait rumination was related to greater IPA among those in the alcohol and rumination condition and those in the placebo and uninstructed condition. In general, results were consistent with I³ theory, suggesting that alcohol disinhibits, rumination impels, and trait reappraisal inhibits IPA. The theoretical and clinical implications of these findings are discussed in the context of current knowledge about the influence of alcohol intoxication and emotion regulation strategies on IPA perpetration. PMID:25844831

  3. GLABROUS INFLORESCENCE STEMS regulates trichome branching by genetically interacting with SIM in Arabidopsis.

    PubMed

    Sun, Li-Li; Zhou, Zhong-Jing; An, Li-Jun; An, Yan; Zhao, Yong-Qin; Meng, Xiao-Fang; Steele-King, Clare; Gan, Yin-Bo

    2013-07-01

    Arabidopsis trichomes are large branched single cells that protrude from the epidermis. The first morphological indication of trichome development is an increase in nuclear content resulting from an initial cycle of endoreduplication. Our previous study has shown that the C2H2 zinc finger protein GLABROUS INFLORESCENCE STEMS (GIS) is required for trichome initiation in the inflorescence organ and for trichome branching in response to gibberellic acid signaling, although GIS gene does not play a direct role in regulating trichome cell division. Here, we describe a novel role of GIS, controlling trichome cell division indirectly by interacting genetically with a key endoreduplication regulator SIAMESE (SIM). Our molecular and genetic studies have shown that GIS might indireclty control cell division and trichome branching by acting downstream of SIM. A loss of function mutation of SIM signficantly reduced the expression of GIS. Futhermore, the overexpression of GIS rescued the trichome cluster cell phenotypes of sim mutant. The gain or loss of function of GIS had no significant effect on the expression of SIM. These results suggest that GIS may play an indirect role in regulating trichome cell division by genetically interacting with SIM. PMID:23825141

  4. The adipogenic transcriptional cofactor ZNF638 interacts with splicing regulators and influences alternative splicing

    PubMed Central

    Du, Chen; Ma, Xinran; Meruvu, Sunitha; Hugendubler, Lynne; Mueller, Elisabetta

    2014-01-01

    Increasing evidence indicates that transcription and alternative splicing are coordinated processes; however, our knowledge of specific factors implicated in both functions during the process of adipocyte differentiation is limited. We have previously demonstrated that the zinc finger protein ZNF638 plays a role as a transcriptional coregulator of adipocyte differentiation via induction of PPARγ in cooperation with CCAAT/enhancer binding proteins (C/EBPs). Here we provide new evidence that ZNF638 is localized in nuclear bodies enriched with splicing factors, and through biochemical purification of ZNF638’s interacting proteins in adipocytes and mass spectrometry analysis, we show that ZNF638 interacts with splicing regulators. Functional analysis of the effects of ectopic ZNF638 expression on a minigene reporter demonstrated that ZNF638 is sufficient to promote alternative splicing, a function enhanced through its recruitment to the minigene promoter at C/EBP responsive elements via C/EBP proteins. Structure-function analysis revealed that the arginine/serine-rich motif and the C-terminal zinc finger domain required for speckle localization are necessary for the adipocyte differentiation function of ZNF638 and for the regulation of the levels of alternatively spliced isoforms of lipin1 and nuclear receptor co-repressor 1. Overall, our data demonstrate that ZNF638 participates in splicing decisions and that it may control adipogenesis through regulation of the relative amounts of differentiation-specific isoforms. PMID:25024404

  5. Modeling Cell–Cell Interactions in Regulating Multiple Myeloma Initiating Cell Fate

    PubMed Central

    Peng, Tao; Peng, Huiming; Choi, Dong Soon; Su, Jing; (Jeff) Chang, Chung-Che

    2014-01-01

    Cancer initiating cells have been documented in multiple myeloma and believed to be a key factor that initiates and drives tumor growth, differentiation, metastasis, and recurrence of the diseases. Although myeloma initiating cells (MICs) are likely to share many properties of normal stem cells, the underlying mechanisms regulating the fate of MICs are largely unknown. Studies designed to explore such communication are urgently needed to enhance our ability to predict the fate decisions of MICs (self-renewal, differentiation, and proliferation). In this study, we developed a novel system to understand the intercellular communication between MICs and their niche by seamlessly integrating experimental data and mathematical model. We first designed dynamic cell culture experiments and collected three types of cells (side population cells, progenitor cells, and mature myeloma cells) under various cultural conditions with flow cytometry. Then we developed a lineage model with ordinary differential equations by considering secreted factors, self-renewal, differentiation, and other biological functions of those cells, to model the cell–cell interactions among the three cell types. Particle swarm optimization was employed to estimate the model parameters by fitting the experimental data to the lineage model. The theoretical results show that the correlation coefficient analysis can reflect the feedback loops among the three cell types, the intercellular feedback signaling can regulate cell population dynamics, and the culture strategies can decide cell growth. This study provides a basic framework of studying cell–cell interactions in regulating MICs fate. PMID:24058033

  6. The Vac14-interaction network is linked to regulators of the endolysosomal and autophagic pathway.

    PubMed

    Schulze, Ulf; Vollenbröker, Beate; Braun, Daniela A; Van Le, Truc; Granado, Daniel; Kremerskothen, Joachim; Fränzel, Benjamin; Klosowski, Rafael; Barth, Johannes; Fufezan, Christian; Wolters, Dirk A; Pavenstädt, Hermann; Weide, Thomas

    2014-06-01

    The scaffold protein Vac14 acts in a complex with the lipid kinase PIKfyve and its counteracting phosphatase FIG4, regulating the interconversion of phosphatidylinositol-3-phosphate to phosphatidylinositol-3,5-bisphosphate. Dysfunctional Vac14 mutants, a deficiency of one of the Vac14 complex components, or inhibition of PIKfyve enzymatic activity results in the formation of large vacuoles in cells. How these vacuoles are generated and which processes are involved are only poorly understood. Here we show that ectopic overexpression of wild-type Vac14 as well as of the PIKfyve-binding deficient Vac14 L156R mutant causes vacuoles. Vac14-dependent vacuoles and PIKfyve inhibitor-dependent vacuoles resulted in elevated levels of late endosomal, lysosomal, and autophagy-associated proteins. However, only late endosomal marker proteins were bound to the membranes of these enlarged vacuoles. In order to decipher the linkage between the Vac14 complex and regulators of the endolysosomal pathway, a protein affinity approach combined with multidimensional protein identification technology was conducted, and novel molecular links were unraveled. We found and verified the interaction of Rab9 and the Rab7 GAP TBC1D15 with Vac14. The identified Rab-related interaction partners support the theory that the regulation of vesicular transport processes and phosphatidylinositol-modifying enzymes are tightly interconnected.

  7. Regulation of GPCR activity, trafficking and localization by GPCR-interacting proteins

    PubMed Central

    Magalhaes, Ana C; Dunn, Henry; Ferguson, Stephen SG

    2012-01-01

    GPCRs represent the largest family of integral membrane proteins and were first identified as receptor proteins that couple via heterotrimeric G-proteins to regulate a vast variety of effector proteins to modulate cellular function. It is now recognized that GPCRs interact with a myriad of proteins that not only function to attenuate their signalling but also function to couple these receptors to heterotrimeric G-protein-independent signalling pathways. In addition, intracellular and transmembrane proteins associate with GPCRs and regulate their processing in the endoplasmic reticulum, trafficking to the cell surface, compartmentalization to plasma membrane microdomains, endocytosis and trafficking between intracellular membrane compartments. The present review will overview the functional consequence of β-arrestin, receptor activity-modifying proteins (RAMPS), regulators of G-protein signalling (RGS), GPCR-associated sorting proteins (GASPs), Homer, small GTPases, PSD95/Disc Large/Zona Occludens (PDZ), spinophilin, protein phosphatases, calmodulin, optineurin and Src homology 3 (SH3) containing protein interactions with GPCRs. LINKED ARTICLES This article is part of a themed section on the Molecular Pharmacology of G Protein-Coupled Receptors (GPCRs). To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-6. To view the 2010 themed section on the same topic visit http://onlinelibrary.wiley.com/doi/10.1111/bph.2010.159.issue-5/issuetoc PMID:21699508

  8. GLABROUS INFLORESCENCE STEMS regulates trichome branching by genetically interacting with SIM in Arabidopsis.

    PubMed

    Sun, Li-Li; Zhou, Zhong-Jing; An, Li-Jun; An, Yan; Zhao, Yong-Qin; Meng, Xiao-Fang; Steele-King, Clare; Gan, Yin-Bo

    2013-07-01

    Arabidopsis trichomes are large branched single cells that protrude from the epidermis. The first morphological indication of trichome development is an increase in nuclear content resulting from an initial cycle of endoreduplication. Our previous study has shown that the C2H2 zinc finger protein GLABROUS INFLORESCENCE STEMS (GIS) is required for trichome initiation in the inflorescence organ and for trichome branching in response to gibberellic acid signaling, although GIS gene does not play a direct role in regulating trichome cell division. Here, we describe a novel role of GIS, controlling trichome cell division indirectly by interacting genetically with a key endoreduplication regulator SIAMESE (SIM). Our molecular and genetic studies have shown that GIS might indireclty control cell division and trichome branching by acting downstream of SIM. A loss of function mutation of SIM signficantly reduced the expression of GIS. Futhermore, the overexpression of GIS rescued the trichome cluster cell phenotypes of sim mutant. The gain or loss of function of GIS had no significant effect on the expression of SIM. These results suggest that GIS may play an indirect role in regulating trichome cell division by genetically interacting with SIM.

  9. REGULATION OF FMN SUBDOMAIN INTERACTIONS AND FUNCTION IN NEURONAL NITRIC OXIDE SYNTHASE‡

    PubMed Central

    Ilagan, Robielyn P.; Tejero, Jesús; Aulak, Kulwant S.; Sinha Ray, Sougata; Hemann, Craig; Wang, Zhi-Qiang; Gangoda, Mahinda; Zweier, Jay L.; Stuehr, Dennis J.

    2009-01-01

    Nitric oxide synthases (NOS) are modular, calmodulin (CaM)-dependent, flavo-heme enzymes that catalyze oxidation of L-arginine to generate nitric oxide (NO) and citrulline. During catalysis, the FMN subdomain cycles between interaction with an NADPH-FAD subdomain to receive electrons, and interaction with an oxygenase domain to deliver electrons to the NOS heme. This process can be described by a three-state, two equilibrium model for the conformation of the FMN subdomain, in which it exists in two distinct bound states (FMN-shielded), and one common unbound state (FMN-deshielded). We studied how each partner subdomain, the FMN redox state, and CaM binding may regulate the conformational equilibria of the FMN module in rat neuronal NOS (nNOS). We utilized four nNOS protein constructs of different subdomain composition, including the isolated FMN subdomain, and determined changes in the conformational state by measuring the degree of FMN shielding by fluorescence, electron paramagnetic resonance, or stopped-flow spectroscopic techniques. Our results suggest: (i) The NADPH-FAD subdomain has a far greater capacity to interact with the FMN subdomain than does the oxygenase domain. (ii) CaM binding has no direct effects on the FMN subdomain. (iii) CaM destabilizes interaction of the FMN subdomain with the NADPH-FAD subdomain but does not measurably increase its interaction with the oxygenase domain. Our results imply that a different set point and CaM regulation exists for either conformational equilibrium of the FMN subdomain. This helps to explain the unique electron transfer and catalytic behaviors of nNOS, relative to other dual-flavin enzymes. PMID:19290671

  10. Phosphorylation of the amino-terminal region of X11L regulates its interaction with APP

    PubMed Central

    Sakuma, Megumi; Tanaka, Emi; Taru, Hidenori; Tomita, Susumu; Gandy, Sam; Nairn, Angus C.; Nakaya, Tadashi; Yamamoto, Tohru; Suzuki, Toshiharu

    2013-01-01

    Summary X11-like (X11L) is neuronal adaptor protein that interacts with the amyloid β-protein precursor (APP) and regulates its metabolism. The phosphotyrosine interaction/binding (PI/PTB) domain of X11L interacts with the cytoplasmic region of APP695. We found that X11L-APP interaction is enhanced in osmotically stressed cells and X11L modification is required for the enhancement. Amino acids 221-250 (X11L221-250) are required for the enhanced association with APP in osmotically stressed cells; this motif is 118 amino acids closer to the amino-terminal end of the protein than the PI/PTB domain (amino acids 368-555). We identified two phosphorylatable seryl residues, Ser236 and Ser238, in X11L221-250 and alanyl substitution of either seryl residue diminished the enhanced association with APP. In brain Ser238 was found to be phosphorylated and phosphorylation of X11L was required for the interaction of X11L and APP. Both seryl residues in X11L221-250 are conserved in neuronal X11, but not in X11L2, a non-neuronal X11 family member that did not exhibit enhanced APP association in osmotically stressed cells. These findings indicate that the region of X11L that regulates association with APP is located outside of, and amino-terminal to, the PI/PTB domain. Modification of this regulatory region may alter the conformation of the PI/PTB domain to modulate APP binding. PMID:19222704

  11. Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo.

    PubMed

    Xue, Binghua; Li, Yan; He, Yilong; Wei, Renyue; Sun, Ruizhen; Yin, Zhi; Bou, Gerelchimeg; Liu, Zhonghua

    2016-01-01

    Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs.

  12. Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo.

    PubMed

    Xue, Binghua; Li, Yan; He, Yilong; Wei, Renyue; Sun, Ruizhen; Yin, Zhi; Bou, Gerelchimeg; Liu, Zhonghua

    2016-01-01

    Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs. PMID:26991423

  13. Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo

    PubMed Central

    Xue, Binghua; Li, Yan; He, Yilong; Wei, Renyue; Sun, Ruizhen; Yin, Zhi; Bou, Gerelchimeg; Liu, Zhonghua

    2016-01-01

    Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs. PMID:26991423

  14. Secreted HHIP1 interacts with heparan sulfate and regulates Hedgehog ligand localization and function.

    PubMed

    Holtz, Alexander M; Griffiths, Samuel C; Davis, Samantha J; Bishop, Benjamin; Siebold, Christian; Allen, Benjamin L

    2015-06-01

    Vertebrate Hedgehog (HH) signaling is controlled by several ligand-binding antagonists including Patched-1 (PTCH1), PTCH2, and HH-interacting protein 1 (HHIP1), whose collective action is essential for proper HH pathway activity. However, the molecular mechanisms used by these inhibitors remain poorly understood. In this paper, we investigated the mechanisms underlying HHIP1 antagonism of HH signaling. Strikingly, we found evidence that HHIP1 non-cell-autonomously inhibits HH-dependent neural progenitor patterning and proliferation. Furthermore, this non-cell-autonomous antagonism of HH signaling results from the secretion of HHIP1 that is modulated by cell type-specific interactions with heparan sulfate (HS). These interactions are mediated by an HS-binding motif in the cysteine-rich domain of HHIP1 that is required for its localization to the neuroepithelial basement membrane (BM) to effectively antagonize HH pathway function. Our data also suggest that endogenous, secreted HHIP1 localization to HS-containing BMs regulates HH ligand distribution. Overall, the secreted activity of HHIP1 represents a novel mechanism to regulate HH ligand localization and function during embryogenesis. PMID:26056142

  15. Tmem2 regulates cell-matrix interactions that are essential for muscle fiber attachment.

    PubMed

    Ryckebüsch, Lucile; Hernandez, Lydia; Wang, Carole; Phan, Jenny; Yelon, Deborah

    2016-08-15

    Skeletal muscle morphogenesis depends upon interactions between developing muscle fibers and the extracellular matrix (ECM) that anchors fibers to the myotendinous junction (MTJ). The pathways that organize the ECM and regulate its engagement by cell-matrix adhesion complexes (CMACs) are therefore essential for muscle integrity. Here, we demonstrate the impact of transmembrane protein 2 (tmem2) on cell-matrix interactions during muscle morphogenesis in zebrafish. Maternal-zygotic tmem2 mutants (MZtmem2) exhibit muscle fiber detachment, in association with impaired laminin organization and ineffective fibronectin degradation at the MTJ. Similarly, disorganized laminin and fibronectin surround MZtmem2 cardiomyocytes, which could account for their hindered movement during cardiac morphogenesis. In addition to ECM defects, MZtmem2 mutants display hypoglycosylation of α-dystroglycan within the CMAC, which could contribute to the observed fiber detachment. Expression of the Tmem2 ectodomain can rescue aspects of the MZtmem2 phenotype, consistent with a possible extracellular function of Tmem2. Together, our results suggest that Tmem2 regulates cell-matrix interactions by affecting both ECM organization and CMAC activity. These findings evoke possible connections between the functions of Tmem2 and the etiologies of congenital muscular dystrophies, particularly dystroglycanopathies. PMID:27471259

  16. Interaction of Munc18 and Syntaxin in the regulation of insulin secretion

    SciTech Connect

    Dong, Yongming; Wan, Qunfang; Yang, Xiaofei; Bai, Li; Xu, Pingyong . E-mail: pyxu@moon.ibp.ac.cn

    2007-08-31

    Syntaxin1A and Munc18-1 play essential roles in exocytosis. However, the molecular mechanism and the functional roles of their interaction in insulin secretion remain to be explored. Using membrane capacitance measurement, we examine effect of overexpressing Munc18-1 on exocytosis in pancreatic {beta} cells. The results show that Munc18-1 negatively regulates vesicle fusion. To probe the interaction between Munc18-1 and Syntaxin1A, Munc18-1-Tdimer2 and EGFP-Syntaxin1A were co-transfected into INS-1 cells. FRET measurement confirmed that Munc18-1 interacted with wild type Syntaxin 1A, but not the constitutively open form (DM) of Syntaxin1A. Overexpressing DM in primary pancreatic {beta} cells augmented insulin secretion, and this effect can overcome the inhibitory effect of Munc18-1 overexpression. We propose that Munc18-1 inhibitis the SNARE complex assembly by stabilizing Syntaxin1A in a closed conformation in vesicle priming process, therefore negatively regulates insulin secretion.

  17. OsMADS32 interacts with PI-like proteins and regulates rice flower development.

    PubMed

    Wang, Huanhuan; Zhang, Liang; Cai, Qiang; Hu, Yun; Jin, Zhenming; Zhao, Xiangxiang; Fan, Wei; Huang, Qianming; Luo, Zhijing; Chen, Mingjiao; Zhang, Dabing; Yuan, Zheng

    2015-05-01

    OsMADS32 is a monocot specific MIKC(c) type MADS-box gene that plays an important role in regulating rice floral meristem and organs identity, a crucial process for reproductive success and rice yield. However, its underlying mechanism of action remains to be clarified. Here, we characterized a hypomorphic mutant allele of OsMADS32/CFO1, cfo1-3 and identified its function in controlling rice flower development by bioinformatics and protein-protein interaction analysis. The cfo1-3 mutant produces defective flowers, including loss of lodicule identity, formation of ectopic lodicule or hull-like organs and decreased stamen number, mimicking phenotypes related to the mutation of B class genes. Molecular characterization indicated that mis-splicing of OsMADS32 transcripts in the cfo1-3 mutant resulted in an extra eight amino acids in the K-domain of OsMADS32 protein. By yeast two hybrid and bimolecular fluorescence complementation assays, we revealed that the insertion of eight amino acids or deletion of the internal region in the K1 subdomain of OsMADS32 affects the interaction between OsMADS32 with PISTILLATA (PI)-like proteins OsMADS2 and OsMADS4. This work provides new insight into the mechanism by which OsMADS32 regulates rice lodicule and stamen identity, by interaction with two PI-like proteins via its K domain. PMID:25081486

  18. Regulation of calreticulin-major histocompatibility complex (MHC) class I interactions by ATP.

    PubMed

    Wijeyesakere, Sanjeeva Joseph; Gagnon, Jessica K; Arora, Karunesh; Brooks, Charles L; Raghavan, Malini

    2015-10-13

    The MHC class I peptide loading complex (PLC) facilitates the assembly of MHC class I molecules with peptides, but factors that regulate the stability and dynamics of the assembly complex are largely uncharacterized. Based on initial findings that ATP, in addition to MHC class I-specific peptide, is able to induce MHC class I dissociation from the PLC, we investigated the interaction of ATP with the chaperone calreticulin, an endoplasmic reticulum (ER) luminal, calcium-binding component of the PLC that is known to bind ATP. We combined computational and experimental measurements to identify residues within the globular domain of calreticulin, in proximity to the high-affinity calcium-binding site, that are important for high-affinity ATP binding and for ATPase activity. High-affinity calcium binding by calreticulin is required for optimal nucleotide binding, but both ATP and ADP destabilize enthalpy-driven high-affinity calcium binding to calreticulin. ATP also selectively destabilizes the interaction of calreticulin with cellular substrates, including MHC class I molecules. Calreticulin mutants that affect ATP or high-affinity calcium binding display prolonged associations with monoglucosylated forms of cellular MHC class I, delaying MHC class I dissociation from the PLC and their transit through the secretory pathway. These studies reveal central roles for ATP and calcium binding as regulators of calreticulin-substrate interactions and as key determinants of PLC dynamics.

  19. Tamoxifen induces a pluripotency signature in breast cancer cells and human tumors.

    PubMed

    Notas, George; Pelekanou, Vassiliki; Kampa, Marilena; Alexakis, Konstantinos; Sfakianakis, Stelios; Laliotis, Aggelos; Askoxilakis, John; Tsentelierou, Eleftheria; Tzardi, Maria; Tsapis, Andreas; Castanas, Elias

    2015-11-01

    Tamoxifen is the treatment of choice in estrogen receptor alpha breast cancer patients that are eligible for adjuvant endocrine therapy. However, ∼50% of ERα-positive tumors exhibit intrinsic or rapidly acquire resistance to endocrine treatment. Unfortunately, prediction of de novo resistance to endocrine therapy and/or assessment of relapse likelihood remain difficult. While several mechanisms regulating the acquisition and the maintenance of endocrine resistance have been reported, there are several aspects of this phenomenon that need to be further elucidated. Altered metabolic fate of tamoxifen within patients and emergence of tamoxifen-resistant clones, driven by evolution of the disease phenotype during treatment, appear as the most compelling hypotheses so far. In addition, tamoxifen was reported to induce pluripotency in breast cancer cell lines, in vitro. In this context, we have performed a whole transcriptome analysis of an ERα-positive (T47D) and a triple-negative breast cancer cell line (MDA-MB-231), exposed to tamoxifen for a short time frame (hours), in order to identify how early pluripotency-related effects of tamoxifen may occur. Our ultimate goal was to identify whether the transcriptional actions of tamoxifen related to induction of pluripotency are mediated through specific ER-dependent or independent mechanisms. We report that even as early as 3 hours after the exposure of breast cancer cells to tamoxifen, a subset of ERα-dependent genes associated with developmental processes and pluripotency are induced and this is accompanied by specific phenotypic changes (expression of pluripotency-related proteins). Furthermore we report an association between the increased expression of pluripotency-related genes in ERα-positive breast cancer tissues samples and disease relapse after tamoxifen therapy. Finally we describe that in a small group of ERα-positive breast cancer patients, with disease relapse after surgery and tamoxifen treatment, ALDH

  20. What can pluripotent stem cells teach us about neurodegenerative diseases?

    PubMed

    Wichterle, Hynek; Przedborski, Serge

    2010-07-01

    Neurodegenerative diseases represent a growing public health challenge. Current medications treat symptoms, but none halt or retard neurodegeneration. The recent advent of pluripotent cell biology has opened new avenues for neurodegenerative disease research. The greatest potential for induced pluripotent cells derived from affected individuals is likely to be their utility for modeling and understanding the mechanisms underlying neurodegenerative processes, and for searching for new treatments, including cell replacement therapies. However, much work remains to be done before pluripotent cells can be used for preclinical and clinical applications. Here we discuss the challenges of generating specific neural cell subtypes from pluripotent stem cells, the use of pluripotent stem cells to model both cell-autonomous and non-cell-autonomous mechanisms of neurodegeneration, whether adult-onset neurodegeneration can be emulated in short-term cultures and the hurdles of cell replacement therapy. Progress in these four areas will substantially accelerate effective application of pluripotent stem cells.

  1. Sall4 controls differentiation of pluripotent cells independently of the Nucleosome Remodelling and Deacetylation (NuRD) complex

    PubMed Central

    Miller, Anzy; Ralser, Meryem; Kloet, Susan L.; Loos, Remco; Nishinakamura, Ryuichi; Bertone, Paul; Vermeulen, Michiel

    2016-01-01

    Sall4 is an essential transcription factor for early mammalian development and is frequently overexpressed in cancer. Although it is reported to play an important role in embryonic stem cell (ESC) self-renewal, whether it is an essential pluripotency factor has been disputed. Here, we show that Sall4 is dispensable for mouse ESC pluripotency. Sall4 is an enhancer-binding protein that prevents precocious activation of the neural gene expression programme in ESCs but is not required for maintenance of the pluripotency gene regulatory network. Although a proportion of Sall4 protein physically associates with the Nucleosome Remodelling and Deacetylase (NuRD) complex, Sall4 neither recruits NuRD to chromatin nor influences transcription via NuRD; rather, free Sall4 protein regulates transcription independently of NuRD. We propose a model whereby enhancer binding by Sall4 and other pluripotency-associated transcription factors is responsible for maintaining the balance between transcriptional programmes in pluripotent cells. PMID:27471257

  2. Protein-protein interactions among ion channels regulate ion transport in the kidney.

    PubMed

    Boulpaep, E

    2009-01-01

    Epithelial ion transport in various organs has long been known to be controlled by extracellular agonists acting via membrane receptors or by intracellular messengers. Evidence is mounting for regulation of transport by direct interaction among membrane proteins or between a membrane transport protein and membrane-attached proteins. The membrane protein CFTR (Cystic Fibrosis Transmembrane Regulator) is widely expressed along the length of the nephron, but its role as a chloride channel does not appear to be critical for renal handling of salt and water. It is well established that the inward rectifying K channels (ROMK = Kir 1.1) in the thick ascending limb of Henle and in principal cells of the collecting duct are inhibited by millimolar concentrations of cytosolic Mg-ATP. However, the mechanism of this inhibition has been an enigma. We propose that the ATP-Binding Cassette (ABC) protein CFTR is a cofactor for Kir 1.1 regulation. Indeed, Mg-ATP sensitivity of Kir 1.1 is completely absent in two different mouse models of cystic fibrosis. In addition, the open-closed state of CFTR appears to provide a molecular gating switch that prevents or facilitates the ATP sensing of Kir 1.1. Does Mg-ATP sensing by the CFTR- Kir 1.1 complex play a role in coupling metabolism to ion transport? Physiological intracellular ATP concentrations in tubule cells are in the millimolar range, a saturating concentration for the gating of Kir 1.1 by Mg-ATP. Therefore, Kir 1.1 channels would be closed and unable to contribute to regulation of potassium secretion unless some other process modulated the CFTR-dependent ATP-sensitivity of Kir 1.1. The third component of the metabolic sensor-effector complex for Kir 1.1 regulation is most likely the AMP-regulated serine-threonine kinase, AMP kinase (AMPK). Changing levels in AMP rather than in ATP constitute the metabolic signal "sensed" by tubule cells. Because AMPK inhibits CFTR by modulating CFTR channel gating, we propose that renal K

  3. Generation of Avian Induced Pluripotent Stem Cells.

    PubMed

    Lu, Yangqing; West, Franklin D; Jordan, Brian J; Beckstead, Robert B; Jordan, Erin T; Stice, Steven L

    2015-01-01

    Avian species are among the most diverse vertebrates on our planet and significantly contribute to the balance of the ecology. They are also important food source and serve as a central animal model to decipher developmental biology and disease principles. Derivation of induced pluripotent stem cells (iPSCs) from avian species would enable conservation of genetic diversity as well as offer a valuable cell source that facilitates the use of avian models in many areas of basic and applied research. In this chapter, we describe methods used to successfully reprogram quail fibroblasts into iPSCs by using human transcription factors and the techniques critical to the characterization of their pluripotency. PMID:26621592

  4. Reprogramming of germ cells into pluripotency

    PubMed Central

    Sekita, Yoichi; Nakamura, Toshinobu; Kimura, Tohru

    2016-01-01

    Primordial germ cells (PGCs) are precursors of all gametes, and represent the founder cells of the germline. Although developmental potency is restricted to germ-lineage cells, PGCs can be reprogrammed into a pluripotent state. Specifically, PGCs give rise to germ cell tumors, such as testicular teratomas, in vivo, and to pluripotent stem cells known as embryonic germ cells in vitro. In this review, we highlight the current knowledge on signaling pathways, transcriptional controls, and post-transcriptional controls that govern germ cell differentiation and de-differentiation. These regulatory processes are common in the reprogramming of germ cells and somatic cells, and play a role in the pathogenesis of human germ cell tumors.

  5. Reprogramming of germ cells into pluripotency.

    PubMed

    Sekita, Yoichi; Nakamura, Toshinobu; Kimura, Tohru

    2016-08-26

    Primordial germ cells (PGCs) are precursors of all gametes, and represent the founder cells of the germline. Although developmental potency is restricted to germ-lineage cells, PGCs can be reprogrammed into a pluripotent state. Specifically, PGCs give rise to germ cell tumors, such as testicular teratomas, in vivo, and to pluripotent stem cells known as embryonic germ cells in vitro. In this review, we highlight the current knowledge on signaling pathways, transcriptional controls, and post-transcriptional controls that govern germ cell differentiation and de-differentiation. These regulatory processes are common in the reprogramming of germ cells and somatic cells, and play a role in the pathogenesis of human germ cell tumors. PMID:27621759

  6. Reprogramming of germ cells into pluripotency

    PubMed Central

    Sekita, Yoichi; Nakamura, Toshinobu; Kimura, Tohru

    2016-01-01

    Primordial germ cells (PGCs) are precursors of all gametes, and represent the founder cells of the germline. Although developmental potency is restricted to germ-lineage cells, PGCs can be reprogrammed into a pluripotent state. Specifically, PGCs give rise to germ cell tumors, such as testicular teratomas, in vivo, and to pluripotent stem cells known as embryonic germ cells in vitro. In this review, we highlight the current knowledge on signaling pathways, transcriptional controls, and post-transcriptional controls that govern germ cell differentiation and de-differentiation. These regulatory processes are common in the reprogramming of germ cells and somatic cells, and play a role in the pathogenesis of human germ cell tumors. PMID:27621759

  7. Induced pluripotency: history, mechanisms, and applications

    PubMed Central

    Stadtfeld, Matthias; Hochedlinger, Konrad

    2010-01-01

    The generation of induced pluripotent stem cells (iPSCs) from somatic cells demonstrated that adult mammalian cells can be reprogrammed to a pluripotent state by the enforced expression of a few embryonic transcription factors. This discovery has raised fundamental questions about the mechanisms by which transcription factors influence the epigenetic conformation and differentiation potential of cells during reprogramming and normal development. In addition, iPSC technology has provided researchers with a unique tool to derive disease-specific stem cells for the study and possible treatment of degenerative disorders with autologous cells. In this review, we summarize the progress that has been made in the iPSC field over the last 4 years, with an emphasis on understanding the mechanisms of cellular reprogramming and its potential applications in cell therapy. PMID:20952534

  8. Photosynthetic photon flux density and phytochrome B interact to regulate branching in Arabidopsis.

    PubMed

    Su, Hongwen; Abernathy, Scott D; White, Richard H; Finlayson, Scott A

    2011-11-01

    Branching is regulated by environmental signals including phytochrome B (phyB)-mediated responses to the ratio of red to far red light. While the mechanisms associated with phytochrome regulation of branching are beginning to be elucidated, there is little information regarding other light signals, including photosynthetic photon flux density (PPFD) and how it influences phytochrome-mediated responses. This study shows that Arabidopsis (Arabidopsis thaliana) branching is modified by both varying PPFD and phyB status and that significant interactions occur between these variables. While phyB deficiency decreased branching when the PPFD was low, the effect was suppressed by high PPFD and some branching aspects were actually promoted. Photosynthesis measurements showed that PPFD may influence branching in phyB-deficient plants at least partially through a specific signalling pathway rather than directly through energy effects on the shoot. The expression of various genes in unelongated buds of phyB-deficient and phyB-sufficient plants grown under high and low PPFD demonstrated potential roles for several hormones, including auxin, cytokinins and ABA, and also showed imperfect correlation between expression of the branching regulators BRC1 and BRC2 and bud fate. These results may implicate additional undiscovered bud autonomous mechanisms and/or components contributing to bud outgrowth regulation by environmental signals.

  9. Metal interaction with redox regulation: an integrating concept in metal carcinogenesis?

    PubMed

    Hartwig, Andrea

    2013-02-01

    The carcinogenicity of cadmium, arsenic, and chromium(VI) compounds has been recognized for some decades. However, the underlying molecular mechanisms seem to be complex and are not completely understood at present. Although, with the exception of chromium(VI), direct DNA damage seems to be of minor importance, interactions with DNA repair processes, tumor suppressor functions, and signal transduction pathways have been described in diverse biological systems. In addition to the induction of damage to cellular macromolecules by reactive oxygen species, the interference with cellular redox regulation by reaction with redox-sensitive protein domains or amino acids may provide one plausible mechanism involved in metal carcinogenicity. Consequences are the distortion of zinc-binding structures and the activation or inactivation of redox-regulated signal transduction pathways, provoking metal-induced genomic instability. Nevertheless, the relevance of the respective mechanisms depends on the actual metal or metal species under consideration and more research is needed to further strengthen this hypothesis.

  10. Charge regulation in ionic solutions: thermal fluctuations and Kirkwood-Schumaker interactions.

    PubMed

    Adžić, Nataša; Podgornik, Rudolf

    2015-02-01

    We study the behavior of two macroions with dissociable charge groups, regulated by local variables such as pH and electrostatic potential, immersed in a monovalent salt solution, considering cases where the net charge can either change sign or remain of the same sign depending on these local parameters. The charge regulation in both cases is described by the proper free-energy function for each of the macroions, while the coupling between the charges is evaluated on the approximate Debye-Hückel level. The charge correlation functions and the ensuing charge fluctuation forces are calculated analytically and numerically. Strong attraction between like-charged macroions is found close to the point of zero charge, specifically due to asymmetric, anticorrelated charge fluctuations of the macroion charges. The general theory is then implemented for a system of two proteinlike macroions, generalizing the form and magnitude of the Kirkwood-Schumaker interaction. PMID:25768539

  11. Interactions of cellular proteins involved in the transcriptional regulation of the human immunodeficiency virus.

    PubMed Central

    Garcia, J A; Wu, F K; Mitsuyasu, R; Gaynor, R B

    1987-01-01

    The human immunodeficiency virus (HIV) is a human retrovirus which is the etiologic agent of the acquired immunodeficiency syndrome. To study the cellular factors involved in the transcriptional regulation of this virus, we performed DNase I footprinting of the viral LTR using partially purified HeLa cell extracts. Five regions of the viral LTR appear critical for DNA binding of cellular proteins. These include the negative regulatory, enhancer, SP1, TATA and untranslated regions. Deletion mutagenesis of these binding domains has significant effects on the basal level of transcription and the ability to be induced by the viral tat protein. Mutations of either the negative regulatory or untranslated regions affect factor binding to the enhancer region. In addition, oligonucleotides complementary to several of the binding domains specifically compete for factor binding. These results suggest that interactions between several distinct cellular proteins are required for HIV transcriptional regulation. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 6. PMID:3428273

  12. Interactions of cellular proteins involved in the transcriptional regulation of the human immunodeficiency virus.

    PubMed

    Garcia, J A; Wu, F K; Mitsuyasu, R; Gaynor, R B

    1987-12-01

    The human immunodeficiency virus (HIV) is a human retrovirus which is the etiologic agent of the acquired immunodeficiency syndrome. To study the cellular factors involved in the transcriptional regulation of this virus, we performed DNase I footprinting of the viral LTR using partially purified HeLa cell extracts. Five regions of the viral LTR appear critical for DNA binding of cellular proteins. These include the negative regulatory, enhancer, SP1, TATA and untranslated regions. Deletion mutagenesis of these binding domains has significant effects on the basal level of transcription and the ability to be induced by the viral tat protein. Mutations of either the negative regulatory or untranslated regions affect factor binding to the enhancer region. In addition, oligonucleotides complementary to several of the binding domains specifically compete for factor binding. These results suggest that interactions between several distinct cellular proteins are required for HIV transcriptional regulation.

  13. Pluripotent stem cells and livestock genetic engineering.

    PubMed

    Soto, Delia A; Ross, Pablo J

    2016-06-01

    The unlimited proliferative ability and capacity to contribute to germline chimeras make pluripotent embryonic stem cells (ESCs) perfect candidates for complex genetic engineering. The utility of ESCs is best exemplified by the numerous genetic models that have been developed in mice, for which such cells are readily available. However, the traditional systems for mouse genetic engineering may not be practical for livestock species, as it requires several generations of mating and selection in order to establish homozygous founders. Nevertheless, the self-renewal and pluripotent characteristics of ESCs could provide advantages for livestock genetic engineering such as ease of genetic manipulation and improved efficiency of cloning by nuclear transplantation. These advantages have resulted in many attempts to isolate livestock ESCs, yet it has been generally concluded that the culture conditions tested so far are not supportive of livestock ESCs self-renewal and proliferation. In contrast, there are numerous reports of derivation of livestock induced pluripotent stem cells (iPSCs), with demonstrated capacity for long term proliferation and in vivo pluripotency, as indicated by teratoma formation assay. However, to what extent these iPSCs represent fully reprogrammed PSCs remains controversial, as most livestock iPSCs depend on continuous expression of reprogramming factors. Moreover, germline chimerism has not been robustly demonstrated, with only one successful report with very low efficiency. Therefore, even 34 years after derivation of mouse ESCs and their extensive use in the generation of genetic models, the livestock genetic engineering field can stand to gain enormously from continued investigations into the derivation and application of ESCs and iPSCs.

  14. Interaction between ROCK II and nucleophosmin/B23 in the regulation of centrosome duplication.

    PubMed

    Ma, Zhiyong; Kanai, Masayuki; Kawamura, Kenji; Kaibuchi, Kozo; Ye, Keqiang; Fukasawa, Kenji

    2006-12-01

    Nucleophosmin (NPM)/B23 has been implicated in the regulation of centrosome duplication. NPM/B23 localizes between two centrioles in the unduplicated centrosome. Upon phosphorylation on Thr(199) by cyclin-dependent kinase 2 (CDK2)/cyclin E, the majority of centrosomal NPM/B23 dissociates from centrosomes, but some NPM/B23 phosphorylated on Thr(199) remains at centrosomes. It has been shown that Thr(199) phosphorylation of NPM/B23 is critical for the physical separation of the paired centrioles, an initial event of the centrosome duplication process. Here, we identified ROCK II kinase, an effector of Rho small GTPase, as a protein that localizes to centrosomes and physically interacts with NPM/B23. Expression of the constitutively active form of ROCK II promotes centrosome duplication, while down-regulation of ROCK II expression results in the suppression of centrosome duplication, especially delaying the initiation of centrosome duplication during the cell cycle. Moreover, ROCK II regulates centrosome duplication in its kinase and centrosome localization activity-dependent manner. We further found that ROCK II kinase activity is significantly enhanced by binding to NPM/B23 and that NPM/B23 acquires a higher binding affinity to ROCK II upon phosphorylation on Thr(199). Moreover, physical interaction between ROCK II and NPM/B23 in vivo occurs in association with CDK2/cyclin E activation and the emergence of Thr(199)-phosphorylated NPM/B23. All these findings point to ROCK II as the effector of the CDK2/cyclin E-NPM/B23 pathway in the regulation of centrosome duplication.

  15. Modelling human disease with pluripotent stem cells

    PubMed Central

    Park, In-Hyun; Sullivan, Gareth J.

    2013-01-01

    Recent progress in the field of cellular reprogramming has opened up the doors to a new era of disease modelling, as pluripotent stem cells representing a myriad of genetic diseases can now be produced from patient tissue. These cells can be expanded and differentiated to produce a potentially limitless supply of the affected cell type, which can then be used as a tool to improve understanding of disease mechanisms and test therapeutic interventions. This process requires high levels of scrutiny and validation at every stage, but international standards for the characterisation of pluripotent cells and their progeny have yet to be established. Here we discuss the current state of the art with regard to modelling diseases affecting the ectodermal, mesodermal and endodermal lineages, focussing on studies which have demonstrated a disease phenotype in the tissue of interest. We also discuss the utility of pluripotent cell technology for the modelling of cancer and infectious disease. Finally, we spell out the technical and scientific challenges which must be addressed if the field is to deliver on its potential and produce improved patient outcomes in the clinic. PMID:23444871

  16. SlbHLH068 interacts with FER to regulate the iron-deficiency response in tomato

    PubMed Central

    Du, Juan; Huang, Zongan; Wang, Biao; Sun, Hua; Chen, Chunlin; Ling, Hong-Qing; Wu, Huilan

    2015-01-01

    Background and Aims Iron is an essential micronutrient for all organisms and its uptake, translocation, distribution and utilization are regulated in a complex manner in plants. FER, isolated from tomato (Solanum lycopersicum), was the first transcription factor involved in the iron homeostasis of higher plants to be identified. A FER defect in the T3238fer mutant drastically downregulates the expression of iron uptake genes, such as ferric-chelate reductase 1 (LeFRO1) and iron-regulated transporter 1 (LeIRT1); however, the molecular mechanism by which FER regulates genes downstream remains unknown. The aim of this work was therefore to identify the gene that interacts with FER to regulate the iron-deficiency response in tomato. Methods The homologue of the Arabidopsis Ib subgroup of the basic helix–loop–helix (bHLH) proteins, SlbHLH068, was identified by using the program BLASTP against the AtbHLH39 amino acid sequence in the tomato genome. The interaction between SlbHLH068 and FER was detected using yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. In addition, virus-induced gene silencing (VIGS) was used to generate tomato plants in which SlbHLH068 expression was downregulated. The expression of genes was analysed using northern blot hybridization and multiple RT-PCR analysis. Seedlings of wild-type and mutant plants were grown under conditions of different nutrient deficiency. Key Results SlbHLH068 is highly upregulated in roots, leaves and stems in response to iron deficiency. An interaction between SlbHLH068 and FER was demonstrated using yeast two-hybrid and BiFC assays. The heterodimer formed by FER with SlbHLH068 directly bound to the promoter of LeFRO1 and activated the expression of its reporter gene in the yeast assay. The downregulation of SlbHLH068 expression by VIGS resulted in a reduction of LeFRO1 and LeIRT1 expression and iron accumulation in leaves and roots. Conclusions The results indicate that SlbHLH068, as a

  17. PKC regulates the delta2 glutamate receptor interaction with S-SCAM/MAGI-2 protein.

    PubMed

    Yap, Chan Choo; Muto, Yuko; Kishida, Haruo; Hashikawa, Tsutomu; Yano, Ryoji

    2003-02-21

    Inside cells, membrane proteins are localized at particular surface domains to perform their precise functions. Various kinds of PDZ domain proteins have been shown to play important roles in the intracellular trafficking and anchoring of membrane proteins. In this study, we show that delta2 glutamate receptor is interacting with S-SCAM/MAGI-2, a PDZ domain protein localized in the perinuclear region and postsynaptic sites of cerebellar Purkinje cells. The binding is regulated by PKC (protein kinase-C) mediated phosphorylation of the receptor with a unique repetitive structure in S-SCAM/MAGI-2. Co-expression of both proteins resulted in drastic changes of the receptor localization in COS7 cells. These results show a novel regulatory mechanism for the binding of PDZ domain proteins and suggest that the interaction between delta2 receptor and S-SCAM/MAGI-2 may be important for intracellular trafficking of the receptor.

  18. Learning Biomarkers of Pluripotent Stem Cells in Mouse

    PubMed Central

    Scheubert, Lena; Schmidt, Rainer; Repsilber, Dirk; Luštrek, Mitja; Fuellen, Georg

    2011-01-01

    Pluripotent stem cells are able to self-renew, and to differentiate into all adult cell types. Many studies report data describing these cells, and characterize them in molecular terms. Machine learning yields classifiers that can accurately identify pluripotent stem cells, but there is a lack of studies yielding minimal sets of best biomarkers (genes/features). We assembled gene expression data of pluripotent stem cells and non-pluripotent cells from the mouse. After normalization and filtering, we applied machine learning, classifying samples into pluripotent and non-pluripotent with high cross-validated accuracy. Furthermore, to identify minimal sets of best biomarkers, we used three methods: information gain, random forests and a wrapper of genetic algorithm and support vector machine (GA/SVM). We demonstrate that the GA/SVM biomarkers work best in combination with each other; pathway and enrichment analyses show that they cover the widest variety of processes implicated in pluripotency. The GA/SVM wrapper yields best biomarkers, no matter which classification method is used. The consensus best biomarker based on the three methods is Tet1, implicated in pluripotency just recently. The best biomarker based on the GA/SVM wrapper approach alone is Fam134b, possibly a missing link between pluripotency and some standard surface markers of unknown function processed by the Golgi apparatus. PMID:21791477

  19. ELF3-PIF4 interaction regulates plant growth independently of the Evening Complex.

    PubMed

    Nieto, Cristina; López-Salmerón, Vadir; Davière, Jean-Michel; Prat, Salomé

    2015-01-19

    The circadian clock plays a pivotal role in the control of Arabidopsis hypocotyl elongation by regulating rhythmic expression of the bHLH factors PHYTOCHROME INTERACTING FACTOR 4 and 5 (PIF4 and 5). Coincidence of increased PIF4/PIF5 transcript levels with the dark period allows nuclear accumulation of these factors, and in short days it phases maximal hypocotyl growth at dawn. During early night, PIF4 and PIF5 transcription is repressed by the Evening Complex (EC) proteins EARLY FLOWERING3 (ELF3), EARLY FLOWERING4 (ELF4), and LUX ARRHYTHMO (LUX). While ELF3 has an essential role in EC complex assembly, several lines of evidence indicate that this protein controls plant growth via other mechanisms that are presently unknown. Here, we show that the ELF3 and PIF4 proteins interact in an EC-independent manner, and that this interaction prevents PIF4 from activating its transcriptional targets. We also show that PIF4 overexpression leads to ELF3 protein destabilization, and that this effect is mediated indirectly by negative feedback regulation of photoactive PHYTOCHROME B (phyB). Physical interaction of the phyB photoreceptor with ELF3 has been reported, but its functional relevance remains poorly understood. Our findings establish that phyB is needed for ELF3 accumulation in the light, most likely by competing for CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)-mediated ubiquitination and the proteasomal degradation of ELF3. Our results explain the short hypocotyl phenotype of ELF3 overexpressors, despite their normal clock function, and provide a molecular framework for understanding how warm temperatures promote hypocotyl elongation and affect the endogenous clock.

  20. Syndecan 4 interacts genetically with Vangl2 to regulate neural tube closure and planar cell polarity

    PubMed Central

    Escobedo, Noelia; Contreras, Osvaldo; Muñoz, Rosana; Farías, Marjorie; Carrasco, Héctor; Hill, Charlotte; Tran, Uyen; Pryor, Sophie E.; Wessely, Oliver; Copp, Andrew J.; Larraín, Juan

    2013-01-01

    Syndecan 4 (Sdc4) is a cell-surface heparan sulfate proteoglycan (HSPG) that regulates gastrulation, neural tube closure and directed neural crest migration in Xenopus development. To determine whether Sdc4 participates in Wnt/PCP signaling during mouse development, we evaluated a possible interaction between a null mutation of Sdc4 and the loop-tail allele of Vangl2. Sdc4 is expressed in multiple tissues, but particularly in the non-neural ectoderm, hindgut and otic vesicles. Sdc4;Vangl2Lp compound mutant mice have defective spinal neural tube closure, disrupted orientation of the stereocilia bundles in the cochlea and delayed wound healing, demonstrating a strong genetic interaction. In Xenopus, co-injection of suboptimal amounts of Sdc4 and Vangl2 morpholinos resulted in a significantly greater proportion of embryos with defective neural tube closure than each individual morpholino alone. To probe the mechanism of this interaction, we overexpressed or knocked down Vangl2 function in HEK293 cells. The Sdc4 and Vangl2 proteins colocalize, and Vangl2, particularly the Vangl2Lp mutant form, diminishes Sdc4 protein levels. Conversely, Vangl2 knockdown enhances Sdc4 protein levels. Overall HSPG steady-state levels were regulated by Vangl2, suggesting a molecular mechanism for the genetic interaction in which Vangl2Lp/+ enhances the Sdc4-null phenotype. This could be mediated via heparan sulfate residues, as Vangl2Lp/+ embryos fail to initiate neural tube closure and develop craniorachischisis (usually seen only in Vangl2Lp/Lp) when cultured in the presence of chlorate, a sulfation inhibitor. These results demonstrate that Sdc4 can participate in the Wnt/PCP pathway, unveiling its importance during neural tube closure in mammalian embryos. PMID:23760952

  1. Hepatocyte Factor JMJD5 Regulates Hepatitis B Virus Replication through Interaction with HBx

    PubMed Central

    Kouwaki, Takahisa; Okamoto, Toru; Ito, Ayano; Sugiyama, Yukari; Yamashita, Kazuo; Suzuki, Tatsuya; Kusakabe, Shinji; Hirano, Junki; Fukuhara, Takasuke; Yamashita, Atsuya; Saito, Kazunobu; Okuzaki, Daisuke; Watashi, Koichi; Sugiyama, Masaya; Yoshio, Sachiyo; Standley, Daron M.; Kanto, Tatsuya; Mizokami, Masashi; Moriishi, Kohji

    2016-01-01

    ABSTRACT Hepatitis B virus (HBV) is a causative agent for chronic liver diseases such as hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). HBx protein encoded by the HBV genome plays crucial roles not only in pathogenesis but also in replication of HBV. Although HBx has been shown to bind to a number of host proteins, the molecular mechanisms by which HBx regulates HBV replication are largely unknown. In this study, we identified jumonji C-domain-containing 5 (JMJD5) as a novel binding partner of HBx interacting in the cytoplasm. DNA microarray analysis revealed that JMJD5-knockout (JMJD5KO) Huh7 cells exhibited a significant reduction in the expression of transcriptional factors involved in hepatocyte differentiation, such as HNF4A, CEBPA, and FOXA3. We found that hydroxylase activity of JMJD5 participates in the regulation of these transcriptional factors. Moreover, JMJD5KO Huh7 cells exhibited a severe reduction in HBV replication, and complementation of HBx expression failed to rescue replication of a mutant HBV deficient in HBx, suggesting that JMJD5 participates in HBV replication through an interaction with HBx. We also found that replacing Gly135 with Glu in JMJD5 abrogates binding with HBx and replication of HBV. Moreover, the hydroxylase activity of JMJD5 was crucial for HBV replication. Collectively, these results suggest that direct interaction of JMJD5 with HBx facilitates HBV replication through the hydroxylase activity of JMJD5. IMPORTANCE HBx protein encoded by hepatitis B virus (HBV) plays important roles in pathogenesis and replication of HBV. We identified jumonji C-domain-containing 5 (JMJD5) as a novel binding partner to HBx. JMJD5 was shown to regulate several transcriptional factors to maintain hepatocyte function. Although HBx had been shown to support HBV replication, deficiency of JMJD5 abolished contribution of HBx in HBV replication, suggesting that HBx-mediated HBV replication is largely dependent on JMJD5. We showed that

  2. Interactions of the algicidal bacterium Kordia algicida with diatoms: regulated protease excretion for specific algal lysis.

    PubMed

    Paul, Carsten; Pohnert, Georg

    2011-01-01

    Interactions of planktonic bacteria with primary producers such as diatoms have great impact on plankton population dynamics. Several studies described the detrimental effect of certain bacteria on diatoms but the biochemical nature and the regulation mechanism involved in the production of the active compounds remained often elusive. Here, we investigated the interactions of the algicidal bacterium Kordia algicida with the marine diatoms Skeletonema costatum, Thalassiosira weissflogii, Phaeodactylum tricornutum, and Chaetoceros didymus. Algicidal activity was only observed towards the first three of the tested diatom species while C. didymus proved to be not susceptible. The cell free filtrate and the >30 kDa fraction of stationary K. algicida cultures is fully active, suggesting a secreted algicidal principle. The active supernatant from bacterial cultures exhibited high protease activity and inhibition experiments proved that these enzymes are involved in the observed algicidal action of the bacteria. Protease mediated interactions are not controlled by the presence of the alga but dependent on the cell density of the K. algicida culture. We show that protease release is triggered by cell free bacterial filtrates suggesting a quorum sensing dependent excretion mechanism of the algicidal protein. The K. algicida / algae interactions in the plankton are thus host specific and under the control of previously unidentified factors. PMID:21695044

  3. The PXDLS linear motif regulates circadian rhythmicity through protein–protein interactions

    PubMed Central

    Shalev, Moran; Aviram, Rona; Adamovich, Yaarit; Kraut-Cohen, Judith; Shamia, Tal; Ben-Dor, Shifra; Golik, Marina; Asher, Gad

    2014-01-01

    The circadian core clock circuitry relies on interlocked transcription-translation feedback loops that largely count on multiple protein interactions. The molecular mechanisms implicated in the assembly of these protein complexes are relatively unknown. Our bioinformatics analysis of short linear motifs, implicated in protein interactions, reveals an enrichment of the Pro-X-Asp-Leu-Ser (PXDLS) motif within circadian transcripts. We show that the PXDLS motif can bind to BMAL1/CLOCK and disrupt circadian oscillations in a cell-autonomous manner. Remarkably, the motif is evolutionary conserved in the core clock protein REV-ERBα, and additional proteins implicated in the clock's function (NRIP1, CBP). In this conjuncture, we uncover a novel cross talk between the two principal core clock feedback loops and show that BMAL/CLOCK and REV-ERBα interact and that the PXDLS motif of REV-ERBα participates in their binding. Furthermore, we demonstrate that the PXDLS motifs of NRIP1 and CBP are involved in circadian rhythmicity. Our findings suggest that the PXDLS motif plays an important role in circadian rhythmicity through regulation of protein interactions within the clock circuitry and that short linear motifs can be employed to modulate circadian oscillations. PMID:25260595

  4. A bipartite interaction between Hsp70 and CHIP regulates ubiquitination of chaperoned client proteins

    PubMed Central

    Zhang, Huaqun; Amick, Joseph; Chakravarti, Ritu; Santarriaga, Stephanie; Schlanger, Simon; McGlone, Cameron; Dare, Michelle; Nix, Jay C.; Scaglione, K. Matthew; Stuehr, Dennis J.; Misra, Saurav; Page, Richard C.

    2015-01-01

    Summary The ubiquitin ligase CHIP plays an important role in cytosolic protein quality control by ubiquitinating proteins chaperoned by Hsp70/Hsc70 and Hsp90, thereby targeting such substrate proteins for degradation. We present a 2.91 Å resolution structure of the TPR domain of CHIP in complex with the α-helical “lid” subdomain and unstructured “tail” of Hsc70. Surprisingly, the CHIP-TPR interacts with determinants within both the Hsc70-lid subdomain and the C-terminal PTIEEVD motif of the tail, exhibiting a novel mode of interaction between chaperones and TPR domains. We demonstrate that the interaction between CHIP and the Hsc70-lid subdomain is required for proper ubiquitination of Hsp70/Hsc70 or Hsp70/Hsc70-bound substrate proteins. Post-translational modifications of the Hsc70 lid and tail disrupt key contacts with the CHIP-TPR and may regulate CHIP-mediated ubiquitination. Our study shows how CHIP docks onto Hsp70/Hsc70 and defines a new bipartite mode of interaction between TPR domains and their binding partners. PMID:25684577

  5. Prohibitin/annexin 2 interaction regulates fatty acid transport in adipose tissue

    PubMed Central

    Salameh, Ahmad; Daquinag, Alexes C.; Staquicini, Daniela I.; An, Zhiqiang; Pasqualini, Renata; Kolonin, Mikhail G.

    2016-01-01

    We have previously identified prohibitin (PHB) and annexin A2 (ANX2) as proteins interacting on the surface of vascular endothelial cells in white adipose tissue (WAT) of humans and mice. Here, we demonstrate that ANX2 and PHB also interact in adipocytes. Mice lacking ANX2 have normal WAT vascularization, adipogenesis, and glucose metabolism but display WAT hypotrophy due to reduced fatty acid uptake by WAT endothelium and adipocytes. By using cell culture systems in which ANX2/PHB binding is disrupted either genetically or through treatment with a blocking peptide, we show that fatty acid transport efficiency relies on this protein complex. We also provide evidence that the interaction between ANX2 and PHB mediates fatty acid transport from the endothelium into adipocytes. Moreover, we demonstrate that ANX2 and PHB form a complex with the fatty acid transporter CD36. Finally, we show that the colocalization of PHB and CD36 on adipocyte surface is induced by extracellular fatty acids. Together, our results suggest that an unrecognized biochemical interaction between ANX2 and PHB regulates CD36-mediated fatty acid transport in WAT, thus revealing a new potential pathway for intervention in metabolic diseases. PMID:27468426

  6. Interactions of the Algicidal Bacterium Kordia algicida with Diatoms: Regulated Protease Excretion for Specific Algal Lysis

    PubMed Central

    Paul, Carsten; Pohnert, Georg

    2011-01-01

    Interactions of planktonic bacteria with primary producers such as diatoms have great impact on plankton population dynamics. Several studies described the detrimental effect of certain bacteria on diatoms but the biochemical nature and the regulation mechanism involved in the production of the active compounds remained often elusive. Here, we investigated the interactions of the algicidal bacterium Kordia algicida with the marine diatoms Skeletonema costatum, Thalassiosira weissflogii, Phaeodactylum tricornutum, and Chaetoceros didymus. Algicidal activity was only observed towards the first three of the tested diatom species while C. didymus proved to be not susceptible. The cell free filtrate and the >30 kDa fraction of stationary K. algicida cultures is fully active, suggesting a secreted algicidal principle. The active supernatant from bacterial cultures exhibited high protease activity and inhibition experiments proved that these enzymes are involved in the observed algicidal action of the bacteria. Protease mediated interactions are not controlled by the presence of the alga but dependent on the cell density of the K. algicida culture. We show that protease release is triggered by cell free bacterial filtrates suggesting a quorum sensing dependent excretion mechanism of the algicidal protein. The K. algicida / algae interactions in the plankton are thus host specific and under the control of previously unidentified factors. PMID:21695044

  7. Non-coding RNA interact to regulate neuronal development and function

    PubMed Central

    Iyengar, Bharat R.; Choudhary, Ashwani; Sarangdhar, Mayuresh A.; Venkatesh, K. V.; Gadgil, Chetan J.; Pillai, Beena

    2014-01-01

    The human brain is one of the most complex biological systems, and the cognitive abilities have greatly expanded compared to invertebrates without much expansion in the number of protein coding genes. This suggests that gene regulation plays a very important role in the development and function of nervous system, by acting at multiple levels such as transcription and translation. In this article we discuss the regulatory roles of three classes of non-protein coding RNAs (ncRNAs)—microRNAs (miRNAs), piwi-interacting RNA (piRNAs) and long-non-coding RNA (lncRNA), in the process of neurogenesis and nervous function including control of synaptic plasticity and potential roles in neurodegenerative diseases. miRNAs are involved in diverse processes including neurogenesis where they channelize the cellular physiology toward neuronal differentiation. miRNAs can also indirectly influence neurogenesis by regulating the proliferation and self renewal of neural stem cells and are dysregulated in several neurodegenerative diseases. miRNAs are also known to regulate synaptic plasticity and are usually found to be co-expressed with their targets. The dynamics of gene regulation is thus dependent on the local architecture of the gene regulatory network (GRN) around the miRNA and its targets. piRNAs had been classically known to regulate transposons in the germ cells. However, piRNAs have been, recently, found to be expressed in the brain and possibly function by imparting epigenetic changes by DNA methylation. piRNAs are known to be maternally inherited and we assume that they may play a role in early development. We also explore the possible function of piRNAs in regulating the expansion of transposons in the brain. Brain is known to express several lncRNA but functional roles in brain development are attributed to a few lncRNA while functions of most of the them remain unknown. We review the roles of some known lncRNA and explore the other possible functions of lnc

  8. DNA Repair in Human Pluripotent Stem Cells Is Distinct from That in Non-Pluripotent Human Cells

    PubMed Central

    Luo, Li Z.; Park, Sang-Won; Bates, Steven E.; Zeng, Xianmin; Iverson, Linda E.; O'Connor, Timothy R.

    2012-01-01

    The potential for human disease treatment using human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells (iPSCs), also carries the risk of added genomic instability. Genomic instability is most often linked to DNA repair deficiencies, which indicates that screening/characterization of possible repair deficiencies in pluripotent human stem cells should be a necessary step prior to their clinical and research use. In this study, a comparison of DNA repair pathways in pluripotent cells, as compared to those in non-pluripotent cells, demonstrated that DNA repair capacities of pluripotent cell lines were more heterogeneous than those of differentiated lines examined and were generally greater. Although pluripotent cells had high DNA repair capacities for nucleotide excision repair, we show that ultraviolet radiation at low fluxes induced an apoptotic response in these cells, while differentiated cells lacked response to this stimulus, and note that pluripotent cells had a similar apoptotic response to alkylating agent damage. This sensitivity of pluripotent cells to damage is notable since viable pluripotent cells exhibit less ultraviolet light-induced DNA damage than do differentiated cells that receive the same flux. In addition, the importance of screening pluripotent cells for DNA repair defects was highlighted by an iPSC line that demonstrated a normal spectral karyotype, but showed both microsatellite instability and reduced DNA repair capacities in three out of four DNA repair pathways examined. Together, these results demonstrate a need to evaluate DNA repair capacities in pluripotent cell lines, in order to characterize their genomic stability, prior to their pre-clinical and clinical use. PMID:22412831

  9. Manganese Superoxide Dismutase Gene Expression Is Induced by Nanog and Oct4, Essential Pluripotent Stem Cells’ Transcription Factors

    PubMed Central

    Solari, Claudia; Vázquez Echegaray, Camila; Cosentino, María Soledad; Petrone, María Victoria; Waisman, Ariel; Luzzani, Carlos; Francia, Marcos; Villodre, Emilly; Lenz, Guido; Miriuka, Santiago; Barañao, Lino; Guberman, Alejandra

    2015-01-01

    Pluripotent stem cells possess complex systems that protect them from oxidative stress and ensure genomic stability, vital for their role in development. Even though it has been reported that antioxidant activity diminishes along stem cell differentiation, little is known about the transcriptional regulation of the involved genes. The reported modulation of some of these genes led us to hypothesize that some of them could be regulated by the transcription factors critical for self-renewal and pluripotency in embryonic stem cells (ESCs) and in induced pluripotent stem cells (iPSCs). In this work, we studied the expression profile of multiple genes involved in antioxidant defense systems in both ESCs and iPSCs. We found that Manganese superoxide dismutase gene (Mn-Sod/Sod2) was repressed during diverse differentiation protocols showing an expression pattern similar to Nanog gene. Moreover, Sod2 promoter activity was induced by Oct4 and Nanog when we performed a transactivation assay using two different reporter constructions. Finally, we studied Sod2 gene regulation by modulating the expression of Oct4 and Nanog in ESCs by shRNAs and found that downregulation of any of them reduced Sod2 expression. Our results indicate that pluripotency transcription factors positively modulate Sod2 gene transcription. PMID:26642061

  10. Manganese Superoxide Dismutase Gene Expression Is Induced by Nanog and Oct4, Essential Pluripotent Stem Cells' Transcription Factors.

    PubMed

    Solari, Claudia; Vázquez Echegaray, Camila; Cosentino, María Soledad; Petrone, María Victoria; Waisman, Ariel; Luzzani, Carlos; Francia, Marcos; Villodre, Emilly; Lenz, Guido; Miriuka, Santiago; Barañao, Lino; Guberman, Alejandra

    2015-01-01

    Pluripotent stem cells possess complex systems that protect them from oxidative stress and ensure genomic stability, vital for their role in development. Even though it has been reported that antioxidant activity diminishes along stem cell differentiation, little is known about the transcriptional regulation of the involved genes. The reported modulation of some of these genes led us to hypothesize that some of them could be regulated by the transcription factors critical for self-renewal and pluripotency in embryonic stem cells (ESCs) and in induced pluripotent stem cells (iPSCs). In this work, we studied the expression profile of multiple genes involved in antioxidant defense systems in both ESCs and iPSCs. We found that Manganese superoxide dismutase gene (Mn-Sod/Sod2) was repressed during diverse differentiation protocols showing an expression pattern similar to Nanog gene. Moreover, Sod2 promoter activity was induced by Oct4 and Nanog when we performed a transactivation assay using two different reporter constructions. Finally, we studied Sod2 gene regulation by modulating the expression of Oct4 and Nanog in ESCs by shRNAs and found that downregulation of any of them reduced Sod2 expression. Our results indicate that pluripotency transcription factors positively modulate Sod2 gene transcription. PMID:26642061

  11. Manganese Superoxide Dismutase Gene Expression Is Induced by Nanog and Oct4, Essential Pluripotent Stem Cells' Transcription Factors.

    PubMed

    Solari, Claudia; Vázquez Echegaray, Camila; Cosentino, María Soledad; Petrone, María Victoria; Waisman, Ariel; Luzzani, Carlos; Francia, Marcos; Villodre, Emilly; Lenz, Guido; Miriuka, Santiago; Barañao, Lino; Guberman, Alejandra

    2015-01-01

    Pluripotent stem cells possess complex systems that protect them from oxidative stress and ensure genomic stability, vital for their role in development. Even though it has been reported that antioxidant activity diminishes along stem cell differentiation, little is known about the transcriptional regulation of the involved genes. The reported modulation of some of these genes led us to hypothesize that some of them could be regulated by the transcription factors critical for self-renewal and pluripotency in embryonic stem cells (ESCs) and in induced pluripotent stem cells (iPSCs). In this work, we studied the expression profile of multiple genes involved in antioxidant defense systems in both ESCs and iPSCs. We found that Manganese superoxide dismutase gene (Mn-Sod/Sod2) was repressed during diverse differentiation protocols showing an expression pattern similar to Nanog gene. Moreover, Sod2 promoter activity was induced by Oct4 and Nanog when we performed a transactivation assay using two different reporter constructions. Finally, we studied Sod2 gene regulation by modulating the expression of Oct4 and Nanog in ESCs by shRNAs and found that downregulation of any of them reduced Sod2 expression. Our results indicate that pluripotency transcription factors positively modulate Sod2 gene transcription.

  12. Characterization of megakaryocyte GATA1-interacting proteins: the corepressor ETO2 and GATA1 interact to regulate terminal megakaryocyte maturation.

    PubMed

    Hamlett, Isla; Draper, Julia; Strouboulis, John; Iborra, Francisco; Porcher, Catherine; Vyas, Paresh

    2008-10-01

    The transcription factor GATA1 coordinates timely activation and repression of megakaryocyte gene expression. Loss of GATA1 function results in excessive megakaryocyte proliferation and disordered terminal platelet maturation, leading to thrombocytopenia and leukemia in patients. The mechanisms by which GATA1 does this are unclear. We have used in vivo biotinylated GATA1 to isolate megakaryocyte GATA1-partner proteins. Here, several independent approaches show that GATA1 interacts with several proteins in the megakaryocyte cell line L8057 and in primary megakaryocytes. They include FOG1, the NURD complex, the pentameric complex containing SCL/TAL-1, the zinc-finger regulators GFI1B and ZFP143, and the corepressor ETO2. Knockdown of ETO2 expression promotes megakaryocyte differentiation and enhances expression of select genes expressed in terminal megakaryocyte maturation, eg, platelet factor 4 (Pf4). ETO2-dependent direct repression of the Pf4 proximal promoter is mediated by GATA-binding sites and an E-Box motif. Consistent with this, endogenous ETO2, GATA1, and the SCL pentameric complex all specifically bind the promoter in vivo. Finally, as ETO2 expression is restricted to immature megakaryocytes, these data suggest that ETO2 directly represses inappropriate early expression of a subset of terminally expressed megakaryocyte genes by binding to GATA1 and SCL. PMID:18625887

  13. Fluorescent reversible regulation based on the interactions of topotecan hydrochloride, neutral red and quantum dots

    NASA Astrophysics Data System (ADS)

    Wang, Linlin; Shen, Yizhong; Liu, Shaopu; Yang, Jidong; Liang, Wanjun; Li, Dan; He, Youqiu

    2015-02-01

    The interactions of topotecan hydrochloride (THC), neutral red (NR) and thioglycolic acid (TGA) capped CdTe/CdS quantum dots (QDs) built a solid base for the controlling of the fluorescent reversible regulation of the system. This study was developed by means of ultraviolet-visible (UV-vis) absorption, fluorescence (FL), resonance Rayleigh scattering (RRS) spectroscopy and transmission electron microscopy (TEM). Corresponding experimental results revealed that the fluorescence of TGA-CdTe/CdS QDs could be effectively quenched by NR, while the RRS of the QDs enhanced gradually with the each increment of NR concentration. After the addition of THC, the strong covalent conjugation between NR and THC which was in carboxylate state enabled NR to be dissociated from the surface of TGA-CdTe/CdS QDs to form more stable complex with THC, thereby enhancing the fluorescence of the TGA-CdTe/CdS QDs-NR system. What is more, through analyzing the optical properties and experimental data of the reaction between TGA-CdTe/CdS QDs and NR, the possible reaction mechanism of the whole system was discussed. This combination of multiple spectroscopic techniques could contribute to the investigation for the fluorescent reversible regulation of QDs and a method could also be established to research the interactions between camptothecin drugs and dyes.

  14. Arabidopsis MYC2 Interacts with DELLA Proteins in Regulating Sesquiterpene Synthase Gene Expression[W][OA

    PubMed Central

    Hong, Gao-Jie; Xue, Xue-Yi; Mao, Ying-Bo; Wang, Ling-Jian; Chen, Xiao-Ya

    2012-01-01

    Arabidopsis thaliana flowers emit volatile terpenes, which may function in plant–insect interactions. Here, we report that Arabidopsis MYC2, a basic helix-loop-helix transcription factor, directly binds to promoters of the sesquiterpene synthase genes TPS21 and TPS11 and activates their expression. Expression of TPS21 and TPS11 can be induced by the phytohormones gibberellin (GA) and jasmonate (JA), and both inductions require MYC2. The induction of TPS21 and TPS11 results in increased emission of sesquiterpene, especially (E)-β-caryophyllene. DELLAs, the GA signaling repressors, negatively affect sesquiterpene biosynthesis, as the sesquiterpene synthase genes were repressed in plants overaccumulating REPRESSOR OF GA1-3 (RGA), one of the Arabidopsis DELLAs, and upregulated in a penta DELLA-deficient mutant. Yeast two-hybrid and coimmunoprecipitation assays demonstrated that DELLAs, represented by RGA, directly interact with MYC2. In yeast cells, the N terminus of MYC2 was responsible for binding to RGA. MYC2 has been proposed as a major mediator of JA signaling and crosstalk with abscisic acid, ethylene, and light signaling pathways. Our results demonstrate that MYC2 is also connected to GA signaling in regulating a subset of genes. In Arabidopsis inflorescences, it integrates both GA and JA signals into transcriptional regulation of sesquiterpene synthase genes and promotes sesquiterpene production. PMID:22669881

  15. Axon-to-Glia Interaction Regulates GABAA Receptor Expression in Oligodendrocytes.

    PubMed

    Arellano, Rogelio O; Sánchez-Gómez, María Victoria; Alberdi, Elena; Canedo-Antelo, Manuel; Chara, Juan Carlos; Palomino, Aitor; Pérez-Samartín, Alberto; Matute, Carlos

    2016-01-01

    Myelination requires oligodendrocyte-neuron communication, and both neurotransmitters and contact interactions are essential for this process. Oligodendrocytes are endowed with neurotransmitter receptors whose expression levels and properties may change during myelination. However, only scant information is available about the extent and timing of these changes or how they are regulated by oligodendrocyte-neuron interactions. Here, we used electrophysiology to study the expression of ionotropic GABA, glutamate, and ATP receptors in oligodendrocytes derived from the optic nerve and forebrain cultured either alone or in the presence of dorsal root ganglion neurons. We observed that oligodendrocytes from both regions responded to these transmitters at 1 day in culture. After the first day in culture, however, GABA sensitivity diminished drastically to less than 10%, while that of glutamate and ATP remained constant. In contrast, the GABA response amplitude was sustained and remained stable in oligodendrocytes cocultured with dorsal root ganglion neurons. Immunochemistry and pharmacological properties of the responses indicated that they were mediated by distinctive GABAA receptors and that in coculture with neurons, the oligodendrocytes bearing the receptors were those in direct contact with axons. These results reveal that GABAA receptor regulation in oligodendrocytes is driven by axonal cues and that GABA signaling may play a role in myelination and/or during axon-glia recognition.

  16. Functional interactions between the Moses corepressor and DHR78 nuclear receptor regulate growth in Drosophila.

    PubMed

    Baker, Keith D; Beckstead, Robert B; Mangelsdorf, David J; Thummel, Carl S

    2007-02-15

    Expression of the Drosophila orphan nuclear receptor DHR78 is regulated by the steroid hormone ecdysone and is required for growth and viability during larval stages. In contrast to our understanding of its biological functions, however, relatively little is known about how DHR78 acts as a transcription factor. Here we show that DHR78 is an obligate partner for Moses (Middleman of seventy-eight signaling), a SAM (sterile alpha motif) domain-containing cofactor that requires DHR78 for its stability. Unlike other nuclear receptor cofactors, Moses has no obvious interaction domains and displays a unique binding specificity for DHR78. Moses acts as a corepressor, inhibiting DHR78 transcriptional activity independently of histone deacetylation. Consistent with their close association, DHR78 and Moses proteins are coexpressed during development and colocalize to specific genomic targets in chromatin. Moses mutants progress normally through early larval stages, like DHR78 mutants, but display an opposite overgrowth phenotype, with hypertrophy of adult tissues. Genetic interactions between DHR78 and moses result in a similar phenotype, suggesting that the relative dose of Moses and DHR78 regulates growth and prevents cancer. The tight functional association between DHR78 and Moses provides a new paradigm for understanding the molecular mechanisms by which cofactors modulate nuclear receptor signaling pathways. PMID:17322404

  17. PAK–PIX interactions regulate adhesion dynamics and membrane protrusion to control neurite outgrowth

    PubMed Central

    Santiago-Medina, Miguel; Gregus, Kelly A.; Gomez, Timothy M.

    2013-01-01

    Summary The roles of P21-activated kinase (PAK) in the regulation of axon outgrowth downstream of extracellular matrix (ECM) proteins are poorly understood. Here we show that PAK1–3 and PIX are expressed in the developing spinal cord and differentially localize to point contacts and filopodial tips within motile growth cones. Using a specific interfering peptide called PAK18, we found that axon outgrowth is robustly stimulated on laminin by partial inhibition of PAK–PIX interactions and PAK function, whereas complete inhibition of PAK function stalls axon outgrowth. Furthermore, modest inhibition of PAK–PIX stimulates the assembly and turnover of growth cone point contacts, whereas strong inhibition over-stabilizes adhesions. Point mutations within PAK confirm the importance of PIX binding. Together our data suggest that regulation of PAK–PIX interactions in growth cones controls neurite outgrowth by influencing the activity of several important mediators of actin filament polymerization and retrograde flow, as well as integrin-dependent adhesion to laminin. PMID:23321640

  18. Antibody probe study of Ca2+ channel regulation by interdomain interaction within the ryanodine receptor.

    PubMed Central

    Kobayashi, Shigeki; Yamamoto, Takeshi; Parness, Jerome; Ikemoto, Noriaki

    2004-01-01

    N-terminal and central domains of ryanodine receptor 1 (RyR1), where many reported malignant hyperthermia (MH) mutations are localized, represent putative channel regulatory domains. Recent domain peptide (DP) probe studies led us to the hypothesis that these domains interact to stabilize the closed state of channel (zipping), while weakening of domain-domain interactions (unzipping) by mutation de-stabilizes the channel, making it leaky to Ca2+ or sensitive to the agonists of RyR1. As shown previously, DP1 (N-terminal domain peptide) and DP4 (central domain peptide) produced MH-like channel activation/sensitization effects, presumably by peptide binding to sites critical to stabilizing domain-domain interactions and resultant loss of conformational constraints. Here we report that polyclonal anti-DP1 and anti-DP4 antibodies also produce MH-like channel activation and sensitization effects as evidenced by about 4-fold enhancement of high affinity [3H]ryanodine binding to RyR1 and by a significant left-shift of the concentration-dependence of activation of sarcoplasmic reticulum Ca2+ release by polylysine. Fluorescence quenching experiments demonstrate that the accessibility of a DP4-directed, conformationally sensitive fluorescence probe linked to the RyR1 N-terminal domain is increased in the presence of domain-specific antibodies, consistent with the view that these antibodies produce unzipping of interacting domains that are of hindered accessibility to the surrounding aqueous environment. Our results suggest that domain-specific antibody binding induces a conformational change resulting in channel activation, and are consistent with the hypothesis that interacting N-terminal and central domains are intimately involved in the regulation of RyR1 channel function. PMID:15027895

  19. Quorum-sensing regulation in rhizobia and its role in symbiotic interactions with legumes.

    PubMed

    Sanchez-Contreras, Maria; Bauer, Wolfgang D; Gao, Mengsheng; Robinson, Jayne B; Allan Downie, J

    2007-07-29

    Legume-nodulating bacteria (rhizobia) usually produce N-acyl homoserine lactones, which regulate the induction of gene expression in a quorum-sensing (or population-density)-dependent manner. There is significant diversity in the types of quorum-sensing regulatory systems that are present in different rhizobia and no two independent isolates worked on in detail have the same complement of quorum-sensing genes. The genes regulated by quorum sensing appear to be rather diverse and many are associated with adaptive aspects of physiology that are probably important in the rhizosphere. It is evident that some aspects of rhizobial physiology related to the interaction between rhizobia and legumes are influenced by quorum sensing. However, it also appears that the legumes play an active role, both in terms of interfering with the rhizobial quorum-sensing systems and responding to the signalling molecules made by the bacteria. In this article, we review the diversity of quorum-sensing regulation in rhizobia and the potential role of legumes in influencing and responding to this signalling system.

  20. Roles of plant hormones in the regulation of host-virus interactions.

    PubMed

    Alazem, Mazen; Lin, Na-Sheng

    2015-06-01

    Hormones are tuners of plant responses to biotic and abiotic stresses. They are involved in various complicated networks, through which they modulate responses to different stimuli. Four hormones primarily regulate plant defence to pathogens: salicylic acid (SA), jasmonic acid (JA), ethylene (Et) and abscisic acid (ABA). In susceptible plants, viral infections result in hormonal disruption, which manifests as the simultaneous induction of several antagonistic hormones. However, these antagonistic hormones may exhibit some sequential accumulation in resistant lines. Virus propagation is usually restricted by the activation of the small interfering RNA (siRNA) antiviral machinery and/or SA signalling pathway. Several studies have investigated these two systems, using different model viruses. However, the roles of hormones other than SA, especially those with antagonistic properties, such as ABA, have been neglected. Increasing evidence indicates that hormones control components of the small RNA system, which regulates many processes (including the siRNA antiviral machinery and the microRNA system) at the transcriptional or post-transcriptional level. Consequently, cross-talk between the antagonistic SA and ABA pathways modulates plant responses at multiple levels. In this review, we summarize recent findings on the different roles of hormones in the regulation of plant-virus interactions, which are helping us to elucidate the fine tuning of viral and plant systems by hormones.

  1. Plant microRNAs: key regulators of root architecture and biotic interactions.

    PubMed

    Couzigou, Jean-Malo; Combier, Jean-Philippe

    2016-10-01

    Contents 22 I. 22 II. 24 III. 25 IV. 27 V. 29 VI. 10 31 References 32 SUMMARY: Plants have evolved a remarkable faculty of adaptation to deal with various and changing environmental conditions. In this context, the roots have taken over nutritional aspects and the root system architecture can be modulated in response to nutrient availability or biotic interactions with soil microorganisms. This adaptability requires a fine tuning of gene expression. Indeed, root specification and development are highly complex processes requiring gene regulatory networks involved in hormonal regulations and cell identity. Among the different molecular partners governing root development, microRNAs (miRNAs) are key players for the fast regulation of gene expression. miRNAs are small RNAs involved in most developmental processes and are required for the normal growth of organisms, by the negative regulation of key genes, such as transcription factors and hormone receptors. Here, we review the known roles of miRNAs in root specification and development, from the embryonic roots to the establishment of root symbioses, highlighting the major roles of miRNAs in these processes. PMID:27292927

  2. Endophilin A1 regulates dendritic spine morphogenesis and stability through interaction with p140Cap

    PubMed Central

    Yang, Yanrui; Wei, Mengping; Xiong, Ying; Du, Xiangyang; Zhu, Shaoxia; Yang, Lin; Zhang, Chen; Liu, Jia-Jia

    2015-01-01

    Dendritic spines are actin-rich membrane protrusions that are the major sites of excitatory synaptic input in the mammalian brain, and their morphological plasticity provides structural basis for learning and memory. Here we report that endophilin A1, with a well-established role in clathrin-mediated synaptic vesicle endocytosis at the presynaptic terminal, also localizes to dendritic spines and is required for spine morphogenesis, synapse formation and synaptic function. We identify p140Cap, a regulator of cytoskeleton reorganization, as a downstream effector of endophilin A1 and demonstrate that disruption of their interaction impairs spine formation and maturation. Moreover, we demonstrate that knockdown of endophilin A1 or p140Cap impairs spine stabilization and synaptic function. We further show that endophilin A1 regulates the distribution of p140Cap and its downstream effector, the F-actin-binding protein cortactin as well as F-actin enrichment in dendritic spines. Together, these results reveal a novel function of postsynaptic endophilin A1 in spine morphogenesis, stabilization and synaptic function through the regulation of p140Cap. PMID:25771685

  3. FLCN, a novel autophagy component, interacts with GABARAP and is regulated by ULK1 phosphorylation.

    PubMed

    Dunlop, Elaine A; Seifan, Sara; Claessens, Tijs; Behrends, Christian; Kamps, Miriam Af; Rozycka, Ewelina; Kemp, Alain J; Nookala, Ravi K; Blenis, John; Coull, Barry J; Murray, James T; van Steensel, Maurice Am; Wilkinson, Simon; Tee, Andrew R

    2014-10-01

    Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterized by benign hair follicle tumors, pneumothorax, and renal cancer. Folliculin (FLCN), the protein product of the FLCN gene, is a poorly characterized tumor suppressor protein, currently linked to multiple cellular pathways. Autophagy maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase ULK1 drives autophagy, the underlying mechanisms are still being unraveled and few ULK1 substrates have been identified to date. Here, we identify that loss of FLCN moderately impairs basal autophagic flux, while re-expression of FLCN rescues autophagy. We reveal that the FLCN complex is regulated by ULK1 and elucidate 3 novel phosphorylation sites (Ser406, Ser537, and Ser542) within FLCN, which are induced by ULK1 overexpression. In addition, our findings demonstrate that FLCN interacts with a second integral component of the autophagy machinery, GABA(A) receptor-associated protein (GABARAP). The FLCN-GABARAP association is modulated by the presence of either folliculin-interacting protein (FNIP)-1 or FNIP2 and further regulated by ULK1. As observed by elevation of GABARAP, sequestome 1 (SQSTM1) and microtubule-associated protein 1 light chain 3 (MAP1LC3B) in chromophobe and clear cell tumors from a BHD patient, we found that autophagy is impaired in BHD-associated renal tumors. Consequently, this work reveals a novel facet of autophagy regulation by ULK1 and substantially contributes to our understanding of FLCN function by linking it directly to autophagy through GABARAP and ULK1.

  4. MicroRNA-Regulated Protein-Protein Interaction Networks and Their Functions in Breast Cancer

    PubMed Central

    Lee, Chia-Hsien; Kuo, Wen-Hong; Lin, Chen-Ching; Oyang, Yen-Jen; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

    2013-01-01

    MicroRNAs, which are small endogenous RNA regulators, have been associated with various types of cancer. Breast cancer is a major health threat for women worldwide. Many miRNAs were reported to be associated with the progression and carcinogenesis of breast cancer. In this study, we aimed to discover novel breast cancer-related miRNAs and to elucidate their functions. First, we identified confident miRNA-target pairs by combining data from miRNA target prediction databases and expression profiles of miRNA and mRNA. Then, miRNA-regulated protein interaction networks (PINs) were constructed with confident pairs and known interaction data in the human protein reference database (HPRD). Finally, the functions of miRNA-regulated PINs were elucidated by functional enrichment analysis. From the results, we identified some previously reported breast cancer-related miRNAs and functions of the PINs, e.g., miR-125b, miR-125a, miR-21, and miR-497. Some novel miRNAs without known association to breast cancer were also found, and the putative functions of their PINs were also elucidated. These include miR-139 and miR-383. Furthermore, we validated our results by receiver operating characteristic (ROC) curve analysis using our miRNA expression profile data, gene expression-based outcome for breast cancer online (GOBO) survival analysis, and a literature search. Our results may provide new insights for research in breast cancer-associated miRNAs. PMID:23722663

  5. Notch1 Regulates Hippocampal Plasticity Through Interaction with the Reelin Pathway, Glutamatergic Transmission and CREB Signaling

    PubMed Central

    Brai, Emanuele; Marathe, Swananda; Astori, Simone; Fredj, Naila Ben; Perry, Elisabeth; Lamy, Christophe; Scotti, Alessandra; Alberi, Lavinia

    2015-01-01

    Notch signaling plays a crucial role in adult brain function such as synaptic plasticity, memory and olfaction. Several reports suggest an involvement of this pathway in neurodegenerative dementia. Yet, to date, the mechanism underlying Notch activity in mature neurons remains unresolved. In this work, we investigate how Notch regulates synaptic potentiation and contributes to the establishment of memory in mice. We observe that Notch1 is a postsynaptic receptor with functional interactions with the Reelin receptor, apolipoprotein E receptor 2 (ApoER2) and the ionotropic receptor, N-methyl-D-aspartate receptor (NMDAR). Targeted loss of Notch1 in the hippocampal CA fields affects Reelin signaling by influencing Dab1 expression and impairs the synaptic potentiation achieved through Reelin stimulation. Further analysis indicates that loss of Notch1 affects the expression and composition of the NMDAR but not AMPAR. Glutamatergic signaling is further compromised through downregulation of CamKII and its secondary and tertiary messengers resulting in reduced cAMP response element-binding (CREB) signaling. Our results identify Notch1 as an important regulator of mechanisms involved in synaptic plasticity and memory formation. These findings emphasize the possible involvement of this signaling receptor in dementia. Highlights In this paper, we propose a mechanism for Notch1-dependent plasticity that likely underlies the function of Notch1 in memory formation: Notch1 interacts with another important developmental pathway, the Reelin cascade. Notch1 regulates both NMDAR expression and composition. Notch1 influences a cascade of cellular events culminating in CREB activation. PMID:26635527

  6. N6-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions

    PubMed Central

    Liu, Nian; Dai, Qing; Zheng, Guanqun; He, Chuan; Parisien, Marc; Pan, Tao

    2015-01-01

    RNA-binding proteins control many aspects of cellular biology through binding single-stranded RNA binding motifs (RBM)1-3. However, RBMs can be buried within their local RNA structures4-7, thus inhibiting RNA-protein interactions. N6-methyladenosine (m6A), the most abundant and dynamic internal modification in eukaryotic messenger RNA8-19, can be selectively recognized by the YTHDF2 protein to affect the stability of cytoplasmic mRNAs15, but how m6A achieves wide-ranging physiological significance needs further exploration. Here we show that m6A controls the RNA-structure-dependent accessibility of RBMs to affect RNA-protein interactions for biological regulation; we term this mechanism “m6A-switch”. We found that m6A alters the local structure in mRNA and long non-coding RNA (lncRNA) to facilitate binding of heterogeneous nuclear ribonucleoprotein C (hnRNP C), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing20-24. Combining PAR-CLIP and m6A/MeRIP approaches enabled us to identify 39,060 m6A-switches among hnRNP C binding sites; and global m6A reduction decreased hnRNP C binding at 2,798 high confidence m6A-switches. We determined that these m6A-switch-regulated hnRNP C binding activities affect the abundance as well as alternative splicing of target mRNAs, demonstrating the regulatory role of m6A-switches on gene expression and RNA maturation. Our results illustrate how RNA-binding proteins gain regulated access to their RBMs through m6A-dependent RNA structural remodeling, and provide a new direction for investigating RNA-modification-coded cellular biology. PMID:25719671

  7. Coupling of X-chromosome reactivation with the pluripotent stem cell state.

    PubMed

    Payer, Bernhard; Lee, Jeannie T

    2014-01-01

    X-chromosome inactivation (XCI) in female mammals is a dramatic example of epigenetic gene regulation, which entails the silencing of an entire chromosome through a wide range of mechanisms involving noncoding RNAs, chromatin-modifications, and DNA-methylation. While XCI is associated with the differentiated cell state, it is reversed by X-chromosome reactivation (XCR) ex vivo in pluripotent stem cells and in vivo in the early mouse embryo and the germline. Critical in the regulation of XCI vs. XCR is the X-inactivation center, a multigene locus on the X-chromosome harboring several long noncoding RNA genes including, most prominently, Xist and Tsix. These genes, which sit at the top of the XCI hierarchy, are by themselves controlled by pluripotency factors, coupling XCR with the naïve pluripotent stem cell state. In this point-of-view article we review the latest findings regarding this intricate relationship between cell differentiation state and epigenetic control of the X-chromosome. In particular, we discuss the emerging picture of complex multifactorial regulatory mechanisms, ensuring both a fine-tuned and robust X-reactivation process.

  8. Analysis of the Mitochondrial DNA and Its Replicative Capacity in Induced Pluripotent Stem Cells.

    PubMed

    Cagnone, Gael; Vaghjiani, Vijesh; Lee, William; Sun, Claire; Johnson, Jacqueline; Yeung, Ka-Yu; St John, Justin C

    2016-01-01

    The mitochondrial genome resides in the mitochondrion of nearly all mammalian cells. It is important for energy production as it encodes 13 of the key subunits of the electron transfer chain, which generates the vast majority of cellular ATP through the process of oxidative phosphorylation. As cells establish pluripotency, they regulate their mtDNA copy number so that they possess few copies but sufficient that they can be replicated to match the differentiated cell-specific requirements for ATP derived through oxidative phosphorylation. However, the failure to strictly regulate this process prevents pluripotent cells from differentiating. We describe a series of protocols that analyze mtDNA copy number, DNA methylation within the nuclear-encoded mtDNA-specific polymerase, and gene expression of the other factors that drive replication of the mitochondrial genome. We demonstrate how to measure ATP-generating capacity through oxygen respiratory capacity and total cellular ATP and lactate levels. Finally, we also describe how to detect mtDNA variants in pluripotent and differentiating cells using next-generation sequencing protocols and how the variants can be confirmed by high-resolution melt analysis.

  9. Progresses and challenges in optimization of human pluripotent stem cell culture.

    PubMed

    Lin, Ge; Xu, Ren-He

    2010-09-01

    The pressing demand to elucidate the biology of human embryonic stem (ES) cells and to realize their therapeutic potential has greatly promoted the progresses in the optimization of the culture systems used for this highly promising cell type. These progresses include the characterization of exogenous regulators of pluripotency and differentiation, the development of animal-free, defined, and scalable culture systems, and some pioneering efforts to establish good manufactory practice facilities to derive and expand clinical-grade human ES cells and their derivatives. All of these advancements appear to be also applicable to the derivation and culture of human induced pluripotent stem cells, an ES cell-like cell type derived from somatic cells via reprogramming. This review attempts to summarize these progresses and discuss some of the remaining challenges.

  10. Lumen Formation Is an Intrinsic Property of Isolated Human Pluripotent Stem Cells.

    PubMed

    Taniguchi, Kenichiro; Shao, Yue; Townshend, Ryan F; Tsai, Yu-Hwai; DeLong, Cynthia J; Lopez, Shawn A; Gayen, Srimonta; Freddo, Andrew M; Chue, Deming J; Thomas, Dennis J; Spence, Jason R; Margolis, Benjamin; Kalantry, Sundeep; Fu, Jianping; O'Shea, K Sue; Gumucio, Deborah L

    2015-12-01

    We demonstrate that dissociated human pluripotent stem cells (PSCs) are intrinsically programmed to form lumens. PSCs form two-cell cysts with a shared apical domain within 20 hr of plating; these cysts collapse to form monolayers after 5 days. Expression of pluripotency markers is maintained throughout this time. In two-cell cysts, an apical domain, marked by EZRIN and atypical PKCζ, is surrounded by apically targeted organelles (early endosomes and Golgi). Molecularly, actin polymerization, regulated by ARP2/3 and mammalian diaphanous-related formin 1 (MDIA), promotes lumen formation, whereas actin contraction, mediated by MYOSIN-II, inhibits this process. Finally, we show that lumenal shape can be manipulated in bioengineered micro-wells. Since lumen formation is an indispensable step in early mammalian development, this system can provide a powerful model for investigation of this process in a controlled environment. Overall, our data establish that lumenogenesis is a fundamental cell biological property of human PSCs. PMID:26626176

  11. Interaction of surfactant protein A with peroxiredoxin 6 regulates phospholipase A2 activity.

    PubMed

    Wu, Yong-Zheng; Manevich, Yefim; Baldwin, James L; Dodia, Chandra; Yu, Kevin; Feinstein, Sheldon I; Fisher, Aron B

    2006-03-17

    Peroxiredoxin 6 (Prdx6) is a "moonlighting" protein with both GSH peroxidase and phospholipase A(2) (PLA(2)) activities. This protein is responsible for degradation of internalized dipalmitoylphosphatidylcholine, the major phospholipid component of lung surfactant. The PLA(2) activity is inhibited by surfactant protein A (SP-A). We postulate that SP-A regulates the PLA(2) activity of Prdx6 through direct protein-protein interaction. Recombinant human Prdx6 and SP-A isolated from human alveolar proteinosis fluid were studied. Measurement of kinetic constants at pH 4.0 (maximal PLA(2) activity) showed K(m)0.35 mm and V(max) 138 nmol/min/mg of protein. SP-A inhibited PLA(2) activity non-competitively with K(i) 10 mug/ml and was Ca(2+) -independent. Activity at pH 7.4 was approximately 50% less, and inhibition by SP-A was partially dependent on Ca(2+). Interaction of SP-A and Prdx6 at pH 7.4 was shown by Prdx6-mediated inhibition of SP-A binding to agarose beads, a pull-down assay using His-tagged Prdx6 and Ni(2) -chelating beads, co-immunoprecipitation from lung epithelial cells and from a binary mixture of the two proteins, binding after treatment with a trifunctional cross-linker, and size-exclusion chromatography. Analysis by static light scattering and surface plasmon resonance showed calcium-independent SP-A binding to Prdx6 at pH 4.0 and partial Ca(2+) dependence of binding at pH 7.4. These results indicate a direct interaction between SP-A and Prdx6, which provides a mechanism for regulation of the PLA(2) activity of Prdx6 by SP-A. PMID:16330552

  12. Axo-Glia Interaction Preceding CNS Myelination Is Regulated by Bidirectional Eph-Ephrin Signaling.

    PubMed

    Linneberg, Cecilie; Harboe, Mette; Laursen, Lisbeth S

    2015-01-01

    In the central nervous system, myelination of axons is required to ensure fast saltatory conduction and for survival of neurons. However, not all axons are myelinated, and the molecular mechanisms involved in guiding the oligodendrocyte processes toward the axons to be myelinated are not well understood. Only a few negative or positive guidance clues that are involved in regulating axo-glia interaction prior to myelination have been identified. One example is laminin, known to be required for early axo-glia interaction, which functions through α6β1 integrin. Here, we identify the Eph-ephrin family of guidance receptors as novel regulators of the initial axo-glia interaction, preceding myelination. We demonstrate that so-called forward and reverse signaling, mediated by members of both Eph and ephrin subfamilies, has distinct and opposing effects on processes extension and myelin sheet formation. EphA forward signaling inhibits oligodendrocyte process extension and myelin sheet formation, and blocking of bidirectional signaling through this receptor enhances myelination. Similarly, EphB forward signaling also reduces myelin membrane formation, but in contrast to EphA forward signaling, this occurs in an integrin-dependent manner, which can be reversed by overexpression of a constitutive active β1-integrin. Furthermore, ephrin-B reverse signaling induced by EphA4 or EphB1 enhances myelin sheet formation. Combined, this suggests that the Eph-ephrin receptors are important mediators of bidirectional signaling between axons and oligodendrocytes. It further implies that balancing Eph-ephrin forward and reverse signaling is important in the selection process of axons to be myelinated.

  13. Introduction to Hair-Follicle-Associated Pluripotent Stem Cells.

    PubMed

    Hoffman, Robert M

    2016-01-01

    Nestin-expressing stem cells of the hair follicle, discovered by our laboratory, have been shown to be able to form outer-root sheaths of the follicle as well as neurons and many other non-follicle cell types. We have termed the nestin-expressing stem cells of the hair follicle as hair-follicle-associated pluripotent (HAP) stem cells. We have shown that the HAP stem cells from the hair follicle can effect the repair of peripheral nerve and spinal cord injury. The hair follicle stem cells differentiate into neuronal and glial cells after transplantation to the injured peripheral nerve and spinal cord, and enhance injury repair and locomotor recovery. When the excised hair follicle with its nerve stump was placed in Gelfoam(®) 3D histoculture, HAP stem cells grew and extended the hair follicle nerve which consisted of βIII-tubulin-positive fibers with F-actin expression at the tip. These findings indicate that βIII-tubulin-positive fibers elongating from the whisker follicle sensory nerve stump were growing axons. The growing whisker sensory nerve was highly enriched in HAP stem cells, which appeared to play a major role in its elongation and interaction with other nerves in 3D Gelfoam(®) histoculture, including the sciatic nerve, the trigeminal nerve, and the trigeminal nerve ganglion. These results suggest that a major function of the HAP stem cells in the hair follicle is for growth of the follicle sensory nerve. Recently, we have shown that HAP stem cells can differentiate into beating cardiac muscle cells. HAP stem cells have critical advantages for regenerative medicine over embryonic stem (ES) cells and induced pluripotent stem (iPS) cells in that they are highly accessible from each patient, thereby eliminating immunological issues since they are autologous, require no genetic manipulation, are non-tumorigenic, and do not present ethical issues.

  14. Nitric oxide regulates cell behavior on an interactive cell-derived extracellular matrix scaffold.

    PubMed

    Xing, Qi; Zhang, Lijun; Redman, Travis; Qi, Shaohai; Zhao, Feng

    2015-12-01

    During tissue injury and wound healing process, there are dynamic reciprocal interactions among cells, extracellular matrix (ECM), and mediating molecules which are crucial for functional tissue repair. Nitric oxide (NO) is one of the key mediating molecules that can positively regulate various biological activities involved in wound healing. Various ECM components serve as binding sites for cells and mediating molecules, and the interactions further stimulate cellular activities. Human mesenchymal stem cells (hMSCs) can migrate to the wound site and contribute to tissue regeneration through differentiation and paracrine signaling. The objective of this work was to investigate the regulatory effect of NO on hMSCs in an interactive ECM-rich microenvironment. In order to mimic the in vivo stromal environment in wound site, a cell-derived ECM scaffold that was able to release NO within the range of in vivo wound fluid NO level was fabricated. Results showed that the micro-molar level of NO released from the ECM scaffold had an inhibitory effect on cellular activities of hMSCs. The NO impaired cell growth, altered cell morphology, disrupted the F-actin organization, also decreased the expression of focal adhesion related molecules integrin α5 and paxillin. These results may contribute to the elucidation of how NO acts on hMSCs in wound healing process.

  15. The Fanconi Anemia Proteins FANCD2 and FANCJ Interact and Regulate Each Other's Chromatin Localization*

    PubMed Central

    Chen, Xiaoyong; Wilson, James B.; McChesney, Patricia; Williams, Stacy A.; Kwon, Youngho; Longerich, Simonne; Marriott, Andrew S.; Sung, Patrick; Jones, Nigel J.; Kupfer, Gary M.

    2014-01-01

    Fanconi anemia is a genetic disease resulting in bone marrow failure, birth defects, and cancer that is thought to encompass a defect in maintenance of genomic stability. Mutations in 16 genes (FANCA, B, C, D1, D2, E, F, G, I, J, L, M, N, O, P, and Q) have been identified in patients, with the Fanconi anemia subtype J (FA-J) resulting from homozygous mutations in the FANCJ gene. Here, we describe the direct interaction of FANCD2 with FANCJ. We demonstrate the interaction of FANCD2 and FANCJ in vivo and in vitro by immunoprecipitation in crude cell lysates and from fractions after gel filtration and with baculovirally expressed proteins. Mutation of the monoubiquitination site of FANCD2 (K561R) preserves interaction with FANCJ constitutively in a manner that impedes proper chromatin localization of FANCJ. FANCJ is necessary for FANCD2 chromatin loading and focus formation in response to mitomycin C treatment. Our results suggest not only that FANCD2 regulates FANCJ chromatin localization but also that FANCJ is necessary for efficient loading of FANCD2 onto chromatin following DNA damage caused by mitomycin C treatment. PMID:25070891

  16. Species interactions regulate the collapse of biodiversity and ecosystem function in tropical forest fragments.

    PubMed

    Bregman, Tom P; Lees, Alexander C; Seddon, Nathalie; Macgregor, Hannah E A; Darski, Bianca; Aleixo, Alexandre; Bonsall, Michael B; Tobias, Joseph A

    2015-10-01

    Competitive interactions among species with similar ecological niches are known to regulate the assembly of biological communities. However, it is not clear whether such forms of competition can predict the collapse of communities and associated shifts in ecosystem function in the face of environmental change. Here, we use phylogenetic and functional trait data to test whether communities of two ecologically important guilds of tropical birds (frugivores and insectivores) are structured by species interactions in a fragmented Amazonian forest landscape. In both guilds, we found that forest patch size, quality, and degree of isolation influence the phylogenetic and functional trait structure of communities, with small, degraded, or isolated forest patches having an increased signature of competition (i.e., phylogenetic and functional trait overdispersion in relation to null models). These results suggest that local extinctions in the context of fragmentation are nonrandom, with a consistent bias toward more densely occupied regions of niche space. We conclude that the loss of biodiversity in fragmented landscapes is mediated by niche-based competitive interactions among species, with potentially far-reaching implications for key ecosystem processes, including seed dispersal and plant damage by phytophagous insects. PMID:26649390

  17. NPR1 Protein Regulates Pathogenic and Symbiotic Interactions between Rhizobium and Legumes and Non-Legumes

    PubMed Central

    Peleg-Grossman, Smadar; Golani, Yael; Kaye, Yuval; Melamed-Book, Naomi; Levine, Alex

    2009-01-01

    Background Legumes are unique in their ability to establish symbiotic interaction with rhizobacteria from Rhizobium genus, which provide them with available nitrogen. Nodulation factors (NFs) produced by Rhizobium initiate legume root hair deformation and curling that entrap the bacteria, and allow it to grow inside the plant. In contrast, legumes and non-legumes activate defense responses when inoculated with pathogenic bacteria. One major defense pathway is mediated by salicylic acid (SA). SA is sensed and transduced to downstream defense components by a redox-regulated protein called NPR1. Methodology/Principal Findings We used Arabidopsis mutants in SA defense pathway to test the role of NPR1 in symbiotic interactions. Inoculation of Sinorhizobium meliloti or purified NF on Medicago truncatula or nim1/npr1 A. thaliana mutants induced root hair deformation and transcription of early and late nodulins. Application of S. meliloti or NF on M. truncatula or A. thaliana roots also induced a strong oxidative burst that lasted much longer than in plants inoculated with pathogenic or mutualistic bacteria. Transient overexpression of NPR1 in M. truncatula suppressed root hair curling, while inhibition of NPR1 expression by RNAi accelerated curling. Conclusions/Significance We show that, while NPR1 has a positive effect on pathogen resistance, it has a negative effect on symbiotic interactions, by inhibiting root hair deformation and nodulin expression. Our results also show that basic plant responses to Rhizobium inoculation are conserved in legumes and non-legumes. PMID:20027302

  18. Species interactions regulate the collapse of biodiversity and ecosystem function in tropical forest fragments.

    PubMed

    Bregman, Tom P; Lees, Alexander C; Seddon, Nathalie; Macgregor, Hannah E A; Darski, Bianca; Aleixo, Alexandre; Bonsall, Michael B; Tobias, Joseph A

    2015-10-01

    Competitive interactions among species with similar ecological niches are known to regulate the assembly of biological communities. However, it is not clear whether such forms of competition can predict the collapse of communities and associated shifts in ecosystem function in the face of environmental change. Here, we use phylogenetic and functional trait data to test whether communities of two ecologically important guilds of tropical birds (frugivores and insectivores) are structured by species interactions in a fragmented Amazonian forest landscape. In both guilds, we found that forest patch size, quality, and degree of isolation influence the phylogenetic and functional trait structure of communities, with small, degraded, or isolated forest patches having an increased signature of competition (i.e., phylogenetic and functional trait overdispersion in relation to null models). These results suggest that local extinctions in the context of fragmentation are nonrandom, with a consistent bias toward more densely occupied regions of niche space. We conclude that the loss of biodiversity in fragmented landscapes is mediated by niche-based competitive interactions among species, with potentially far-reaching implications for key ecosystem processes, including seed dispersal and plant damage by phytophagous insects.

  19. Lamin A reassembly at the end of mitosis is regulated by its SUMO-interacting motif.

    PubMed

    Moriuchi, Takanobu; Kuroda, Masaki; Kusumoto, Fumiya; Osumi, Takashi; Hirose, Fumiko

    2016-03-01

    Modification of proteins with small ubiquitin-related modifier (SUMO; SUMOylation) is involved in the regulation of various biological processes. Recent studies have demonstrated that noncovalent associations between SUMOylated proteins and co-operative proteins containing SUMO-interacting motifs (SIMs) are important for the spatiotemporal organization of many protein complexes. In this study, we demonstrate that interactions between lamin A, a major component of the nuclear lamina, and SUMO isoforms are dependent on one of the four SIMs (SIM3) resided in lamin A polypeptide in vitro. Live cell imaging and immunofluorescence staining showed that SIM3 is required for accumulation of lamin A on the chromosomes during telophase, and subsequent evaluation of a panel of deletion mutants determined that a 156-amino acid region spanning the carboxyl-terminal Ig-fold domain of lamin A is sufficient for this accumulation. Notably, mutation of SIM3 abrogated the dephosphorylation of mitosis-specific phosphorylation at Ser-22 of lamin A, which normally occurs during telophase, and the subsequent nuclear lamina reorganization. Furthermore, expression of a conjugation-defective SUMO2 mutant, which was previously shown to inhibit endogenous SUMOylation in a dominant-negative manner, also impaired the accumulation of wild type lamin A on telophase chromosomes. These findings suggest that interactions between SIM3 of lamin A and a putative SUMO2-modified protein plays an important role in the reorganization of the nuclear lamina at the end of mitosis.

  20. Nitric oxide regulates cell behavior on an interactive cell-derived extracellular matrix scaffold.

    PubMed

    Xing, Qi; Zhang, Lijun; Redman, Travis; Qi, Shaohai; Zhao, Feng

    2015-12-01

    During tissue injury and wound healing process, there are dynamic reciprocal interactions among cells, extracellular matrix (ECM), and mediating molecules which are crucial for functional tissue repair. Nitric oxide (NO) is one of the key mediating molecules that can positively regulate various biological activities involved in wound healing. Various ECM components serve as binding sites for cells and mediating molecules, and the interactions further stimulate cellular activities. Human mesenchymal stem cells (hMSCs) can migrate to the wound site and contribute to tissue regeneration through differentiation and paracrine signaling. The objective of this work was to investigate the regulatory effect of NO on hMSCs in an interactive ECM-rich microenvironment. In order to mimic the in vivo stromal environment in wound site, a cell-derived ECM scaffold that was able to release NO within the range of in vivo wound fluid NO level was fabricated. Results showed that the micro-molar level of NO released from the ECM scaffold had an inhibitory effect on cellular activities of hMSCs. The NO impaired cell growth, altered cell morphology, disrupted the F-actin organization, also decreased the expression of focal adhesion related molecules integrin α5 and paxillin. These results may contribute to the elucidation of how NO acts on hMSCs in wound healing process. PMID:26074441

  1. Diacylglycerol kinase-zeta localization in skeletal muscle is regulated by phosphorylation and interaction with syntrophins.

    PubMed

    Abramovici, Hanan; Hogan, Angela B; Obagi, Christopher; Topham, Matthew K; Gee, Stephen H

    2003-11-01

    Syntrophins are scaffolding proteins that link signaling molecules to dystrophin and the cytoskeleton. We previously reported that syntrophins interact with diacylglycerol kinase-zeta (DGK-zeta), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show syntrophins and DGK-zeta form a complex in skeletal muscle whose translocation from the cytosol to the plasma membrane is regulated by protein kinase C-dependent phosphorylation of the DGK-zeta MARCKS domain. DGK-zeta mutants that do not bind syntrophins were mislocalized, and an activated mutant of this sort induced atypical changes in the actin cytoskeleton, indicating syntrophins are important for localizing DGK-zeta and regulating its activity. Consistent with a role in actin organization, DGK-zeta and syntrophins were colocalized with filamentous (F)-actin and Rac in lamellipodia and ruffles. Moreover, extracellular signal-related kinase-dependent phosphorylation of DGK-zeta regulated its association with the cytoskeleton. In adult muscle, DGK-zeta was colocalized with syntrophins on the sarcolemma and was concentrated at neuromuscular junctions (NMJs), whereas in type IIB fibers it was found exclusively at NMJs. DGK-zeta was reduced at the sarcolemma of dystrophin-deficient mdx mouse myofibers but was specifically retained at NMJs, indicating that dystrophin is important for the sarcolemmal but not synaptic localization of DGK-zeta. Together, our findings suggest syntrophins localize DGK-zeta signaling complexes at specialized domains of muscle cells, which may be critical for the proper control of lipid-signaling pathways regulating actin organization. In dystrophic muscle, mislocalized DGK-zeta may cause abnormal cytoskeletal changes that contribute to disease pathogenesis. PMID:14551255

  2. Diacylglycerol kinase-zeta localization in skeletal muscle is regulated by phosphorylation and interaction with syntrophins.

    PubMed

    Abramovici, Hanan; Hogan, Angela B; Obagi, Christopher; Topham, Matthew K; Gee, Stephen H

    2003-11-01

    Syntrophins are scaffolding proteins that link signaling molecules to dystrophin and the cytoskeleton. We previously reported that syntrophins interact with diacylglycerol kinase-zeta (DGK-zeta), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show syntrophins and DGK-zeta form a complex in skeletal muscle whose translocation from the cytosol to the plasma membrane is regulated by protein kinase C-dependent phosphorylation of the DGK-zeta MARCKS domain. DGK-zeta mutants that do not bind syntrophins were mislocalized, and an activated mutant of this sort induced atypical changes in the actin cytoskeleton, indicating syntrophins are important for localizing DGK-zeta and regulating its activity. Consistent with a role in actin organization, DGK-zeta and syntrophins were colocalized with filamentous (F)-actin and Rac in lamellipodia and ruffles. Moreover, extracellular signal-related kinase-dependent phosphorylation of DGK-zeta regulated its association with the cytoskeleton. In adult muscle, DGK-zeta was colocalized with syntrophins on the sarcolemma and was concentrated at neuromuscular junctions (NMJs), whereas in type IIB fibers it was found exclusively at NMJs. DGK-zeta was reduced at the sarcolemma of dystrophin-deficient mdx mouse myofibers but was specifically retained at NMJs, indicating that dystrophin is important for the sarcolemmal but not synaptic localization of DGK-zeta. Together, our findings suggest syntrophins localize DGK-zeta signaling complexes at specialized domains of muscle cells, which may be critical for the proper control of lipid-signaling pathways regulating actin organization. In dystrophic muscle, mislocalized DGK-zeta may cause abnormal cytoskeletal changes that contribute to disease pathogenesis.

  3. Computational Biology Methods for Characterization of Pluripotent Cells.

    PubMed

    Araúzo-Bravo, Marcos J

    2016-01-01

    Pluripotent cells are a powerful tool for regenerative medicine and drug discovery. Several techniques have been developed to induce pluripotency, or to extract pluripotent cells from different tissues and biological fluids. However, the characterization of pluripotency requires tedious, expensive, time-consuming, and not always reliable wet-lab experiments; thus, an easy, standard quality-control protocol of pluripotency assessment remains to be established. Here to help comes the use of high-throughput techniques, and in particular, the employment of gene expression microarrays, which has become a complementary technique for cellular characterization. Research has shown that the transcriptomics comparison with an Embryonic Stem Cell (ESC) of reference is a good approach to assess the pluripotency. Under the premise that the best protocol is a computer software source code, here I propose and explain line by line a software protocol coded in R-Bioconductor for pluripotency assessment based on the comparison of transcriptomics data of pluripotent cells with an ESC of reference. I provide advice for experimental design, warning about possible pitfalls, and guides for results interpretation.

  4. Regulatory and functional interactions of plant growth regulators and plant glutathione S-transferases (GSTs).

    PubMed

    Moons, Ann

    2005-01-01

    Plant glutathioneS-transferases (GSTs) are a heterogeneous superfamily of multifunctional proteins, grouped into six classes. The tau (GSTU) and phi (GSTF) class GSTs are the most represented ones and are plant-specific, whereas the smaller theta (GSTT) and zeta (GSTZ) classes are also found in animals. The lambda GSTs (GSTL) and the dehydroascorbate reductases (DHARs) are more distantly related. Plant GSTs perform a variety of pivotal catalytic and non-enzymatic functions in normal plant development and plant stress responses, roles that are only emerging. Catalytic functions include glutathione (GSH)-conjugation in the metabolic detoxification of herbicides and natural products. GSTs can also catalyze GSH-dependent peroxidase reactions that scavenge toxic organic hydroperoxides and protect from oxidative damage. GSTs can furthermore catalyze GSH-dependent isomerizations in endogenous metabolism, exhibit GSH-dependent thioltransferase safeguarding protein function from oxidative damage and DHAR activity functioning in redox homeostasis. Plant GSTs can also function as ligandins or binding proteins for phytohormones (i.e., auxins and cytokinins) or anthocyanins, thereby facilitating their distribution and transport. Finally, GSTs are also indirectly involved in the regulation of apoptosis and possibly also in stress signaling. Plant GST genes exhibit a diversity of expression patterns during biotic and abiotic stresses. Stress-induced plant growth regulators (i.e., jasmonic acid [JA], salicylic acid [SA], ethylene [ETH], and nitric oxide [NO] differentially activate GST gene expression. It is becoming increasingly evident that unique combinations of multiple, often interactive signaling pathways from various phytohormones and reactive oxygen species or antioxidants render the distinct transcriptional activation patterns of individual GSTs during stress. Underestimated post-transcriptional regulations of individual GSTs are becoming increasingly evident and roles

  5. Small RNA Regulators of Plant-Hemipteran Interactions: Micromanagers with Versatile Roles.

    PubMed

    Sattar, Sampurna; Thompson, Gary A

    2016-01-01

    Non-coding small RNAs (sRNAs) in plants have important roles in regulating biological processes, including development, reproduction, and stress responses. Recent research indicates significant roles for sRNA-mediated gene silencing during plant-hemipteran interactions that involve all three of these biological processes. Plant responses to hemipteran feeding are determined by changes in the host transcriptome that appear to be fine-tuned by sRNAs. The role of sRNA in plant defense responses is complex. Different forms of sRNAs, with specific modes of action, regulate changes in the host transcriptome primarily through post-transcriptional gene silencing and occasionally through translational repression. Plant genetic resistance against hemipterans provides a model to explore the regulatory roles of sRNAs in plant defense. Aphid-induced sRNA expression in resistance genotypes delivers a new paradigm in understanding the regulation of R gene-mediated resistance in host plants. Unique sRNA profiles, including changes in sRNA biogenesis and expression can also provide insights into susceptibility to insect herbivores. Activation of phytohormone-mediated defense responses against insect herbivory is another hallmark of this interaction, and recent studies have shown that regulation of phytohormone signaling is under the control of sRNAs. Hemipterans feeding on resistant plants also show changes in insect sRNA profiles, possibly influencing insect development and reproduction. Changes in insect traits such as fecundity, host range, and resistance to insecticides are impacted by sRNAs and can directly contribute to the success of certain insect biotypes. In addition to causing direct damage to the host plant, hemipteran insects are often vectors of viral pathogens. Insect anti-viral RNAi machinery is activated to limit virus accumulation, suggesting a role in insect immunity. Virus-derived long sRNAs strongly resemble insect piRNAs, leading to the speculation that the pi

  6. Regulated Norepinephrine Transporter Interaction with the Neurokinin-1 Receptor Establishes Transporter Subcellular Localization*

    PubMed Central

    Arapulisamy, Obulakshmi; Mannangatti, Padmanabhan; Jayanthi, Lankupalle D.

    2013-01-01

    Neurokinin-1 receptor (NK1R) mediates down-regulation of human norepinephrine (NE) transporter (hNET) via protein kinase C (PKC). However, native NET regulation by NK1R and the mechanism by which NK1R targets NET among other potential effectors are unknown. Effect of NK1R activation on native NET regulation and NET/NK1R interaction were studied using rat brain synaptosomes expressing native NET and NK1R as well as human placental trophoblast (HTR) cells coexpressing WT-hNET or NK1R/PKC-resistant hNET-T258A,S259A double mutant (NET-DM) and hNK1R. The selective NK1R agonist, GR73632, and Substance-P (SP) inhibited NE transport and reduced plasma membrane expression of NET and NK1R. Pretreatment with the NK1R antagonist, EMEND (aprepitant) prevented these NK1R-mediated effects. Immunoprecipitation experiments showed that NET forms stable complexes with NK1R. In HTR cells, combined biotinylation and immunoprecipitation studies revealed plasma membrane localization of NET·NK1R complexes. Receptor activation resulted in the internalization of NET·NK1R complexes. Lipid raft and immunoprecipitation analyses revealed the presence of NET·NK1R complexes exclusively in non-raft membrane fractions under basal/unstimulated conditions. However, NK1R activation led to translocation of NET·NK1R complexes to raft-rich membrane fractions. Importantly, PKCα was found in association with raft-localized NET following SP treatment. Similar to WT-NET, PKC-resistant NET-DM was found in association with NK1R exclusively in non-raft fractions. However, SP treatment failed to translocate NET-DM·NK1R complexes from non-raft fractions to raft fractions. Collectively, these results suggest that NK1R forms physical complexes with NET and that the receptor-mediated Thr258 + Ser259 motif-dependent translocation of NET·NK1R complexes into raft-rich microdomains facilitates NET/NK1R interaction with PKCα to coordinate spatially restricted NET regulation. PMID:23979140

  7. Small RNA Regulators of Plant-Hemipteran Interactions: Micromanagers with Versatile Roles

    PubMed Central

    Sattar, Sampurna; Thompson, Gary A.

    2016-01-01

    Non-coding small RNAs (sRNAs) in plants have important roles in regulating biological processes, including development, reproduction, and stress responses. Recent research indicates significant roles for sRNA-mediated gene silencing during plant-hemipteran interactions that involve all three of these biological processes. Plant responses to hemipteran feeding are determined by changes in the host transcriptome that appear to be fine-tuned by sRNAs. The role of sRNA in plant defense responses is complex. Different forms of sRNAs, with specific modes of action, regulate changes in the host transcriptome primarily through post-transcriptional gene silencing and occasionally through translational repression. Plant genetic resistance against hemipterans provides a model to explore the regulatory roles of sRNAs in plant defense. Aphid-induced sRNA expression in resistance genotypes delivers a new paradigm in understanding the regulation of R gene-mediated resistance in host plants. Unique sRNA profiles, including changes in sRNA biogenesis and expression can also provide insights into susceptibility to insect herbivores. Activation of phytohormone-mediated defense responses against insect herbivory is another hallmark of this interaction, and recent studies have shown that regulation of phytohormone signaling is under the control of sRNAs. Hemipterans feeding on resistant plants also show changes in insect sRNA profiles, possibly influencing insect development and reproduction. Changes in insect traits such as fecundity, host range, and resistance to insecticides are impacted by sRNAs and can directly contribute to the success of certain insect biotypes. In addition to causing direct damage to the host plant, hemipteran insects are often vectors of viral pathogens. Insect anti-viral RNAi machinery is activated to limit virus accumulation, suggesting a role in insect immunity. Virus-derived long sRNAs strongly resemble insect piRNAs, leading to the speculation that the pi

  8. Small RNA Regulators of Plant-Hemipteran Interactions: Micromanagers with Versatile Roles

    PubMed Central

    Sattar, Sampurna; Thompson, Gary A.

    2016-01-01

    Non-coding small RNAs (sRNAs) in plants have important roles in regulating biological processes, including development, reproduction, and stress responses. Recent research indicates significant roles for sRNA-mediated gene silencing during plant-hemipteran interactions that involve all three of these biological processes. Plant responses to hemipteran feeding are determined by changes in the host transcriptome that appear to be fine-tuned by sRNAs. The role of sRNA in plant defense responses is complex. Different forms of sRNAs, with specific modes of action, regulate changes in the host transcriptome primarily through post-transcriptional gene silencing and occasionally through translational repression. Plant genetic resistance against hemipterans provides a model to explore the regulatory roles of sRNAs in plant defense. Aphid-induced sRNA expression in resistance genotypes delivers a new paradigm in understanding the regulation of R gene-mediated resistance in host plants. Unique sRNA profiles, including changes in sRNA biogenesis and expression can also provide insights into susceptibility to insect herbivores. Activation of phytohormone-mediated defense responses against insect herbivory is another hallmark of this interaction, and recent studies have shown that regulation of phytohormone signaling is under the control of sRNAs. Hemipterans feeding on resistant plants also show changes in insect sRNA profiles, possibly influencing insect development and reproduction. Changes in insect traits such as fecundity, host range, and resistance to insecticides are impacted by sRNAs and can directly contribute to the success of certain insect biotypes. In addition to causing direct damage to the host plant, hemipteran insects are often vectors of viral pathogens. Insect anti-viral RNAi machinery is activated to limit virus accumulation, suggesting a role in insect immunity. Virus-derived long sRNAs strongly resemble insect piRNAs, leading to the speculation that the pi

  9. Arabidopsis thaliana AUCSIA-1 Regulates Auxin Biology and Physically Interacts with a Kinesin-Related Protein

    PubMed Central

    Pii, Youry; Korte, Arthur; Spena, Angelo

    2012-01-01

    Aucsia is a green plant gene family encoding 44–54 amino acids long miniproteins. The sequenced genomes of most land plants contain two Aucsia genes. RNA interference of both tomato (Solanum lycopersicum) Aucsia genes (SlAucsia-1 and SlAucsia-2) altered auxin sensitivity, auxin transport and distribution; it caused parthenocarpic development of the fruit and other auxin-related morphological changes. Here we present data showing that the Aucsia-1 gene of Arabidopsis thaliana alters, by itself, root auxin biology and that the AtAUCSIA-1 miniprotein physically interacts with a kinesin-related protein. The AtAucsia-1 gene is ubiquitously expressed, although its expression is higher in roots and inflorescences in comparison to stems and leaves. Two allelic mutants for AtAucsia-1 gene did not display visible root morphological alterations; however both basipetal and acropetal indole-3-acetic acid (IAA) root transport was reduced as compared with wild-type plants. The transcript steady state levels of the auxin efflux transporters ATP BINDING CASSETTE subfamily B (ABCB) ABCB1, ABCB4 and ABCB19 were reduced in ataucsia-1 plants. In ataucsia-1 mutant, lateral root growth showed an altered response to i) exogenous auxin, ii) an inhibitor of polar auxin transport and iii) ethylene. Overexpression of AtAucsia-1 inhibited primary root growth. In vitro and in vivo protein-protein interaction experiments showed that AtAUCSIA-1 interacts with a 185 amino acids long fragment belonging to a 2712 amino acids long protein of unknown function (At4g31570). Bioinformatics analysis indicates that the AtAUCSIA-1 interacting protein (AtAUCSIA-1IP) clusters with a group of CENP-E kinesin-related proteins. Gene ontology predictions for the two proteins are consistent with the hypothesis that the AtAUCSIA-1/AtAUCSIA-1IP complex is involved in the regulation of the cytoskeleton dynamics underlying auxin biology. PMID:22911780

  10. Interactions with the bifunctional interface of the transcriptional coactivator DCoH1 are kinetically regulated.

    PubMed

    Wang, Dongli; Coco, Matthew W; Rose, Robert B

    2015-02-13

    Pterin-4a-carbinolamine dehydratase (PCD) is a highly conserved enzyme that evolved a second, unrelated function in mammals, as a transcriptional coactivator. As a coactivator, PCD is known as DCoH or dimerization cofactor of the transcription factor HNF-1. These two activities are associated with a change in oligomeric state: from two dimers interacting as an enzyme in the cytoplasm to a dimer interacting with a dimer of HNF-1 in the nucleus. The same interface of DCoH forms both complexes. To determine how DCoH partitions between its two functions, we studied the folding and stability of the DCoH homotetramer. We show that the DCoH1 homotetramer is kinetically trapped, meaning once it forms it will not dissociate to interact with HNF-1. In contrast, DCoH2, a paralog of DCoH1, unfolds within hours. A simple mutation in the interface of DCoH2 from Ser-51 to Thr, as found in DCoH1, increases the kinetic stability by 9 orders of magnitude, to τ(½) ∼ 2 million years. This suggests that the DCoH1·HNF-1 complex must co-fold to interact. We conclude that simple mutations can dramatically affect the dissociation kinetics of a complex. Residue 51 represents a "kinetic hot spot" instead of a "thermodynamic hot spot." Kinetic regulation allows PCD to adopt two distinct functions. Mutations in DCoH1 associated with diabetes affect both functions of DCoH1, perhaps by disrupting the balance between the two DCoH complexes.

  11. Interactions with the Bifunctional Interface of the Transcriptional Coactivator DCoH1 Are Kinetically Regulated*

    PubMed Central

    Wang, Dongli; Coco, Matthew W.; Rose, Robert B.

    2015-01-01

    Pterin-4a-carbinolamine dehydratase (PCD) is a highly conserved enzyme that evolved a second, unrelated function in mammals, as a transcriptional coactivator. As a coactivator, PCD is known as DCoH or dimerization cofactor of the transcription factor HNF-1. These two activities are associated with a change in oligomeric state: from two dimers interacting as an enzyme in the cytoplasm to a dimer interacting with a dimer of HNF-1 in the nucleus. The same interface of DCoH forms both complexes. To determine how DCoH partitions between its two functions, we studied the folding and stability of the DCoH homotetramer. We show that the DCoH1 homotetramer is kinetically trapped, meaning once it forms it will not dissociate to interact with HNF-1. In contrast, DCoH2, a paralog of DCoH1, unfolds within hours. A simple mutation in the interface of DCoH2 from Ser-51 to Thr, as found in DCoH1, increases the kinetic stability by 9 orders of magnitude, to τ½ ∼2 million years. This suggests that the DCoH1·HNF-1 complex must co-fold to interact. We conclude that simple mutations can dramatically affect the dissociation kinetics of a complex. Residue 51 represents a “kinetic hot spot” instead of a “thermodynamic hot spot.” Kinetic regulation allows PCD to adopt two distinct functions. Mutations in DCoH1 associated with diabetes affect both functions of DCoH1, perhaps by disrupting the balance between the two DCoH complexes. PMID:25538247

  12. Reactivation of Endogenous Genes and Epigenetic Remodeling Are Barriers for Generating Transgene-Free Induced Pluripotent Stem Cells in Pig

    PubMed Central

    Choi, Kwang-Hwan; Park, Jin-Kyu; Son, Dongchan; Hwang, Jae Yeon; Lee, Dong-Kyung; Ka, Hakhyun; Park, Joonghoon; Lee, Chang-Kyu

    2016-01-01

    Cellular reprogramming of committed cells into a pluripotent state can be induced by ectopic expression of genes such as OCT4, SOX2, KLF4, and MYC. Reprogrammed cells can be maintained by activating endogenous pluripotent networks without transgene expression. Although various research groups have attempted to generate pig induced pluripotent stem cells (iPSCs), authentic iPSCs have not be obtained, instead showing dependence on transgene expression. In this study, iPSCs were derived from porcine fetal fibroblasts via drug-inducible vectors carrying human transcription factors (OCT4, SOX2, KLF4, and MYC). Therefore, this study investigated characteristics of iPSCs and reprogramming mechanisms in pig. The iPSCs were stably maintained over an extended period with potential in vitro differentiation into three germ layers. In addition, the pluripotent state of iPSCs was regulated by modulating culture conditions. They showed naive- or primed-like pluripotent states in LIF or bFGF supplemented culture conditions, respectively. However, iPSCs could not be maintained without ectopic expression of transgenes. The cultured iPSCs expressed endogenous transcription factors such as OCT4 and SOX2, but not NANOG (a known gateway to complete reprogramming). Endogenous genes related to mesenchymal-to-epithelial transition (DPPA2, CDH1, EPCAM, and OCLN) were not sufficiently reactivated, as measured by qPCR. DNA methylation analysis for promoters of OCT4, NANOG, and XIST showed that epigenetic reprogramming did not occur in female iPSCs. Based on our results, expression of exogenous genes could not sufficiently activate the essential endogenous genes and remodel the epigenetic milieu to achieve faithful pluripotency in pig. Accordingly, investigating iPSCs could help us improve and develop reprogramming methods by understanding reprogramming mechanisms in pig. PMID:27336671

  13. Reactivation of Endogenous Genes and Epigenetic Remodeling Are Barriers for Generating Transgene-Free Induced Pluripotent Stem Cells in Pig.

    PubMed

    Choi, Kwang-Hwan; Park, Jin-Kyu; Son, Dongchan; Hwang, Jae Yeon; Lee, Dong-Kyung; Ka, Hakhyun; Park, Joonghoon; Lee, Chang-Kyu

    2016-01-01

    Cellular reprogramming of committed cells into a pluripotent state can be induced by ectopic expression of genes such as OCT4, SOX2, KLF4, and MYC. Reprogrammed cells can be maintained by activating endogenous pluripotent networks without transgene expression. Although various research groups have attempted to generate pig induced pluripotent stem cells (iPSCs), authentic iPSCs have not be obtained, instead showing dependence on transgene expression. In this study, iPSCs were derived from porcine fetal fibroblasts via drug-inducible vectors carrying human transcription factors (OCT4, SOX2, KLF4, and MYC). Therefore, this study investigated characteristics of iPSCs and reprogramming mechanisms in pig. The iPSCs were stably maintained over an extended period with potential in vitro differentiation into three germ layers. In addition, the pluripotent state of iPSCs was regulated by modulating culture conditions. They showed naive- or primed-like pluripotent states in LIF or bFGF supplemented culture conditions, respectively. However, iPSCs could not be maintained without ectopic expression of transgenes. The cultured iPSCs expressed endogenous transcription factors such as OCT4 and SOX2, but not NANOG (a known gateway to complete reprogramming). Endogenous genes related to mesenchymal-to-epithelial transition (DPPA2, CDH1, EPCAM, and OCLN) were not sufficiently reactivated, as measured by qPCR. DNA methylation analysis for promoters of OCT4, NANOG, and XIST showed that epigenetic reprogramming did not occur in female iPSCs. Based on our results, expression of exogenous genes could not sufficiently activate the essential endogenous genes and remodel the epigenetic milieu to achieve faithful pluripotency in pig. Accordingly, investigating iPSCs could help us improve and develop reprogr