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Sample records for cell type-specific mirna

  1. Epigenetic regulation of normal human mammary cell type-specific miRNAs

    SciTech Connect

    Vrba, Lukas; Garbe, James C.; Stampfer, Martha R.; Futscher, Bernard W.

    2011-08-26

    Epigenetic mechanisms are important regulators of cell type–specific genes, including miRNAs. In order to identify cell type-specific miRNAs regulated by epigenetic mechanisms, we undertook a global analysis of miRNA expression and epigenetic states in three isogenic pairs of human mammary epithelial cells (HMEC) and human mammary fibroblasts (HMF), which represent two differentiated cell types typically present within a given organ, each with a distinct phenotype and a distinct epigenotype. While miRNA expression and epigenetic states showed strong interindividual concordance within a given cell type, almost 10% of the expressed miRNA showed a cell type–specific pattern of expression that was linked to the epigenetic state of their promoter. The tissue-specific miRNA genes were epigenetically repressed in nonexpressing cells by DNA methylation (38%) and H3K27me3 (58%), with only a small set of miRNAs (21%) showing a dual epigenetic repression where both DNA methylation and H3K27me3 were present at their promoters, such as MIR10A and MIR10B. Individual miRNA clusters of closely related miRNA gene families can each display cell type–specific repression by the same or complementary epigenetic mechanisms, such as the MIR200 family, and MIR205, where fibroblasts repress MIR200C/141 by DNA methylation, MIR200A/200B/429 by H3K27me3, and MIR205 by both DNA methylation and H3K27me3. Since deregulation of many of the epigenetically regulated miRNAs that we identified have been linked to disease processes such as cancer, it is predicted that compromise of the epigenetic control mechanisms is important for this process. Overall, these results highlight the importance of epigenetic regulation in the control of normal cell type–specific miRNA expression.

  2. Neuron type-specific miRNA represses two broadly expressed genes to modulate an avoidance behavior in C. elegans

    PubMed Central

    Drexel, Tanja; Mahofsky, Katharina; Latham, Richard; Zimmer, Manuel

    2016-01-01

    Two broad gene classes are distinguished within multicellular organisms: cell type-specific genes, which confer particular cellular properties, and ubiquitous genes that support general cellular functions. However, certain so-called ubiquitous genes show functionally relevant cell type-specific repression. How such repression is achieved is poorly understood. MicroRNAs (miRNAs) are repressors, many of which are expressed with high cell type specificity. Here we show that mir-791, expressed exclusively in the CO2-sensing neurons in Caenorhabditis elegans, represses two otherwise broadly expressed genes. This repression is necessary for normal neuronal function and behavior of the animals toward CO2. miRNA-mediated repression of broadly transcribed genes is a previously unappreciated strategy for cellular specialization. PMID:27688400

  3. A versatile Multisite Gateway-compatible promoter and transgenic line collection for cell type-specific functional genomics in Arabidopsis.

    PubMed

    Marquès-Bueno, Maria Mar; Morao, Ana K; Cayrel, Anne; Platre, Matthieu P; Barberon, Marie; Caillieux, Erwann; Colot, Vincent; Jaillais, Yvon; Roudier, François; Vert, Grégory

    2016-01-01

    Multicellular organisms are composed of many cell types that acquire their specific fate through a precisely controlled pattern of gene expression in time and space dictated in part by cell type-specific promoter activity. Understanding the contribution of highly specialized cell types in the development of a whole organism requires the ability to isolate or analyze different cell types separately. We have characterized and validated a large collection of root cell type-specific promoters and have generated cell type-specific marker lines. These benchmarked promoters can be readily used to evaluate cell type-specific complementation of mutant phenotypes, or to knockdown gene expression using targeted expression of artificial miRNA. We also generated vectors and characterized transgenic lines for cell type-specific induction of gene expression and cell type-specific isolation of nuclei for RNA and chromatin profiling. Vectors and seeds from transgenic Arabidopsis plants will be freely available, and will promote rapid progress in cell type-specific functional genomics. We demonstrate the power of this promoter set for analysis of complex biological processes by investigating the contribution of root cell types in the IRT1-dependent root iron uptake. Our findings revealed the complex spatial expression pattern of IRT1 in both root epidermis and phloem companion cells and the requirement for IRT1 to be expressed in both cell types for proper iron homeostasis.

  4. A versatile Multisite Gateway-compatible promoter and transgenic line collection for cell type-specific functional genomics in Arabidopsis

    PubMed Central

    Platre, Matthieu Pierre; Barberon, Marie; Caillieux, Erwann; Colot, Vincent

    2016-01-01

    Summary Multicellular organisms are composed of many cell types that acquire their specific fate through a precisely controlled pattern of gene expression in time and space dictated in part by cell type-specific promoter activity. Understanding the contribution of highly specialized cell types in the development of a whole organism requires the ability to isolate or analyze different cell types separately. We have characterized and validated a large collection of root cell type-specific promoters and have generated cell type-specific marker lines. These benchmarked promoters can be readily used to evaluate cell type-specific complementation of mutant phenotypes, or to knockdown gene expression using targeted expression of artificial miRNA. We also generated vectors and characterized transgenic lines for cell type-specific induction of gene expression and cell type-specific isolation of nuclei for RNA and chromatin profiling. Vectors and seeds from transgenic Arabidopsis plants will be freely available, and will promote rapid progress in cell type-specific functional genomics. We demonstrate the power of this promoter set for analysis of complex biological processes by investigating the contribution of root cell types in the IRT1-dependent root iron uptake. Our findings revealed the complex spatial expression pattern of IRT1 in both root epidermis and phloem companion cells and the requirement for IRT1 to be expressed in both cell types for proper iron homeostasis. PMID:26662936

  5. Expression Profiling of Human Immune Cell Subsets Identifies miRNA-mRNA Regulatory Relationships Correlated with Cell Type Specific Expression

    PubMed Central

    Bergauer, Tobias; Ravindran, Palanikumar; Rossier, Michel F.; Ebeling, Martin; Badi, Laura; Reis, Bernhard; Bitter, Hans; D'Asaro, Matilde; Chiappe, Alberto; Sridhar, Sriram; Pacheco, Gonzalo Duran; Burczynski, Michael E.; Hochstrasser, Denis; Vonderscher, Jacky; Matthes, Thomas

    2012-01-01

    Blood consists of different cell populations with distinct functions and correspondingly, distinct gene expression profiles. In this study, global miRNA expression profiling was performed across a panel of nine human immune cell subsets (neutrophils, eosinophils, monocytes, B cells, NK cells, CD4 T cells, CD8 T cells, mDCs and pDCs) to identify cell-type specific miRNAs. mRNA expression profiling was performed on the same samples to determine if miRNAs specific to certain cell types down-regulated expression levels of their target genes. Six cell-type specific miRNAs (miR-143; neutrophil specific, miR-125; T cells and neutrophil specific, miR-500; monocyte and pDC specific, miR-150; lymphoid cell specific, miR-652 and miR-223; both myeloid cell specific) were negatively correlated with expression of their predicted target genes. These results were further validated using an independent cohort where similar immune cell subsets were isolated and profiled for both miRNA and mRNA expression. miRNAs which negatively correlated with target gene expression in both cohorts were identified as candidates for miRNA/mRNA regulatory pairs and were used to construct a cell-type specific regulatory network. miRNA/mRNA pairs formed two distinct clusters in the network corresponding to myeloid (nine miRNAs) and lymphoid lineages (two miRNAs). Several myeloid specific miRNAs targeted common genes including ABL2, EIF4A2, EPC1 and INO80D; these common targets were enriched for genes involved in the regulation of gene expression (p<9.0E-7). Those miRNA might therefore have significant further effect on gene expression by repressing the expression of genes involved in transcriptional regulation. The miRNA and mRNA expression profiles reported in this study form a comprehensive transcriptome database of various human blood cells and serve as a valuable resource for elucidating the role of miRNA mediated regulation in the establishment of immune cell identity. PMID:22276136

  6. Strand and Cell Type-specific Function of microRNA-126 in Angiogenesis.

    PubMed

    Zhou, Qinbo; Anderson, Chastain; Hanus, Jakub; Zhao, Fangkun; Ma, Jing; Yoshimura, Akihiko; Wang, Shusheng

    2016-10-01

    microRNAs or miRs have been shown to be pivotal modulators of vascular development. The strand and cell type-specific function of miR-126 in angiogenesis, especially pathological angiogenesis, remains poorly defined. We characterized the retinal vascular phenotype of miR-126(-/-) mice, and tested the function of miR-126 strands (miR-126-3p and -5p) using in vitro angiogenesis models and a mouse model of neovascular age-related macular degeneration. We found that miR-126 is critical for retinal vascular development but has dual function in pathological angiogenesis. miR-126(-/-) mice showed defective postnatal retinal vascular development and remodeling, which is partially rescued by genetic knockout of its target gene Spred-1. Surprisingly, either silencing miR-126-3p by LNA-antimiR or overexpressing miR-126-3p by miRNA mimic repressed laser-induced choroidal neovascularization. To dissect the underlying mechanism, we found in endothelial cells, silencing of miR-126-3p repressed angiogenesis, while overexpression of miR-126-5p enhanced angiogenesis. However, in retinal pigment epithelial cells, miR-126-3p repressed vascular endothelial growth factor (VEGF-A) expression via a novel mechanism of regulating αB-Crystallin promoter activity and by directly targeting VEGF-A 3'-untranslated region. These findings provide first genetic evidence that miR-126 is required for the development of different retinal vascular layers, and also uncover a strand and cell type-specific function of miR-126 in ocular pathological angiogenesis.

  7. Cell-type-specific miR-431 dysregulation in a motor neuron model of spinal muscular atrophy.

    PubMed

    Wertz, Mary H; Winden, Kellen; Neveu, Pierre; Ng, Shi-Yan; Ercan, Ebru; Sahin, Mustafa

    2016-06-01

    Spinal muscular atrophy (SMA) is an autosomal-recessive pediatric neurodegenerative disease characterized by selective loss of spinal motor neurons. It is caused by mutation in the survival of motor neuron 1, SMN1, gene and leads to loss of function of the full-length SMN protein. microRNAs (miRNAs) are small RNAs that are involved in post-transcriptional regulation of gene expression. Prior studies have implicated miRNAs in the pathogenesis of motor neuron disease. We hypothesized that motor neuron-specific miRNA expression changes are involved in their selective vulnerability in SMA. Therefore, we sought to determine the effect of SMN loss on miRNAs and their target mRNAs in spinal motor neurons. We used microarray and RNAseq to profile both miRNA and mRNA expression in primary spinal motor neuron cultures after acute SMN knockdown. By integrating the miRNA:mRNA profiles, a number of dysregulated miRNAs were identified with enrichment in differentially expressed putative mRNA targets. miR-431 expression was highly increased, and a number of its putative mRNA targets were significantly downregulated in motor neurons after SMN loss. Further, we found that miR-431 regulates motor neuron neurite length by targeting several molecules previously identified to play a role in motor neuron axon outgrowth, including chondrolectin. Together, our findings indicate that cell-type-specific dysregulation of miR-431 plays a role in the SMA motor neuron phenotype. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Cell-Type Specific Four-Component Hydrogel

    PubMed Central

    Aberle, Timo; Franke, Katrin; Rist, Elke; Benz, Karin; Schlosshauer, Burkhard

    2014-01-01

    In the field of regenerative medicine we aim to develop implant matrices for specific tissue needs. By combining two per se, cell-permissive gel systems with enzymatic crosslinkers (gelatin/transglutaminase and fibrinogen/thrombin) to generate a blend (technical term: quattroGel), an unexpected cell-selectivity evolved. QuattroGels were porous and formed cavities in the cell diameter range, possessed gelation kinetics in the minute range, viscoelastic properties and a mechanical strength appropriate for general cell adhesion, and restricted diffusion. Cell proliferation of endothelial cells, chondrocytes and fibroblasts was essentially unaffected. In contrast, on quattroGels neither endothelial cells formed vascular tubes nor did primary neurons extend neurites in significant amounts. Only chondrocytes differentiated properly as judged by collagen isoform expression. The biophysical quattroGel characteristics appeared to leave distinct cell processes such as mitosis unaffected and favored differentiation of sessile cells, but hampered differentiation of migratory cells. This cell-type selectivity is of interest e.g. during articular cartilage or invertebral disc repair, where pathological innervation and angiogenesis represent adverse events in tissue engineering. PMID:24475174

  9. Cation Type Specific Cell Remodeling Regulates Attachment Strength

    PubMed Central

    Fuhrmann, Alexander; Li, Julie; Chien, Shu; Engler, Adam J.

    2014-01-01

    Single-molecule experiments indicate that integrin affinity is cation-type-dependent, but in spread cells integrins are engaged in complex focal adhesions (FAs), which can also regulate affinity. To better understand cation-type-dependent adhesion in fully spread cells, we investigated attachment strength by application of external shear. While cell attachment strength is indeed modulated by cations, the regulation of integrin-mediated adhesion is also exceedingly complex, cell specific, and niche dependent. In the presence of magnesium only, fibroblasts and fibrosarcoma cells remodel their cytoskeleton to align in the direction of applied shear in an α5-integrin/fibronectin-dependent manner, which allows them to withstand higher shear. In the presence of calcium or on collagen in modest shear, fibroblasts undergo piecewise detachment but fibrosarcoma cells exhibit increased attachment strength. These data augment the current understanding of force-mediated detachment by suggesting a dynamic interplay in situ between cell adhesion and integrins depending on local niche cation conditions. PMID:25014042

  10. Cell Type-Specific Modulation of Respiratory Chain Supercomplex Organization

    PubMed Central

    Sun, Dayan; Li, Bin; Qiu, Ruyi; Fang, Hezhi; Lyu, Jianxin

    2016-01-01

    Respiratory chain complexes are organized into large supercomplexes among which supercomplex In + IIIn + IVn is the only one that can directly transfer electrons from NADH to oxygen. Recently, it was reported that the formation of supercomplex In + IIIn + IVn in mice largely depends on their genetic background. However, in this study, we showed that the composition of supercomplex In + IIIn + IVn is well conserved in various mouse and human cell lines. Strikingly, we found that a minimal supercomplex In + IIIn, termed “lowest supercomplex” (LSC) in this study because of its migration at the lowest position close to complex V dimers in blue native polyacrylamide gel electrophoresis, was associated with complex IV to form a supercomplex In + IIIn + IVn in some, but not all of the human and mouse cells. In addition, we observed that the 3697G>A mutation in mitochondrial-encoded NADH dehydrogenase 1 (ND1) in one patient with Leigh’s disease specifically affected the assembly of supercomplex In + IIIn + IVn containing LSC, leading to decreased cellular respiration and ATP generation. In conclusion, we showed the existence of LSC In + IIIn + IVn and impairment of this supercomplex causes disease. PMID:27338358

  11. PathCellNet: Cell-type specific pathogen-response network explorer.

    PubMed

    Katanic, Dejan; Khan, Atif; Thakar, Juilee

    2016-12-01

    Pathogen specific immune response is a complex interplay between several innate and adaptive immune cell-types. Innate immune cells play a critical role in pathogen recognition and initiating the antigen specific adaptive immune response. Despite specific functional roles of the innate immune cells, they share several anti-viral pathways. The question then becomes, what is the overlap in the transcriptional changes induced upon viral infections across different cell-types? Here we investigate the extent to which gene signatures are conserved across innate immune cell-types by performing a comparative analysis of transcriptomic data. Particularly, we integrate transcriptomic datasets measuring response of two innate immune cells (epithelial and dendritic cells) to influenza virus. The study reveals cell-type specific regulatory genes and a conserved network between the two cell-types. Additionally, novel functionally associated gene clusters are identified which are robustly defined across multiple independent studies. These gene clusters can be used in future investigation, and to facilitate their use we release PathCellNet (version 0), a cloud based tool to explore cell-type specific connectivity of user-defined genes. In the future, expansion of PathCellNet will allow exploration of cell-type specific responses across a variety of pathogens and cell-types. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Isolation of cell type-specific apoptotic bodies by fluorescence-activated cell sorting

    PubMed Central

    Atkin-Smith, Georgia K.; Paone, Stephanie; Zanker, Damien J.; Duan, Mubing; Phan, Than K.; Chen, Weisan; Hulett, Mark D.; Poon, Ivan K. H.

    2017-01-01

    Apoptotic bodies (ApoBDs) are membrane-bound extracellular vesicles that can mediate intercellular communication in physiological and pathological settings. By combining recently developed analytical strategies with fluorescence-activated cell sorting (FACS), we have developed a method that enables the isolation of ApoBDs from cultured cells to 99% purity. In addition, this approach also enables the identification and isolation of cell type-specific ApoBDs from tissue, bodily fluid and blood-derived samples. PMID:28057919

  13. Single-Cell mRNA Profiling Reveals Cell-Type Specific Expression of Neurexin Isoforms

    PubMed Central

    Fuccillo, Marc V.; Földy, Csaba; Gökce, Özgün; Rothwell, Patrick E.; Sun, Gordon L.; Malenka, Robert C.; Südhof, Thomas C.

    2016-01-01

    Summary Neurexins are considered central organizers of synapse architecture that are implicated in neuropsychiatric disorders. Expression of neurexins in hundreds of alternatively spliced isoforms suggested that individual neurons might exhibit a cell type-specific neurexin expression pattern (a neurexin code). To test this hypothesis, we quantified the single-cell levels of neurexin isoforms and other trans-synaptic cell-adhesion molecules by microfluidics-based RT-PCR. We show that the neurexin repertoire displays pronounced cell-type specificity that is remarkably consistent within each type of neuron. Furthermore, we uncovered region-specific regulation of neurexin transcription and splice-site usage. Finally, we demonstrate that the transcriptional profiles of neurexins can be altered in an experience-dependent fashion by exposure to a drug of abuse. Our data provide evidence of cell type-specific expression patterns of multiple neurexins at the single-cell level, and suggest that expression of synaptic cell-adhesion molecules overlaps with other key features of cellular identity and diversity. PMID:26182417

  14. p53 shapes genome-wide and cell type-specific changes in microRNA expression during the human DNA damage response.

    PubMed

    Hattori, Hiroyoshi; Janky, Rekin's; Nietfeld, Wilfried; Aerts, Stein; Madan Babu, M; Venkitaraman, Ashok R

    2014-01-01

    The human DNA damage response (DDR) triggers profound changes in gene expression, whose nature and regulation remain uncertain. Although certain micro-(mi)RNA species including miR34, miR-18, miR-16 and miR-143 have been implicated in the DDR, there is as yet no comprehensive description of genome-wide changes in the expression of miRNAs triggered by DNA breakage in human cells. We have used next-generation sequencing (NGS), combined with rigorous integrative computational analyses, to describe genome-wide changes in the expression of miRNAs during the human DDR. The changes affect 150 of 1523 miRNAs known in miRBase v18 from 4-24 h after the induction of DNA breakage, in cell-type dependent patterns. The regulatory regions of the most-highly regulated miRNA species are enriched in conserved binding sites for p53. Indeed, genome-wide changes in miRNA expression during the DDR are markedly altered in TP53-/- cells compared to otherwise isogenic controls. The expression levels of certain damage-induced, p53-regulated miRNAs in cancer samples correlate with patient survival. Our work reveals genome-wide and cell type-specific alterations in miRNA expression during the human DDR, which are regulated by the tumor suppressor protein p53. These findings provide a genomic resource to identify new molecules and mechanisms involved in the DDR, and to examine their role in tumor suppression and the clinical outcome of cancer patients.

  15. p53 shapes genome-wide and cell type-specific changes in microRNA expression during the human DNA damage response

    PubMed Central

    Hattori, Hiroyoshi; Janky, Rekin’s; Nietfeld, Wilfried; Aerts, Stein; Madan Babu, M; Venkitaraman, Ashok R

    2014-01-01

    The human DNA damage response (DDR) triggers profound changes in gene expression, whose nature and regulation remain uncertain. Although certain micro-(mi)RNA species including miR34, miR-18, miR-16 and miR-143 have been implicated in the DDR, there is as yet no comprehensive description of genome-wide changes in the expression of miRNAs triggered by DNA breakage in human cells. We have used next-generation sequencing (NGS), combined with rigorous integrative computational analyses, to describe genome-wide changes in the expression of miRNAs during the human DDR. The changes affect 150 of 1523 miRNAs known in miRBase v18 from 4–24 h after the induction of DNA breakage, in cell-type dependent patterns. The regulatory regions of the most-highly regulated miRNA species are enriched in conserved binding sites for p53. Indeed, genome-wide changes in miRNA expression during the DDR are markedly altered in TP53-/- cells compared to otherwise isogenic controls. The expression levels of certain damage-induced, p53-regulated miRNAs in cancer samples correlate with patient survival. Our work reveals genome-wide and cell type-specific alterations in miRNA expression during the human DDR, which are regulated by the tumor suppressor protein p53. These findings provide a genomic resource to identify new molecules and mechanisms involved in the DDR, and to examine their role in tumor suppression and the clinical outcome of cancer patients. PMID:25486198

  16. Differential microRNA expression signatures and cell type-specific association with Taxol resistance in ovarian cancer cells

    PubMed Central

    Kim, Yong-Wan; Kim, Eun Young; Jeon, Doin; Liu, Juinn-Lin; Kim, Helena Suhyun; Choi, Jin Woo; Ahn, Woong Shick

    2014-01-01

    Paclitaxel (Taxol) resistance remains a major obstacle for the successful treatment of ovarian cancer. MicroRNAs (miRNAs) have oncogenic and tumor suppressor activity and are associated with poor prognosis phenotypes. miRNA screenings for this drug resistance are needed to estimate the prognosis of the disease and find better drug targets. miRNAs that were differentially expressed in Taxol-resistant ovarian cancer cells, compared with Taxol-sensitive cells, were screened by Illumina Human MicroRNA Expression BeadChips. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to identify target genes of selected miRNAs. Kaplan–Meier survival analysis was applied to identify dysregulated miRNAs in ovarian cancer patients using data from The Cancer Genome Atlas. A total of 82 miRNAs were identified in ovarian carcinoma cells compared to normal ovarian cells. miR-141, miR-106a, miR-200c, miR-96, and miR-378 were overexpressed, and miR-411, miR-432, miR-494, miR-409-3p, and miR-655 were underexpressed in ovarian cancer cells. Seventeen miRNAs were overexpressed in Taxol-resistant cells, including miR-663, miR-622, and HS_188. Underexpressed miRNAs in Taxol-sensitive cells included miR-497, miR-187, miR-195, and miR-107. We further showed miR-663 and miR-622 as significant prognosis markers of the chemo-resistant patient group. In particular, the downregulation of the two miRNAs was associated with better survival, perhaps increasing the sensitivity of cancer cells to Taxol. In the chemo-sensitive patient group, only miR-647 could be a prognosis marker. These miRNAs inhibit several interacting genes of p53 networks, especially in TUOS-3 and TUOS-4, and showed cell line-specific inhibition effects. Taken together, the data indicate that the three miRNAs are closely associated with Taxol resistance and potentially better prognosis factors. Our results suggest that these miRNAs were successfully and reliably identified and would be used in the

  17. Differential microRNA expression signatures and cell type-specific association with Taxol resistance in ovarian cancer cells.

    PubMed

    Kim, Yong-Wan; Kim, Eun Young; Jeon, Doin; Liu, Juinn-Lin; Kim, Helena Suhyun; Choi, Jin Woo; Ahn, Woong Shick

    2014-01-01

    Paclitaxel (Taxol) resistance remains a major obstacle for the successful treatment of ovarian cancer. MicroRNAs (miRNAs) have oncogenic and tumor suppressor activity and are associated with poor prognosis phenotypes. miRNA screenings for this drug resistance are needed to estimate the prognosis of the disease and find better drug targets. miRNAs that were differentially expressed in Taxol-resistant ovarian cancer cells, compared with Taxol-sensitive cells, were screened by Illumina Human MicroRNA Expression BeadChips. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to identify target genes of selected miRNAs. Kaplan-Meier survival analysis was applied to identify dysregulated miRNAs in ovarian cancer patients using data from The Cancer Genome Atlas. A total of 82 miRNAs were identified in ovarian carcinoma cells compared to normal ovarian cells. miR-141, miR-106a, miR-200c, miR-96, and miR-378 were overexpressed, and miR-411, miR-432, miR-494, miR-409-3p, and miR-655 were underexpressed in ovarian cancer cells. Seventeen miRNAs were overexpressed in Taxol-resistant cells, including miR-663, miR-622, and HS_188. Underexpressed miRNAs in Taxol-sensitive cells included miR-497, miR-187, miR-195, and miR-107. We further showed miR-663 and miR-622 as significant prognosis markers of the chemo-resistant patient group. In particular, the downregulation of the two miRNAs was associated with better survival, perhaps increasing the sensitivity of cancer cells to Taxol. In the chemo-sensitive patient group, only miR-647 could be a prognosis marker. These miRNAs inhibit several interacting genes of p53 networks, especially in TUOS-3 and TUOS-4, and showed cell line-specific inhibition effects. Taken together, the data indicate that the three miRNAs are closely associated with Taxol resistance and potentially better prognosis factors. Our results suggest that these miRNAs were successfully and reliably identified and would be used in the

  18. Monitoring Astrocytic Proteome Dynamics by Cell Type-Specific Protein Labeling

    PubMed Central

    Müller, Anke; Stellmacher, Anne; Freitag, Christine E.; Landgraf, Peter; Dieterich, Daniela C.

    2015-01-01

    The ability of the nervous system to undergo long-term plasticity is based on changes in cellular and synaptic proteomes. While many studies have explored dynamic alterations in neuronal proteomes during plasticity, there has been less attention paid to the astrocytic counterpart. Indeed, progress in identifying cell type-specific proteomes is limited owing to technical difficulties. Here, we present a cell type-specific metabolic tagging technique for a mammalian coculture model based on the bioorthogonal amino acid azidonorleucine and the mutated Mus musculus methionyl-tRNA synthetaseL274G enabling azidonorleucine introduction into de novo synthesized proteins. Azidonorleucine incorporation resulted in cell type-specific protein labeling and retained neuronal or astrocytic cell viability. Furthermore, we were able to label astrocytic de novo synthesized proteins and identified both Connexin-43 and 60S ribosomal protein L10a upregulated upon treatment with Brain-derived neurotrophic factor in astrocytes of a neuron-glia coculture. Taken together, we demonstrate the successful dissociation of astrocytic from neuronal proteomes by cell type-specific metabolic labeling offering new possibilities for the analyses of cell type-specific proteome dynamics. PMID:26690742

  19. Quantitative analysis of cell-type specific gene expression in the green alga Volvox carteri

    PubMed Central

    Nematollahi, Ghazaleh; Kianianmomeni, Arash; Hallmann, Armin

    2006-01-01

    Background The multicellular alga Volvox carteri possesses only two cell types: mortal, motile somatic cells and potentially immortal, immotile reproductive cells. It is therefore an attractive model system for studying how cell-autonomous cytodifferentiation is programmed within a genome. Moreover, there are ongoing genome projects both in Volvox carteri and in the closely related unicellular alga Chlamydomonas reinhardtii. However, gene sequencing is only the beginning. To identify cell-type specific expression and to determine relative expression rates, we evaluate the potential of real-time RT-PCR for quantifying gene transcript levels. Results Here we analyze a diversified pool of 39 target genes by real-time RT-PCR for each cell type. This gene pool contains previously known genes with unknown localization of cellular expression, 28 novel genes which are described in this study for the first time, and a few known, cell-type specific genes as a control. The respective gene products are, for instance, part of photosynthesis, cellular regulation, stress response, or transport processes. We provide expression data for all these genes. Conclusion The results show that quantitative real-time RT-PCR is a favorable approach to analyze cell-type specific gene expression in Volvox, which can be extended to a much larger number of genes or to developmental or metabolic mutants. Our expression data also provide a basis for a detailed analysis of individual, previously unknown, cell-type specifically expressed genes. PMID:17184518

  20. High content screening application for cell-type specific behaviour in heterogeneous primary breast epithelial subpopulations.

    PubMed

    Johnston, Rebecca L; Wockner, Leesa; McCart Reed, Amy E; Wiegmans, Adrian; Chenevix-Trench, Georgia; Khanna, Kum Kum; Lakhani, Sunil R; Smart, Chanel E

    2016-02-09

    The complex interaction between multiple cell types and the microenvironment underlies the diverse pathways to carcinogenesis and necessitates sophisticated approaches to in vitro hypotheses testing. The combination of mixed culture format with high content immunofluorescence screening technology provides a powerful platform for observation of cell type specific behavior. We have developed a versatile, high-throughput method for assessing cell-type specific responses. In addition to the specificity and sensitivity offered traditionally by immunofluorescent detection in flow cytometry, the 'in-cell' analysis method we describe provides the added benefits of higher throughput and the ability to analyse protein subcellular localisation in situ. Furthermore, elimination of the cell dissociation step allows for more immediate analysis of responses to specific extrinsic stimuli. We applied this method to investigate ionising radiation treatment response in normal breast epithelial cells, measuring growth rate, cell cycle response and double-strand DNA breaks. The 'in-cell' analysis approach elucidated several interesting donor and cell-type specific differences. Notably, in response to ionizing radiation we observed differential expression in luminal and basal-like cells of a member of the APOBEC enzyme family, recently identified as a critical driver of an oncogenic signature. Our findings suggest that this enzyme is active in the normal breast epithelium during DNA damage response. We demonstrate the practical application of a new method for assessing cell-type specific change in mixed cultures, especially the analysis of normal primary cultures, overcoming a major technical issue of dissecting the response of multiple cell types in a heterogeneous population.

  1. Cell-type specific gene expression profiles of leukocytes in human peripheral blood

    PubMed Central

    Palmer, Chana; Diehn, Maximilian; Alizadeh, Ash A; Brown, Patrick O

    2006-01-01

    Background Blood is a complex tissue comprising numerous cell types with distinct functions and corresponding gene expression profiles. We attempted to define the cell type specific gene expression patterns for the major constituent cells of blood, including B-cells, CD4+ T-cells, CD8+ T-cells, lymphocytes and granulocytes. We did this by comparing the global gene expression profiles of purified B-cells, CD4+ T-cells, CD8+ T-cells, granulocytes, and lymphocytes using cDNA microarrays. Results Unsupervised clustering analysis showed that similar cell populations from different donors share common gene expression profiles. Supervised analyses identified gene expression signatures for B-cells (427 genes), T-cells (222 genes), CD8+ T-cells (23 genes), granulocytes (411 genes), and lymphocytes (67 genes). No statistically significant gene expression signature was identified for CD4+ cells. Genes encoding cell surface proteins were disproportionately represented among the genes that distinguished among the lymphocyte subpopulations. Lymphocytes were distinguishable from granulocytes based on their higher levels of expression of genes encoding ribosomal proteins, while granulocytes exhibited characteristic expression of various cell surface and inflammatory proteins. Conclusion The genes comprising the cell-type specific signatures encompassed many of the genes already known to be involved in cell-type specific processes, and provided clues that may prove useful in discovering the functions of many still unannotated genes. The most prominent feature of the cell type signature genes was the enrichment of genes encoding cell surface proteins, perhaps reflecting the importance of specialized systems for sensing the environment to the physiology of resting leukocytes. PMID:16704732

  2. Locus- and cell type-specific epigenetic switching during cellular differentiation in mammals

    PubMed Central

    Zhao, Ying-Tao; Fasolino, Maria; Zhou, Zhaolan

    2016-01-01

    BACKGROUND Epigenomic reconfiguration, including changes in DNA methylation and histone modifications, is crucial for the differentiation of embryonic stem cells (ESCs) into somatic cells. However, the extent to which the epigenome is reconfigured and the interplay between components of the epigenome during cellular differentiation remain poorly defined. METHODS We systematically analyzed and compared DNA methylation, various histone modification, and transcriptome profiles in ESCs with those of two distinct types of somatic cells from human and mouse. RESULTS We found that global DNA methylation levels are lower in somatic cells compared to ESCs in both species. We also found that 80% of regions with histone modification occupancy differ between human ESCs and the two human somatic cell types. Approximately 70% of the reconfigurations in DNA methylation and histone modifications are locus- and cell type-specific. Intriguingly, the loss of DNA methylation is accompanied by the gain of different histone modifications in a locus- and cell type-specific manner. Further examination of transcriptional changes associated with epigenetic reconfiguration at promoter regions revealed an epigenetic switching for gene regulation—a transition from stable gene silencing mediated by DNA methylation in ESCs to flexible gene repression facilitated by repressive histone modifications in somatic cells. CONCLUSIONS Our findings demonstrate that the epigenome is reconfigured in a locus- and cell type-specific manner and epigenetic switching is common during cellular differentiation in both human and mouse. PMID:28261266

  3. Cell type-specific glycoconjugates of collecting duct cells during maturation of the rat kidney.

    PubMed

    Holthöfer, H

    1988-08-01

    The ontogeny of lectin-positive epithelial cell types and the maturation of polarized expression of the glycocalyx of the collecting ducts (CD) of the rat kidney were studied from samples of 18th-day fetal and neonatal kidneys of various ages. Lectins from Dolichos biflorus (DBA) and Vicia villosa (VVA), with preferential affinity to principal cells, stained virtually all CD cells of the fetal kidneys. However, within two days postnatally, the number of cells positive for DBA and VVA decreased to amounts found in the adult kidneys. Moreover, a characteristic change occurred rapidly after birth in the intracellular polarization of the reactive glycoconjugates, from a uniform plasmalemmal to a preferentially apical staining. In contrast, lectins from Arachis hypogaea (PNA), Maclura pomifera (MPA) and Lotus tetragonolobus (LTA), reacting indiscriminatively with principal and intercalated cells of adult kidneys, stained most CD cells in the fetal kidneys, and failed to show any postnatal change in the amount of positive cells or in the intracellular polarization. The immunocytochemical tests for (Na + K)-ATPase and carbonic anhydrase (CA II) revealed the characteristic postnatal decrease in the amount of principal cells and simultaneous increase in the amount of CA II rich intercalated cells. DBA and VVA reactive cells also decreased postnatally, paralleling the changes observed in the (Na + K)-ATPase positive principal cells. The present results suggest that the expression of the cell type-specific glycocalyx of principal and intercalated cells is developmentally regulated, undergoes profound changes during maturation, and is most likely associated with electrolyte transport phenomena.

  4. TU-tagging: cell type specific RNA isolation from intact complex tissues

    PubMed Central

    Miller, Michael R.; Robinson, Kristin J.; Cleary, Michael D.; Doe, Chris Q.

    2009-01-01

    We show that the combination of spatially restricted uracil phosphoribosyltransferase (UPRT) expression with 4-thiouracil (4TU) delivery can be used to label and purify cell type specific RNA from intact complex tissues in Drosophila melanogaster. This method is useful for isolating RNA from cell types that are difficult to isolate by dissection or dissociation methods and should work in many organisms, including mammals and other vertebrates. PMID:19430475

  5. miRConnect: Identifying Effector Genes of miRNAs and miRNA Families in Cancer Cells

    PubMed Central

    Larsen, Niels; Kjems, Jørgen; Lund, Anders H.; Peter, Marcus E.

    2011-01-01

    micro(mi)RNAs are small non-coding RNAs that negatively regulate expression of most mRNAs. They are powerful regulators of various differentiation stages, and the expression of genes that either negatively or positively correlate with expressed miRNAs is expected to hold information on the biological state of the cell and, hence, of the function of the expressed miRNAs. We have compared the large amount of available gene array data on the steady state system of the NCI60 cell lines to two different data sets containing information on the expression of 583 individual miRNAs. In addition, we have generated custom data sets containing expression information of 54 miRNA families sharing the same seed match. We have developed a novel strategy for correlating miRNAs with individual genes based on a summed Pearson Correlation Coefficient (sPCC) that mimics an in silico titration experiment. By focusing on the genes that correlate with the expression of miRNAs without necessarily being direct targets of miRNAs, we have clustered miRNAs into different functional groups. This has resulted in the identification of three novel miRNAs that are linked to the epithelial-to-mesenchymal transition (EMT) in addition to the known EMT regulators of the miR-200 miRNA family. In addition, an analysis of gene signatures associated with EMT, c-MYC activity, and ribosomal protein gene expression allowed us to assign different activities to each of the functional clusters of miRNAs. All correlation data are available via a web interface that allows investigators to identify genes whose expression correlates with the expression of single miRNAs or entire miRNA families. miRConnect.org will aid in identifying pathways regulated by miRNAs without requiring specific knowledge of miRNA targets. PMID:22046300

  6. Cell-Type-Specific mRNA Purification by Translating Ribosome Affinity Purification (TRAP)

    PubMed Central

    Heiman, Myriam; Kulicke, Ruth; Fenster, Robert J.; Greengard, Paul; Heintz, Nathaniel

    2014-01-01

    Cellular diversity and architectural complexity create barriers to understanding the function of the mammalian central nervous system (CNS) at a molecular level. To address this problem, we recently developed a methodology that provides the ability to profile the entire translated mRNA complement of any genetically defined cell population. This methodology, which we termed translating ribosome affinity purification, or TRAP, combines cell-type-specific transgene expression with affinity purification of translating ribosomes. TRAP can be used to study the cell-type-specific mRNA profiles of any genetically defined cell type, and has been successfully used to date in organisms ranging from D. melanogaster to mice and human cultured cells. Unlike other methodologies that rely upon micro-dissection, cell panning, or cell sorting, the TRAP methodology bypasses the need for tissue fixation or single-cell suspensions (and potential artifacts these treatments introduce), and reports on mRNAs in the entire cell body. This protocol provides a step-by-step guide to implementing the TRAP methodology, which takes two days to complete once all materials are in hand. PMID:24810037

  7. Ribosome Profiling Reveals a Cell-Type-Specific Translational Landscape in Brain Tumors

    PubMed Central

    Gonzalez, Christian; Sims, Jennifer S.; Hornstein, Nicholas; Mela, Angeliki; Garcia, Franklin; Lei, Liang; Gass, David A.; Amendolara, Benjamin; Bruce, Jeffrey N.

    2014-01-01

    Glioma growth is driven by signaling that ultimately regulates protein synthesis. Gliomas are also complex at the cellular level and involve multiple cell types, including transformed and reactive cells in the brain tumor microenvironment. The distinct functions of the various cell types likely lead to different requirements and regulatory paradigms for protein synthesis. Proneural gliomas can arise from transformation of glial progenitors that are driven to proliferate via mitogenic signaling that affects translation. To investigate translational regulation in this system, we developed a RiboTag glioma mouse model that enables cell-type-specific, genome-wide ribosome profiling of tumor tissue. Infecting glial progenitors with Cre-recombinant retrovirus simultaneously activates expression of tagged ribosomes and delivers a tumor-initiating mutation. Remarkably, we find that although genes specific to transformed cells are highly translated, their translation efficiencies are low compared with normal brain. Ribosome positioning reveals sequence-dependent regulation of ribosomal activity in 5′-leaders upstream of annotated start codons, leading to differential translation in glioma compared with normal brain. Additionally, although transformed cells express a proneural signature, untransformed tumor-associated cells, including reactive astrocytes and microglia, express a mesenchymal signature. Finally, we observe the same phenomena in human disease by combining ribosome profiling of human proneural tumor and non-neoplastic brain tissue with computational deconvolution to assess cell-type-specific translational regulation. PMID:25122893

  8. Cell-Type Specific Modulation of Pituitary Cells by Activin, Inhibin and Follistatin

    PubMed Central

    Bilezikjian, Louise M.; Justice, Nicholas J.; Blackler, Alissa N.; Wiater, Ezra; Vale, Wylie W.

    2012-01-01

    Activins are multifunctional proteins and members of the TGF-β superfamily. Activins are expressed locally in most tissues and, analogous to the actions of other members of this large family of pleiotropic factors, play prominent roles in the regulation of diverse biological processes in both differentiated and embryonic stem cells. They have an essential role in maintaining tissue homeostasis in the adult and are known to contribute to the developmental programs in the embryo. Activins are further implicated in the growth and metastasis of tumor cells. Through distinct modes of action, inhibins and follistatins function as antagonists of activin and several other TGF-β family members, including a subset of BMPs/GDFs, and modulate cellular responses and the signaling cascades downstream of these ligands. In the pituitary, the activin pathway is known to regulate key aspects of gonadotrope functions and also exert effects on other pituitary cell types. As in other tissues, activin is produced locally by pituitary cells and acts locally by exerting cell-type specific actions on gonadotropes. These local actions of activin on gonadotropes are modulated by the autocrine/paracrine actions of locally secreted follistatin and by the feedback actions of gonadal inhibin. Knowledge about the mechanism of activin, inhibin and follistatin actions is providing information about their importance for pituitary function as well as their contribution to the pathophysiology of pituitary adenomas. The aim of this review is to highlight recent findings and summarize the evidence that supports the important functions of activin, inhibin and follistatin in the pituitary. PMID:22330643

  9. Cell-type-specific, Aptamer-functionalized Agents for Targeted Disease Therapy

    PubMed Central

    Zhou, Jiehua; Rossi, John J.

    2014-01-01

    One hundred years ago, Dr. Paul Ehrlich popularized the “magic bullet” concept for cancer therapy in which an ideal therapeutic agent would only kill the specific tumor cells it targeted. Since then, “targeted therapy” that specifically targets the molecular defects responsible for a patient's condition has become a long-standing goal for treating human disease. However, safe and efficient drug delivery during the treatment of cancer and infectious disease remains a major challenge for clinical translation and the development of new therapies. The advent of SELEX technology has inspired many groundbreaking studies that successfully adapted cell-specific aptamers for targeted delivery of active drug substances in both in vitro and in vivo models. By covalently linking or physically functionalizing the cell-specific aptamers with therapeutic agents, such as siRNA, microRNA, chemotherapeutics or toxins, or delivery vehicles, such as organic or inorganic nanocarriers, the targeted cells and tissues can be specifically recognized and the therapeutic compounds internalized, thereby improving the local concentration of the drug and its therapeutic efficacy. Currently, many cell-type-specific aptamers have been developed that can target distinct diseases or tissues in a cell-type-specific manner. In this review, we discuss recent advances in the use of cell-specific aptamers for targeted disease therapy, as well as conjugation strategies and challenges. PMID:24936916

  10. Transition to chaos in random networks with cell-type-specific connectivity

    PubMed Central

    Aljadeff, Johnatan; Stern, Merav; Sharpee, Tatyana

    2015-01-01

    In neural circuits, statistical connectivity rules strongly depend on cell-type identity. We study dynamics of neural networks with cell-type specific connectivity by extending the dynamic mean field method, and find that these networks exhibit a phase transition between silent and chaotic activity. By analyzing the locus of this transition, we derive a new result in random matrix theory: the spectral radius of a random connectivity matrix with block-structured variances. We apply our results to show how a small group of hyper-excitable neurons within the network can significantly increase the network’s computational capacity by bringing it into the chaotic regime. PMID:25768781

  11. Cell type-specific affinity purification of nuclei for chromatin profiling in whole animals.

    PubMed

    Steiner, Florian A; Henikoff, Steven

    2015-01-01

    Analyzing cell differentiation during development in a complex organism requires the analysis of expression and chromatin profiles in individual cell types. Our laboratory has developed a simple and generally applicable strategy to purify specific cell types from whole organisms for simultaneous analysis of chromatin and expression. The method, termed INTACT for Isolation of Nuclei TAgged in specific Cell Types, depends on the expression of an affinity-tagged nuclear envelope protein in the cell type of interest. These nuclei can be affinity-purified from the total pool of nuclei and used as a source for RNA and chromatin. The method serves as a simple and scalable alternative to FACS sorting or laser capture microscopy to circumvent the need for expensive equipment and specialized skills. This chapter provides detailed protocols for the cell-type specific purification of nuclei from Caenorhabditis elegans.

  12. L1 regularization facilitates detection of cell type-specific parameters in dynamical systems

    PubMed Central

    Steiert, Bernhard; Timmer, Jens; Kreutz, Clemens

    2016-01-01

    Motivation: A major goal of drug development is to selectively target certain cell types. Cellular decisions influenced by drugs are often dependent on the dynamic processing of information. Selective responses can be achieved by differences between the involved cell types at levels of receptor, signaling, gene regulation or further downstream. Therefore, a systematic approach to detect and quantify cell type-specific parameters in dynamical systems becomes necessary. Results: Here, we demonstrate that a combination of nonlinear modeling with L1 regularization is capable of detecting cell type-specific parameters. To adapt the least-squares numerical optimization routine to L1 regularization, sub-gradient strategies as well as truncation of proposed optimization steps were implemented. Likelihood-ratio tests were used to determine the optimal regularization strength resulting in a sparse solution in terms of a minimal number of cell type-specific parameters that is in agreement with the data. By applying our implementation to a realistic dynamical benchmark model of the DREAM6 challenge we were able to recover parameter differences with an accuracy of 78%. Within the subset of detected differences, 91% were in agreement with their true value. Furthermore, we found that the results could be improved using the profile likelihood. In conclusion, the approach constitutes a general method to infer an overarching model with a minimum number of individual parameters for the particular models. Availability and Implementation: A MATLAB implementation is provided within the freely available, open-source modeling environment Data2Dynamics. Source code for all examples is provided online at http://www.data2dynamics.org/. Contact: bernhard.steiert@fdm.uni-freiburg.de PMID:27587694

  13. Innate immune response to pulmonary contusion: Identification of cell-type specific inflammatory responses

    PubMed Central

    Hoth, J. Jason; Wells, Jonathan D.; Yoza, Barbara K.; McCall, Charles E.

    2012-01-01

    Lung injury from pulmonary contusion is a common traumatic injury, predominantly seen after blunt chest trauma such as in vehicular accidents. The local and systemic inflammatory response to injury includes activation of innate immune receptors, elaboration of a variety inflammatory mediators, and recruitment of inflammatory cells to the injured lung. Using a mouse model of pulmonary contusion, we had previously shown that innate immune Toll like receptors 2 and 4 (TLR2 and TLR4) mediate the inflammatory response to lung injury. In this study, we used chimeric mice generated by adoptive bone marrow (BM) transfer between TLR2−/− or TLR4−/− and wild-type (WT) mice. We found that in the lung, both BM-derived and non-myeloid cells contribute to TLR-dependent inflammatory responses after injury in a cell type specific manner. We also show a novel TLR2 dependent injury mechanism that is associated with enhanced airway epithelial cell apoptosis and increased pulmonary FasL and Fas expression in the lungs from injured mice. Thus, in addition to cardiopulmonary physiological dysfunction, cell type specific TLR and their differential response to injury may provide novel specific targets for management of patients with pulmonary contusion. PMID:22293596

  14. Innate immune response to pulmonary contusion: identification of cell type-specific inflammatory responses.

    PubMed

    Hoth, J Jason; Wells, Jonathan D; Yoza, Barbara K; McCall, Charles E

    2012-04-01

    Lung injury from pulmonary contusion is a common traumatic injury, predominantly seen after blunt chest trauma, such as in vehicular accidents. The local and systemic inflammatory response to injury includes activation of innate immune receptors, elaboration of a variety of inflammatory mediators, and recruitment of inflammatory cells to the injured lung. Using a mouse model of pulmonary contusion, we had previously shown that innate immune Toll-like receptors 2 and 4 (TLR2 and TLR4) mediate the inflammatory response to lung injury. In this study, we used chimeric mice generated by adoptive bone marrow transfer between TLR2 or TLR4 and wild-type mice. We found that, in the lung, both bone marrow-derived and nonmyeloid cells contribute to TLR-dependent inflammatory responses after injury in a cell type-specific manner. We also show a novel TLR2-dependent injury mechanism that is associated with enhanced airway epithelial cell apoptosis and increased pulmonary FasL and Fas expression in the lungs from injured mice. Thus, in addition to cardiopulmonary physiological dysfunction, cell type-specific TLR and their differential response to injury may provide novel specific targets for management of patients with pulmonary contusion.

  15. Integrating and mining the chromatin landscape of cell-type specificity using self-organizing maps

    PubMed Central

    Mortazavi, Ali; Pepke, Shirley; Jansen, Camden; Marinov, Georgi K.; Ernst, Jason; Kellis, Manolis; Hardison, Ross C.; Myers, Richard M.; Wold, Barbara J.

    2013-01-01

    We tested whether self-organizing maps (SOMs) could be used to effectively integrate, visualize, and mine diverse genomics data types, including complex chromatin signatures. A fine-grained SOM was trained on 72 ChIP-seq histone modifications and DNase-seq data sets from six biologically diverse cell lines studied by The ENCODE Project Consortium. We mined the resulting SOM to identify chromatin signatures related to sequence-specific transcription factor occupancy, sequence motif enrichment, and biological functions. To highlight clusters enriched for specific functions such as transcriptional promoters or enhancers, we overlaid onto the map additional data sets not used during training, such as ChIP-seq, RNA-seq, CAGE, and information on cis-acting regulatory modules from the literature. We used the SOM to parse known transcriptional enhancers according to the cell-type-specific chromatin signature, and we further corroborated this pattern on the map by EP300 (also known as p300) occupancy. New candidate cell-type-specific enhancers were identified for multiple ENCODE cell types in this way, along with new candidates for ubiquitous enhancer activity. An interactive web interface was developed to allow users to visualize and custom-mine the ENCODE SOM. We conclude that large SOMs trained on chromatin data from multiple cell types provide a powerful way to identify complex relationships in genomic data at user-selected levels of granularity. PMID:24170599

  16. Identifying and mapping cell-type-specific chromatin programming of gene expression.

    PubMed

    Marstrand, Troels T; Storey, John D

    2014-02-11

    A problem of substantial interest is to systematically map variation in chromatin structure to gene-expression regulation across conditions, environments, or differentiated cell types. We developed and applied a quantitative framework for determining the existence, strength, and type of relationship between high-resolution chromatin structure in terms of DNaseI hypersensitivity and genome-wide gene-expression levels in 20 diverse human cell types. We show that ∼25% of genes show cell-type-specific expression explained by alterations in chromatin structure. We find that distal regions of chromatin structure (e.g., ±200 kb) capture more genes with this relationship than local regions (e.g., ±2.5 kb), yet the local regions show a more pronounced effect. By exploiting variation across cell types, we were capable of pinpointing the most likely hypersensitive sites related to cell-type-specific expression, which we show have a range of contextual uses. This quantitative framework is likely applicable to other settings aimed at relating continuous genomic measurements to gene-expression variation.

  17. Cell-type specific cis-regulatory networks: insights from Hox transcription factors.

    PubMed

    Polychronidou, Maria; Lohmann, Ingrid

    2013-01-01

    Hox proteins are a prominent class of transcription factors that specify cell and tissue identities in animal embryos. In sharp contrast to tissue-specifically expressed transcription factors, which coordinate regulatory pathways leading to the differentiation of a selected tissue, Hox proteins are active in many different cell types but are nonetheless able to differentially regulate gene expression in a context-dependent manner. This particular feature makes Hox proteins ideal candidates for elucidating the mechanisms employed by transcription factors to achieve tissue-specific functions in multi-cellular organisms. Here we discuss how the recent genome-wide identification and characterization of Hox cis-regulatory elements has provided insight concerning the molecular mechanisms underlying the high spatiotemporal specificity of Hox proteins. In particular, it was shown that Hox transcriptional outputs depend on the cell-type specific interplay of the different Hox proteins with co-regulatory factors as well as with epigenetic modifiers. Based on these observations it becomes clear that cell-type specific approaches are required for dissecting the tissue-specific Hox regulatory code. Identification and comparative analysis of Hox cis-regulatory elements driving target gene expression in different cell types in combination with analyses on how cofactors, epigenetic modifiers and protein-protein interactions mediate context-dependent Hox function will elucidate the mechanistic basis of tissue-specific gene regulation.

  18. Transcription factor co-localization patterns affect human cell type-specific gene expression

    PubMed Central

    2012-01-01

    Background Cellular development requires the precise control of gene expression states. Transcription factors are involved in this regulatory process through their combinatorial binding with DNA. Information about transcription factor binding sites can help determine which combinations of factors work together to regulate a gene, but it is unclear how far the binding data from one cell type can inform about regulation in other cell types. Results By integrating data on co-localized transcription factor binding sites in the K562 cell line with expression data across 38 distinct hematopoietic cell types, we developed regression models to describe the relationship between the expression of target genes and the transcription factors that co-localize nearby. With K562 binding sites identifying the predictors, the proportion of expression explained by the models is statistically significant only for monocytic cells (p-value< 0.001), which are closely related to K562. That is, cell type specific binding patterns are crucial for choosing the correct transcription factors for the model. Comparison of predictors obtained from binding sites in the GM12878 cell line with those from K562 shows that the amount of difference between binding patterns is directly related to the quality of the prediction. By identifying individual genes whose expression is predicted accurately by the binding sites, we are able to link transcription factors FOS, TAF1 and YY1 to a sparsely studied gene LRIG2. We also find that the activity of a transcription factor may be different depending on the cell type and the identity of other co-localized factors. Conclusion Our approach shows that gene expression can be explained by a modest number of co-localized transcription factors, however, information on cell-type specific binding is crucial for understanding combinatorial gene regulation. PMID:22721266

  19. miRNAs in mtDNA-less cell mitochondria

    PubMed Central

    Dasgupta, N; Peng, Y; Tan, Z; Ciraolo, G; Wang, D; Li, R

    2015-01-01

    The novel regulation mechanism in mtDNA-less cells was investigated. Very low mtDNA copy in mtDNA-less 206 ρ° cells was identified. But no 13 mitochondria-specific proteins were translated in 206 ρ° cells. Their mitochondrial respiration complexes V, III and II were 86.5, 29.4 and 49.6% of 143B cells, respectively. Complexes I and IV completely lack in 206 ρ° cells. Non-mitochondrial respiration to generate ATP in 206 ρ° cells was discovered. The expression levels of some mitochondrial RNAs including 12S rRNA, COX1, COX2, COX3, ND4 and ND5 were low. However, ND1, ND3 and Cyto b were not expressed in 206 ρ° cells. Unequal transcription of mitochondrial RNAs indicated the post-transcriptional cleavage and processing mechanisms in the regulation of mitochondrial gene expression in 206 ρ° cells. MicroRNAs (miRNAs) may modulate these mitochondrial RNA expression in these cells. RNA-induced silencing complex indeed within 206 ρ° cell mitochondria indicated miRNAs in 206 ρ° cell mitochondria. miRNA profile in mtDNA-less 206 ρ° cells was studied by next-generation sequencing of small RNAs. Several mitochondria-enriched miRNAs such as miR-181c-5p and miR-146a-5p were identified in 206 ρ° cell mitochondria. miR-181c-5p and miR-146a-5p had 23 and 19 potential targets on mitochondrial RNAs respectively, and these two miRNAs had multiple targets on mitochondria-associated messenger RNAs encoded by nuclear genes. These data provided the first direct evidence that miRNAs were imported into mitochondria and regulated mitochondrial RNA expressions. PMID:27551440

  20. Cell-type-specific signatures of microRNAs on target mRNA expression.

    PubMed

    Sood, Pranidhi; Krek, Azra; Zavolan, Mihaela; Macino, Giuseppe; Rajewsky, Nikolaus

    2006-02-21

    Although it is known that the human genome contains hundreds of microRNA (miRNA) genes and that each miRNA can regulate a large number of mRNA targets, the overall effect of miRNAs on mRNA tissue profiles has not been systematically elucidated. Here, we show that predicted human mRNA targets of several highly tissue-specific miRNAs are typically expressed in the same tissue as the miRNA but at significantly lower levels than in tissues where the miRNA is not present. Conversely, highly expressed genes are often enriched in mRNAs that do not have the recognition motifs for the miRNAs expressed in these tissues. Together, our data support the hypothesis that miRNA expression broadly contributes to tissue specificity of mRNA expression in many human tissues. Based on these insights, we apply a computational tool to directly correlate 3' UTR motifs with changes in mRNA levels upon miRNA overexpression or knockdown. We show that this tool can identify functionally important 3' UTR motifs without cross-species comparison.

  1. An Efficient Antipodal Cell Isolation Method for Screening of Cell Type-Specific Genes in Arabidopsis thaliana

    PubMed Central

    Sun, Meng-xiang

    2016-01-01

    In flowering plants, the mature embryo sac consists of seven cells, namely two synergid cells and an egg cell at the micropylar end, one central cell, and three antipodal cells at the chalazal end. Excluding the antipodal cell, as a model for the study of cell fate determination and cell type specification, the roles of these embryo sac component cells in fertilization and seed formation have been widely investigated. At this time, little is known regarding the function of antipodal cells and their cell type-specific gene expression patterns. One reason for this is difficulties related to the observation and isolation of cells for detailed functional analyses. Here, we report a method for antipodal cell isolation and transcriptome analysis. We identified antipodal cell-specific marker line K44-1, and based on this marker line, established a procedure allowing us to isolate antipodal cells with both high quality and quantity. PCR validation of antipodal-specific genes from antipodal cell cDNA showed that the isolated cells are qualified and can be used for transcriptome analysis and screening of cell type-specific marker genes. The isolated cells could keep viable for a week in culture condition. This method can be used to efficiently isolate antipodal cells of high quality and will promote the functional investigation of antipodal cells in Arabidopsis thaliana. This increases our understanding of the molecular regulatory mechanism of antipodal cell specification. PMID:27875553

  2. Ligation-free ribosome profiling of cell type-specific translation in the brain.

    PubMed

    Hornstein, Nicholas; Torres, Daniela; Das Sharma, Sohani; Tang, Guomei; Canoll, Peter; Sims, Peter A

    2016-07-05

    Ribosome profiling has emerged as a powerful tool for genome-wide measurements of translation, but library construction requires multiple ligation steps and remains cumbersome relative to more conventional deep-sequencing experiments. We report a new, ligation-free approach to ribosome profiling that does not require ligation. Library construction for ligation-free ribosome profiling can be completed in one day with as little as 1 ng of purified RNA footprints. We apply ligation-free ribosome profiling to mouse brain tissue to identify new patterns of cell type-specific translation and test its ability to identify translational targets of mTOR signaling in the brain.

  3. Cell Type-Specific Effects of Mutant DISC1: A Proteomics Study.

    PubMed

    Xia, Meng; Broek, Jantine A C; Jouroukhin, Yan; Schoenfelder, Jeannine; Abazyan, Sofya; Jaaro-Peled, Hanna; Sawa, Akira; Bahn, Sabine; Pletnikov, Mikhail

    2016-05-01

    Despite the recent progress in psychiatric genetics, very few studies have focused on genetic risk factors in glial cells that, compared to neurons, can manifest different molecular pathologies underlying psychiatric disorders. In order to address this issue, we studied the effects of mutant disrupted in schizophrenia 1 (DISC1), a genetic risk factor for schizophrenia, in cultured primary neurons and astrocytes using an unbiased mass spectrometry-based proteomic approach. We found that selective expression of mutant DISC1 in neurons affects a wide variety of proteins predominantly involved in neuronal development (e.g., SOX1) and vesicular transport (Rab proteins), whereas selective expression of mutant DISC1 in astrocytes produces changes in the levels of mitochondrial (GDPM), nuclear (TMM43) and cell adhesion (ECM2) proteins. The present study demonstrates that DISC1 variants can perturb distinct molecular pathways in a cell type-specific fashion to contribute to psychiatric disorders through heterogenic effects in diverse brain cells.

  4. Cell Type-Specific Effects of Mutant DISC1: A Proteomics Study

    PubMed Central

    Xia, Meng; Broek, Jantine A.C.; Jouroukhin, Yan; Schoenfelder, Jeannine; Abazyan, Sofya; Jaaro-Peled, Hanna; Sawa, Akira; Bahn, Sabine; Pletnikov, Mikhail

    2016-01-01

    Despite the recent progress in psychiatric genetics, very few studies have focused on genetic risk factors in glial cells that, compared to neurons, can manifest different molecular pathologies underlying psychiatric disorders. In order to address this issue, we studied the effects of mutant disrupted in schizophrenia 1 (DISC1), a genetic risk factor for schizophrenia, in cultured primary neurons and astrocytes using an unbiased mass spectrometry-based proteomic approach. We found that selective expression of mutant DISC1 in neurons affects a wide variety of proteins predominantly involved in neuronal development (e.g., SOX1) and vesicular transport (Rab proteins), whereas selective expression of mutant DISC1 in astrocytes produces changes in the levels of mitochondrial (GDPM), nuclear (TMM43) and cell adhesion (ECM2) proteins. The present study demonstrates that DISC1 variants can perturb distinct molecular pathways in a cell type-specific fashion to contribute to psychiatric disorders through heterogenic effects in diverse brain cells. PMID:27606318

  5. Systematic and Cell Type-Specific Telomere Length Changes in Subsets of Lymphocytes

    PubMed Central

    Lin, Jue; Cheon, Joshua; Brown, Rashida; Coccia, Michael; Puterman, Eli; Aschbacher, Kirstin; Sinclair, Elizabeth; Epel, Elissa; Blackburn, Elizabeth H.

    2016-01-01

    Telomeres, the protective DNA-protein complexes at the ends of linear chromosomes, are important for genome stability. Leukocyte or peripheral blood mononuclear cell (PBMC) telomere length is a potential biomarker for human aging that integrates genetic, environmental, and lifestyle factors and is associated with mortality and risks for major diseases. However, only a limited number of studies have examined longitudinal changes of telomere length and few have reported data on sorted circulating immune cells. We examined the average telomere length (TL) in CD4+, CD8+CD28+, and CD8+CD28− T cells, B cells, and PBMCs, cross-sectionally and longitudinally, in a cohort of premenopausal women. We report that TL changes over 18 months were correlated among these three T cell types within the same participant. Additionally, PBMC TL change was also correlated with those of all three T cell types, and B cells. The rate of shortening for B cells was significantly greater than for the three T cell types. CD8+CD28− cells, despite having the shortest TL, showed significantly more rapid attrition when compared to CD8+CD28+ T cells. These results suggest systematically coordinated, yet cell type-specific responses to factors and pathways contribute to telomere length regulation. PMID:26977417

  6. Cell type-specific glycosylation of Orai1 modulates store-operated Ca2+ entry.

    PubMed

    Dörr, Kathrin; Kilch, Tatiana; Kappel, Sven; Alansary, Dalia; Schwär, Gertrud; Niemeyer, Barbara A; Peinelt, Christine

    2016-03-08

    N-glycosylation of cell surface proteins affects protein function, stability, and interaction with other proteins. Orai channels, which mediate store-operated Ca(2+) entry (SOCE), are composed of N-glycosylated subunits. Upon activation by Ca(2+) sensor proteins (stromal interaction molecules STIM1 or STIM2) in the endoplasmic reticulum, Orai Ca(2+) channels in the plasma membrane mediate Ca(2+) influx. Lectins are carbohydrate-binding proteins, and Siglecs are a family of sialic acid-binding lectins with immunoglobulin-like repeats. Using Western blot analysis and lectin-binding assays from various primary human cells and cancer cell lines, we found that glycosylation of Orai1 is cell type-specific. Ca(2+) imaging experiments and patch-clamp experiments revealed that mutation of the only glycosylation site of Orai1 (Orai1N223A) enhanced SOCE in Jurkat T cells. Knockdown of the sialyltransferase ST6GAL1 reduced α-2,6-linked sialic acids in the glycan structure of Orai1 and was associated with increased Ca(2+) entry in Jurkat T cells. In human mast cells, inhibition of sialyl sulfation altered the N-glycan of Orai1 (and other proteins) and increased SOCE. These data suggest that cell type-specific glycosylation influences the interaction of Orai1 with specific lectins, such as Siglecs, which then attenuates SOCE. In summary, the glycosylation state of Orai1 influences SOCE-mediated Ca(2+) signaling and, thus, may contribute to pathophysiological Ca(2+) signaling observed in immune disease and cancer.

  7. Cell type specificity and host genetic polymorphisms influence antibody-dependent enhancement of dengue virus infection.

    PubMed

    Boonnak, Kobporn; Dambach, Kaitlyn M; Donofrio, Gina C; Tassaneetrithep, Boonrat; Marovich, Mary A

    2011-02-01

    Antibody-dependent enhancement (ADE) is implicated in severe, usually secondary, dengue virus (DV) infections. Preexisting heterotypic antibodies, via their Fc-gamma receptor (FcγR) interactions, may increase disease severity through enhanced target cell infection. Greater numbers of infected target cells may contribute to higher viremia and excess cytokine levels often observed in severe disease. Monocytes, macrophages, and immature and mature dendritic cells (DC) are considered major cellular targets of DV. Apheresis of multiple donors allowed isolation of autologous primary myeloid target cell types for head-to-head comparison of infection rates, viral output, and cytokine production under direct infection (without antibody) or ADE conditions (with antibody). All studied cell types except immature DC supported ADE. All cells undergoing ADE secreted proinflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]) at enhancement titers, but distinct cell-type-specific patterns were observed for other relevant proteins (alpha/beta interferon [IFN-α/β] and IL-10). Macrophages produced type I interferons (IFN-α/β) that were modulated by ADE. Mature DC mainly secreted IFN-β. Interestingly, only monocytes secreted IL-10, and only upon antibody-enhanced infection. While ADE infection rates were remarkably consistent in monocytes (10 to 15%) across donors, IL-10 protein levels varied according to previously described regulatory single nucleotide polymorphisms (SNPs) in the IL-10 promoter region. The homozygous GCC haplotype was associated with high-level IL-10 secretion, while the ACC and ATA haplotypes produced intermediate and low levels of IL-10, respectively. Our data suggest that ADE effects are cell type specific, are influenced by host genetics, and, depending on relative infection rates, may further contribute to the complexity of DV pathogenesis.

  8. Cell type-specific transcriptional regulation of the gene encoding importin-{alpha}1

    SciTech Connect

    Kamikawa, Yasunao; Yasuhara, Noriko; Yoneda, Yoshihiro

    2011-08-15

    Importin-{alpha}1 belongs to a receptor family that recognizes classical nuclear localization signals. Encoded by Kpna2, this receptor subtype is highly expressed in mouse embryonic stem (ES) cells. In this study, we identified a critical promoter region in Kpna2 and showed that the expression of this gene is differentially regulated in ES cells and NIH3T3 cells. Conserved CCAAT boxes are required for Kpna2 promoter activity in both ES and NIH3T3 cells. Interestingly, deletion of the region from nucleotide position - 251 to - 179 bp resulted in a drastic reduction in Kpna2 transcriptional activity only in ES cells. This region contains Krueppel-like factor (Klf) binding sequences and is responsible for transactivation of the gene by Klf2 and Klf4. Accordingly, endogenous Kpna2 mRNA levels decreased in response to depletion of Klf2 and Klf4 in ES cells. Our results suggest that Klf2 and Klf4 function redundantly to drive high level of Kpna2 expression in ES cells. -- Research Highlights: {yields} We showed the cell type-specific transcriptional regulation of Kpna2 encoding importin-al. {yields} NF-Y binds the CCAAT boxes to activate Kpna2 transcription in NIH3T3 cells. {yields} Klf2 and Klf4 redundantly activate the expression of Kpna2 in ES cells.

  9. Cell type-specific synaptic dynamics of synchronized bursting in the juvenile CA3 rat hippocampus.

    PubMed

    Aradi, Ildiko; Maccaferri, Gianmaria

    2004-10-27

    Spontaneous synchronous bursting of the CA3 hippocampus in vitro is a widely studied model of physiological and pathological network synchronization. The role of inhibitory conductances during network bursting is not understood in detail, despite the fact that several antiepileptic drugs target GABA(A) receptors. Here, we show that the first manifestation of a burst event is a cell type-specific flurry of GABA(A) receptor-mediated inhibitory input to pyramidal cells, but not to stratum oriens horizontal interneurons. Moreover, GABA(A) receptor-mediated synaptic input is proportionally smaller in these interneurons compared with pyramidal cells. Computational models and dynamic-clamp studies using experimentally derived conductance waveforms indicate that both these factors modulate spike timing during synchronized activity. In particular, the different kinetics and the larger strength of GABAergic input to pyramidal cells defer action potential initiation and contribute to the observed delay of firing, so that the interneuronal activity leads the burst cycle. In contrast, excitatory inputs to both neuronal populations during a burst are kinetically similar, as required to maintain synchronicity. We also show that the natural pattern of activation of inhibitory and excitatory conductances during a synchronized burst cycle is different within the same neuronal population. In particular, GABA(A) receptor-mediated currents activate earlier and outlast the excitatory components driving the bursts. Thus, cell type-specific balance and timing of GABA(A) receptor-mediated input are critical to set the appropriate spike timing in pyramidal cells and interneurons and coordinate additional neurotransmitter release modulating burst strength and network frequency.

  10. Cell type-specific transcriptomics of hypothalamic energy-sensing neuron responses to weight-loss.

    PubMed

    Henry, Fredrick E; Sugino, Ken; Tozer, Adam; Branco, Tiago; Sternson, Scott M

    2015-09-02

    Molecular and cellular processes in neurons are critical for sensing and responding to energy deficit states, such as during weight-loss. Agouti related protein (AGRP)-expressing neurons are a key hypothalamic population that is activated during energy deficit and increases appetite and weight-gain. Cell type-specific transcriptomics can be used to identify pathways that counteract weight-loss, and here we report high-quality gene expression profiles of AGRP neurons from well-fed and food-deprived young adult mice. For comparison, we also analyzed Proopiomelanocortin (POMC)-expressing neurons, an intermingled population that suppresses appetite and body weight. We find that AGRP neurons are considerably more sensitive to energy deficit than POMC neurons. Furthermore, we identify cell type-specific pathways involving endoplasmic reticulum-stress, circadian signaling, ion channels, neuropeptides, and receptors. Combined with methods to validate and manipulate these pathways, this resource greatly expands molecular insight into neuronal regulation of body weight, and may be useful for devising therapeutic strategies for obesity and eating disorders.

  11. Cell type-specific transcriptomics of hypothalamic energy-sensing neuron responses to weight-loss

    PubMed Central

    Henry, Fredrick E; Sugino, Ken; Tozer, Adam; Branco, Tiago; Sternson, Scott M

    2015-01-01

    Molecular and cellular processes in neurons are critical for sensing and responding to energy deficit states, such as during weight-loss. Agouti related protein (AGRP)-expressing neurons are a key hypothalamic population that is activated during energy deficit and increases appetite and weight-gain. Cell type-specific transcriptomics can be used to identify pathways that counteract weight-loss, and here we report high-quality gene expression profiles of AGRP neurons from well-fed and food-deprived young adult mice. For comparison, we also analyzed Proopiomelanocortin (POMC)-expressing neurons, an intermingled population that suppresses appetite and body weight. We find that AGRP neurons are considerably more sensitive to energy deficit than POMC neurons. Furthermore, we identify cell type-specific pathways involving endoplasmic reticulum-stress, circadian signaling, ion channels, neuropeptides, and receptors. Combined with methods to validate and manipulate these pathways, this resource greatly expands molecular insight into neuronal regulation of body weight, and may be useful for devising therapeutic strategies for obesity and eating disorders. DOI: http://dx.doi.org/10.7554/eLife.09800.001 PMID:26329458

  12. Cell type-specific bipolar cell input to ganglion cells in the mouse retina.

    PubMed

    Neumann, S; Hüser, L; Ondreka, K; Auler, N; Haverkamp, S

    2016-03-01

    Many distinct ganglion cell types, which are the output elements of the retina, were found to encode for specific features of a visual scene such as contrast, color information or movement. The detailed composition of retinal circuits leading to this tuning of retinal ganglion cells, however, is apart from some prominent examples, largely unknown. Here we aimed to investigate if ganglion cell types in the mouse retina receive selective input from specific bipolar cell types or if they sample their synaptic input non-selectively from all bipolar cell types stratifying within their dendritic tree. To address this question we took an anatomical approach and immunolabeled retinae of two transgenic mouse lines (GFP-O and JAM-B) with markers for ribbon synapses and type 2 bipolar cells. We morphologically identified all green fluorescent protein (GFP)-expressing ganglion cell types, which co-stratified with type 2 bipolar cells and assessed the total number of bipolar input synapses and the proportion of synapses deriving from type 2 bipolar cells. Only JAM-B ganglion cells received synaptic input preferentially from bipolar cell types other than type 2 bipolar cells whereas the other analyzed ganglion cell types sampled their bipolar input most likely from all bipolar cell terminals within their dendritic arbor. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  13. Cell-free Circulating miRNA Biomarkers in Cancer

    PubMed Central

    Mo, Meng-Hsuan; Chen, Liang; Fu, Yebo; Wang, Wendy; Fu, Sidney W.

    2012-01-01

    Considerable attention and an enormous amount of resources have been dedicated to cancer biomarker discovery and validation. However, there are still a limited number of useful biomarkers available for clinical use. An ideal biomarker should be easily assayed with minimally invasive medical procedures but possess high sensitivity and specificity. Commonly used circulating biomarkers are proteins in serum, most of which require labor-intensive analysis hindered by low sensitivity in early tumor detection. Since the deregulation of microRNA (miRNA) is associated with cancer development and progression, profiling of circulating miRNAs has been used in a number of studies to identify novel minimally invasive miRNA biomarkers. In this review, we discuss the origin of the circulating cell-free miRNAs and their carriers in blood. We summarize the clinical use and function of potentially promising miRNA biomarkers in a variety of different cancers, along with their downstream target genes in tumor initiation and development. Additionally, we analyze some technical challenges in applying miRNA biomarkers to clinical practice. PMID:23074383

  14. The female gametophyte: an emerging model for cell type-specific systems biology in plant development

    PubMed Central

    Schmid, Marc W.; Schmidt, Anja; Grossniklaus, Ueli

    2015-01-01

    Systems biology, a holistic approach describing a system emerging from the interactions of its molecular components, critically depends on accurate qualitative determination and quantitative measurements of these components. Development and improvement of large-scale profiling methods (“omics”) now facilitates comprehensive measurements of many relevant molecules. For multicellular organisms, such as animals, fungi, algae, and plants, the complexity of the system is augmented by the presence of specialized cell types and organs, and a complex interplay within and between them. Cell type-specific analyses are therefore crucial for the understanding of developmental processes and environmental responses. This review first gives an overview of current methods used for large-scale profiling of specific cell types exemplified by recent advances in plant biology. The focus then lies on suitable model systems to study plant development and cell type specification. We introduce the female gametophyte of flowering plants as an ideal model to study fundamental developmental processes. Moreover, the female reproductive lineage is of importance for the emergence of evolutionary novelties such as an unequal parental contribution to the tissue nurturing the embryo or the clonal production of seeds by asexual reproduction (apomixis). Understanding these processes is not only interesting from a developmental or evolutionary perspective, but bears great potential for further crop improvement and the simplification of breeding efforts. We finally highlight novel methods, which are already available or which will likely soon facilitate large-scale profiling of the specific cell types of the female gametophyte in both model and non-model species. We conclude that it may take only few years until an evolutionary systems biology approach toward female gametogenesis may decipher some of its biologically most interesting and economically most valuable processes. PMID:26579157

  15. The female gametophyte: an emerging model for cell type-specific systems biology in plant development.

    PubMed

    Schmid, Marc W; Schmidt, Anja; Grossniklaus, Ueli

    2015-01-01

    Systems biology, a holistic approach describing a system emerging from the interactions of its molecular components, critically depends on accurate qualitative determination and quantitative measurements of these components. Development and improvement of large-scale profiling methods ("omics") now facilitates comprehensive measurements of many relevant molecules. For multicellular organisms, such as animals, fungi, algae, and plants, the complexity of the system is augmented by the presence of specialized cell types and organs, and a complex interplay within and between them. Cell type-specific analyses are therefore crucial for the understanding of developmental processes and environmental responses. This review first gives an overview of current methods used for large-scale profiling of specific cell types exemplified by recent advances in plant biology. The focus then lies on suitable model systems to study plant development and cell type specification. We introduce the female gametophyte of flowering plants as an ideal model to study fundamental developmental processes. Moreover, the female reproductive lineage is of importance for the emergence of evolutionary novelties such as an unequal parental contribution to the tissue nurturing the embryo or the clonal production of seeds by asexual reproduction (apomixis). Understanding these processes is not only interesting from a developmental or evolutionary perspective, but bears great potential for further crop improvement and the simplification of breeding efforts. We finally highlight novel methods, which are already available or which will likely soon facilitate large-scale profiling of the specific cell types of the female gametophyte in both model and non-model species. We conclude that it may take only few years until an evolutionary systems biology approach toward female gametogenesis may decipher some of its biologically most interesting and economically most valuable processes.

  16. Cell type-specific translational repression of Cyclin B during meiosis in males

    PubMed Central

    Baker, Catherine Craig; Gim, Byung Soo; Fuller, Margaret T.

    2015-01-01

    The unique cell cycle dynamics of meiosis are controlled by layers of regulation imposed on core mitotic cell cycle machinery components by the program of germ cell development. Although the mechanisms that regulate Cdk1/Cyclin B activity in meiosis in oocytes have been well studied, little is known about the trans-acting factors responsible for developmental control of these factors in male gametogenesis. During meiotic prophase in Drosophila males, transcript for the core cell cycle protein Cyclin B1 (CycB) is expressed in spermatocytes, but the protein does not accumulate in spermatocytes until just before the meiotic divisions. Here, we show that two interacting proteins, Rbp4 and Fest, expressed at the onset of spermatocyte differentiation under control of the developmental program of male gametogenesis, function to direct cell type- and stage-specific repression of translation of the core G2/M cell cycle component cycB during the specialized cell cycle of male meiosis. Binding of Fest to Rbp4 requires a 31-amino acid region within Rbp4. Rbp4 and Fest are required for translational repression of cycB in immature spermatocytes, with Rbp4 binding sequences in a cell type-specific shortened form of the cycB 3′ UTR. Finally, we show that Fest is required for proper execution of meiosis I. PMID:26443637

  17. Cell type-specific translational repression of Cyclin B during meiosis in males.

    PubMed

    Baker, Catherine Craig; Gim, Byung Soo; Fuller, Margaret T

    2015-10-01

    The unique cell cycle dynamics of meiosis are controlled by layers of regulation imposed on core mitotic cell cycle machinery components by the program of germ cell development. Although the mechanisms that regulate Cdk1/Cyclin B activity in meiosis in oocytes have been well studied, little is known about the trans-acting factors responsible for developmental control of these factors in male gametogenesis. During meiotic prophase in Drosophila males, transcript for the core cell cycle protein Cyclin B1 (CycB) is expressed in spermatocytes, but the protein does not accumulate in spermatocytes until just before the meiotic divisions. Here, we show that two interacting proteins, Rbp4 and Fest, expressed at the onset of spermatocyte differentiation under control of the developmental program of male gametogenesis, function to direct cell type- and stage-specific repression of translation of the core G2/M cell cycle component cycB during the specialized cell cycle of male meiosis. Binding of Fest to Rbp4 requires a 31-amino acid region within Rbp4. Rbp4 and Fest are required for translational repression of cycB in immature spermatocytes, with Rbp4 binding sequences in a cell type-specific shortened form of the cycB 3' UTR. Finally, we show that Fest is required for proper execution of meiosis I.

  18. Cell-Type-Specific Control of Brainstem Locomotor Circuits by Basal Ganglia

    PubMed Central

    Roseberry, Thomas K.; Lee, A. Moses; Lalive, Arnaud L.; Wilbrecht, Linda; Bonci, Antonello; Kreitzer, Anatol C.

    2015-01-01

    Summary The basal ganglia (BG) are critical for adaptive motor control, but the circuit principles underlying their pathway-specific modulation of target regions are not well understood. Here, we dissect the mechanisms underlying BG direct- and indirect-pathway-mediated control of the mesencephalic locomotor region (MLR), a brainstem target of the BG that is critical for locomotion. We optogenetically dissect the locomotor function of the three neurochemically-distinct cell types within the MLR: glutamatergic, GABAergic, and cholinergic neurons. We find that the glutamatergic subpopulation encodes locomotor state and speed, is necessary and sufficient for locomotion, and is selectively innervated by BG. We further show activation and suppression, respectively, of MLR glutamatergic neurons by direct and indirect pathways, which is required for bidirectional control of locomotion by BG circuits. These findings provide a fundamental understanding of how the BG can initiate or suppress a motor program through cell-type-specific regulation of neurons linked to specific actions. PMID:26824660

  19. Cell type specific targeted intracellular delivery into muscle of a monoclonal antibody that binds myosin IIb.

    PubMed

    Weisbart, Richard H; Yang, Fusheng; Chan, Grace; Wakelin, Rika; Ferreri, Kevin; Zack, Debra J; Harrison, Brooke; Leinwand, Leslie A; Cole, Greg M

    2003-03-01

    Methods for cell type specific targeted intracellular delivery of proteins in vivo remain limited. A murine monoclonal anti-dsDNA antibody, mAb 3E10, was selectively transported into skeletal muscle cells in vivo. The antibody bound a 200 kDa protein only found in lysates of skeletal muscle by Western blotting. The 200 kDa protein was purified from muscle lysate by antibody affinity chromatography and identified as the skeletal muscle specific heavy chain of myosin IIb by electrospray mass spectrometry. Antibody binding specificity for myosin IIb was demonstrated in Western blots by binding myosin in skeletal muscle lysates from mice null for myosin IId but not in mice null for myosin IIb. Myosin IIb is implicated in the specific targeting of mAb 3E10 to skeletal muscle.

  20. Cell-Type-Specific Epigenetic Editing at the Fosb Gene Controls Susceptibility to Social Defeat Stress.

    PubMed

    Hamilton, Peter J; Burek, Dominika J; Lombroso, Sonia I; Neve, Rachael L; Robison, Alfred J; Nestler, Eric J; Heller, Elizabeth A

    2017-05-02

    Chronic social defeat stress regulates the expression of Fosb in the nucleus accumbens (NAc) to promote the cell-type-specific accumulation of ΔFosB in the two medium spiny neuron (MSN) subtypes in this region. ΔFosB is selectively induced in D1-MSNs in the NAc of resilient mice, and in D2-MSNs of susceptible mice. However, little is known about the consequences of such selective induction, particularly in D2-MSNs. This study examined how cell-type-specific control of the endogenous Fosb gene in NAc regulates susceptibility to social defeat stress. Histone post-translational modifications (HPTMs) were targeted specifically to Fosb using engineered zinc-finger proteins (ZFPs). Fosb-ZFPs were fused to either the transcriptional repressor, G9a, which promotes histone methylation or the transcriptional activator, p65, which promotes histone acetylation. These ZFPs were expressed in D1- vs D2-MSNs using Cre-dependent viral expression in the NAc of mice transgenic for Cre recombinase in these MSN subtypes. We found that stress susceptibility is oppositely regulated by the specific cell type and HPTM targeted. We report that Fosb-targeted histone acetylation in D2-MSNs or histone methylation in D1-MSNs promotes a stress-susceptible, depressive-like phenotype, while histone methylation in D2-MSNs or histone acetylation in D1-MSNs increases resilience to social stress as quantified by social interaction behavior and sucrose preference. This work presents the first demonstration of cell- and gene-specific targeting of histone modifications, which model naturally occurring transcriptional phenomena that control social defeat stress behavior. This epigenetic-editing approach, which recapitulates physiological changes in gene expression, reveals clear differences in the social defeat phenotype induced by Fosb gene manipulation in MSN subtypes.Neuropsychopharmacology advance online publication, 7 June 2017; doi:10.1038/npp.2017.88.

  1. Cell-Type-Specific Cytokinin Distribution within the Arabidopsis Primary Root Apex.

    PubMed

    Antoniadi, Ioanna; Plačková, Lenka; Simonovik, Biljana; Doležal, Karel; Turnbull, Colin; Ljung, Karin; Novák, Ondřej

    2015-07-01

    Cytokinins (CKs) play a crucial role in many physiological and developmental processes at the levels of individual plant components (cells, tissues, and organs) and by coordinating activities across these parts. High-resolution measurements of intracellular CKs in different plant tissues can therefore provide insights into their metabolism and mode of action. Here, we applied fluorescence-activated cell sorting of green fluorescent protein (GFP)-marked cell types, combined with solid-phase microextraction and an ultra-high-sensitivity mass spectrometry (MS) method for analysis of CK biosynthesis and homeostasis at cellular resolution. This method was validated by series of control experiments, establishing that protoplast isolation and cell sorting procedures did not greatly alter endogenous CK levels. The MS-based method facilitated the quantification of all the well known CK isoprenoid metabolites in four different transgenic Arabidopsis thaliana lines expressing GFP in specific cell populations within the primary root apex. Our results revealed the presence of a CK gradient within the Arabidopsis root tip, with a concentration maximum in the lateral root cap, columella, columella initials, and quiescent center cells. This distribution, when compared with previously published auxin gradients, implies that the well known antagonistic interactions between the two hormone groups are cell type specific. © 2015 American Society of Plant Biologists. All rights reserved.

  2. Small RNA zippers lock miRNA molecules and block miRNA function in mammalian cells

    PubMed Central

    Meng, Lingyu; Liu, Cuicui; Lü, Jinhui; Zhao, Qian; Deng, Shengqiong; Wang, Guangxue; Qiao, Jing; Zhang, Chuyi; Zhen, Lixiao; Lu, Ying; Li, Wenshu; Zhang, Yuzhen; Pestell, Richard G.; Fan, Huiming; Chen, Yi-Han; Liu, Zhongmin; Yu, Zuoren

    2017-01-01

    MicroRNAs (miRNAs) loss-of-function phenotypes are mainly induced by chemically modified antisense oligonucleotides. Here we develop an alternative inhibitor for miRNAs, termed ‘small RNA zipper'. It is designed to connect miRNA molecules end to end, forming a DNA–RNA duplex through a complementary interaction with high affinity, high specificity and high stability. Two miRNAs, miR-221 and miR-17, are tested in human breast cancer cell lines, demonstrating the 70∼90% knockdown of miRNA levels by 30–50 nM small RNA zippers. The miR-221 zipper shows capability in rescuing the expression of target genes of miR-221 and reversing the oncogenic function of miR-221 in breast cancer cells. In addition, we demonstrate that the miR-221 zipper attenuates doxorubicin resistance with higher efficiency than anti-miR-221 in human breast cancer cells. Taken together, small RNA zippers are a miRNA inhibitor, which can be used to induce miRNA loss-of-function phenotypes and validate miRNA target genes. PMID:28045030

  3. Cell-type-specific roles for COX-2 in UVB-induced skin cancer

    PubMed Central

    Herschman, Harvey

    2014-01-01

    In human tumors, and in mouse models, cyclooxygenase-2 (COX-2) levels are frequently correlated with tumor development/burden. In addition to intrinsic tumor cell expression, COX-2 is often present in fibroblasts, myofibroblasts and endothelial cells of the tumor microenvironment, and in infiltrating immune cells. Intrinsic cancer cell COX-2 expression is postulated as only one of many sources for prostanoids required for tumor promotion/progression. Although both COX-2 inhibition and global Cox-2 gene deletion ameliorate ultraviolet B (UVB)-induced SKH-1 mouse skin tumorigenesis, neither manipulation can elucidate the cell type(s) in which COX-2 expression is required for tumorigenesis; both eliminate COX-2 activity in all cells. To address this question, we created Cox-2 flox/flox mice, in which the Cox-2 gene can be eliminated in a cell-type-specific fashion by targeted Cre recombinase expression. Cox-2 deletion in skin epithelial cells of SKH-1 Cox-2 flox/flox;K14Cre + mice resulted, following UVB irradiation, in reduced skin hyperplasia and increased apoptosis. Targeted epithelial cell Cox-2 deletion also resulted in reduced tumor incidence, frequency, size and proliferation rate, altered tumor cell differentiation and reduced tumor vascularization. Moreover, Cox-2 flox/flox;K14Cre + papillomas did not progress to squamous cell carcinomas. In contrast, Cox-2 deletion in SKH-1 Cox-2 flox/flox; LysMCre + myeloid cells had no effect on UVB tumor induction. We conclude that (i) intrinsic epithelial COX-2 activity plays a major role in UVB-induced skin cancer, (ii) macrophage/myeloid COX-2 plays no role in UVB-induced skin cancer and (iii) either there may be another COX-2-dependent prostanoid source(s) that drives UVB skin tumor induction or there may exist a COX-2-independent pathway(s) to UVB-induced skin cancer. PMID:24469308

  4. Cell-type-specific roles for COX-2 in UVB-induced skin cancer.

    PubMed

    Jiao, Jing; Mikulec, Carol; Ishikawa, Tomo-o; Magyar, Clara; Dumlao, Darren S; Dennis, Edward A; Fischer, Susan M; Herschman, Harvey

    2014-06-01

    In human tumors, and in mouse models, cyclooxygenase-2 (COX-2) levels are frequently correlated with tumor development/burden. In addition to intrinsic tumor cell expression, COX-2 is often present in fibroblasts, myofibroblasts and endothelial cells of the tumor microenvironment, and in infiltrating immune cells. Intrinsic cancer cell COX-2 expression is postulated as only one of many sources for prostanoids required for tumor promotion/progression. Although both COX-2 inhibition and global Cox-2 gene deletion ameliorate ultraviolet B (UVB)-induced SKH-1 mouse skin tumorigenesis, neither manipulation can elucidate the cell type(s) in which COX-2 expression is required for tumorigenesis; both eliminate COX-2 activity in all cells. To address this question, we created Cox-2(flox/flox) mice, in which the Cox-2 gene can be eliminated in a cell-type-specific fashion by targeted Cre recombinase expression. Cox-2 deletion in skin epithelial cells of SKH-1 Cox-2(flox/flox);K14Cre(+) mice resulted, following UVB irradiation, in reduced skin hyperplasia and increased apoptosis. Targeted epithelial cell Cox-2 deletion also resulted in reduced tumor incidence, frequency, size and proliferation rate, altered tumor cell differentiation and reduced tumor vascularization. Moreover, Cox-2(flox/flox);K14Cre(+) papillomas did not progress to squamous cell carcinomas. In contrast, Cox-2 deletion in SKH-1 Cox-2(flox/flox); LysMCre(+) myeloid cells had no effect on UVB tumor induction. We conclude that (i) intrinsic epithelial COX-2 activity plays a major role in UVB-induced skin cancer, (ii) macrophage/myeloid COX-2 plays no role in UVB-induced skin cancer and (iii) either there may be another COX-2-dependent prostanoid source(s) that drives UVB skin tumor induction or there may exist a COX-2-independent pathway(s) to UVB-induced skin cancer.

  5. PINK1 is required for timely cell-type specific mitochondrial clearance during Drosophila midgut metamorphosis.

    PubMed

    Liu, Yan; Lin, Jingjing; Zhang, Minjie; Chen, Kai; Yang, Shengxi; Wang, Qun; Yang, Hongqin; Xie, Shusen; Zhou, Yongjian; Zhang, Xi; Chen, Fei; Yang, Yufeng

    2016-11-15

    Mitophagy is the selective degradation of mitochondria by autophagy, which is an important mitochondrial quality and quantity control process. During Drosophila metamorphosis, the degradation of midgut involves a large change in length and organization, which is mediated by autophagy. Here we noticed a cell-type specific mitochondrial clearance process that occurs in enterocytes (ECs), while most mitochondria remain in intestinal stem cells (ISCs) during metamorphosis. Although PINK1/PARKIN represent the canonical pathway for the elimination of impaired mitochondria in varied pathological conditions, their roles in developmental processes or normal physiological conditions have been less studied. To examine the potential contribution of PINK1 in developmental processes, we monitored the dynamic expression pattern of PINK1 in the midgut development by taking advantage of a newly CRISPR/Cas9 generated knock-in fly strain expressing PINK1-mCherry fusion protein that presumably recapitulates the endogenous expression pattern of PINK1. We disclosed a spatiotemporal correlation between the expression pattern of PINK1 and the mitochondrial clearance or persistence in ECs or ISCs respectively. By mosaic genetic analysis, we then demonstrated that PINK1 and PARKIN function epistatically to mediate the specific timely removal of mitochondria, and are involved in global autophagy in ECs during Drosophila midgut metamorphosis, with kinase-dead PINK1 exerting dominant negative effects. Taken together, our studies concluded that the PINK1/PARKIN is crucial for timely cell-type specific mitophagy under physiological conditions and demonstrated again that Drosophila midgut metamorphosis might serve as an elegant in vivo model to study autophagy. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. eFORGE: A Tool for Identifying Cell Type-Specific Signal in Epigenomic Data.

    PubMed

    Breeze, Charles E; Paul, Dirk S; van Dongen, Jenny; Butcher, Lee M; Ambrose, John C; Barrett, James E; Lowe, Robert; Rakyan, Vardhman K; Iotchkova, Valentina; Frontini, Mattia; Downes, Kate; Ouwehand, Willem H; Laperle, Jonathan; Jacques, Pierre-Étienne; Bourque, Guillaume; Bergmann, Anke K; Siebert, Reiner; Vellenga, Edo; Saeed, Sadia; Matarese, Filomena; Martens, Joost H A; Stunnenberg, Hendrik G; Teschendorff, Andrew E; Herrero, Javier; Birney, Ewan; Dunham, Ian; Beck, Stephan

    2016-11-15

    Epigenome-wide association studies (EWAS) provide an alternative approach for studying human disease through consideration of non-genetic variants such as altered DNA methylation. To advance the complex interpretation of EWAS, we developed eFORGE (http://eforge.cs.ucl.ac.uk/), a new standalone and web-based tool for the analysis and interpretation of EWAS data. eFORGE determines the cell type-specific regulatory component of a set of EWAS-identified differentially methylated positions. This is achieved by detecting enrichment of overlap with DNase I hypersensitive sites across 454 samples (tissues, primary cell types, and cell lines) from the ENCODE, Roadmap Epigenomics, and BLUEPRINT projects. Application of eFORGE to 20 publicly available EWAS datasets identified disease-relevant cell types for several common diseases, a stem cell-like signature in cancer, and demonstrated the ability to detect cell-composition effects for EWAS performed on heterogeneous tissues. Our approach bridges the gap between large-scale epigenomics data and EWAS-derived target selection to yield insight into disease etiology.

  7. Unique cell-type-specific patterns of DNA methylation in the root meristem.

    PubMed

    Kawakatsu, Taiji; Stuart, Tim; Valdes, Manuel; Breakfield, Natalie; Schmitz, Robert J; Nery, Joseph R; Urich, Mark A; Han, Xinwei; Lister, Ryan; Benfey, Philip N; Ecker, Joseph R

    2016-04-29

    DNA methylation is an epigenetic modification that differs between plant organs and tissues, but the extent of variation between cell types is not known. Here, we report single-base-resolution whole-genome DNA methylomes, mRNA transcriptomes and small RNA transcriptomes for six cell populations covering the major cell types of the Arabidopsis root meristem. We identify widespread cell-type-specific patterns of DNA methylation, especially in the CHH sequence context, where H is A, C or T. The genome of the columella root cap is the most highly methylated Arabidopsis cell characterized so far. It is hypermethylated within transposable elements (TEs), accompanied by increased abundance of transcripts encoding RNA-directed DNA methylation (RdDM) pathway components and 24-nt small RNAs (smRNAs). The absence of the nucleosome remodeller DECREASED DNA METHYLATION 1 (DDM1), required for maintenance of DNA methylation, and low abundance of histone transcripts involved in heterochromatin formation suggests that a loss of heterochromatin may occur in the columella, thus allowing access of RdDM factors to the whole genome, and producing an excess of 24-nt smRNAs in this tissue. Together, these maps provide new insights into the epigenomic diversity that exists between distinct plant somatic cell types.

  8. Heterochromatin regulates cell type-specific long-range chromatin interactions essential for directed recombination.

    PubMed

    Jia, Songtao; Yamada, Takatomi; Grewal, Shiv I S

    2004-11-12

    Mating-type switching in Schizosaccharomyces pombe involves replacing genetic information at the expressed mat1 locus with sequences copied from one of two silent donor loci, mat2-P or mat3-M, located within a 20-kb heterochromatic domain. Donor selection is dictated by cell type: mat2 is the preferred donor in M cells, and mat3 is the preferred donor in P cells. Here we show that a recombination-promoting complex (RPC) containing Swi2 and Swi5 proteins exhibits cell type-specific localization pattern at the silent mating-type region and this differential localization modulates donor preference during mating-type switching. In P cells, RPC localization is restricted to a recombination enhancer located adjacent to mat3, but in M cells, RPC spreads in cis across the entire silent mating-type interval in a heterochromatin-dependent manner. Our analyses implicate heterochromatin in long-range regulatory interactions and suggest that heterochromatin imposes at the mating-type region structural organization that is important for the donor-choice mechanism.

  9. Different GATA factors dictate CCR3 transcription in allergic inflammatory cells in a cell type-specific manner.

    PubMed

    Kong, Su-Kang; Kim, Byung Soo; Uhm, Tae Gi; Lee, Wonyong; Lee, Gap Ryol; Park, Choon-Sik; Lee, Chul-Hoon; Chung, Il Yup

    2013-06-01

    The chemokine receptor CCR3 is expressed in prominent allergic inflammatory cells, including eosinophils, mast cells, and Th2 cells. We previously identified a functional GATA element within exon 1 of the CCR3 gene that is responsible for GATA-1-mediated CCR3 transcription. Because allergic inflammatory cells exhibit distinct expression patterns of different GATA factors, we investigated whether different GATA factors dictate CCR3 transcription in a cell type-specific manner. GATA-2 was expressed in EoL-1 eosinophilic cells, GATA-1 and GATA-2 were expressed in HMC-1 mast cells, and GATA-3 was preferentially expressed in Jurkat cells. Unlike a wild-type CCR3 reporter, reporters lacking the functional GATA element were not active in any of the three cell types, implying the involvement of different GATA factors in CCR3 transcription. RNA interference assays showed that small interfering RNAs specific for different GATA factors reduced CCR3 reporter activity in a cell type-specific fashion. Consistent with these findings, chromatin immunoprecipitation and EMSA analyses demonstrated cell type-specific binding of GATA factors to the functional GATA site. More importantly, specific inhibition of the CCR3 reporter activity by different GATA small interfering RNAs was well preserved in respective cell types differentiated from cord blood; in particular, GATA-3 was entirely responsible for reporter activity in Th2 cells and replaced the role predominantly played by GATA-1 and GATA-2. These results highlight a mechanistic role of GATA factors in which cell type-specific expression is the primary determinant of transcription of the CCR3 gene in major allergic inflammatory cells.

  10. Cell-type-specific inhibition of the dendritic plateau potential in striatal spiny projection neurons.

    PubMed

    Du, Kai; Wu, Yu-Wei; Lindroos, Robert; Liu, Yu; Rózsa, Balázs; Katona, Gergely; Ding, Jun B; Kotaleski, Jeanette Hellgren

    2017-09-05

    Striatal spiny projection neurons (SPNs) receive convergent excitatory synaptic inputs from the cortex and thalamus. Activation of spatially clustered and temporally synchronized excitatory inputs at the distal dendrites could trigger plateau potentials in SPNs. Such supralinear synaptic integration is crucial for dendritic computation. However, how plateau potentials interact with subsequent excitatory and inhibitory synaptic inputs remains unknown. By combining computational simulation, two-photon imaging, optogenetics, and dual-color uncaging of glutamate and GABA, we demonstrate that plateau potentials can broaden the spatiotemporal window for integrating excitatory inputs and promote spiking. The temporal window of spiking can be delicately controlled by GABAergic inhibition in a cell-type-specific manner. This subtle inhibitory control of plateau potential depends on the location and kinetics of the GABAergic inputs and is achieved by the balance between relief and reestablishment of NMDA receptor Mg(2+) block. These findings represent a mechanism for controlling spatiotemporal synaptic integration in SPNs.

  11. Cell Type Specific Analysis of Human Brain Transcriptome Data to Predict Alterations in Cellular Composition.

    PubMed

    Xu, Xiaoxiao; Nehorai, Arye; Dougherty, Joseph

    2013-07-01

    The central nervous system (CNS) is composed of hundreds of distinct cell types, each expressing different subsets of genes from the genome. High throughput gene expression analysis of the CNS from patients and controls is a common method to screen for potentially pathological molecular mechanisms of psychiatric disease. One mechanism by which gene expression might be seen to vary across samples would be alterations in the cellular composition of the tissue. While the expressions of gene 'markers' for each cell type can provide certain information of cellularity, for many rare cell types markers are not well characterized. Moreover, if only small sets of markers are known, any substantial variation of a marker's expression pattern due to experiment conditions would result in poor sensitivity and specificity. Here, our proposed method combines prior information from mice cell-specific transcriptome profiling experiments with co-expression network analysis, to select large sets of potential cell type-specific gene markers in a systematic and unbiased manner. The method is efficient and robust, and identifies sufficient markers for further cellularity analysis. We then employ the markers to analytically detect changing cellular composition in human brain. Application of our method to temporal human brain microarray data successfully detects changes in cellularity over time that roughly correspond to known epochs of human brain development. Furthermore, application of our method to human brain samples with the neurodevelopmental disorder of autism supports the interpretation that the changes in astrocytes and neurons might contribute to the disorder.

  12. Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction

    SciTech Connect

    Fujii, Hodaka . E-mail: hodaka@med.nyu.edu

    2007-03-16

    Binding of interleukin-2 (IL-2) to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2 receptor (IL-2R)-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling.

  13. Cell Type-Specific Expression Analysis to Identify Putative Cellular Mechanisms for Neurogenetic Disorders

    PubMed Central

    Xu, Xiaoxiao; Wells, Alan B.; O'Brien, David R.; Nehorai, Arye

    2014-01-01

    Recent advances have substantially increased the number of genes that are statistically associated with complex genetic disorders of the CNS such as autism and schizophrenia. It is now clear that there will likely be hundreds of distinct loci contributing to these disorders, underscoring a remarkable genetic heterogeneity. It is unclear whether this genetic heterogeneity indicates an equal heterogeneity of cellular mechanisms for these diseases. The commonality of symptoms across patients suggests there could be a functional convergence downstream of these loci upon a limited number of cell types or circuits that mediate the affected behaviors. One possible mechanism for this convergence would be the selective expression of at least a subset of these genes in the cell types that comprise these circuits. Using profiling data from mice and humans, we have developed and validated an approach, cell type-specific expression analysis, for identifying candidate cell populations likely to be disrupted across sets of patients with distinct genetic lesions. Using human genetics data and postmortem gene expression data, our approach can correctly identify the cell types for disorders of known cellular etiology, including narcolepsy and retinopathies. Applying this approach to autism, a disease where the cellular mechanism is unclear, indicates there may be multiple cellular routes to this disorder. Our approach may be useful for identifying common cellular mechanisms arising from distinct genetic lesions. PMID:24453331

  14. Flotillin-mediated endocytic events dictate cell-type specific responses to Semaphorin 3A

    PubMed Central

    Carcea, Ioana; Ma'ayan, Avi; Mesias, Roxana; Sepulveda, Bryan; Salton, Stephen R.; Benson, Deanna L.

    2010-01-01

    Cortical efferents growing in the same environment diverge early in development. The expression of particular transcription factors dictates the trajectories taken presumably by regulating responsiveness to guidance cues via cellular mechanisms that are not yet known. Here we show that cortical neurons that are dissociated and grown in culture maintain their cell-type specific identities defined by the expression of transcription factors. Using this model system we sought to identify and characterize mechanisms that are recruited to produce cell-type specific responses to Semaphorin 3A (Sema3A), a guidance cue that would be presented similarly to cortical axons in vivo. Axons from presumptive corticofugal neurons lacking the transcription factor Satb2 and expressing Ctip2 or Tbr1 respond far more robustly to Sema3A than those from presumptive callosal neurons expressing Satb2. Both populations of axons express similar levels of Sema3A receptors (Neuropilin-1, L1CAM and PlexinA4), but significantly, axons from neurons lacking Satb2 internalize more Sema3A and they do so via a raft-mediated endocytic pathway. We used an in silico approach to identify the endocytosis effector Flotillin-1 as a Sema3A signaling candidate. We tested the contributions of Flotillin-1 to Sema3A endocytosis and signaling, and show that raft-mediated Sema3A endocytosis is defined by and depends on the recruitment of Flotillin-1, which mediates LIMK activation, and regulates axon responsiveness to Sema3A in presumptive corticofugal axons. PMID:21068336

  15. Cell type specificity of a diffusible inducer is determined by a GATA family transcription factor.

    PubMed

    Keller, Thomas; Thompson, Christopher R L

    2008-05-01

    One poorly understood mechanism of developmental patterning involves the intermingled differentiation of different cell types that then sort out to generate pattern. Examples of this are known in nematodes and vertebrates, and in Dictyostelium it is the major mechanism. However, a general problem with this mechanism is the possibility that different inducers are required for each cell type that arises independently of positional information. Consistent with this idea, in Dictyostelium the signalling molecule DIF acts as a position-independent signal and was thought only to regulate the differentiation of a single cell type (pstO). The results presented here challenge this idea. In a novel genetic selection to isolate genes required for DIF signal transduction, we found a mutant (dimC(-)) that is a hypomorphic allele of a GATA family transcription factor (gtaC). gtaC expression is directly regulated by DIF, and GtaC rapidly translocates to the nucleus in response to DIF. gtaC(-) null cells showed some hallmark DIF signalling defects. Surprisingly, other aspects of the mutant were distinct from those of other DIF signalling mutants, suggesting that gtaC regulates a subset of DIF responses. For example, pstO cell differentiation appeared normal. However, we found that pstB cells were mislocalised and the pstB-derived basal disc was much reduced or missing. These defects are due to a failure to respond to DIF as they are phenocopied in other DIF signalling mutants. These findings therefore identify a novel small-molecule-activated GATA factor that is required to regulate the cell type-specific effects of DIF. They also reveal that a non-positional signal can regulate the differentiation of multiple cell types through differential interpretation in receiving cells.

  16. Cell-Type-Specific Chromatin States Differentially Prime Squamous Cell Carcinoma Tumor-Initiating Cells for Epithelial to Mesenchymal Transition.

    PubMed

    Latil, Mathilde; Nassar, Dany; Beck, Benjamin; Boumahdi, Soufiane; Wang, Li; Brisebarre, Audrey; Dubois, Christine; Nkusi, Erwin; Lenglez, Sandrine; Checinska, Agnieszka; Vercauteren Drubbel, Alizée; Devos, Michael; Declercq, Wim; Yi, Rui; Blanpain, Cédric

    2017-02-02

    Epithelial to mesenchymal transition (EMT) in cancer cells has been associated with metastasis, stemness, and resistance to therapy. Some tumors undergo EMT while others do not, which may reflect intrinsic properties of their cell of origin. However, this possibility is largely unexplored. By targeting the same oncogenic mutations to discrete skin compartments, we show that cell-type-specific chromatin and transcriptional states differentially prime tumors to EMT. Squamous cell carcinomas (SCCs) derived from interfollicular epidermis (IFE) are generally well differentiated, while hair follicle (HF) stem cell-derived SCCs frequently exhibit EMT, efficiently form secondary tumors, and possess increased metastatic potential. Transcriptional and epigenomic profiling revealed that IFE and HF tumor-initiating cells possess distinct chromatin landscapes and gene regulatory networks associated with tumorigenesis and EMT that correlate with accessibility of key epithelial and EMT transcription factor binding sites. These findings highlight the importance of chromatin states and transcriptional priming in dictating tumor phenotypes and EMT.

  17. Cell type-specific termination of transcription by transposable element sequences.

    PubMed

    Conley, Andrew B; Jordan, I King

    2012-09-30

    Transposable elements (TEs) encode sequences necessary for their own transposition, including signals required for the termination of transcription. TE sequences within the introns of human genes show an antisense orientation bias, which has been proposed to reflect selection against TE sequences in the sense orientation owing to their ability to terminate the transcription of host gene transcripts. While there is evidence in support of this model for some elements, the extent to which TE sequences actually terminate transcription of human gene across the genome remains an open question. Using high-throughput sequencing data, we have characterized over 9,000 distinct TE-derived sequences that provide transcription termination sites for 5,747 human genes across eight different cell types. Rarefaction curve analysis suggests that there may be twice as many TE-derived termination sites (TE-TTS) genome-wide among all human cell types. The local chromatin environment for these TE-TTS is similar to that seen for 3' UTR canonical TTS and distinct from the chromatin environment of other intragenic TE sequences. However, those TE-TTS located within the introns of human genes were found to be far more cell type-specific than the canonical TTS. TE-TTS were much more likely to be found in the sense orientation than other intragenic TE sequences of the same TE family and TE-TTS in the sense orientation terminate transcription more efficiently than those found in the antisense orientation. Alu sequences were found to provide a large number of relatively weak TTS, whereas LTR elements provided a smaller number of much stronger TTS. TE sequences provide numerous termination sites to human genes, and TE-derived TTS are particularly cell type-specific. Thus, TE sequences provide a powerful mechanism for the diversification of transcriptional profiles between cell types and among evolutionary lineages, since most TE-TTS are evolutionarily young. The extent of transcription

  18. Cell type-specific termination of transcription by transposable element sequences

    PubMed Central

    2012-01-01

    Background Transposable elements (TEs) encode sequences necessary for their own transposition, including signals required for the termination of transcription. TE sequences within the introns of human genes show an antisense orientation bias, which has been proposed to reflect selection against TE sequences in the sense orientation owing to their ability to terminate the transcription of host gene transcripts. While there is evidence in support of this model for some elements, the extent to which TE sequences actually terminate transcription of human gene across the genome remains an open question. Results Using high-throughput sequencing data, we have characterized over 9,000 distinct TE-derived sequences that provide transcription termination sites for 5,747 human genes across eight different cell types. Rarefaction curve analysis suggests that there may be twice as many TE-derived termination sites (TE-TTS) genome-wide among all human cell types. The local chromatin environment for these TE-TTS is similar to that seen for 3′ UTR canonical TTS and distinct from the chromatin environment of other intragenic TE sequences. However, those TE-TTS located within the introns of human genes were found to be far more cell type-specific than the canonical TTS. TE-TTS were much more likely to be found in the sense orientation than other intragenic TE sequences of the same TE family and TE-TTS in the sense orientation terminate transcription more efficiently than those found in the antisense orientation. Alu sequences were found to provide a large number of relatively weak TTS, whereas LTR elements provided a smaller number of much stronger TTS. Conclusions TE sequences provide numerous termination sites to human genes, and TE-derived TTS are particularly cell type-specific. Thus, TE sequences provide a powerful mechanism for the diversification of transcriptional profiles between cell types and among evolutionary lineages, since most TE-TTS are evolutionarily young. The

  19. Fluorescence-Activated Cell Sorting for Analysis of Cell Type-Specific Responses to Salinity Stress in Arabidopsis and Rice

    PubMed Central

    Evrard, Aurelie; Bargmann, Bastiaan O.R.; Birnbaum, Kenneth D.; Tester, Mark; Baumann, Ute; Johnson, Alexander A.T.

    2014-01-01

    Fluorescence-activated cell sorting (FACS) provides a rapid means of isolating large numbers of fluorescently tagged cells from a heterogeneous mixture of cells. Collections of transgenic plants with cell type-specific expression of fluorescent marker genes such as green fluorescent protein (GFP) are ideally suited for FACS-assisted studies of individual cell types. Here we describe the use of Arabidopsis and rice enhancer trap lines with tissue-specific GFP expression patterns in the root to isolate specific cell types of root tissues using FACS. Additionally, protocols are provided to impose a ramped salinity stress for 48 h prior to cell sorting. PMID:22895766

  20. Cell-type-specific resonances shape the responses of striatal neurons to synaptic input

    PubMed Central

    Beatty, Joseph A.; Song, Soomin C.

    2014-01-01

    Neurons respond to synaptic inputs in cell-type-specific ways. Each neuron type may thus respond uniquely to shared patterns of synaptic input. We applied statistically identical barrages of artificial synaptic inputs to four striatal cell types to assess differences in their responses to a realistic input pattern. Each interneuron type fired in phase with a specific input-frequency component. The fast-spiking interneuron fired in relation to the gamma-band (and higher) frequencies, the low-threshold spike interneuron to the beta-band frequencies, and the cholinergic neurons to the delta-band frequencies. Low-threshold spiking and cholinergic interneurons showed input impedance resonances at frequencies matching their spiking resonances. Fast-spiking interneurons showed resonance of input impedance but at lower than gamma frequencies. The spiny projection neuron's frequency preference did not have a fixed frequency but instead tracked its own firing rate. Spiny cells showed no input impedance resonance. Striatal interneurons are each tuned to a specific frequency band corresponding to the major frequency components of local field potentials. Their influence in the circuit may fluctuate along with the contribution of that frequency band to the input. In contrast, spiny neurons may tune to any of the frequency bands by a change in firing rate. PMID:25411465

  1. A Cell Type-Specific Expression Signature Predicts Haploinsufficient Autism-Susceptibility Genes.

    PubMed

    Zhang, Chaolin; Shen, Yufeng

    2017-02-01

    Recent studies have identified many genes with rare de novo mutations in autism, but a limited number of these have been conclusively established as disease-susceptibility genes due to the lack of recurrence and confounding background mutations. Such extreme genetic heterogeneity severely limits recurrence-based statistical power even in studies with a large sample size. Here, we use cell-type specific expression profiles to differentiate mutations in autism patients from those in unaffected siblings. We report a gene expression signature in different neuronal cell types shared by genes with likely gene-disrupting (LGD) mutations in autism cases. This signature reflects haploinsufficiency of risk genes enriched in transcriptional and post-transcriptional regulators, with the strongest positive associations with specific types of neurons in different brain regions, including cortical neurons, cerebellar granule cells, and striatal medium spiny neurons. When used to prioritize genes with a single LGD mutation in cases, a D-score derived from the signature achieved a precision of 40% as compared with the 15% baseline with a minimal loss in sensitivity. An ensemble model combining D-score with mutation intolerance metrics from Exome Aggregation Consortium further improved the precision to 60%, resulting in 117 high-priority candidates. These prioritized lists can facilitate identification of additional autism-susceptibility genes.

  2. Cell type-specific genetic and optogenetic tools reveal novel hippocampal CA2 circuits

    PubMed Central

    Kohara, Keigo; Pignatelli, Michele; Rivest, Alexander J.; Jung, Hae-Yoon; Kitamura, Takashi; Suh, Junghyup; Frank, Dominic; Kajikawa, Koichiro; Mise, Nathan; Obata, Yuichi; Wickersham, Ian R.; Tonegawa, Susumu

    2014-01-01

    The formation and recall of episodic memory requires precise information processing by the entorhinal-hippocampal network. For several decades, the trisynaptic circuit, entorhinal cortex layer II (ECII)→dentate gyrus (DG)→CA3→CA1 and the monosynaptic circuit ECIII→CA1 have been considered the main substrates of the network responsible for learning and memory. Circuits linked to another hippocampal region, CA2, have only recently come to light. Here, by using highly cell type-specific transgenic mouse lines, optogenetics, and patch-clamp recordings, we show that DG cells, long believed not to project to CA2, send functional monosynaptic inputs to CA2 pyramidal cells, through abundant longitudinal projections. CA2 innervates CA1 to complete an alternate trisynaptic circuit but, unlike CA3, projects preferentially to the deep rather than superficial sublayer of CA1. Furthermore, contrary to the current knowledge, ECIII does not project to CA2. These new anatomical results will allow for a deeper understanding of the biology of learning and memory. PMID:24336151

  3. Vasopressin-regulated miRNAs and AQP2-targeting miRNAs in kidney collecting duct cells.

    PubMed

    Kim, Jae-Eun; Jung, Hyun Jun; Lee, Yu-Jung; Kwon, Tae-Hwan

    2015-04-01

    Mature microRNA (miRNA) acts as an important posttranscriptional regulator. We aimed to profile vasopressin-responsive miRNAs in kidney inner medullary collecting duct cells and to identify aquaporin-2 (AQP2)-targeting miRNAs. Microarray chip assay was carried out in inner medullary collecting duct tubule suspensions from rat kidneys in the absence or presence of desmopressin (dDAVP) stimulation (10(-9) M, 2 h). The results demonstrated 19 miRNAs, including both precursor and mature miRNAs, as potential candidates that showed significant changes in expression after dDAVP stimulation (P < 0.05). Nine mature miRNAs exhibiting >1.3-fold changes in expression on the microarray (miR-127, miR-1, miR-873, miR-16, miR-206, miR-678, miR-496, miR-298, and miR-463) were further examined by quantitative real-time PCR, and target genes of the selected miRNAs were predicted. Next, to identify AQP2-targeting miRNAs, in silico analysis was performed. Four miRNAs (miR-32, miR-137, miR-216a, and miR-216b) target the 3'-untranslated region of rat AQP2 mRNA. Target seed regions of miR-32 and miR-137 were also conserved in the 3'-untranslated region of mouse AQP2 mRNA. Quantitative real-time PCR and immunoblot analysis demonstrated that dDAVP-induced AQP2 expression was significantly attenuated in mpkCCDc14 cells when cells were transfected with miRNA mimics of miR-32 or miR-137. Moreover, luciferase reporter assay demonstrated a significant decrease of AQP2 translation in mpkCCDc14 cells transfected with miRNA mimics of miR-32 or miR-137. The present study provides novel insights into the regulation of AQP2 by RNA interference; however, vasopressin-regulated miRNAs did not include miR-32 or miR-137, indicating that the interaction of miRNAs with the AQP2 regulatory pathway requires further analysis. Copyright © 2015 the American Physiological Society.

  4. Partitioning heritability of regulatory and cell-type-specific variants across 11 common diseases.

    PubMed

    Gusev, Alexander; Lee, S Hong; Trynka, Gosia; Finucane, Hilary; Vilhjálmsson, Bjarni J; Xu, Han; Zang, Chongzhi; Ripke, Stephan; Bulik-Sullivan, Brendan; Stahl, Eli; Kähler, Anna K; Hultman, Christina M; Purcell, Shaun M; McCarroll, Steven A; Daly, Mark; Pasaniuc, Bogdan; Sullivan, Patrick F; Neale, Benjamin M; Wray, Naomi R; Raychaudhuri, Soumya; Price, Alkes L

    2014-11-06

    Regulatory and coding variants are known to be enriched with associations identified by genome-wide association studies (GWASs) of complex disease, but their contributions to trait heritability are currently unknown. We applied variance-component methods to imputed genotype data for 11 common diseases to partition the heritability explained by genotyped SNPs (hg(2)) across functional categories (while accounting for shared variance due to linkage disequilibrium). Extensive simulations showed that in contrast to current estimates from GWAS summary statistics, the variance-component approach partitions heritability accurately under a wide range of complex-disease architectures. Across the 11 diseases DNaseI hypersensitivity sites (DHSs) from 217 cell types spanned 16% of imputed SNPs (and 24% of genotyped SNPs) but explained an average of 79% (SE = 8%) of hg(2) from imputed SNPs (5.1× enrichment; p = 3.7 × 10(-17)) and 38% (SE = 4%) of hg(2) from genotyped SNPs (1.6× enrichment, p = 1.0 × 10(-4)). Further enrichment was observed at enhancer DHSs and cell-type-specific DHSs. In contrast, coding variants, which span 1% of the genome, explained <10% of hg(2) despite having the highest enrichment. We replicated these findings but found no significant contribution from rare coding variants in independent schizophrenia cohorts genotyped on GWAS and exome chips. Our results highlight the value of analyzing components of heritability to unravel the functional architecture of common disease.

  5. Partitioning Heritability of Regulatory and Cell-Type-Specific Variants across 11 Common Diseases

    PubMed Central

    Gusev, Alexander; Lee, S. Hong; Trynka, Gosia; Finucane, Hilary; Vilhjálmsson, Bjarni J.; Xu, Han; Zang, Chongzhi; Ripke, Stephan; Bulik-Sullivan, Brendan; Stahl, Eli; Ripke, Stephan; Neale, Benjamin M.; Corvin, Aiden; Walters, James T.R.; Farh, Kai-How; Holmans, Peter A.; Lee, Phil; Bulik-Sullivan, Brendan; Collier, David A.; Huang, Hailiang; Pers, Tune H.; Agartz, Ingrid; Agerbo, Esben; Albus, Margot; Alexander, Madeline; Amin, Farooq; Bacanu, Silviu A.; Begemann, Martin; Belliveau, Richard A.; Bene, Judit; Bergen, Sarah E.; Bevilacqua, Elizabeth; Bigdeli, Tim B.; Black, Donald W.; Børglum, Anders D.; Bruggeman, Richard; Buccola, Nancy G.; Buckner, Randy L.; Byerley, William; Cahn, Wiepke; Cai, Guiqing; Campion, Dominique; Cantor, Rita M.; Carr, Vaughan J.; Carrera, Noa; Catts, Stanley V.; Chambert, Kimberly D.; Chan, Raymond C.K.; Chen, Ronald Y.L.; Chen, Eric Y.H.; Cheng, Wei; Cheung, Eric F.C.; Chong, Siow Ann; Cloninger, C. Robert; Cohen, David; Cohen, Nadine; Cormican, Paul; Craddock, Nick; Crowley, James J.; Curtis, David; Davidson, Michael; Davis, Kenneth L.; Degenhardt, Franziska; Del Favero, Jurgen; DeLisi, Lynn E.; Demontis, Ditte; Dikeos, Dimitris; Dinan, Timothy; Djurovic, Srdjan; Donohoe, Gary; Drapeau, Elodie; Duan, Jubao; Dudbridge, Frank; Durmishi, Naser; Eichhammer, Peter; Eriksson, Johan; Escott-Price, Valentina; Essioux, Laurent; Fanous, Ayman H.; Farrell, Martilias S.; Frank, Josef; Franke, Lude; Freedman, Robert; Freimer, Nelson B.; Friedl, Marion; Friedman, Joseph I.; Fromer, Menachem; Genovese, Giulio; Georgieva, Lyudmila; Gershon, Elliot S.; Giegling, Ina; Giusti-Rodrguez, Paola; Godard, Stephanie; Goldstein, Jacqueline I.; Golimbet, Vera; Gopal, Srihari; Gratten, Jacob; Grove, Jakob; de Haan, Lieuwe; Hammer, Christian; Hamshere, Marian L.; Hansen, Mark; Hansen, Thomas; Haroutunian, Vahram; Hartmann, Annette M.; Henskens, Frans A.; Herms, Stefan; Hirschhorn, Joel N.; Hoffmann, Per; Hofman, Andrea; Hollegaard, Mads V.; Hougaard, David M.; Ikeda, Masashi; Joa, Inge; Julià, Antonio; Kahn, René S.; Kalaydjieva, Luba; Karachanak-Yankova, Sena; Karjalainen, Juha; Kavanagh, David; Keller, Matthew C.; Kelly, Brian J.; Kennedy, James L.; Khrunin, Andrey; Kim, Yunjung; Klovins, Janis; Knowles, James A.; Konte, Bettina; Kucinskas, Vaidutis; Kucinskiene, Zita Ausrele; Kuzelova-Ptackova, Hana; Kähler, Anna K.; Laurent, Claudine; Keong, Jimmy Lee Chee; Lee, S. Hong; Legge, Sophie E.; Lerer, Bernard; Li, Miaoxin; Li, Tao; Liang, Kung-Yee; Lieberman, Jeffrey; Limborska, Svetlana; Loughland, Carmel M.; Lubinski, Jan; Lnnqvist, Jouko; Macek, Milan; Magnusson, Patrik K.E.; Maher, Brion S.; Maier, Wolfgang; Mallet, Jacques; Marsal, Sara; Mattheisen, Manuel; Mattingsdal, Morten; McCarley, Robert W.; McDonald, Colm; McIntosh, Andrew M.; Meier, Sandra; Meijer, Carin J.; Melegh, Bela; Melle, Ingrid; Mesholam-Gately, Raquelle I.; Metspalu, Andres; Michie, Patricia T.; Milani, Lili; Milanova, Vihra; Mokrab, Younes; Morris, Derek W.; Mors, Ole; Mortensen, Preben B.; Murphy, Kieran C.; Murray, Robin M.; Myin-Germeys, Inez; Mller-Myhsok, Bertram; Nelis, Mari; Nenadic, Igor; Nertney, Deborah A.; Nestadt, Gerald; Nicodemus, Kristin K.; Nikitina-Zake, Liene; Nisenbaum, Laura; Nordin, Annelie; O’Callaghan, Eadbhard; O’Dushlaine, Colm; O’Neill, F. Anthony; Oh, Sang-Yun; Olincy, Ann; Olsen, Line; Van Os, Jim; Pantelis, Christos; Papadimitriou, George N.; Papiol, Sergi; Parkhomenko, Elena; Pato, Michele T.; Paunio, Tiina; Pejovic-Milovancevic, Milica; Perkins, Diana O.; Pietilinen, Olli; Pimm, Jonathan; Pocklington, Andrew J.; Powell, John; Price, Alkes; Pulver, Ann E.; Purcell, Shaun M.; Quested, Digby; Rasmussen, Henrik B.; Reichenberg, Abraham; Reimers, Mark A.; Richards, Alexander L.; Roffman, Joshua L.; Roussos, Panos; Ruderfer, Douglas M.; Salomaa, Veikko; Sanders, Alan R.; Schall, Ulrich; Schubert, Christian R.; Schulze, Thomas G.; Schwab, Sibylle G.; Scolnick, Edward M.; Scott, Rodney J.; Seidman, Larry J.; Shi, Jianxin; Sigurdsson, Engilbert; Silagadze, Teimuraz; Silverman, Jeremy M.; Sim, Kang; Slominsky, Petr; Smoller, Jordan W.; So, Hon-Cheong; Spencer, Chris C.A.; Stahl, Eli A.; Stefansson, Hreinn; Steinberg, Stacy; Stogmann, Elisabeth; Straub, Richard E.; Strengman, Eric; Strohmaier, Jana; Stroup, T. Scott; Subramaniam, Mythily; Suvisaari, Jaana; Svrakic, Dragan M.; Szatkiewicz, Jin P.; Sderman, Erik; Thirumalai, Srinivas; Toncheva, Draga; Tooney, Paul A.; Tosato, Sarah; Veijola, Juha; Waddington, John; Walsh, Dermot; Wang, Dai; Wang, Qiang; Webb, Bradley T.; Weiser, Mark; Wildenauer, Dieter B.; Williams, Nigel M.; Williams, Stephanie; Witt, Stephanie H.; Wolen, Aaron R.; Wong, Emily H.M.; Wormley, Brandon K.; Wu, Jing Qin; Xi, Hualin Simon; Zai, Clement C.; Zheng, Xuebin; Zimprich, Fritz; Wray, Naomi R.; Stefansson, Kari; Visscher, Peter M.; Adolfsson, Rolf; Andreassen, Ole A.; Blackwood, Douglas H.R.; Bramon, Elvira; Buxbaum, Joseph D.; Brglum, Anders D.; Cichon, Sven; Darvasi, Ariel; Domenici, Enrico; Ehrenreich, Hannelore; Esko, Tõnu; Gejman, Pablo V.; Gill, Michael; Gurling, Hugh; Hultman, Christina M.; Iwata, Nakao; Jablensky, Assen V.; Jönsson, Erik G.; Kendler, Kenneth S.; Kirov, George; Knight, Jo; Lencz, Todd; Levinson, Douglas F.; Li, Qingqin S.; Liu, Jianjun; Malhotra, Anil K.; McCarroll, Steven A.; McQuillin, Andrew; Moran, Jennifer L.; Mortensen, Preben B.; Mowry, Bryan J.; Nthen, Markus M.; Ophoff, Roel A.; Owen, Michael J.; Palotie, Aarno; Pato, Carlos N.; Petryshen, Tracey L.; Posthuma, Danielle; Rietschel, Marcella; Riley, Brien P.; Rujescu, Dan; Sham, Pak C.; Sklar, Pamela; St. Clair, David; Weinberger, Daniel R.; Wendland, Jens R.; Werge, Thomas; Daly, Mark J.; Sullivan, Patrick F.; O’Donovan, Michael C.; Ripke, Stephan; O’Dushlaine, Colm; Chambert, Kimberly; Moran, Jennifer L.; Kähler, Anna K.; Akterin, Susanne; Bergen, Sarah; Magnusson, Patrik K.E.; Neale, Benjamin M.; Ruderfer, Douglas; Scolnick, Edward; Purcell, Shaun; McCarroll, Steve; Sklar, Pamela; Hultman, Christina M.; Sullivan, Patrick F.; Kähler, Anna K.; Hultman, Christina M.; Purcell, Shaun M.; McCarroll, Steven A.; Daly, Mark; Pasaniuc, Bogdan; Sullivan, Patrick F.; Neale, Benjamin M.; Wray, Naomi R.; Raychaudhuri, Soumya; Price, Alkes L.

    2014-01-01

    Regulatory and coding variants are known to be enriched with associations identified by genome-wide association studies (GWASs) of complex disease, but their contributions to trait heritability are currently unknown. We applied variance-component methods to imputed genotype data for 11 common diseases to partition the heritability explained by genotyped SNPs (hg2) across functional categories (while accounting for shared variance due to linkage disequilibrium). Extensive simulations showed that in contrast to current estimates from GWAS summary statistics, the variance-component approach partitions heritability accurately under a wide range of complex-disease architectures. Across the 11 diseases DNaseI hypersensitivity sites (DHSs) from 217 cell types spanned 16% of imputed SNPs (and 24% of genotyped SNPs) but explained an average of 79% (SE = 8%) of hg2 from imputed SNPs (5.1× enrichment; p = 3.7 × 10−17) and 38% (SE = 4%) of hg2 from genotyped SNPs (1.6× enrichment, p = 1.0 × 10−4). Further enrichment was observed at enhancer DHSs and cell-type-specific DHSs. In contrast, coding variants, which span 1% of the genome, explained <10% of hg2 despite having the highest enrichment. We replicated these findings but found no significant contribution from rare coding variants in independent schizophrenia cohorts genotyped on GWAS and exome chips. Our results highlight the value of analyzing components of heritability to unravel the functional architecture of common disease. PMID:25439723

  6. Using fluorescence activated cell sorting to examine cell-type-specific gene expression in rat brain tissue.

    PubMed

    Schwarz, Jaclyn M

    2015-05-28

    The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

  7. Cell-type-specific expression pattern of ceramide synthase 2 protein in mouse tissues.

    PubMed

    Kremser, Christiane; Klemm, Anna-Lena; van Uelft, Martina; Imgrund, Silke; Ginkel, Christina; Hartmann, Dieter; Willecke, Klaus

    2013-11-01

    Ceramide synthase 2 (CerS2) catalyzes the synthesis of dihydroceramides from dihydrosphingosine and very long fatty acyl (C22-C24)-CoAs. CerS2-deficient (gene trap) mice were reported to exhibit myelin and behavioral abnormalities, associated with the expression of CerS2 in oligodendrocytes and neurons based on expression of lacZ reporter cDNA instead of the cers2 gene in these mice. In order to clarify the cell-type-specific expression of CerS2 protein, we have raised antibodies that specifically recognize the glycosylated and non-glycosylated CerS2 protein in wild-type but not in CerS2-deficient mouse tissues. In early postnatal, juvenile and adult mouse brain, the new antibodies detect CerS2 protein only in oligodendrocytes but not in neurons, suggesting that the gene trap vector in CerS2-deficient mice led to ectopic expression of the lacZ reporter gene in neurons. In liver, the CerS2 protein is expressed in hepatocytes but not in Ito cells or Kupffer cells. We conclude that the behavioral abnormalities observed in CerS2-deficient mice originate primarily in oligodendrocytes and not in neurons. The identification of specific cell types in which CerS2 protein is expressed is prerequisite to further mechanistic characterization of phenotypic abnormalities exhibited by CerS2-deficient mice. The amount of CerS2 protein detected in different tissues by immunoblot analyses does not strictly correspond to the activity of the CerS2 enzyme. Disproportional results are likely due to post-translational regulation of the CerS2 protein.

  8. Neurophysiology of space travel: energetic solar particles cause cell type-specific plasticity of neurotransmission.

    PubMed

    Lee, Sang-Hun; Dudok, Barna; Parihar, Vipan K; Jung, Kwang-Mook; Zöldi, Miklós; Kang, Young-Jin; Maroso, Mattia; Alexander, Allyson L; Nelson, Gregory A; Piomelli, Daniele; Katona, István; Limoli, Charles L; Soltesz, Ivan

    2016-11-30

    In the not too distant future, humankind will embark on one of its greatest adventures, the travel to distant planets. However, deep space travel is associated with an inevitable exposure to radiation fields. Space-relevant doses of protons elicit persistent disruptions in cognition and neuronal structure. However, whether space-relevant irradiation alters neurotransmission is unknown. Within the hippocampus, a brain region crucial for cognition, perisomatic inhibitory control of pyramidal cells (PCs) is supplied by two distinct cell types, the cannabinoid type 1 receptor (CB1)-expressing basket cells (CB1BCs) and parvalbumin (PV)-expressing interneurons (PVINs). Mice subjected to low-dose proton irradiation were analyzed using electrophysiological, biochemical and imaging techniques months after exposure. In irradiated mice, GABA release from CB1BCs onto PCs was dramatically increased. This effect was abolished by CB1 blockade, indicating that irradiation decreased CB1-dependent tonic inhibition of GABA release. These alterations in GABA release were accompanied by decreased levels of the major CB1 ligand 2-arachidonoylglycerol. In contrast, GABA release from PVINs was unchanged, and the excitatory connectivity from PCs to the interneurons also underwent cell type-specific alterations. These results demonstrate that energetic charged particles at space-relevant low doses elicit surprisingly selective long-term plasticity of synaptic microcircuits in the hippocampus. The magnitude and persistent nature of these alterations in synaptic function are consistent with the observed perturbations in cognitive performance after irradiation, while the high specificity of these changes indicates that it may be possible to develop targeted therapeutic interventions to decrease the risk of adverse events during interplanetary travel.

  9. Isolation and purification of a type-specific antigen from Chlamydia trachomatis propagated in cell culture utilizing molecular shift chromatography.

    PubMed

    Hourihan, J T; Rota, T R; MacDonald, A B

    1980-05-01

    Various techniques have been utilized for antigen solubilization, isolation, and purification. This report is the first to describe the isolation and purification of a type-specific antigen from Chlamydia trachomatis serotype A grown in cell culture. The type-specific antigen was prepared from Chlamydia trachomatis serotype A organisms grown in baby hamster kidney cells (BHK21). The extraction process employed a combination of both pH change and Triton X-100 solubilization. The soluble extract was radioiodinated and subjected to ion exchange and gel filtration chromatography. The fractions eluted were tested for type specificity utilizing the IgG prepared from exhaustively cross-absorbed hyperimmune sera from rabbits immunized with homologous organisms. Molecular shift chromatography was employed for analysis. Small samples of the isolated antigen were later used as markers for preparation of larger quantities necessary for antigenic characterization. The purified type-specific antigen has a m.w. of 30,000 to 32,000.

  10. Cell-type specific requirements for thiol/disulfide exchange during HIV-1 entry and infection.

    PubMed

    Stantchev, Tzanko S; Paciga, Mark; Lankford, Carla R; Schwartzkopff, Franziska; Broder, Christopher C; Clouse, Kathleen A

    2012-12-03

    The role of disulfide bond remodeling in HIV-1 infection is well described, but the process still remains incompletely characterized. At present, the data have been predominantly obtained using established cell lines and/or CXCR4-tropic laboratory-adapted virus strains. There is also ambiguity about which disulfide isomerases/reductases play a major role in HIV-1 entry, as protein disulfide isomerase (PDI) and/or thioredoxin (Trx) have emerged as the two enzymes most often implicated in this process. We have extended our previous findings and those of others by focusing on CCR5-using HIV-1 strains and their natural targets--primary human macrophages and CD4+ T lymphocytes. We found that the nonspecific thiol/disulfide exchange inhibitor, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), significantly reduced HIV-1 entry and infection in cell lines, human monocyte-derived macrophages (MDM), and also phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). Subsequent studies were performed using specific anti-PDI or Trx monoclonal antibodies (mAb) in HIV-1 envelope pseudotyped and wild type (wt) virus infection systems. Although human donor-to-donor variability was observed as expected, Trx appeared to play a greater role than PDI in HIV-1 infection of MDM. In contrast, PDI, but not Trx, was predominantly involved in HIV-1 entry and infection of the CD4+/CCR5+ T cell line, PM-1, and PHA-stimulated primary human T lymphocytes. Intriguingly, both PDI and Trx were present on the surface of MDM, PM-1 and PHA-stimulated CD4+ T cells. However, considerably lower levels of Trx were detected on freshly isolated CD4+ lymphocytes, compared to PHA-stimulated cells. Our findings clearly demonstrate the role of thiol/disulfide exchange in HIV-1 entry in primary T lymphocytes and MDM. They also establish a cell-type specificity regarding the involvement of particular disulfide isomerases/reductases in this process and may provide an explanation for differences

  11. Cell-type specific requirements for thiol/disulfide exchange during HIV-1 entry and infection

    PubMed Central

    2012-01-01

    Background The role of disulfide bond remodeling in HIV-1 infection is well described, but the process still remains incompletely characterized. At present, the data have been predominantly obtained using established cell lines and/or CXCR4-tropic laboratory-adapted virus strains. There is also ambiguity about which disulfide isomerases/ reductases play a major role in HIV-1 entry, as protein disulfide isomerase (PDI) and/or thioredoxin (Trx) have emerged as the two enzymes most often implicated in this process. Results We have extended our previous findings and those of others by focusing on CCR5-using HIV-1 strains and their natural targets - primary human macrophages and CD4+ T lymphocytes. We found that the nonspecific thiol/disulfide exchange inhibitor, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), significantly reduced HIV-1 entry and infection in cell lines, human monocyte-derived macrophages (MDM), and also phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). Subsequent studies were performed using specific anti-PDI or Trx monoclonal antibodies (mAb) in HIV-1 envelope pseudotyped and wild type (wt) virus infection systems. Although human donor-to-donor variability was observed as expected, Trx appeared to play a greater role than PDI in HIV-1 infection of MDM. In contrast, PDI, but not Trx, was predominantly involved in HIV-1 entry and infection of the CD4+/CCR5+ T cell line, PM-1, and PHA-stimulated primary human T lymphocytes. Intriguingly, both PDI and Trx were present on the surface of MDM, PM-1 and PHA-stimulated CD4+ T cells. However, considerably lower levels of Trx were detected on freshly isolated CD4+ lymphocytes, compared to PHA-stimulated cells. Conclusions Our findings clearly demonstrate the role of thiol/disulfide exchange in HIV-1 entry in primary T lymphocytes and MDM. They also establish a cell-type specificity regarding the involvement of particular disulfide isomerases/reductases in this process and may provide an

  12. Lim homeobox genes in the Ctenophore Mnemiopsis leidyi: the evolution of neural cell type specification.

    PubMed

    Simmons, David K; Pang, Kevin; Martindale, Mark Q

    2012-01-13

    Nervous systems are thought to be important to the evolutionary success and diversification of metazoans, yet little is known about the origin of simple nervous systems at the base of the animal tree. Recent data suggest that ctenophores, a group of macroscopic pelagic marine invertebrates, are the most ancient group of animals that possess a definitive nervous system consisting of a distributed nerve net and an apical statocyst. This study reports on details of the evolution of the neural cell type specifying transcription factor family of LIM homeobox containing genes (Lhx), which have highly conserved functions in neural specification in bilaterian animals. Using next generation sequencing, the first draft of the genome of the ctenophore Mnemiopsis leidyi has been generated. The Lhx genes in all animals are represented by seven subfamilies (Lhx1/5, Lhx3/4, Lmx, Islet, Lhx2/9, Lhx6/8, and LMO) of which four were found to be represented in the ctenophore lineage (Lhx1/5, Lhx3/4, Lmx, and Islet). Interestingly, the ctenophore Lhx gene complement is more similar to the sponge complement (sponges do not possess neurons) than to either the cnidarian-bilaterian or placozoan Lhx complements. Using whole mount in situ hybridization, the Lhx gene expression patterns were examined and found to be expressed around the blastopore and in cells that give rise to the apical organ and putative neural sensory cells. This research gives us a first look at neural cell type specification in the ctenophore M. leidyi. Within M. leidyi, Lhx genes are expressed in overlapping domains within proposed neural cellular and sensory cell territories. These data suggest that Lhx genes likely played a conserved role in the patterning of sensory cells in the ancestor of sponges and ctenophores, and may provide a link to the expression of Lhx orthologs in sponge larval photoreceptive cells. Lhx genes were later co-opted into patterning more diversified complements of neural and non-neural cell

  13. Lim homeobox genes in the Ctenophore Mnemiopsis leidyi: the evolution of neural cell type specification

    PubMed Central

    2012-01-01

    Background Nervous systems are thought to be important to the evolutionary success and diversification of metazoans, yet little is known about the origin of simple nervous systems at the base of the animal tree. Recent data suggest that ctenophores, a group of macroscopic pelagic marine invertebrates, are the most ancient group of animals that possess a definitive nervous system consisting of a distributed nerve net and an apical statocyst. This study reports on details of the evolution of the neural cell type specifying transcription factor family of LIM homeobox containing genes (Lhx), which have highly conserved functions in neural specification in bilaterian animals. Results Using next generation sequencing, the first draft of the genome of the ctenophore Mnemiopsis leidyi has been generated. The Lhx genes in all animals are represented by seven subfamilies (Lhx1/5, Lhx3/4, Lmx, Islet, Lhx2/9, Lhx6/8, and LMO) of which four were found to be represented in the ctenophore lineage (Lhx1/5, Lhx3/4, Lmx, and Islet). Interestingly, the ctenophore Lhx gene complement is more similar to the sponge complement (sponges do not possess neurons) than to either the cnidarian-bilaterian or placozoan Lhx complements. Using whole mount in situ hybridization, the Lhx gene expression patterns were examined and found to be expressed around the blastopore and in cells that give rise to the apical organ and putative neural sensory cells. Conclusion This research gives us a first look at neural cell type specification in the ctenophore M. leidyi. Within M. leidyi, Lhx genes are expressed in overlapping domains within proposed neural cellular and sensory cell territories. These data suggest that Lhx genes likely played a conserved role in the patterning of sensory cells in the ancestor of sponges and ctenophores, and may provide a link to the expression of Lhx orthologs in sponge larval photoreceptive cells. Lhx genes were later co-opted into patterning more diversified complements of

  14. Scaffolds for Artificial miRNA Expression in Animal Cells.

    PubMed

    Calloni, Raquel; Bonatto, Diego

    2015-10-01

    Artificial miRNAs (amiRNAs) are molecules that have been developed to promote gene silencing in a similar manner to naturally occurring miRNAs. amiRNAs are generally constructed by replacing the mature miRNA sequence in the pre-miRNA stem-loop with a sequence targeting a gene of interest. These molecules offer an interesting alternative to silencing approaches that are based on shRNAs and siRNAs because they present the same efficiency as these options and are less cytotoxic. amiRNAs have mostly been applied to gene knockdown in plants; they have been examined to a lesser extent in animal cells. Therefore, this article reviews the amiRNAs that have been developed for animal cells and focuses on the miRNA scaffolds that can already be applied to construct the artificial counterparts, as well as on the different approaches that have been described to promote amiRNA expression and silencing efficiency. Furthermore, the availability of amiRNA libraries and other tools that can be used to design and construct these molecules is briefly discussed, along with an overview of the therapeutic applications for which amiRNAs have already been evaluated.

  15. Identification of Cell Type-Specific Differences in Erythropoietin Receptor Signaling in Primary Erythroid and Lung Cancer Cells

    PubMed Central

    Salopiata, Florian; Depner, Sofia; Wäsch, Marvin; Böhm, Martin E.; Mücke, Oliver; Plass, Christoph; Lehmann, Wolf D.; Kreutz, Clemens; Timmer, Jens; Klingmüller, Ursula

    2016-01-01

    Lung cancer, with its most prevalent form non-small-cell lung carcinoma (NSCLC), is one of the leading causes of cancer-related deaths worldwide, and is commonly treated with chemotherapeutic drugs such as cisplatin. Lung cancer patients frequently suffer from chemotherapy-induced anemia, which can be treated with erythropoietin (EPO). However, studies have indicated that EPO not only promotes erythropoiesis in hematopoietic cells, but may also enhance survival of NSCLC cells. Here, we verified that the NSCLC cell line H838 expresses functional erythropoietin receptors (EPOR) and that treatment with EPO reduces cisplatin-induced apoptosis. To pinpoint differences in EPO-induced survival signaling in erythroid progenitor cells (CFU-E, colony forming unit-erythroid) and H838 cells, we combined mathematical modeling with a method for feature selection, the L1 regularization. Utilizing an example model and simulated data, we demonstrated that this approach enables the accurate identification and quantification of cell type-specific parameters. We applied our strategy to quantitative time-resolved data of EPO-induced JAK/STAT signaling generated by quantitative immunoblotting, mass spectrometry and quantitative real-time PCR (qRT-PCR) in CFU-E and H838 cells as well as H838 cells overexpressing human EPOR (H838-HA-hEPOR). The established parsimonious mathematical model was able to simultaneously describe the data sets of CFU-E, H838 and H838-HA-hEPOR cells. Seven cell type-specific parameters were identified that included for example parameters for nuclear translocation of STAT5 and target gene induction. Cell type-specific differences in target gene induction were experimentally validated by qRT-PCR experiments. The systematic identification of pathway differences and sensitivities of EPOR signaling in CFU-E and H838 cells revealed potential targets for intervention to selectively inhibit EPO-induced signaling in the tumor cells but leave the responses in erythroid

  16. Cell-type specific regulation of cortical excitability through the allatostatin receptor system

    PubMed Central

    Ikrar, Taruna; Shi, Yulin; Velasquez, Tomoko; Goulding, Martyn; Xu, Xiangmin

    2012-01-01

    Recent technical advances enable the regulation of neuronal circuit activity with high spatial and temporal resolution through genetic delivery of molecular activation or inactivation systems.Among them, the allatostatin receptor (AlstR)/ligand system has been developed for selective and quickly reversible silencing of mammalian neurons. However, targeted AlstR-mediated inactivation of specific neuronal types, particularly diverse types of inhibitory interneurons, remains to be established. In the present study, we achieved Cre-directed expression of AlstRs to excitatory and inhibitory cell-types in the cortex, and found that the AlstR-mediated inactivation was specific and robust at single-cell and neuronal population levels. Bath application of the allatostatin peptide markedly reduced spiking activity of AlstR-expressing excitatory and inhibitory neurons in response to intrasomatic current injections and laser photostimulation via glutamate uncaging, but control neurons without AlstR expression were not affected. As for the cortical network activity, the peptide application constrained photostimulation-evoked excitatory activity propagation detected by fast voltage-sensitive dye (VSD) imaging of the slices expressing AlstRs selectively in excitatory neurons, while it augmented excitatory activity in those slices with inhibitory neurons expressing AlstRs. In addition, AlstR-mediated inactivation effectively suppressed pharmacologically induced seizure activity in the slices targeting AlstRs to excitatory neurons. Taken together, our work demonstrated that the genetic delivery of AlstRs can be used for regulation of cortical excitability in a cell-type specific manner, and suggested that the AlstR system can be potentially used for fast seizure control. PMID:22319474

  17. Cell-Type-Specific Effects of RNase L on Viral Induction of Beta Interferon

    PubMed Central

    Banerjee, Shuvojit; Chakrabarti, Arindam; Jha, Babal Kant; Weiss, Susan R.; Silverman, Robert H.

    2014-01-01

    ABSTRACT The interferon (IFN)-inducible antiviral state is mediated in part by the 2′,5′-oligoadenylate (2-5A) synthetase (OAS)/RNase L system. 2-5A, produced from ATP by OAS proteins in response to viral double-stranded RNA, binds to and activates RNase L. RNase L restricts viral infections by degrading viral and cellular RNA, inducing autophagy and apoptosis, and producing RNA degradation products that amplify production of type I interferons (IFNs) through RIG-I-like receptors. However, the effects of the OAS/RNase L pathway on IFN induction in different cell types that vary in basal levels of these proteins have not been previously reported. Here we report higher basal expression of both RNase L and OAS in mouse macrophages in comparison to mouse embryonic fibroblasts (MEFs). In MEFs, RNase L gene knockout decreased induction of IFN-β by encephalomyocarditis virus infection or poly(rI):poly(rC) (pIC) transfection. In contrast, in macrophages, RNase L deletion increased (rather than decreased) induction of IFN-β by virus or pIC. RNA damage from RNase L in virus-infected macrophages is likely responsible for reducing IFN-β production. Similarly, direct activation of RNase L by transfection with 2-5A induced IFN-β in MEFs but not in macrophages. Also, viral infection or pIC transfection caused RNase L-dependent apoptosis of macrophages but not of MEFs. Our results suggest that cell-type-specific differences in basal levels of OAS and RNase L are determinants of IFN-β induction that could affect tissue protection and survival during viral infections. PMID:24570368

  18. Identification of cell-type-specific mutations in nodal T-cell lymphomas

    PubMed Central

    Nguyen, T B; Sakata-Yanagimoto, M; Asabe, Y; Matsubara, D; Kano, J; Yoshida, K; Shiraishi, Y; Chiba, K; Tanaka, H; Miyano, S; Izutsu, K; Nakamura, N; Takeuchi, K; Miyoshi, H; Ohshima, K; Minowa, T; Ogawa, S; Noguchi, M; Chiba, S

    2017-01-01

    Recent genetic analysis has identified frequent mutations in ten-eleven translocation 2 (TET2), DNA methyltransferase 3A (DNMT3A), isocitrate dehydrogenase 2 (IDH2) and ras homolog family member A (RHOA) in nodal T-cell lymphomas, including angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified. We examined the distribution of mutations in these subtypes of mature T-/natural killer cell neoplasms to determine their clonal architecture. Targeted sequencing was performed for 71 genes in tumor-derived DNA of 87 cases. The mutations were then analyzed in a programmed death-1 (PD1)-positive population enriched with tumor cells and CD20-positive B cells purified by laser microdissection from 19 cases. TET2 and DNMT3A mutations were identified in both the PD1+ cells and the CD20+ cells in 15/16 and 4/7 cases, respectively. All the RHOA and IDH2 mutations were confined to the PD1+ cells, indicating that some, including RHOA and IDH2 mutations, being specific events in tumor cells. Notably, we found that all NOTCH1 mutations were detected only in the CD20+ cells. In conclusion, we identified both B- as well as T-cell-specific mutations, and mutations common to both T and B cells. These findings indicate the expansion of a clone after multistep and multilineal acquisition of gene mutations. PMID:28157189

  19. Coordinating cell proliferation and differentiation: Antagonism between cell cycle regulators and cell type-specific gene expression

    PubMed Central

    Ruijtenberg, Suzan; van den Heuvel, Sander

    2016-01-01

    ABSTRACT Cell proliferation and differentiation show a remarkable inverse relationship. Precursor cells continue division before acquiring a fully differentiated state, while terminal differentiation usually coincides with proliferation arrest and permanent exit from the division cycle. Mechanistic insight in the temporal coordination between cell cycle exit and differentiation has come from studies of cells in culture and genetic animal models. As initially described for skeletal muscle differentiation, temporal coordination involves mutual antagonism between cyclin-dependent kinases that promote cell cycle entry and transcription factors that induce tissue-specific gene expression. Recent insights highlight the contribution of chromatin-regulating complexes that act in conjunction with the transcription factors and determine their activity. In particular SWI/SNF chromatin remodelers contribute to dual regulation of cell cycle and tissue-specific gene expression during terminal differentiation. We review the concerted regulation of the cell cycle and cell type-specific transcription, and discuss common mutations in human cancer that emphasize the clinical importance of proliferation versus differentiation control. PMID:26825227

  20. Coordinating cell proliferation and differentiation: Antagonism between cell cycle regulators and cell type-specific gene expression.

    PubMed

    Ruijtenberg, Suzan; van den Heuvel, Sander

    2016-01-01

    Cell proliferation and differentiation show a remarkable inverse relationship. Precursor cells continue division before acquiring a fully differentiated state, while terminal differentiation usually coincides with proliferation arrest and permanent exit from the division cycle. Mechanistic insight in the temporal coordination between cell cycle exit and differentiation has come from studies of cells in culture and genetic animal models. As initially described for skeletal muscle differentiation, temporal coordination involves mutual antagonism between cyclin-dependent kinases that promote cell cycle entry and transcription factors that induce tissue-specific gene expression. Recent insights highlight the contribution of chromatin-regulating complexes that act in conjunction with the transcription factors and determine their activity. In particular SWI/SNF chromatin remodelers contribute to dual regulation of cell cycle and tissue-specific gene expression during terminal differentiation. We review the concerted regulation of the cell cycle and cell type-specific transcription, and discuss common mutations in human cancer that emphasize the clinical importance of proliferation versus differentiation control.

  1. Establishment of human cell type-specific iPS cells with enhanced chondrogenic potential.

    PubMed

    Guzzo, Rosa M; Scanlon, Vanessa; Sanjay, Archana; Xu, Ren-He; Drissi, Hicham

    2014-12-01

    The propensity of induced pluripotent stem (iPS) cells to differentiate into specific lineages may be influenced by a number of factors, including the selection of the somatic cell type used for reprogramming. Herein we report the generation of new iPS cells, which we derived from human articular chondrocytes and from cord blood mononucleocytes via lentiviral-mediated delivery of Oct4, Klf4, Sox2, and cMyc. Molecular, cytochemical, and cytogenic analyses confirmed the acquisition of hallmark features of pluripotency, as well as the retention of normal karyotypes following reprogramming of both the human articular chondrocytes (AC) and the cord blood (CB) cells. In vitro and in vivo functional analyses formally established the pluripotent differentiation capacity of all cell lines. Chondrogenic differentiation assays comparing iPS cells derived from AC, CB, and a well established dermal fibroblast cell line (HDFa-Yk26) identified enhanced proteoglycan-rich matrix formation and cartilage-associated gene expression from AC-derived iPS cells. These findings suggest that the tissue of origin may impact the fate potential of iPS cells for differentiating into specialized cell types, such as chondrocytes. Thus, we generated new cellular tools for the identification of inherent features driving high chondrogenic potential of reprogrammed cells.

  2. Cell-type-specific effects of RNase L on viral induction of beta interferon.

    PubMed

    Banerjee, Shuvojit; Chakrabarti, Arindam; Jha, Babal Kant; Weiss, Susan R; Silverman, Robert H

    2014-02-25

    The interferon (IFN)-inducible antiviral state is mediated in part by the 2',5'-oligoadenylate (2-5A) synthetase (OAS)/RNase L system. 2-5A, produced from ATP by OAS proteins in response to viral double-stranded RNA, binds to and activates RNase L. RNase L restricts viral infections by degrading viral and cellular RNA, inducing autophagy and apoptosis, and producing RNA degradation products that amplify production of type I interferons (IFNs) through RIG-I-like receptors. However, the effects of the OAS/RNase L pathway on IFN induction in different cell types that vary in basal levels of these proteins have not been previously reported. Here we report higher basal expression of both RNase L and OAS in mouse macrophages in comparison to mouse embryonic fibroblasts (MEFs). In MEFs, RNase L gene knockout decreased induction of IFN-β by encephalomyocarditis virus infection or poly(rI):poly(rC) (pIC) transfection. In contrast, in macrophages, RNase L deletion increased (rather than decreased) induction of IFN-β by virus or pIC. RNA damage from RNase L in virus-infected macrophages is likely responsible for reducing IFN-β production. Similarly, direct activation of RNase L by transfection with 2-5A induced IFN-β in MEFs but not in macrophages. Also, viral infection or pIC transfection caused RNase L-dependent apoptosis of macrophages but not of MEFs. Our results suggest that cell-type-specific differences in basal levels of OAS and RNase L are determinants of IFN-β induction that could affect tissue protection and survival during viral infections. IMPORTANCE Type I interferons (IFNs) such as IFN-β are essential antiviral cytokines that are often required for animal survival following infections by highly pathogenic viruses. Therefore, host factors that regulate type I IFN production are critically important for animal and human health. Previously we reported that the OAS/RNase L pathway amplifies antiviral innate immunity by enhancing IFN-β production in mouse

  3. Cell-type specific posttranslational processing of peptides by different pituitary cell lines.

    PubMed

    Dickerson, I M; Mains, R E

    1990-07-01

    In order to compare prohormone processing in two distinct pituitary cell types, somatomammotrope cells (GH3) and corticotrope cells (AtT-20) were stably transfected with vectors encoding preproneuropeptide Y (preproNPY) containing four different pairs of basic amino acids at the single endoproteolytic cleavage site: wildtype or KR (lysine-arginine), RR, RK, and KK. The GH-NPY cell lines cleaved proNPY to a similar extent, regardless of the sequence of the basic amino acids at the cleavage site (KR = RR = RK = KK). AtT-20-NPY cells are known to exhibit a strong hierarchy of cleavage site preference when processing wildtype and mutated proNPY forms (KR = RR greater than RK much greater than KK). All four types of GH-NPY and AtT-NPY cells faithfully produced NPY (1-36) NH2 from proNPY (1-69), regardless of the amino acid sequence at the cleavage site. All four types of GH-NPY cells produced some of the expected proNPY-COOH-terminal peptide with Ser40 at its NH2-terminal [proNPY (40-69)]. GH3 cells expressing the RR, RK, and KK forms of proNPY yielded in addition some proNPY-COOH-terminal peptide retaining the amino terminals Lys39 or Arg39 residue. In contrast, AtT-NPY-RK cells produced only the Lys39 form of proNPY-COOH-terminal peptide while the other three AtT-NPY lines (KR, RR, and KK) produced only the Ser40 form of proNPY-COOH-terminal peptide. The residence time of proNPY and NPY in GH3 cells was dramatically increased by treatment with insulin, estradiol, and epidermal growth factor, in concert with the expected increase in PRL synthesis and decrease in GH synthesis; increased residence time in the cells did not result in an increase in the extent of cleavage of proNPY to NPY. AtT-20 cells did not respond to the somatomammotrope-specific set of hormones. Thus, there are several important differences in the posttranslational processing and storage of peptide hormones in corticotropes and somatomammotropes.

  4. Comparative DNA methylation analysis to decipher common and cell type-specific patterns among multiple cell types.

    PubMed

    Yang, Xiaofei; Shao, Xiaojian; Gao, Lin; Zhang, Shihua

    2016-11-01

    DNA methylation has been proved to play important roles in cell development and complex diseases through comparative studies of DNA methylation profiles across different tissues and samples. Current studies indicate that the regulation of DNA methylation to gene expression depends on the genomic locations of CpGs. Common DNA methylation patterns shared across different cell types and tissues are abundant, and they are likely involved in the basic functions of cell development, such as housekeeping functions. By way of contrast, cell type-specific DNA methylation patterns show distinct functional relevance with cell type specificity. Additionally, abnormal DNA methylation patterns are extensively involved in tumour development. Pan-cancer methylation patterns reveal common mechanisms and new similarities of different cancers, while cancer-specific patterns are relating to tumour heterogeneity and patient survival. Moreover, DNA methylation patterns in specific cancer are relevant with diverse regulatory elements such as enhancers and long non-coding RNAs. In this review, we survey the recent advances on DNA methylation patterns in normal or tumour states to illustrate their potential roles in cell development and cell canceration. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  5. Cell-Type Specific Inactivation of Hippocampal CA1 Disrupts Location-Dependent Object Recognition in the Mouse

    ERIC Educational Resources Information Center

    Haettig, Jakob; Sun, Yanjun; Wood, Marcelo A.; Xu, Xiangmin

    2013-01-01

    The allatostatin receptor (AlstR)/ligand inactivation system enables potent regulation of neuronal circuit activity. To examine how different cell types participate in memory formation, we have used this system through Cre-directed, cell-type specific expression in mouse hippocampal CA1 in vivo and examined functional effects of inactivation of…

  6. Cell-Type Specific Inactivation of Hippocampal CA1 Disrupts Location-Dependent Object Recognition in the Mouse

    ERIC Educational Resources Information Center

    Haettig, Jakob; Sun, Yanjun; Wood, Marcelo A.; Xu, Xiangmin

    2013-01-01

    The allatostatin receptor (AlstR)/ligand inactivation system enables potent regulation of neuronal circuit activity. To examine how different cell types participate in memory formation, we have used this system through Cre-directed, cell-type specific expression in mouse hippocampal CA1 in vivo and examined functional effects of inactivation of…

  7. Differential expression of miRNA between the monolayer and three dimensional cells after ionizing radiation

    NASA Astrophysics Data System (ADS)

    Pan, Dong; Ren, Zhenxin; Hu, Burong

    2014-04-01

    We detect the expression of miRNA in 2D and 3D human lung epithelial cells (3KT). And our primary experimental results showed that more miRNA in 3D 3KT down regulated than in 2D 3KT cells after not only X-ray but also C-beam irradiation using the miRNA chip assay. Meanwhile, X-ray induced more significantly differential expression of miRNA when the relative expression value of miRNA in 3D cells were compared to 2D cells after irradiation.

  8. Cell type-specific expression of JC virus early promoter is determined by positive and negative regulation.

    PubMed

    Tada, H; Lashgari, M; Rappaport, J; Khalili, K

    1989-01-01

    We analyzed control sequences of the human papovavirus JC virus (JCV) to define the cis-acting elements that regulate specific expression of the viral early region genes in glial cells. Nuclear run-on transcription, S1 analysis, and chloramphenicol acetyltransferase enzyme activity in a transient transfection assay established that the cell type-specific expression of JCV early genes is determined at the transcriptional level. Using DNase footprinting analysis of nuclear proteins prepared from glial and nonglial cells, we located four regions within the JCV control sequences that specifically interacted with the proteins. In glial cells, all four domains contributed to the specific expression of a heterologous promoter, whereas in nonglial cells, two protein-binding regions showed no effect on basal transcriptional activity and the other two domains significantly downregulated transcription of the promoter. We conclude that cell type-specific transcription of the JCV early promoter is under both positive and negative regulation in eucaryotic cells.

  9. Increased miRNA-22 expression sensitizes esophageal squamous cell carcinoma to irradiation

    PubMed Central

    Wang, Xiao-chun; Zhang, Zhu-Bo; Wang, Yue-Ying; Wu, Hong-Ying; Li, De-Guan; Meng, Ai-Min; Fan, Fei-Yue

    2013-01-01

    miRNA-22 was previously reported to be a tumor suppressor. The aim of this study was to explore the expression and function of miRNA-22 in esophageal squamous cell carcinoma (ESCC). Expression of miRNA-22 in 100 ESCC tissues was examined by q-PCR. The correlation between miRNA-22 level and clinicopathological features was analyzed using SPSS16.0 statistical software. Moreover, the effect of miRNA-22 expression on radiosensitivity of ESCC cells was examined. miRNA-22 expression decreased in ESCC tissues, and statistical analyses showed that the expression of miRNA-22 was associated with the stage of clinical classification. No correlation was found between miRNA-22 expression and the overall survival of ESCC patients. However, significant positive correlation was found between miRNA-22 expression and the survival of patients who received radiotherapy (P < 0.05). Increased expression of miRNA-22 sensitized ESCC cells to γ-ray radiation and promoted the apoptosis of ESCC cells induced by γ-ray radiation. Increased expression level of miRNA-22 had effects on Rad51 expression after irradiation. These results demonstrate for the first time that decreased miRNA-22 expression correlates with increased radiotherapy resistance of ESCC, and that this effect is mediated, at least in part, by the Rad51 pathway. PMID:23188185

  10. Cell Type-specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex

    PubMed Central

    Zhang, Xiaochang; Chen, Ming Hui; Wu, Xuebing; Kodani, Andrew; Fan, Jean; Doan, Ryan; Ozawa, Manabu; Ma, Jacqueline; Yoshida, Nobuaki; Reiter, Jeremy F.; Black, Douglas L.; Kharchenko, Peter V.; Sharp, Phillip A.; Walsh, Christopher A.

    2017-01-01

    SUMMARY Alternative splicing is prevalent in the mammalian brain. To interrogate the functional role of alternative splicing in neural development, we analyzed purified neural progenitor cells (NPCs) and neurons from developing cerebral cortices, revealing hundreds of differentially spliced exons that preferentially alter key protein domains—especially in cytoskeletal proteins—and can harbor disease-causing mutations. We show that Ptbp1 and Rbfox proteins antagonistically govern the NPC-to-neuron transition by regulating neuron-specific exons. While Ptbp1 maintains apical progenitors partly through suppressing a poison exon of Flna in NPCs, Rbfox proteins promote neuronal differentiation by switching Ninein from a centrosomal splice form in NPCs to a non-centrosomal isoform in neurons. We further uncover an intronic human mutation within a PTBP1 binding site that disrupts normal skipping of the FLNA poison exon in NPCs and causes a brain-specific malformation. Our study indicates that dynamic control of alternative splicing governs cell fate in cerebral cortical development. PMID:27565344

  11. Role of the chromatin landscape and sequence in determining cell type-specific genomic glucocorticoid receptor binding and gene regulation

    PubMed Central

    Huska, Matthew R.; Jurk, Marcel; Schöpflin, Robert; Starick, Stephan R.; Schwahn, Kevin; Cooper, Samantha B.; Yamamoto, Keith R.; Thomas-Chollier, Morgane; Vingron, Martin

    2017-01-01

    Abstract The genomic loci bound by the glucocorticoid receptor (GR), a hormone-activated transcription factor, show little overlap between cell types. To study the role of chromatin and sequence in specifying where GR binds, we used Bayesian modeling within the universe of accessible chromatin. Taken together, our results uncovered that although GR preferentially binds accessible chromatin, its binding is biased against accessible chromatin located at promoter regions. This bias can only be explained partially by the presence of fewer GR recognition sequences, arguing for the existence of additional mechanisms that interfere with GR binding at promoters. Therefore, we tested the role of H3K9ac, the chromatin feature with the strongest negative association with GR binding, but found that this correlation does not reflect a causative link. Finally, we find a higher percentage of promoter–proximal GR binding for genes regulated by GR across cell types than for cell type-specific target genes. Given that GR almost exclusively binds accessible chromatin, we propose that cell type-specific regulation by GR preferentially occurs via distal enhancers, whose chromatin accessibility is typically cell type-specific, whereas ubiquitous target gene regulation is more likely to result from binding to promoter regions, which are often accessible regardless of cell type examined. PMID:27903902

  12. Invariant TAD Boundaries Constrain Cell-Type-Specific Looping Interactions between Promoters and Distal Elements around the CFTR Locus

    PubMed Central

    Smith, Emily M.; Lajoie, Bryan R.; Jain, Gaurav; Dekker, Job

    2016-01-01

    Three-dimensional genome structure plays an important role in gene regulation. Globally, chromosomes are organized into active and inactive compartments while, at the gene level, looping interactions connect promoters to regulatory elements. Topologically associating domains (TADs), typically several hundred kilobases in size, form an intermediate level of organization. Major questions include how TADs are formed and how they are related to looping interactions between genes and regulatory elements. Here we performed a focused 5C analysis of a 2.8 Mb chromosome 7 region surrounding CFTR in a panel of cell types. We find that the same TAD boundaries are present in all cell types, indicating that TADs represent a universal chromosome architecture. Furthermore, we find that these TAD boundaries are present irrespective of the expression and looping of genes located between them. In contrast, looping interactions between promoters and regulatory elements are cell-type specific and occur mostly within TADs. This is exemplified by the CFTR promoter that in different cell types interacts with distinct sets of distal cell-type-specific regulatory elements that are all located within the same TAD. Finally, we find that long-range associations between loci located in different TADs are also detected, but these display much lower interaction frequencies than looping interactions within TADs. Interestingly, interactions between TADs are also highly cell-type-specific and often involve loci clustered around TAD boundaries. These data point to key roles of invariant TAD boundaries in constraining as well as mediating cell-type-specific long-range interactions and gene regulation. PMID:26748519

  13. miRNA in situ hybridization in circulating tumor cells--MishCTC.

    PubMed

    Ortega, Francisco G; Lorente, Jose A; Garcia Puche, Jose L; Ruiz, Maria P; Sanchez-Martin, Rosario M; de Miguel-Pérez, Diego; Diaz-Mochon, Juan J; Serrano, Maria J

    2015-03-17

    Circulating tumor cells (CTCs) must be phenotypically and genetically characterized before they can be utilized in clinical applications. Here, we present the first protocol for the detection of miRNAs in CTCs using in situ hybridization (ISH) combined with immunomagnetic selection based on cytokeratin (CK) expression and immunocytochemistry. Locked-Nucleic Acid (LNA) probes associated with an enzyme-labeled fluorescence (ELF) signal amplification approach were used to detect miRNA-21 in CTCs. This protocol was optimized using both epithelial tumor (MDA-MB468) and epithelial non-tumor (MCF-10A) cell lines, and miRNA-21 was selected as the target miRNA because of its known role as an onco-miRNA. Hematopoietic cells do not express miRNA-21; thus, miRNA-21 is an ideal marker for detecting CTCs. Peripheral blood samples were taken from 25 cancer patients and these samples were analyzed using our developed protocol. Of the 25 samples, 11 contained CTCs. For all 11 CTC-positive samples, the isolated CTCs expressed both CK and miRNA-21. Finally, the protocol was applied to monitor miRNA-21 expression in epithelial to mesenchymal transition (EMT)-induced MCF-7 cells, an epithelial tumor cell line. CK expression was lost in these cells, whereas miRNA-21 was still expressed, suggesting that miRNA-21 might be a good marker for detecting CTCs with an EMT phenotype.

  14. miRNA in situ hybridization in circulating tumor cells - MishCTC

    PubMed Central

    Ortega, Francisco G.; Lorente, Jose A.; Garcia Puche, Jose L.; Ruiz, Maria P.; Sanchez-Martin, Rosario M.; de Miguel-Pérez, Diego; Diaz-Mochon, Juan J.; Serrano, Maria J.

    2015-01-01

    Circulating tumor cells (CTCs) must be phenotypically and genetically characterized before they can be utilized in clinical applications. Here, we present the first protocol for the detection of miRNAs in CTCs using in situ hybridization (ISH) combined with immunomagnetic selection based on cytokeratin (CK) expression and immunocytochemistry. Locked-Nucleic Acid (LNA) probes associated with an enzyme-labeled fluorescence (ELF) signal amplification approach were used to detect miRNA-21 in CTCs. This protocol was optimized using both epithelial tumor (MDA-MB468) and epithelial non-tumor (MCF-10A) cell lines, and miRNA-21 was selected as the target miRNA because of its known role as an onco-miRNA. Hematopoietic cells do not express miRNA-21; thus, miRNA-21 is an ideal marker for detecting CTCs. Peripheral blood samples were taken from 25 cancer patients and these samples were analyzed using our developed protocol. Of the 25 samples, 11 contained CTCs. For all 11 CTC-positive samples, the isolated CTCs expressed both CK and miRNA-21. Finally, the protocol was applied to monitor miRNA-21 expression in epithelial to mesenchymal transition (EMT)-induced MCF-7 cells, an epithelial tumor cell line. CK expression was lost in these cells, whereas miRNA-21 was still expressed, suggesting that miRNA-21 might be a good marker for detecting CTCs with an EMT phenotype. PMID:25777797

  15. Mechanisms of epigenetic and cell-type specific regulation of Hey target genes in ES cells and cardiomyocytes.

    PubMed

    Weber, David; Heisig, Julia; Kneitz, Susanne; Wolf, Elmar; Eilers, Martin; Gessler, Manfred

    2015-02-01

    Hey bHLH transcription factors are critical effectors of Notch signaling. During mammalian heart development they are expressed in atrial and ventricular cardiomyocytes and in the developing endocardium. Hey knockout mice suffer from lethal cardiac defects, such as ventricular septum defects, valve defects and cardiomyopathy. Despite this functional relevance, little is known about the regulation of downstream targets in relevant cell types. The objective of this study was to elucidate the regulatory mechanisms by which Hey proteins affect gene expression in a cell type specific manner. We used an in vitro cardiomyocyte differentiation system with inducible Hey1 or Hey2 expression to study target gene regulation in cardiomyocytes (CM) generated from murine embryonic stem cells (ESC). The effects of Hey1 and Hey2 are largely redundant, but cell type specific. The number of regulated genes is comparable between ESC and CM, but the total number of binding sites is much higher, especially in ESC, targeting mainly genes involved in transcriptional regulation and developmental processes. Repression by Hey proteins generally correlates with the extent of Hey-binding to target promoters, Hdac recruitment and lower histone acetylation. Functionally, treatment with the Hdac inhibitor TSA abolished Hey target gene regulation. However, in CM the repressive effect of Hey-binding is lost for a subset of genes. These also lack Hey-dependent histone deacetylation in CM and are enriched for binding sites of cardiac specific activators like Srf, Nkx2-5, and Gata4. Ectopic Nkx2-5 overexpression in ESC blocks Hey-mediated repression of these genes. Thus, Hey proteins mechanistically repress target genes via Hdac recruitment and histone deacetylation. In CM Hey-repression is counteracted by cardiac activators, which recruit histone acetylases and prevent Hey mediated deacetylation and subsequent repression for a subset of genes.

  16. Identification of a Wnt/Dvl/beta-Catenin --> Pitx2 pathway mediating cell-type-specific proliferation during development.

    PubMed

    Kioussi, Chrissa; Briata, Paola; Baek, Sung Hee; Rose, David W; Hamblet, Natasha S; Herman, Thomas; Ohgi, Kenneth A; Lin, Chijen; Gleiberman, Anatoli; Wang, Jianbo; Brault, Veronique; Ruiz-Lozano, Pilar; Nguyen, H D; Kemler, Rolf; Glass, Christopher K; Wynshaw-Boris, Anthony; Rosenfeld, Michael G

    2002-11-27

    Understanding the cell type-specific molecular mechanisms by which distinct signaling pathways combinatorially control proliferation during organogenesis is a central issue in development and disease. Here, we report that the bicoid-related transcription factor Pitx2 is rapidly induced by the Wnt/Dvl/beta-catenin pathway and is required for effective cell-type-specific proliferation by directly activating specific growth-regulating genes. Regulated exchange of HDAC1/beta-catenin converts Pitx2 from repressor to activator, analogous to control of TCF/LEF1. Pitx2 then serves as a competence factor required for the temporally ordered and growth factor-dependent recruitment of a series of specific coactivator complexes that prove necessary for Cyclin D2 gene induction. The molecular strategy underlying interactions between the Wnt and growth factor-dependent signaling pathways in cardiac outflow tract and pituitary proliferation is likely to be prototypic of cell-specific proliferation strategies in other tissues.

  17. Comprehensive Identification and Annotation of Cell Type-Specific and Ubiquitous CTCF-Binding Sites in the Human Genome

    PubMed Central

    Shu, Wenjie; Bo, Xiaochen; Wang, Shengqi

    2012-01-01

    Chromatin insulators are DNA elements that regulate the level of gene expression either by preventing gene silencing through the maintenance of heterochromatin boundaries or by preventing gene activation by blocking interactions between enhancers and promoters. CCCTC-binding factor (CTCF), a ubiquitously expressed 11-zinc-finger DNA-binding protein, is the only protein implicated in the establishment of insulators in vertebrates. While CTCF has been implicated in diverse regulatory functions, CTCF has only been studied in a limited number of cell types across human genome. Thus, it is not clear whether the identified cell type-specific differences in CTCF-binding sites are functionally significant. Here, we identify and characterize cell type-specific and ubiquitous CTCF-binding sites in the human genome across 38 cell types designated by the Encyclopedia of DNA Elements (ENCODE) consortium. These cell type-specific and ubiquitous CTCF-binding sites show uniquely versatile transcriptional functions and characteristic chromatin features. In addition, we confirm the insulator barrier function of CTCF-binding and explore the novel function of CTCF in DNA replication. These results represent a critical step toward the comprehensive and systematic understanding of CTCF-dependent insulators and their versatile roles in the human genome. PMID:22829947

  18. Induced pluripotent stem cell models of Zellweger spectrum disorder show impaired peroxisome assembly and cell type-specific lipid abnormalities.

    PubMed

    Wang, Xiao-Ming; Yik, Wing Yan; Zhang, Peilin; Lu, Wange; Huang, Ning; Kim, Bo Ram; Shibata, Darryl; Zitting, Madison; Chow, Robert H; Moser, Ann B; Steinberg, Steven J; Hacia, Joseph G

    2015-08-29

    Zellweger spectrum disorder (PBD-ZSD) is a disease continuum caused by mutations in a subset of PEX genes required for normal peroxisome assembly and function. They highlight the importance of peroxisomes in the development and functions of the central nervous system, liver, and other organs. To date, the underlying bases for the cell-type specificity of disease are not fully elucidated. Primary skin fibroblasts from seven PBD-ZSD patients with biallelic PEX1, PEX10, PEX12, or PEX26 mutations and three healthy donors were transduced with retroviral vectors expressing Yamanaka reprogramming factors. Candidate induced pluripotent stem cells (iPSCs) were subject to global gene expression, DNA methylation, copy number variation, genotyping, in vitro differentiation and teratoma formation assays. Confirmed iPSCs were differentiated into neural progenitor cells (NPCs), neurons, oligodendrocyte precursor cells (OPCs), and hepatocyte-like cell cultures with peroxisome assembly evaluated by microscopy. Saturated very long chain fatty acid (sVLCFA) and plasmalogen levels were determined in primary fibroblasts and their derivatives. iPSCs were derived from seven PBD-ZSD patient-derived fibroblasts with mild to severe peroxisome assembly defects. Although patient and control skin fibroblasts had similar gene expression profiles, genes related to mitochondrial functions and organelle cross-talk were differentially expressed among corresponding iPSCs. Mitochondrial DNA levels were consistent among patient and control fibroblasts, but varied among all iPSCs. Relative to matching controls, sVLCFA levels were elevated in patient-derived fibroblasts, reduced in patient-derived iPSCs, and not significantly different in patient-derived NPCs. All cell types derived from donors with biallelic null mutations in a PEX gene showed plasmalogen deficiencies. Reporter gene assays compatible with high content screening (HCS) indicated patient-derived OPC and hepatocyte-like cell cultures had

  19. Modelling epigenetic regulation of gene expression in 12 human cell types reveals combinatorial patterns of cell-type-specific genes.

    PubMed

    Lu, Yiming; Qu, Wubin; Min, Bo; Liu, Zheyan; Chen, Changsheng; Zhang, Chenggang

    2014-06-01

    The maintenance of the diverse cell types in a multicellular organism is one of the fundamental mysteries of biology. Modelling the dynamic regulatory relationships between the histone modifications and the gene expression across the diverse cell types is essential for the authors to understand the mechanisms of the epigenetic regulation. Here, the authors thoroughly assessed the histone modification enrichment profiles at the promoters and constructed quantitative models between the histone modification abundances and the gene expression in 12 human cell types. The author's results showed that the histone modifications at the promoters exhibited remarkably cell-type-dependent variability in the cell-type-specific (CTS) genes. They demonstrated that the variable profiles of the modifications are highly predictive for the dynamic changes of the gene expression across all the cell types. Their findings revealed the close relationship between the combinatorial patterns of the histone modifications and the CTS gene expression. They anticipate that the findings and the methods they used in this study could provide useful information for the future studies of the regulatory roles of the histone modifications in the CTS genes.

  20. miRNA Profiling of Naïve, Effector and Memory CD8 T Cells

    PubMed Central

    Wu, Haoquan; Neilson, Joel R.; Kumar, Priti; Manocha, Monika; Shankar, Premlata; Sharp, Phillip A.; Manjunath, N.

    2007-01-01

    microRNAs have recently emerged as master regulators of gene expression during development and cell differentiation. Although profound changes in gene expression also occur during antigen-induced T cell differentiation, the role of miRNAs in the process is not known. We compared the miRNA expression profiles between antigen-specific naïve, effector and memory CD8+ T cells using 3 different methods-small RNA cloning, miRNA microarray analysis and real-time PCR. Although many miRNAs were expressed in all the T cell subsets, the frequency of 7 miRNAs (miR-16, miR-21, miR-142-3p, miR-142-5p, miR-150, miR-15b and let-7f) alone accounted for ∼60% of all miRNAs, and their expression was several fold higher than the other expressed miRNAs. Global downregulation of miRNAs (including 6/7 dominantly expressed miRNAs) was observed in effector T cells compared to naïve cells and the miRNA expression levels tended to come back up in memory T cells. However, a few miRNAs, notably miR-21 were higher in effector and memory T cells compared to naïve T cells. These results suggest that concomitant with profound changes in gene expression, miRNA profile also changes dynamically during T cell differentiation. Sequence analysis of the cloned mature miRNAs revealed an extensive degree of end polymorphism. While 3′end polymorphisms dominated, heterogeneity at both ends, resembling drosha/dicer processing shift was also seen in miR-142, suggesting a possible novel mechanism to generate new miRNA and/or to diversify miRNA target selection. Overall, our results suggest that dynamic changes in the expression of miRNAs may be important for the regulation of gene expression during antigen-induced T cell differentiation. Our study also suggests possible novel mechanisms for miRNA biogenesis and function. PMID:17925868

  1. Cell Type-Specific Transcriptome of Brassicaceae Stigmatic Papilla Cells From a Combination of Laser Microdissection and RNA Sequencing

    PubMed Central

    Osaka, Masaaki; Matsuda, Tomoki; Sakazono, Satomi; Masuko-Suzuki, Hiromi; Maeda, Shunsuke; Sewaki, Misato; Sone, Mikako; Takahashi, Hirokazu; Nakazono, Mikio; Iwano, Megumi; Takayama, Seiji; Shimizu, Kentaro K.; Yano, Kentaro; Lim, Yong Pyo; Suzuki, Go; Suwabe, Keita; Watanabe, Masao

    2013-01-01

    Pollination is an early and critical step in plant reproduction, leading to successful fertilization. It consists of many sequential processes, including adhesion of pollen grains onto the surface of stigmatic papilla cells, foot formation to strengthen pollen–stigma interaction, pollen hydration and germination, and pollen tube elongation and penetration. We have focused on an examination of the expressed genes in papilla cells, to increase understanding of the molecular systems of pollination. From three representative species of Brassicaceae (Arabidopsis thaliana, A. halleri and Brassica rapa), stigmatic papilla cells were isolated precisely by laser microdissection, and cell type-specific gene expression in papilla cells was determined by RNA sequencing. As a result, 17,240, 19,260 and 21,026 unigenes were defined in papilla cells of A. thaliana, A. halleri and B. rapa, respectively, and, among these, 12,311 genes were common to all three species. Among the17,240 genes predicted in A. thaliana, one-third were papilla specific while approximately half of the genes were detected in all tissues examined. Bioinformatics analysis revealed that genes related to a wide range of reproduction and development functions are expressed in papilla cells, particularly metabolism, transcription and membrane-mediated information exchange. These results reflect the conserved features of general cellular function and also the specific reproductive role of papilla cells, highlighting a complex cellular system regulated by a diverse range of molecules in these cells. This study provides fundamental biological knowledge to dissect the molecular mechanisms of pollination in papilla cells and will shed light on our understanding of plant reproduction mechanisms. PMID:24058146

  2. CAST-ChIP maps cell-type-specific chromatin states in the Drosophila central nervous system.

    PubMed

    Schauer, Tamás; Schwalie, Petra C; Handley, Ava; Margulies, Carla E; Flicek, Paul; Ladurner, Andreas G

    2013-10-17

    Chromatin organization and gene activity are responsive to developmental and environmental cues. Although many genes are transcribed throughout development and across cell types, much of gene regulation is highly cell-type specific. To readily track chromatin features at the resolution of cell types within complex tissues, we developed and validated chromatin affinity purification from specific cell types by chromatin immunoprecipitation (CAST-ChIP), a broadly applicable biochemical procedure. RNA polymerase II (Pol II) CAST-ChIP identifies ~1,500 neuronal and glia-specific genes in differentiated cells within the adult Drosophila brain. In contrast, the histone H2A.Z is distributed similarly across cell types and throughout development, marking cell-type-invariant Pol II-bound regions. Our study identifies H2A.Z as an active chromatin signature that is refractory to changes across cell fates. Thus, CAST-ChIP powerfully identifies cell-type-specific as well as cell-type-invariant chromatin states, enabling the systematic dissection of chromatin structure and gene regulation within complex tissues such as the brain.

  3. Hunger States Control the Directions of Synaptic Plasticity via Switching Cell Type-Specific Subunits of NMDA Receptors.

    PubMed

    Qi, Yong; Yang, Yunlei

    2015-09-23

    It remains largely unknown whether and how hunger states control activity-dependent synaptic plasticity, such as long-term potentiation (LTP) and long-term depression (LTD). We here report that both LTP and LTD of excitatory synaptic strength within the appetite control circuits residing in hypothalamic arcuate nucleus (ARC) behave in a manner of hunger states dependence and cell type specificity. For instance, we find that tetanic stimulation induces LTP at orexigenic agouti-related protein (AgRP) neurons in ad libitum fed mice, whereas it induces LTD in food-deprived mice. In an opposite direction, the same induction protocol induces LTD at anorexigenic pro-opiomelanocortin (POMC) neurons in fed mice but weak LTP in deprived mice. Mechanistically, we also find that food deprivation increases the expressions of NR2C/NR2D/NR3-containing NMDA receptors (NMDARs) at AgRP neurons that contribute to the inductions of LTD, whereas it decreases their expressions at POMC neurons. Collectively, our data reveal that hunger states control the directions of activity-dependent synaptic plasticity by switching NMDA receptor subpopulations in a cell type-specific manner, providing insights into NMDAR-mediated interactions between energy states and associative memory. Significance statement: Based on the experiments performed in this study, we demonstrate that activity-dependent synaptic plasticity is also under the control of energy states by regulating NMDAR subpopulations in a cell type-specific manner. We thus propose a reversible memory configuration constructed from energy states-dependent cell type-specific bidirectional conversions of LTP and LTD. Together with the distinct functional roles played by NMDAR signaling in the control of food intake and energy states, these findings reveal a new reciprocal interaction between energy states and associative memory, one that might serve as a target for therapeutic treatments of the energy-related memory disorders or vice versa.

  4. Plasma miRNA-506 as a Prognostic Biomarker for Esophageal Squamous Cell Carcinoma.

    PubMed

    Li, Shu-Ping; Su, Hong-Xin; Zhao, Da; Guan, Quan-Lin

    2016-06-27

    BACKGROUND MicroRNAs (miRNAs) are responsible for regulating proliferation, differentiation, apoptosis, invasion, and metastasis in tumor cells. miRNA-506 is abnormally expressed in multiple tumors, indicating that it might be oncogenic or tumor-suppressive. However, little is known about the association between miRNA-506 expression and esophageal squamous cell carcinoma (ESCC). MATERIAL AND METHODS We examined the expression of miRNA-506 in the plasma of ESCC patients using quantitative real-time polymerase chain reaction (qRT-PCR) to determine the association between miRNA-506 expression and clinicopathological features of ESCC. ROC curves were produced for ESCC diagnosis by plasma miRNA-506 and the area under curve was calculated to explore its diagnostic value. RESULTS Average miRNA-506 expression levels were remarkably higher in the plasma of ESCC patients than in healthy volunteers (P<0.001). The expression of miRNA-506 in the plasma was closely associated with lymph node status (P=0.004), TNM stage (P=0.031), and tumor length (P<0.001). According to ROC curves, the area under the curve for plasma miRNA-506 was 0.835, indicating statistical significance for ESCC diagnosis by plasma miRNA-506 (P<0.001). Kaplan-Meier analysis showed that patients with high miRNA-506 expression had significantly shorter survival time than those with low miRNA-506 expression. Cox regression analysis demonstrated that T stage, N stage, tumor length, and miRNA-506 expression levels were significantly correlated with prognosis in ESCC patients. CONCLUSIONS miRNA-506 can serve as an important molecular marker for diagnosis and prognostic prediction of ESCC.

  5. Genome-wide exploration of miRNA function in mammalian muscle cell differentiation.

    PubMed

    Polesskaya, Anna; Degerny, Cindy; Pinna, Guillaume; Maury, Yves; Kratassiouk, Gueorgui; Mouly, Vincent; Morozova, Nadya; Kropp, Jeremie; Frandsen, Niels; Harel-Bellan, Annick

    2013-01-01

    MiRNAs impact on the control of cell fate by regulating gene expression at the post-transcriptional level. Here, using mammalian muscle differentiation as a model and a phenotypic loss-of-function screen, we explored the function of miRNAs at the genome-wide level. We found that the depletion of a high number of miRNAs (63) impacted on differentiation of human muscle precursors, underscoring the importance of this post-transcriptional mechanism of gene regulation. Interestingly, a comparison with miRNA expression profiles revealed that most of the hit miRNAs did not show any significant variations of expression during differentiation. These constitutively expressed miRNAs might be required for basic and/or essential cell function, or else might be regulated at the post-transcriptional level. MiRNA inhibition yielded a variety of phenotypes, reflecting the widespread miRNA involvement in differentiation. Using a functional screen (the STarS--Suppressor Target Screen--approach, i. e. concomitant knockdown of miRNAs and of candidate target proteins), we discovered miRNA protein targets that are previously uncharacterized controllers of muscle-cell terminal differentiation. Our results provide a strategy for functional annotation of the human miRnome.

  6. Cell type specific gene expression analysis of prostate needle biopsies resolves tumor tissue heterogeneity

    PubMed Central

    Krönig, Malte; Walter, Max; Drendel, Vanessa; Werner, Martin; Jilg, Cordula A.; Richter, Andreas S.; Backofen, Rolf; McGarry, David; Follo, Marie; Schultze-Seemann, Wolfgang; Schüle, Roland

    2015-01-01

    A lack of cell surface markers for the specific identification, isolation and subsequent analysis of living prostate tumor cells hampers progress in the field. Specific characterization of tumor cells and their microenvironment in a multi-parameter molecular assay could significantly improve prognostic accuracy for the heterogeneous prostate tumor tissue. Novel functionalized gold-nano particles allow fluorescence-based detection of absolute mRNA expression levels in living cells by fluorescent activated flow cytometry (FACS). We use of this technique to separate prostate tumor and benign cells in human prostate needle biopsies based on the expression levels of the tumor marker alpha-methylacyl-CoA racemase (AMACR). We combined RNA and protein detection of living cells by FACS to gate for epithelial cell adhesion molecule (EPCAM) positive tumor and benign cells, EPCAM/CD45 double negative mesenchymal cells and CD45 positive infiltrating lymphocytes. EPCAM positive epithelial cells were further sub-gated into AMACR high and low expressing cells. Two hundred cells from each population and several biopsies from the same patient were analyzed using a multiplexed gene expression profile to generate a cell type resolved profile of the specimen. This technique provides the basis for the clinical evaluation of cell type resolved gene expression profiles as pre-therapeutic prognostic markers for prostate cancer. PMID:25514598

  7. Cell-Type-Specific Genome-wide Expression Profiling after Laser Capture Microdissection of Living Tissue

    SciTech Connect

    Marchetti, F; Manohar, C F

    2005-02-09

    The purpose of this technical feasibility study was to develop and evaluate robust microgenomic tools for investigations of genome-wide expression of very small numbers of cells isolated from whole tissue sections. Tissues contain large numbers of cell-types that play varied roles in organ function and responses to endogenous and exogenous toxicants whether bacterial, viral, chemical or radiation. Expression studies of whole tissue biopsy are severely limited because heterogeneous cell-types result in an averaging of molecular signals masking subtle but important changes in gene expression in any one cell type(s) or group of cells. Accurate gene expression analysis requires the study of specific cell types in their tissue environment but without contamination from surrounding cells. Laser capture microdissection (LCM) is a new technology to isolate morphologically distinct cells from tissue sections. Alternative methods are available for isolating single cells but not yet for their reliable genome-wide expression analyses. The tasks of this feasibility project were to: (1) Develop efficient protocols for laser capture microdissection of cells from tissues identified by antibody label, or morphological stain. (2) Develop reproducible gene-transcript analyses techniques for single cell-types and determine the numbers of cells needed for reliable genome-wide analyses. (3) Validate the technology for epithelial and endothelial cells isolated from the gastrointestinal tract of mice.

  8. Distal Regions of the Human IFNG Locus Direct Cell Type-Specific Expression

    PubMed Central

    Collins, Patrick L.; Chang, Shaojing; Henderson, Melodie; Soutto, Mohammed; Davis, Georgia M.; McLoed, Allyson G.; Townsend, Michael J.; Glimcher, Laurie H.; Mortlock, Douglas P.; Aune, Thomas M.

    2010-01-01

    Genes, such as IFNG, which are expressed in multiple cell lineages of the immune system, may employ a common set of regulatory elements to direct transcription in multiple cell types or individual regulatory elements to direct expression in individual cell lineages. By employing a bacterial artificial chromosome transgenic system, we demonstrate that IFNG employs unique regulatory elements to achieve lineage-specific transcriptional control. Specifically, a one 1-kb element 30 kb upstream of IFNG activates transcription in T cells and NKT cells but not in NK cells. This distal regulatory element is a Runx3 binding site in Th1 cells and is needed for RNA polymerase II recruitment to IFNG, but it is not absolutely required for histone acetylation of the IFNG locus. These results support a model whereby IFNG utilizes cis-regulatory elements with cell type-restricted function. PMID:20574006

  9. Histone-fold domain protein NF-Y promotes chromatin accessibility for cell type-specific master transcription factors

    PubMed Central

    Oldfield, Andrew J.; Yang, Pengyi; Conway, Amanda E; Cinghu, Senthilkumar; Freudenberg, Johannes M.; Yellaboina, Sailu; Jothi, Raja

    2014-01-01

    SUMMARY Cell type-specific master transcription factors (TFs) play vital roles in defining cell identity and function. However, the roles ubiquitous factors play in the specification of cell identity remain underappreciated. Here we show that the ubiquitous CCAAT-binding NF-Y complex is required for the maintenance of embryonic stem cell (ESC) identity and is an essential component of the core pluripotency network. Genome-wide studies in ESCs and neurons reveal that not only does NF-Y regulate genes with housekeeping functions through cell type-invariant promoter-proximal binding, but also genes required for cell identity by binding to cell type-specific enhancers with master TFs. Mechanistically, NF-Y’s distinct DNA-binding mode promotes master/pioneer TF binding at enhancers by facilitating a permissive chromatin conformation. Our studies unearth a conceptually unique function for histone-fold domain (HFD) protein NF-Y in promoting chromatin accessibility and suggest that other HFD proteins with analogous structural and DNA-binding properties may function in similar ways. PMID:25132174

  10. Getting down to specifics: profiling gene expression and protein-DNA interactions in a cell type-specific manner

    PubMed Central

    McClure, Colin D.; Southall, Tony D.

    2015-01-01

    The majority of multicellular organisms are comprised of an extraordinary range of cell types, with different properties and gene expression profiles. Understanding what makes each cell type unique, and how their individual characteristics are attributed, are key questions for both developmental and neurobiologists alike. The brain is an excellent example of the cellular diversity expressed in the majority of eukaryotes. The mouse brain comprises of approximately 75 million neurons varying in morphology, electrophysiology, and preferences for synaptic partners. A powerful process in beginning to pick apart the mechanisms that specify individual characteristics of the cell, as well as their fate, is to profile gene expression patterns, chromatin states, and transcriptional networks in a cell type-specific manner, i.e. only profiling the cells of interest in a particular tissue. Depending on the organism, the questions being investigated, and the material available, certain cell type-specific profiling methods are more suitable than others. This chapter reviews the approaches presently available for selecting and isolating specific cell types and evaluates their key features. PMID:26410031

  11. Cell type-specific response to high intracellular loading of polyacrylic acid-coated magnetic nanoparticles

    PubMed Central

    Lojk, Jasna; Bregar, Vladimir B; Rajh, Maruša; Miš, Katarina; Kreft, Mateja Erdani; Pirkmajer, Sergej; Veranič, Peter; Pavlin, Mojca

    2015-01-01

    Magnetic nanoparticles (NPs) are a special type of NP with a ferromagnetic, electron-dense core that enables several applications such as cell tracking, hyperthermia, and magnetic separation, as well as multimodality. So far, superparamagnetic iron oxide NPs (SPIONs) are the only clinically approved type of metal oxide NPs, but cobalt ferrite NPs have properties suitable for biomedical applications as well. In this study, we analyzed the cellular responses to magnetic cobalt ferrite NPs coated with polyacrylic acid (PAA) in three cell types: Chinese Hamster Ovary (CHO), mouse melanoma (B16) cell line, and primary human myoblasts (MYO). We compared the internalization pathway, intracellular trafficking, and intracellular fate of our NPs using fluorescence and transmission electron microscopy (TEM) as well as quantified NP uptake and analyzed uptake dynamics. We determined cell viability after 24 or 96 hours’ exposure to increasing concentrations of NPs, and quantified the generation of reactive oxygen species (ROS) upon 24 and 48 hours’ exposure. Our NPs have been shown to readily enter and accumulate in cells in high quantities using the same two endocytic pathways; mostly by macropinocytosis and partially by clathrin-mediated endocytosis. The cell types differed in their uptake rate, the dynamics of intracellular trafficking, and the uptake capacity, as well as in their response to higher concentrations of internalized NPs. The observed differences in cell responses stress the importance of evaluation of NP–cell interactions on several different cell types for better prediction of possible toxic effects on different cell and tissue types in vivo. PMID:25733835

  12. Cell type-specific response to high intracellular loading of polyacrylic acid-coated magnetic nanoparticles.

    PubMed

    Lojk, Jasna; Bregar, Vladimir B; Rajh, Maruša; Miš, Katarina; Kreft, Mateja Erdani; Pirkmajer, Sergej; Veranič, Peter; Pavlin, Mojca

    2015-01-01

    Magnetic nanoparticles (NPs) are a special type of NP with a ferromagnetic, electron-dense core that enables several applications such as cell tracking, hyperthermia, and magnetic separation, as well as multimodality. So far, superparamagnetic iron oxide NPs (SPIONs) are the only clinically approved type of metal oxide NPs, but cobalt ferrite NPs have properties suitable for biomedical applications as well. In this study, we analyzed the cellular responses to magnetic cobalt ferrite NPs coated with polyacrylic acid (PAA) in three cell types: Chinese Hamster Ovary (CHO), mouse melanoma (B16) cell line, and primary human myoblasts (MYO). We compared the internalization pathway, intracellular trafficking, and intracellular fate of our NPs using fluorescence and transmission electron microscopy (TEM) as well as quantified NP uptake and analyzed uptake dynamics. We determined cell viability after 24 or 96 hours' exposure to increasing concentrations of NPs, and quantified the generation of reactive oxygen species (ROS) upon 24 and 48 hours' exposure. Our NPs have been shown to readily enter and accumulate in cells in high quantities using the same two endocytic pathways; mostly by macropinocytosis and partially by clathrin-mediated endocytosis. The cell types differed in their uptake rate, the dynamics of intracellular trafficking, and the uptake capacity, as well as in their response to higher concentrations of internalized NPs. The observed differences in cell responses stress the importance of evaluation of NP-cell interactions on several different cell types for better prediction of possible toxic effects on different cell and tissue types in vivo.

  13. Dopamine D1 receptor expression is bipolar cell type-specific in the mouse retina.

    PubMed

    Farshi, Pershang; Fyk-Kolodziej, Bozena; Krolewski, David M; Walker, Paul D; Ichinose, Tomomi

    2016-07-01

    In the retina, dopamine is a key molecule for daytime vision. Dopamine is released by retinal dopaminergic amacrine cells and transmits signaling either by conventional synaptic or by volume transmission. By means of volume transmission, dopamine modulates all layers of retinal neurons; however, it is not well understood how dopamine modulates visual signaling pathways in bipolar cells. Here we analyzed Drd1a-tdTomato BAC transgenic mice and found that the dopamine D1 receptor (D1R) is expressed in retinal bipolar cells in a type-dependent manner. Strong tdTomato fluorescence was detected in the inner nuclear layer and localized to type 1, 3b, and 4 OFF bipolar cells and type 5-2, XBC, 6, and 7 ON bipolar cells. In contrast, type 2, 3a, 5-1, 9, and rod bipolar cells did not express Drd1a-tdTomato. Other interneurons were also found to express tdTomato including horizontal cells and a subset (25%) of AII amacrine cells. Diverse visual processing pathways, such as color or motion-coded pathways, are thought to be initiated in retinal bipolar cells. Our results indicate that dopamine sculpts bipolar cell performance in a type-dependent manner to facilitate daytime vision. J. Comp. Neurol. 524:2059-2079, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  14. Cell-type specific expression of ATP-sensitive potassium channels in the rat hippocampus

    PubMed Central

    Zawar, C; Plant, T D; Schirra, C; Konnerth, A; Neumcke, B

    1999-01-01

    The distribution of ATP-sensitive K+ channels (KATP channels) was investigated in four cell types in hippocampal slices prepared from 10- to 13-day-old rats: CA1 pyramidal cells, interneurones of stratum radiatum in CA1, complex glial cells of the same area and granule cells of the dentate gyrus. The neuronal cell types were identified visually and characterized by the shapes and patterns of their action potentials and by neurobiotin labelling.The patch-clamp technique was used to study the sensitivity of whole-cell currents to diazoxide (0·3 mm), a KATP channel opener, and to tolbutamide (0·5 mm) or glibenclamide (20 μm), two KATP channel inhibitors. The fraction of cells in which whole-cell currents were activated by diazoxide and inhibited by tolbutamide was 26% of pyramidal cells, 89% of interneurones, 100% of glial cells and 89% of granule cells. The reversal potential of the diazoxide-induced current was at the K+ equilibrium potential and a similar current activated spontaneously when cells were dialysed with an ATP-free pipette solution.Using the single-cell RT-PCR method, the presence of mRNA encoding KATP channel subunits (Kir6.1, Kir6.2, SUR1 and SUR2) was examined in CA1 pyramidal cells and interneurones. Subunit mRNA combinations that can result in functional KATP channels (Kir6.1 together with SUR1, Kir6.2 together with SUR1 or SUR2) were detected in only 17% of the pyramidal cells. On the other hand, KATP channelsmay be formed in 75%of the interneurones, mainly by the combination of Kir6.2 with SUR1 (58% of all interneurones).The results of these combined analyses indicate that functional KATP channels are present in principal neurones, interneurones and glial cells of the rat hippocampus, but at highly different densities in the four cell types studied. PMID:9852317

  15. Cell-type-specific enrichment of risk-associated regulatory elements at ovarian cancer susceptibility loci

    PubMed Central

    Coetzee, Simon G.; Shen, Howard C.; Hazelett, Dennis J.; Lawrenson, Kate; Kuchenbaecker, Karoline; Tyrer, Jonathan; Rhie, Suhn K.; Levanon, Keren; Karst, Alison; Drapkin, Ronny; Ramus, Susan J.; Couch, Fergus J.; Offit, Kenneth; Chenevix-Trench, Georgia; Monteiro, Alvaro N.A.; Antoniou, Antonis; Freedman, Matthew; Coetzee, Gerhard A.; Pharoah, Paul D.P.; Noushmehr, Houtan; Gayther, Simon A.

    2015-01-01

    Understanding the regulatory landscape of the human genome is a central question in complex trait genetics. Most single-nucleotide polymorphisms (SNPs) associated with cancer risk lie in non-protein-coding regions, implicating regulatory DNA elements as functional targets of susceptibility variants. Here, we describe genome-wide annotation of regions of open chromatin and histone modification in fallopian tube and ovarian surface epithelial cells (FTSECs, OSECs), the debated cellular origins of high-grade serous ovarian cancers (HGSOCs) and in endometriosis epithelial cells (EECs), the likely precursor of clear cell ovarian carcinomas (CCOCs). The regulatory architecture of these cell types was compared with normal human mammary epithelial cells and LNCaP prostate cancer cells. We observed similar positional patterns of global enhancer signatures across the three different ovarian cancer precursor cell types, and evidence of tissue-specific regulatory signatures compared to non-gynecological cell types. We found significant enrichment for risk-associated SNPs intersecting regulatory biofeatures at 17 known HGSOC susceptibility loci in FTSECs (P = 3.8 × 10−30), OSECs (P = 2.4 × 10−23) and HMECs (P = 6.7 × 10−15) but not for EECs (P = 0.45) or LNCaP cells (P = 0.88). Hierarchical clustering of risk SNPs conditioned on the six different cell types indicates FTSECs and OSECs are highly related (96% of samples using multi-scale bootstrapping) suggesting both cell types may be precursors of HGSOC. These data represent the first description of regulatory catalogues of normal precursor cells for different ovarian cancer subtypes, and provide unique insights into the tissue specific regulatory variation with respect to the likely functional targets of germline genetic susceptibility variants for ovarian cancer. PMID:25804953

  16. Cell-type-specific enrichment of risk-associated regulatory elements at ovarian cancer susceptibility loci.

    PubMed

    Coetzee, Simon G; Shen, Howard C; Hazelett, Dennis J; Lawrenson, Kate; Kuchenbaecker, Karoline; Tyrer, Jonathan; Rhie, Suhn K; Levanon, Keren; Karst, Alison; Drapkin, Ronny; Ramus, Susan J; Couch, Fergus J; Offit, Kenneth; Chenevix-Trench, Georgia; Monteiro, Alvaro N A; Antoniou, Antonis; Freedman, Matthew; Coetzee, Gerhard A; Pharoah, Paul D P; Noushmehr, Houtan; Gayther, Simon A

    2015-07-01

    Understanding the regulatory landscape of the human genome is a central question in complex trait genetics. Most single-nucleotide polymorphisms (SNPs) associated with cancer risk lie in non-protein-coding regions, implicating regulatory DNA elements as functional targets of susceptibility variants. Here, we describe genome-wide annotation of regions of open chromatin and histone modification in fallopian tube and ovarian surface epithelial cells (FTSECs, OSECs), the debated cellular origins of high-grade serous ovarian cancers (HGSOCs) and in endometriosis epithelial cells (EECs), the likely precursor of clear cell ovarian carcinomas (CCOCs). The regulatory architecture of these cell types was compared with normal human mammary epithelial cells and LNCaP prostate cancer cells. We observed similar positional patterns of global enhancer signatures across the three different ovarian cancer precursor cell types, and evidence of tissue-specific regulatory signatures compared to non-gynecological cell types. We found significant enrichment for risk-associated SNPs intersecting regulatory biofeatures at 17 known HGSOC susceptibility loci in FTSECs (P = 3.8 × 10(-30)), OSECs (P = 2.4 × 10(-23)) and HMECs (P = 6.7 × 10(-15)) but not for EECs (P = 0.45) or LNCaP cells (P = 0.88). Hierarchical clustering of risk SNPs conditioned on the six different cell types indicates FTSECs and OSECs are highly related (96% of samples using multi-scale bootstrapping) suggesting both cell types may be precursors of HGSOC. These data represent the first description of regulatory catalogues of normal precursor cells for different ovarian cancer subtypes, and provide unique insights into the tissue specific regulatory variation with respect to the likely functional targets of germline genetic susceptibility variants for ovarian cancer. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  17. Cell Type-Specific Regulation of Immunological Synapse Dynamics by B7 Ligand Recognition

    PubMed Central

    Brzostek, Joanna; Gascoigne, Nicholas R. J.; Rybakin, Vasily

    2016-01-01

    B7 proteins CD80 (B7-1) and CD86 (B7-2) are expressed on most antigen-presenting cells and provide critical co-stimulatory or inhibitory input to T cells via their T-cell-expressed receptors: CD28 and CTLA-4. CD28 is expressed on effector T cells and regulatory T cells (Tregs), and CD28-dependent signals are required for optimum activation of effector T cell functions. CD28 ligation on effector T cells leads to formation of distinct molecular patterns and induction of cytoskeletal rearrangements at the immunological synapse (IS). CD28 plays a critical role in recruitment of protein kinase C (PKC)-θ to the effector T cell IS. CTLA-4 is constitutively expressed on the surface of Tregs, but it is expressed on effector T cells only after activation. As CTLA-4 binds to B7 proteins with significantly higher affinity than CD28, B7 ligand recognition by cells expressing both receptors leads to displacement of CD28 and PKC-θ from the IS. In Tregs, B7 ligand recognition leads to recruitment of CTLA-4 and PKC-η to the IS. CTLA-4 plays a role in regulation of T effector and Treg IS stability and cell motility. Due to their important roles in regulating T-cell-mediated responses, B7 receptors are emerging as important drug targets in oncology. In this review, we present an integrated summary of current knowledge about the role of B7 family receptor–ligand interactions in the regulation of spatial and temporal IS dynamics in effector and Tregs. PMID:26870040

  18. Cell-type-specific neuroanatomy of cliques of autism-related genes in the mouse brain

    PubMed Central

    Grange, Pascal; Menashe, Idan; Hawrylycz, Michael

    2015-01-01

    Two cliques of genes identified computationally for their high co-expression in the mouse brain according to the Allen Brain Atlas, and for their enrichment in genes related to autism spectrum disorder (ASD), have recently been shown to be highly co-expressed in the cerebellar cortex, compared to what could be expected by chance. Moreover, the expression of these cliques of genes is not homogeneous across the cerebellar cortex, and it has been noted that their expression pattern seems to highlight the granular layer. However, this observation was only made by eye, and recent advances in computational neuroanatomy allow to rank cell types in the mouse brain (characterized by their transcriptome profiles) according to the similarity between their spatial density profiles and the spatial expression profiles of the cliques. We establish by Monte Carlo simulation that with probability at least 99%, the expression profiles of the two cliques are more similar to the density profile of granule cells than 99% of the expression of cliques containing the same number of genes (Purkinje cells also score above 99% in one of the cliques). Thresholding the expression profiles shows that the signal is more intense in the granular layer. Finally, we work out pairs of cell types whose combined expression profiles are more similar to the expression profiles of the cliques than any single cell type. These pairs predominantly consist of one cortical pyramidal cell and one cerebellar cell (which can be either a granule cell or a Purkinje cell). PMID:26074809

  19. Mouse TU tagging: a chemical/genetic intersectional method for purifying cell type-specific nascent RNA.

    PubMed

    Gay, Leslie; Miller, Michael R; Ventura, P Britten; Devasthali, Vidusha; Vue, Zer; Thompson, Heather L; Temple, Sally; Zong, Hui; Cleary, Michael D; Stankunas, Kryn; Doe, Chris Q

    2013-01-01

    Transcriptional profiling is a powerful approach for understanding development and disease. Current cell type-specific RNA purification methods have limitations, including cell dissociation trauma or inability to identify all RNA species. Here, we describe "mouse thiouracil (TU) tagging," a genetic and chemical intersectional method for covalent labeling and purification of cell type-specific RNA in vivo. Cre-induced expression of uracil phosphoribosyltransferase (UPRT) provides spatial specificity; injection of 4-thiouracil (4TU) provides temporal specificity. Only UPRT(+) cells exposed to 4TU produce thio-RNA, which is then purified for RNA sequencing (RNA-seq). This method can purify transcripts from spatially complex and rare (<5%) cells, such as Tie2:Cre(+) brain endothelia/microglia (76% validated by expression pattern), or temporally dynamic transcripts, such as those acutely induced by lipopolysaccharide (LPS) injection. Moreover, generating chimeric mice via UPRT(+) bone marrow transplants identifies immune versus niche spleen RNA. TU tagging provides a novel method for identifying actively transcribed genes in specific cells at specific times within intact mice.

  20. Mouse TU tagging: a chemical/genetic intersectional method for purifying cell type-specific nascent RNA

    PubMed Central

    Gay, Leslie; Miller, Michael R.; Ventura, P. Britten; Devasthali, Vidusha; Vue, Zer; Thompson, Heather L.; Temple, Sally; Zong, Hui; Cleary, Michael D.; Stankunas, Kryn; Doe, Chris Q.

    2013-01-01

    Transcriptional profiling is a powerful approach for understanding development and disease. Current cell type-specific RNA purification methods have limitations, including cell dissociation trauma or inability to identify all RNA species. Here, we describe “mouse thiouracil (TU) tagging,” a genetic and chemical intersectional method for covalent labeling and purification of cell type-specific RNA in vivo. Cre-induced expression of uracil phosphoribosyltransferase (UPRT) provides spatial specificity; injection of 4-thiouracil (4TU) provides temporal specificity. Only UPRT+ cells exposed to 4TU produce thio-RNA, which is then purified for RNA sequencing (RNA-seq). This method can purify transcripts from spatially complex and rare (<5%) cells, such as Tie2:Cre+ brain endothelia/microglia (76% validated by expression pattern), or temporally dynamic transcripts, such as those acutely induced by lipopolysaccharide (LPS) injection. Moreover, generating chimeric mice via UPRT+ bone marrow transplants identifies immune versus niche spleen RNA. TU tagging provides a novel method for identifying actively transcribed genes in specific cells at specific times within intact mice. PMID:23307870

  1. A Comprehensive Analysis of Cell Type-Specific Nuclear RNA From Neurons and Glia of the Brain.

    PubMed

    Reddy, Adarsh S; O'Brien, David; Pisat, Nilambari; Weichselbaum, Claire T; Sakers, Kristina; Lisci, Miriam; Dalal, Jasbir S; Dougherty, Joseph D

    2017-02-01

    Studies in psychiatric genetics have identified >100 loci associated with disease risk, yet many of these loci are distant from protein coding genes. Recent characterization of the transcriptional landscape of cell lines and whole tissues has suggested widespread transcription in both coding and noncoding regions of the genome, including differential expression from loci that produce regulatory noncoding RNAs that function within the nucleus; however, the nuclear transcriptome of specific cell types in the brain has not been previously investigated. We defined the nuclear transcriptional landscape of the three major cellular divisions of the nervous system using flow sorting of genetically labeled nuclei from bacTRAP mouse lines. Next, we characterized the unique expression of coding, noncoding, and intergenic RNAs in the mature mouse brain with RNA-Seq and validation with independent methods. We found diverse expression across the cell types of all classes of RNAs, including long noncoding RNAs, several of which were confirmed as highly enriched in the nuclei of specific cell types using anatomic methods. We also discovered several examples of cell type-specific expression of tandem gene fusions, and we report the first cell type-specific expression of circular RNAs-a neuron-specific and nuclear-enriched RNA arising from the gene Hnrnpu. These data provide an important resource for studies evaluating the function of various noncoding RNAs in the brain, including noncoding RNAs that may play a role in psychiatric disease. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  2. Mutant PIK3CA Induces EMT in a Cell Type Specific Manner

    PubMed Central

    Bhagirath, Divya; Zhao, Xiangshan; Mirza, Sameer; West, William W.; Band, Hamid; Band, Vimla

    2016-01-01

    Breast cancer is characterized into different molecular subtypes, and each subtype is characterized by differential gene expression that are associated with distinct survival outcomes in patients. PIK3CA mutations are commonly associated with most breast cancer subtypes. More recently PIK3CA mutations have been shown to induce tumor heterogeneity and are associated with activation of EGFR-signaling and reduced relapse free survival in basal subtype of breast cancer. Thus, understanding what determines PIK3CA induced heterogeneity and oncogenesis, is an important area of investigation. In this study, we assessed the effect of mutant PIK3CA together with mutant Ras plus mutant p53 on oncogenic behavior of two distinct stem/progenitor breast cell lines, designated as K5+/K19- and K5+/K19+. Constructs were ectopically overexpressed in K5+/K19- and K5+/K19+ stem/progenitor cells, followed by various in-vitro and in-vivo analyses. Oncogene combination m-Ras/m-p53/m-PIK3CA efficiently transformed both K5+/K19- and K5+/K19+ cell lines in-vitro, as assessed by anchorage-independent soft agar colony formation assay. Significantly, while this oncogene combination induced a complete epithelial-to-mesenchymal transition (EMT) in K5+/K19- cell line, mostly epithelial phenotype with minor EMT component was seen in K5+/K19+ cell line. However, both K5+/K19- and K5+/K19+ transformed cells exhibited increased invasion and migration abilities. Analyses of CD44 and CD24 expression showed both cell lines had tumor-initiating CD44+/CD24low cell population, however transformed K5+/K19- cells had more proportion of these cells. Significantly, both cell types exhibited in-vivo tumorigenesis, and maintained their EMT and epithelial nature in-vivo in mice tumors. Notably, while both cell types exhibited increase in tumor-initiating cell population, differential EMT phenotype was observed in these cell lines. These results suggest that EMT is a cell type dependent phenomenon and does not

  3. Mutant PIK3CA Induces EMT in a Cell Type Specific Manner.

    PubMed

    Bhagirath, Divya; Zhao, Xiangshan; Mirza, Sameer; West, William W; Band, Hamid; Band, Vimla

    2016-01-01

    Breast cancer is characterized into different molecular subtypes, and each subtype is characterized by differential gene expression that are associated with distinct survival outcomes in patients. PIK3CA mutations are commonly associated with most breast cancer subtypes. More recently PIK3CA mutations have been shown to induce tumor heterogeneity and are associated with activation of EGFR-signaling and reduced relapse free survival in basal subtype of breast cancer. Thus, understanding what determines PIK3CA induced heterogeneity and oncogenesis, is an important area of investigation. In this study, we assessed the effect of mutant PIK3CA together with mutant Ras plus mutant p53 on oncogenic behavior of two distinct stem/progenitor breast cell lines, designated as K5+/K19- and K5+/K19+. Constructs were ectopically overexpressed in K5+/K19- and K5+/K19+ stem/progenitor cells, followed by various in-vitro and in-vivo analyses. Oncogene combination m-Ras/m-p53/m-PIK3CA efficiently transformed both K5+/K19- and K5+/K19+ cell lines in-vitro, as assessed by anchorage-independent soft agar colony formation assay. Significantly, while this oncogene combination induced a complete epithelial-to-mesenchymal transition (EMT) in K5+/K19- cell line, mostly epithelial phenotype with minor EMT component was seen in K5+/K19+ cell line. However, both K5+/K19- and K5+/K19+ transformed cells exhibited increased invasion and migration abilities. Analyses of CD44 and CD24 expression showed both cell lines had tumor-initiating CD44+/CD24low cell population, however transformed K5+/K19- cells had more proportion of these cells. Significantly, both cell types exhibited in-vivo tumorigenesis, and maintained their EMT and epithelial nature in-vivo in mice tumors. Notably, while both cell types exhibited increase in tumor-initiating cell population, differential EMT phenotype was observed in these cell lines. These results suggest that EMT is a cell type dependent phenomenon and does not

  4. Assessment of Membrane Fluidity Fluctuations during Cellular Development Reveals Time and Cell Type Specificity.

    PubMed

    Noutsi, Pakiza; Gratton, Enrico; Chaieb, Sahraoui

    2016-01-01

    Cell membrane is made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as differentiation cell membranes undergo dramatic fluidity changes induced by proteins such as ARC and Cofilin among others. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. Membrane fluidity was measured at 12h, 72h and 92 h. Our results show significant changes in membrane fluidity among all cell types at different time points. GP values tend to increase significantly within 92 h in hN2 cells and 72 h in NIH3T3 cells and only at 92 h in HEK293 cells. L6 showed a marked decrease in membrane fluidity at 72 h and starts to increase at 92 h. As expected, NIH3T3 cells have more rigid membrane at earlier time points. On the other hand, neurons tend to have the highest membrane fluidity at early time points emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines.

  5. Cell Type-Specific Modulation of Cobalamin Uptake by Bovine Serum

    PubMed Central

    Zhao, Hua; Ruberu, Kalani; Li, Hongyun; Garner, Brett

    2016-01-01

    Tracking cellular 57Co-labelled cobalamin (57Co-Cbl) uptake is a well-established method for studying Cbl homeostasis. Previous studies established that bovine serum is not generally permissive for cellular Cbl uptake when used as a supplement in cell culture medium, whereas supplementation with human serum promotes cellular Cbl uptake. The underlying reasons for these differences are not fully defined. In the current study we address this question. We extend earlier observations by showing that fetal calf serum inhibits cellular 57Co-Cbl uptake by HT1080 cells (a fibrosarcoma-derived fibroblast cell line). Furthermore, we discovered that a simple heat-treatment protocol (95°C for 10 min) ameliorates this inhibitory activity for HT1080 cell 57Co-Cbl uptake. We provide evidence that the very high level of haptocorrin in bovine serum (as compared to human serum) is responsible for this inhibitory activity. We suggest that bovine haptocorrin competes with cell-derived transcobalamin for Cbl binding, and that cellular Cbl uptake may be minimised in the presence of large amounts of bovine haptocorrin that are present under routine in vitro cell culture conditions. In experiments conducted with AG01518 cells (a neonatal foreskin-derived fibroblast cell line), overall cellular 57Co-Cbl uptake was 86% lower than for HT1080 cells, cellular TC production was below levels detectable by western blotting, and heat treatment of fetal calf serum resulted in only a modest increase in cellular 57Co-Cbl uptake. We recommend a careful assessment of cell culture protocols should be conducted in order to determine the potential benefits that heat-treated bovine serum may provide for in vitro studies of mammalian cell lines. PMID:27893837

  6. Assessment of Membrane Fluidity Fluctuations during Cellular Development Reveals Time and Cell Type Specificity

    PubMed Central

    Noutsi, Pakiza; Gratton, Enrico; Chaieb, Sahraoui

    2016-01-01

    Cell membrane is made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as differentiation cell membranes undergo dramatic fluidity changes induced by proteins such as ARC and Cofilin among others. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. Membrane fluidity was measured at 12h, 72h and 92 h. Our results show significant changes in membrane fluidity among all cell types at different time points. GP values tend to increase significantly within 92 h in hN2 cells and 72 h in NIH3T3 cells and only at 92 h in HEK293 cells. L6 showed a marked decrease in membrane fluidity at 72 h and starts to increase at 92 h. As expected, NIH3T3 cells have more rigid membrane at earlier time points. On the other hand, neurons tend to have the highest membrane fluidity at early time points emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines. PMID:27362860

  7. A Generic and Cell-Type-Specific Wound Response Precedes Regeneration in Planarians.

    PubMed

    Wurtzel, Omri; Cote, Lauren E; Poirier, Amber; Satija, Rahul; Regev, Aviv; Reddien, Peter W

    2015-12-07

    Regeneration starts with injury. Yet how injuries affect gene expression in different cell types and how distinct injuries differ in gene expression remain unclear. We defined the transcriptomes of major cell types of planarians--flatworms that regenerate from nearly any injury--and identified 1,214 tissue-specific markers across 13 cell types. RNA sequencing on 619 single cells revealed that wound-induced genes were expressed either in nearly all cell types or specifically in one of three cell types (stem cells, muscle, or epidermis). Time course experiments following different injuries indicated that a generic wound response is activated with any injury regardless of the regenerative outcome. Only one gene, notum, was differentially expressed early between anterior- and posterior-facing wounds. Injury-specific transcriptional responses emerged 30 hr after injury, involving context-dependent patterning and stem-cell-specialization genes. The regenerative requirement of every injury is different; however, our work demonstrates that all injuries start with a common transcriptional response.

  8. Mouse Testicular Cell Type-Specific Antiviral Response against Mumps Virus Replication

    PubMed Central

    Wu, Han; Zhao, Xiang; Wang, Fei; Jiang, Qian; Shi, Lili; Gong, Maolei; Liu, Weihua; Gao, Bo; Song, Chengyi; Li, Qihan; Chen, Yongmei; Han, Daishu

    2017-01-01

    Mumps virus (MuV) infection has high tropism to the testis and usually leads to orchitis, an etiological factor in male infertility. However, MuV replication in testicular cells and the cellular antiviral responses against MuV are not fully understood. The present study showed that MuV infected the majority of testicular cells, including Leydig cells (LC), testicular macrophages, Sertoli cells (SC), and male germ cells (GC). MuV was replicated at relatively high efficiencies in SC compared with LC and testicular macrophages. In contrast, MuV did not replicate in male GC. Notably, testicular cells exhibited different innate antiviral responses against MuV replication. We showed that interferon β (IFN-β) inhibited MuV replication in LC, macrophages, and SC, which were associated with the upregulation of major antiviral proteins. We provided primary evidence that autophagy plays a role in blocking MuV replication in male GC. Autophagy was also involved in limiting MuV replication in testicular macrophages but not in Leydig and SC. These findings indicate the involvement of the innate defense against MuV replication in testicular cells. PMID:28239382

  9. Mouse Testicular Cell Type-Specific Antiviral Response against Mumps Virus Replication.

    PubMed

    Wu, Han; Zhao, Xiang; Wang, Fei; Jiang, Qian; Shi, Lili; Gong, Maolei; Liu, Weihua; Gao, Bo; Song, Chengyi; Li, Qihan; Chen, Yongmei; Han, Daishu

    2017-01-01

    Mumps virus (MuV) infection has high tropism to the testis and usually leads to orchitis, an etiological factor in male infertility. However, MuV replication in testicular cells and the cellular antiviral responses against MuV are not fully understood. The present study showed that MuV infected the majority of testicular cells, including Leydig cells (LC), testicular macrophages, Sertoli cells (SC), and male germ cells (GC). MuV was replicated at relatively high efficiencies in SC compared with LC and testicular macrophages. In contrast, MuV did not replicate in male GC. Notably, testicular cells exhibited different innate antiviral responses against MuV replication. We showed that interferon β (IFN-β) inhibited MuV replication in LC, macrophages, and SC, which were associated with the upregulation of major antiviral proteins. We provided primary evidence that autophagy plays a role in blocking MuV replication in male GC. Autophagy was also involved in limiting MuV replication in testicular macrophages but not in Leydig and SC. These findings indicate the involvement of the innate defense against MuV replication in testicular cells.

  10. Phytoestrogens modulate prostaglandin production in bovine endometrium: cell type specificity and intracellular mechanisms.

    PubMed

    Woclawek-Potocka, Izabela; Acosta, Tomas J; Korzekwa, Anna; Bah, Mamadou M; Shibaya, Masami; Okuda, Kiyoshi; Skarzynski, Dariusz J

    2005-05-01

    Prostaglandins (PGs) are known to modulate the proper cyclicity of bovine reproductive organs. The main luteolytic agent in ruminants is PGF2alpha, whereas PGE2 has luteotropic actions. Estradiol 17beta (E2) regulates uterus function by influencing PG synthesis. Phytoestrogens structurally resemble E2 and possess estrogenic activity; therefore, they may mimic the effects of E2 on PG synthesis and influence the reproductive system. Using a cell-culture system of bovine epithelial and stromal cells, we determined cell-specific effects of phytoestrogens (i.e., daidzein, genistein), their metabolites (i.e., equol and para-ethyl-phenol, respectively), and E2 on PGF2alpha and PGE2 synthesis and examined the intracellular mechanisms of their actions. Both PGs produced by stromal and epithelial cells were significantly stimulated by phytoestrogens and their metabolites. However, PGF2alpha synthesis by both kinds of cells was greater stimulated than PGE2 synthesis. Moreover, epithelial cells treated with phytoestrogens synthesized more PGF2alpha than stromal cells, increasing the PGF2alpha to PGE2 ratio. The epithelial and stromal cells were preincubated with an estrogen-receptor (ER) antagonist (i.e., ICI), a translation inhibitor (i.e., actinomycin D), a protein kinase A inhibitor (i.e., staurosporin), and a phospholipase C inhibitor (i.e., U73122) for 0.5 hrs and then stimulated with equol, para-ethyl-phenol, or E2. Although the action of E2 on PGF2alpha synthesis was blocked by all reagents, the stimulatory effect of phytoestrogens was blocked only by ICI and actinomycin D in both cell types. Moreover, in contrast to E2 action, phytoestrogens did not cause intracellular calcium mobilization in either epithelial or stromal cells. Phytoestrogens stimulate both PGF2alpha and PGE2 in both cell types of bovine endometrium via an ER-dependent genomic pathway. However, because phytoestrogens preferentially stimulated PGF2alpha synthesis in epithelial cells of bovine

  11. Cell type specificity and structural determinants of IRES activity from the 5' leaders of different HIV-1 transcripts.

    PubMed

    Plank, Terra-Dawn M; Whitehurst, James T; Kieft, Jeffrey S

    2013-07-01

    Internal ribosome entry site (IRES) RNAs are important regulators of gene expression, but their diverse molecular mechanisms remain partially understood. The HIV-1 gag transcript leader contains an IRES that may be a good model for understanding the function of many other IRESs. We investigated the possibility that this IRES' function is linked to both the structure of the RNA and its cellular environment. We find that in the context of a bicistronic reporter construct, HIV-1 gag IRES' activity is cell type-specific, with higher activity in T-cell culture systems that model the natural target cells for HIV-1 infection. This finding underscores how an IRES may be fine tuned to function in certain cells, perhaps owing to cell type-specific protein factors. Using RNA probing and mutagenesis, we demonstrate that the HIV-1 gag IRES does not use pre-folded RNA structure to drive function, a finding that gives insight into how conformationally dynamic IRESs operate. Furthermore, we find that a common exon drives IRES activity in a diverse set of alternatively spliced transcripts. We propose a mechanism in which a structurally plastic RNA element confers the ability to initiate translation internally, and activity from this common element is modulated by 3' nucleotides added by alternative splicing.

  12. Cell type specificity and structural determinants of IRES activity from the 5′ leaders of different HIV-1 transcripts

    PubMed Central

    Plank, Terra-Dawn M.; Whitehurst, James T.; Kieft, Jeffrey S.

    2013-01-01

    Internal ribosome entry site (IRES) RNAs are important regulators of gene expression, but their diverse molecular mechanisms remain partially understood. The HIV-1 gag transcript leader contains an IRES that may be a good model for understanding the function of many other IRESs. We investigated the possibility that this IRES’ function is linked to both the structure of the RNA and its cellular environment. We find that in the context of a bicistronic reporter construct, HIV-1 gag IRES’ activity is cell type-specific, with higher activity in T-cell culture systems that model the natural target cells for HIV-1 infection. This finding underscores how an IRES may be fine tuned to function in certain cells, perhaps owing to cell type-specific protein factors. Using RNA probing and mutagenesis, we demonstrate that the HIV-1 gag IRES does not use pre-folded RNA structure to drive function, a finding that gives insight into how conformationally dynamic IRESs operate. Furthermore, we find that a common exon drives IRES activity in a diverse set of alternatively spliced transcripts. We propose a mechanism in which a structurally plastic RNA element confers the ability to initiate translation internally, and activity from this common element is modulated by 3′ nucleotides added by alternative splicing. PMID:23661682

  13. Different transmitter transients underlie presynaptic cell type specificity of GABAA,slow and GABAA,fast

    PubMed Central

    Szabadics, János; Tamás, Gábor; Soltesz, Ivan

    2007-01-01

    Phasic (synaptic) and tonic (extrasynaptic) inhibition represent the two most fundamental forms of GABAA receptor-mediated transmission. Inhibitory postsynaptic currents (IPSCs) generated by GABAA receptors are typically extremely rapid synaptic events that do not last beyond a few milliseconds. Although unusually slow GABAA IPSCs, lasting for tens of milliseconds, have been observed in recordings of spontaneous events, their origin and mechanisms are not known. We show that neocortical GABAA,slow IPSCs originate from a specialized interneuron called neurogliaform cells. Compared with classical GABAA,fast IPSCs evoked by basket cells, single spikes in neurogliaform cells evoke extraordinarily prolonged GABAA responses that display tight regulation by transporters, low peak GABA concentration, unusual benzodiazepine modulation, and spillover. These results reveal a form of GABAA receptor mediated communication by a dedicated cell type that produces slow ionotropic responses with properties intermediate between phasic and tonic inhibition. PMID:17785408

  14. From Endoderm to Liver Bud: Paradigms of Cell Type Specification and Tissue Morphogenesis.

    PubMed

    Zaret, Kenneth S

    2016-01-01

    The early specification, rapid growth and morphogenesis, and conserved functions of the embryonic liver across diverse model organisms have made the system an experimentally facile paradigm for understanding basic regulatory mechanisms that govern cell differentiation and organogenesis. This essay highlights concepts that have emerged from studies of the discrete steps of foregut endoderm development into the liver bud, as well as from modeling the steps via embryonic stem cell differentiation. Such concepts include understanding the chromatin basis for the competence of progenitor cells to develop into specific lineages; the importance of combinatorial signaling from different sources to induce cell fates; the impact of inductive signaling on preexisting chromatin states; the ability of separately specified domains of cells to merge into a common tissue; and the marked cell biological dynamics, including interactions with the developing vasculature, which establish the initial morphogenesis and patterning of a tissue. The principles gleaned from these studies, focusing on the 2 days it takes for the endoderm to develop into a liver bud, should be instructive for many other organogenic systems and for manipulating tissues in regenerative contexts for biomedical purposes.

  15. Defining cell-type specificity at the transcriptional level in human disease.

    PubMed

    Ju, Wenjun; Greene, Casey S; Eichinger, Felix; Nair, Viji; Hodgin, Jeffrey B; Bitzer, Markus; Lee, Young-Suk; Zhu, Qian; Kehata, Masami; Li, Min; Jiang, Song; Rastaldi, Maria Pia; Cohen, Clemens D; Troyanskaya, Olga G; Kretzler, Matthias

    2013-11-01

    Cell-lineage-specific transcripts are essential for differentiated tissue function, implicated in hereditary organ failure, and mediate acquired chronic diseases. However, experimental identification of cell-lineage-specific genes in a genome-scale manner is infeasible for most solid human tissues. We developed the first genome-scale method to identify genes with cell-lineage-specific expression, even in lineages not separable by experimental microdissection. Our machine-learning-based approach leverages high-throughput data from tissue homogenates in a novel iterative statistical framework. We applied this method to chronic kidney disease and identified transcripts specific to podocytes, key cells in the glomerular filter responsible for hereditary and most acquired glomerular kidney disease. In a systematic evaluation of our predictions by immunohistochemistry, our in silico approach was significantly more accurate (65% accuracy in human) than predictions based on direct measurement of in vivo fluorescence-tagged murine podocytes (23%). Our method identified genes implicated as causal in hereditary glomerular disease and involved in molecular pathways of acquired and chronic renal diseases. Furthermore, based on expression analysis of human kidney disease biopsies, we demonstrated that expression of the podocyte genes identified by our approach is significantly related to the degree of renal impairment in patients. Our approach is broadly applicable to define lineage specificity in both cell physiology and human disease contexts. We provide a user-friendly website that enables researchers to apply this method to any cell-lineage or tissue of interest. Identified cell-lineage-specific transcripts are expected to play essential tissue-specific roles in organogenesis and disease and can provide starting points for the development of organ-specific diagnostics and therapies.

  16. Defining cell-type specificity at the transcriptional level in human disease

    PubMed Central

    Ju, Wenjun; Greene, Casey S.; Eichinger, Felix; Nair, Viji; Hodgin, Jeffrey B.; Bitzer, Markus; Lee, Young-suk; Zhu, Qian; Kehata, Masami; Li, Min; Jiang, Song; Rastaldi, Maria Pia; Cohen, Clemens D.; Troyanskaya, Olga G.; Kretzler, Matthias

    2013-01-01

    Cell-lineage–specific transcripts are essential for differentiated tissue function, implicated in hereditary organ failure, and mediate acquired chronic diseases. However, experimental identification of cell-lineage–specific genes in a genome-scale manner is infeasible for most solid human tissues. We developed the first genome-scale method to identify genes with cell-lineage–specific expression, even in lineages not separable by experimental microdissection. Our machine-learning–based approach leverages high-throughput data from tissue homogenates in a novel iterative statistical framework. We applied this method to chronic kidney disease and identified transcripts specific to podocytes, key cells in the glomerular filter responsible for hereditary and most acquired glomerular kidney disease. In a systematic evaluation of our predictions by immunohistochemistry, our in silico approach was significantly more accurate (65% accuracy in human) than predictions based on direct measurement of in vivo fluorescence-tagged murine podocytes (23%). Our method identified genes implicated as causal in hereditary glomerular disease and involved in molecular pathways of acquired and chronic renal diseases. Furthermore, based on expression analysis of human kidney disease biopsies, we demonstrated that expression of the podocyte genes identified by our approach is significantly related to the degree of renal impairment in patients. Our approach is broadly applicable to define lineage specificity in both cell physiology and human disease contexts. We provide a user-friendly website that enables researchers to apply this method to any cell-lineage or tissue of interest. Identified cell-lineage–specific transcripts are expected to play essential tissue-specific roles in organogenesis and disease and can provide starting points for the development of organ-specific diagnostics and therapies. PMID:23950145

  17. Cell-Type-Specific Gene Programs of the Normal Human Nephron Define Kidney Cancer Subtypes.

    PubMed

    Lindgren, David; Eriksson, Pontus; Krawczyk, Krzysztof; Nilsson, Helén; Hansson, Jennifer; Veerla, Srinivas; Sjölund, Jonas; Höglund, Mattias; Johansson, Martin E; Axelson, Håkan

    2017-08-08

    Comprehensive transcriptome studies of cancers often rely on corresponding normal tissue samples to serve as a transcriptional reference. In this study, we performed in-depth analyses of normal kidney tissue transcriptomes from the TCGA and demonstrate that the histological variability in cellularity, inherent in the kidney architecture, lead to considerable transcriptional differences between samples. This should be considered when comparing expression profiles of normal and cancerous kidney tissues. We exploited these differences to define renal-cell-specific gene signatures and used these as a framework to analyze renal cell carcinoma (RCC) ontogeny. Chromophobe RCCs express FOXI1-driven genes that define collecting duct intercalated cells, whereas HNF-regulated genes, specific for proximal tubule cells, are an integral part of clear cell and papillary RCC transcriptomes. These networks may be used as a framework for understanding the interplay between genomic changes in RCC subtypes and the lineage-defining regulatory machinery of their non-neoplastic counterparts. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  18. Cell type-specific tuning of hippocampal interneuron firing during gamma oscillations in vivo.

    PubMed

    Tukker, John J; Fuentealba, Pablo; Hartwich, Katja; Somogyi, Peter; Klausberger, Thomas

    2007-08-01

    Cortical gamma oscillations contribute to cognitive processing and are thought to be supported by perisomatic-innervating GABAergic interneurons. We performed extracellular recordings of identified interneurons in the hippocampal CA1 area of anesthetized rats, revealing that the firing patterns of five distinct interneuron types are differentially correlated to spontaneous gamma oscillations. The firing of bistratified cells, which target dendrites of pyramidal cells coaligned with the glutamatergic input from hippocampal area CA3, is strongly phase locked to field gamma oscillations. Parvalbumin-expressing basket, axo-axonic, and cholecystokinin-expressing interneurons exhibit moderate gamma modulation, whereas the spike timing of distal dendrite-innervating oriens-lacunosum moleculare interneurons is not correlated to field gamma oscillations. Cholecystokinin-expressing interneurons fire earliest in the gamma cycle, a finding that is consistent with their suggested function of thresholding individual pyramidal cells. Furthermore, we show that field gamma amplitude correlates with interneuronal spike-timing precision and firing rate. Overall, our recordings suggest that gamma synchronization in vivo is assisted by temporal- and domain-specific GABAergic inputs to pyramidal cells and is initiated in pyramidal cell dendrites in addition to somata and axon initial segments.

  19. Reconstitution of cell-type-specific transcription of the rat prolactin gene in vitro.

    PubMed Central

    Cao, Z D; Barron, E A; Carillo, A J; Sharp, Z D

    1987-01-01

    We present evidence for the existence of prolactin upstream factor 1 (PUF-1) in rat pituitary-derived cells and demonstrate its interaction with a symmetrical DNA element located in the 5' flanking region of the gene. An in vitro expression system developed from pituitary-derived GH3 cells was used to determine that 420 base pairs (bp) of 5' flanking DNA was sufficient for cell-specific, accurate, and efficient RNA polymerase II transcription of the rat prolactin gene. Reconstitution of in vitro transcription with pituitary and nonpituitary nuclear extracts suggested that the presence of GH3 cell-specific factors mediated the activation of prolactin gene expression. We also demonstrated that a functionally stable transcription complex assembled on the prolactin promoter. Using DNase I protection procedures, we have identified the DNA-protein binding area in the prolactin 5' flanking region. GH3 nuclear extracts contain a cell-specific protein (PUF-I) that binds to a 28-bp region (-63 to -36)which contains an 18-bp imperfect palindrome (-63 to -46). The role that the interaction between PUF-I and the imperfect palindrome plays in in vitro pituitary-specific prolactin gene expression is discussed. Images PMID:3683387

  20. Role of Cell-Type-Specific Endoplasmic Reticulum-Associated Degradation in Polyomavirus Trafficking

    PubMed Central

    Bennett, Shauna M.; Jiang, Mengxi

    2013-01-01

    BK polyomavirus (BKPyV) is a widespread human pathogen that establishes a lifelong persistent infection and can cause severe disease in immunosuppressed patients. BKPyV is a nonenveloped DNA virus that must traffic through the endoplasmic reticulum (ER) for productive infection to occur; however, it is unknown how BKPyV exits the ER before nuclear entry. In this study, we elucidated the role of the ER-associated degradation (ERAD) pathway during BKPyV intracellular trafficking in renal proximal tubule epithelial (RPTE) cells, a natural host cell. Using proteasome and ERAD inhibitors, we showed that ERAD is required for productive entry. Altered trafficking and accumulation of uncoated viral intermediates were detected by fluorescence in situ hybridization and indirect immunofluorescence in the presence of an inhibitor. Additionally, we detected a change in localization of partially uncoated virus within the ER during proteasome inhibition, from a BiP-rich area to a calnexin-rich subregion, indicating that BKPyV accumulated in an ER subcompartment. Furthermore, inhibiting ERAD did not prevent entry of capsid protein VP1 into the cytosol from the ER. By comparing the cytosolic entry of the related polyomavirus simian virus 40 (SV40), we found that dependence on the ERAD pathway for cytosolic entry varied between the polyomaviruses and between different cell types, namely, immortalized CV-1 cells and primary RPTE cells. PMID:23740996

  1. Systemic and cell type-specific gene expression patterns in scleroderma skin

    PubMed Central

    Whitfield, Michael L.; Finlay, Deborah R.; Murray, John Isaac; Troyanskaya, Olga G.; Chi, Jen-Tsan; Pergamenschikov, Alexander; McCalmont, Timothy H.; Brown, Patrick O.; Botstein, David; Connolly, M. Kari

    2003-01-01

    We used DNA microarrays representing >12,000 human genes to characterize gene expression patterns in skin biopsies from individuals with a diagnosis of systemic sclerosis with diffuse scleroderma. We found consistent differences in the patterns of gene expression between skin biopsies from individuals with scleroderma and those from normal, unaffected individuals. The biopsies from affected individuals showed nearly indistinguishable patterns of gene expression in clinically affected and clinically unaffected tissue, even though these were clearly distinguishable from the patterns found in similar tissue from unaffected individuals. Genes characteristically expressed in endothelial cells, B lymphocytes, and fibroblasts showed differential expression between scleroderma and normal biopsies. Analysis of lymphocyte populations in scleroderma skin biopsies by immunohistochemistry suggest the B lymphocyte signature observed on our arrays is from CD20+ B cells. These results provide evidence that scleroderma has systemic manifestations that affect multiple cell types and suggests genes that could be used as potential markers for the disease. PMID:14530402

  2. Mouse models of fear-related disorders: Cell-type-specific manipulations in amygdala.

    PubMed

    Gafford, G M; Ressler, K J

    2016-05-03

    Fear conditioning is a model system used to study threat responses, fear memory and their dysregulation in a variety of organisms. Newly developed tools such as optogenetics, Cre recombinase and DREADD technologies have allowed researchers to manipulate anatomically or molecularly defined cell subtypes with a high degree of temporal control and determine the effect of this manipulation on behavior. These targeted molecular techniques have opened up a new appreciation for the critical contributions different subpopulations of cells make to fear behavior and potentially to treatment of fear and anxiety disorders. Here we review progress to date across a variety of techniques to understand fear-related behavior through the manipulation of different cell subtypes within the amygdala.

  3. Cell-type specific synchronization of neural activity in FEF with V4 during attention

    PubMed Central

    Gregoriou, Georgia G.; Gotts, Stephen J.; Desimone, Robert

    2012-01-01

    Summary Shifts of gaze and shifts of attention are closely linked and it is debated whether they result from the same neural mechanisms. Both processes involve the frontal eye fields (FEF), an area which is also a source of top-down feedback to area V4 during covert attention. To test the relative contributions of oculomotor and attention-related FEF signals to such feedback, we recorded simultaneously from both areas in a covert attention task and in a saccade task. In the attention task, only visual and visuomovement FEF neurons showed enhanced responses, whereas movement cells were unchanged. Importantly, visual, but not movement or visuomovement cells, showed enhanced gamma frequency synchronization with activity in V4 during attention. Within FEF, beta synchronization was increased for movement cells during attention but was suppressed in the saccade task. These findings support the idea that the attentional modulation of visual processing is not mediated by movement neurons. PMID:22325208

  4. Triplet repeat mutation length gains correlate with cell-type specific vulnerability in Huntington disease brain.

    PubMed

    Shelbourne, Peggy F; Keller-McGandy, Christine; Bi, Wenya Linda; Yoon, Song-Ro; Dubeau, Louis; Veitch, Nicola J; Vonsattel, Jean Paul; Wexler, Nancy S; Arnheim, Norman; Augood, Sarah J

    2007-05-15

    Huntington disease is caused by the expansion of a CAG repeat encoding an extended glutamine tract in a protein called huntingtin. Here, we provide evidence supporting the hypothesis that somatic increases of mutation length play a role in the progressive nature and cell-selective aspects of HD pathogenesis. Results from micro-dissected tissue and individual laser-dissected cells obtained from human HD cases and knock-in HD mice indicate that the CAG repeat is unstable in all cell types tested although neurons tend to have longer mutation length gains than glia. Mutation length gains occur early in the disease process and continue to accumulate as the disease progresses. In keeping with observed patterns of cell loss, neuronal mutation length gains tend to be more prominent in the striatum than in the cortex of low-grade human HD cases, less so in more advanced cases. Interestingly, neuronal sub-populations of HD mice appear to have different propensities for mutation length gains; in particular, smaller mutation length gains occur in nitric oxide synthase-positive striatal interneurons (a relatively spared cell type in HD) compared with the pan-striatal neuronal population. More generally, the data demonstrate that neuronal changes in HD repeat length can be at least as great, if not greater, than those observed in the germline. The fact that significant CAG repeat length gains occur in non-replicating cells also argues that processes such as inappropriate mismatch repair rather than DNA replication are involved in generating mutation instability in HD brain tissue.

  5. Unique Cell Type-Specific Junctional Complexes in Vascular Endothelium of Human and Rat Liver Sinusoids

    PubMed Central

    Straub, Beate K.; Peitsch, Wiebke K.; Demory, Alexandra; Dörflinger, Yvette; Schledzewski, Kai; Schmieder, Astrid; Schemmer, Peter; Augustin, Hellmut G.; Schirmacher, Peter; Goerdt, Sergij

    2012-01-01

    Liver sinusoidal endothelium is strategically positioned to control access of fluids, macromolecules and cells to the liver parenchyma and to serve clearance functions upstream of the hepatocytes. While clearance of macromolecular debris from the peripheral blood is performed by liver sinusoidal endothelial cells (LSECs) using a delicate endocytic receptor system featuring stabilin-1 and -2, the mannose receptor and CD32b, vascular permeability and cell trafficking are controlled by transcellular pores, i.e. the fenestrae, and by intercellular junctional complexes. In contrast to blood vascular and lymphatic endothelial cells in other organs, the junctional complexes of LSECs have not yet been consistently characterized in molecular terms. In a comprehensive analysis, we here show that LSECs express the typical proteins found in endothelial adherens junctions (AJ), i.e. VE-cadherin as well as α-, β-, p120-catenin and plakoglobin. Tight junction (TJ) transmembrane proteins typical of endothelial cells, i.e. claudin-5 and occludin, were not expressed by rat LSECs while heterogenous immunreactivity for claudin-5 was detected in human LSECs. In contrast, junctional molecules preferentially associating with TJ such as JAM-A, B and C and zonula occludens proteins ZO-1 and ZO-2 were readily detected in LSECs. Remarkably, among the JAMs JAM-C was considerably over-expressed in LSECs as compared to lung microvascular endothelial cells. In conclusion, we show here that LSECs form a special kind of mixed-type intercellular junctions characterized by co-occurrence of endothelial AJ proteins, and of ZO-1 and -2, and JAMs. The distinct molecular architecture of the intercellular junctional complexes of LSECs corroborates previous ultrastructural findings and provides the molecular basis for further analyses of the endothelial barrier function of liver sinusoids under pathologic conditions ranging from hepatic inflammation to formation of liver metastasis. PMID:22509281

  6. Unexpected Roles for Core Promoter Recognition Factors in Cell-type Specific Transcription and Gene Regulation

    PubMed Central

    Goodrich, James A.

    2010-01-01

    Until recently, the eukaryotic core promoter recognition complex was generally thought to play an essential but passive role in the regulation of gene expression. However, recent evidence indicates that core-promoter recognition complexes in conjunction with “non-prototypic” subunits may play a critical regulatory role in driving cell specific programs of transcription during development. Furthermore, new roles for components of these complexes have been identified beyond development, for example in mediating interactions with chromatin and in maintaining active gene expression across cell divisions. PMID:20628347

  7. Cell type-specific interactions of transcription factors with a housekeeping promoter in vivo.

    PubMed Central

    Stapleton, G; Somma, M P; Lavia, P

    1993-01-01

    Mammalian housekeeping promoters represent a class of regulatory elements different from those of tissues-specific genes, lacking a TATA box and associated with CG-rich DNA. We have compared the organization of the housekeeping Htf9 promoter in different cell types by genomic footprinting. The sites of in vivo occupancy clearly reflected local combinations of tissue-specific and ubiquitous binding factors. The flexibility of the Htf9 promoter in acting as the target of cell-specific combinations of factors may ensure ubiquitous expression of the Htf9-associated genes. Images PMID:8389443

  8. Highly efficient cell-type-specific gene inactivation reveals a key function for the Drosophila FUS homolog cabeza in neurons.

    PubMed

    Frickenhaus, Marie; Wagner, Marina; Mallik, Moushami; Catinozzi, Marica; Storkebaum, Erik

    2015-03-16

    To expand the rich genetic toolkit of Drosophila melanogaster, we evaluated whether introducing FRT or LoxP sites in endogenous genes could allow for cell-type-specific gene inactivation in both dividing and postmitotic cells by GAL4-driven expression of FLP or Cre recombinase. For proof of principle, conditional alleles were generated for cabeza (caz), the Drosophila homolog of human FUS, a gene implicated in the neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Upon selective expression in neurons or muscle, both FLP and Cre mediated caz inactivation in all neurons or muscle cells, respectively. Neuron-selective caz inactivation resulted in failure of pharate adult flies to eclose from the pupal case, and adult escapers displayed motor performance defects and reduced life span. Due to Cre-toxicity, FLP/FRT is the preferred system for cell-type-specific gene inactivation, and this strategy outperforms RNAi-mediated knock-down. Furthermore, the GAL80 target system allowed for temporal control over gene inactivation, as induction of FLP expression from the adult stage onwards still inactivated caz in >99% of neurons. Remarkably, selective caz inactivation in adult neurons did not affect motor performance and life span, indicating that neuronal caz is required during development, but not for maintenance of adult neuronal function.

  9. Cell Type-Specific Epigenomic Analysis Reveals a Uniquely Closed Chromatin Architecture in Mouse Rod Photoreceptors

    PubMed Central

    Hughes, Andrew E. O.; Enright, Jennifer M.; Myers, Connie A.; Shen, Susan Q.; Corbo, Joseph C.

    2017-01-01

    Rod photoreceptors are specialized neurons that mediate vision in dim light and are the predominant photoreceptor type in nocturnal mammals. The rods of nocturnal mammals are unique among vertebrate cell types in having an ‘inverted’ nuclear architecture, with a dense mass of heterochromatin in the center of the nucleus rather than dispersed clumps at the periphery. To test if this unique nuclear architecture is correlated with a unique epigenomic landscape, we performed ATAC-seq on mouse rods and their most closely related cell type, cone photoreceptors. We find that thousands of loci are selectively closed in rods relative to cones as well as >60 additional cell types. Furthermore, we find that the open chromatin profile of photoreceptors lacking the rod master regulator Nrl is nearly indistinguishable from that of native cones, indicating that Nrl is required for selective chromatin closure in rods. Finally, we identified distinct enrichments of transcription factor binding sites in rods and cones, revealing key differences in the cis-regulatory grammar of these cell types. Taken together, these data provide insight into the development and maintenance of photoreceptor identity, and highlight rods as an attractive system for studying the relationship between nuclear organization and local changes in gene regulation. PMID:28256534

  10. Cell-Type Specific Responses to DNA Replication Stress in Early C. elegans Embryos

    PubMed Central

    Stevens, Holly; Williams, Ashley B.

    2016-01-01

    To better understand how the cellular response to DNA replication stress is regulated during embryonic development, we and others have established the early C. elegans embryo as a model system to study this important problem. As is the case in most eukaryotic cell types, the replication stress response is controlled by the ATR kinase in early worm embryos. In this report we use RNAi to systematically characterize ATR pathway components for roles in promoting cell cycle delay during a replication stress response, and we find that these genetic requirements vary, depending on the source of stress. We also examine how individual cell types within the embryo respond to replication stress, and we find that the strength of the response, as defined by duration of cell cycle delay, varies dramatically within blastomeres of the early embryo. Our studies shed light on how the replication stress response is managed in the context of embryonic development and show that this pathway is subject to developmental regulation. PMID:27727303

  11. Haploinsufficiency for BRCA1 leads to cell-type-specific genomic instability and premature senescence.

    PubMed

    Sedic, Maja; Skibinski, Adam; Brown, Nelson; Gallardo, Mercedes; Mulligan, Peter; Martinez, Paula; Keller, Patricia J; Glover, Eugene; Richardson, Andrea L; Cowan, Janet; Toland, Amanda E; Ravichandran, Krithika; Riethman, Harold; Naber, Stephen P; Näär, Anders M; Blasco, Maria A; Hinds, Philip W; Kuperwasser, Charlotte

    2015-06-24

    Although BRCA1 function is essential for maintaining genomic integrity in all cell types, it is unclear why increased risk of cancer in individuals harbouring deleterious mutations in BRCA1 is restricted to only a select few tissues. Here we show that human mammary epithelial cells (HMECs) from BRCA1-mutation carriers (BRCA1(mut/+)) exhibit increased genomic instability and rapid telomere erosion in the absence of tumour-suppressor loss. Furthermore, we uncover a novel form of haploinsufficiency-induced senescence (HIS) specific to epithelial cells, which is triggered by pRb pathway activation rather than p53 induction. HIS and telomere erosion in HMECs correlate with misregulation of SIRT1 leading to increased levels of acetylated pRb as well as acetylated H4K16 both globally and at telomeric regions. These results identify a novel form of cellular senescence and provide a potential molecular basis for the rapid cell- and tissue- specific predisposition of breast cancer development associated with BRCA1 haploinsufficiency.

  12. Cell type-specific dependence of muscarinic signalling in mouse hippocampal stratum oriens interneurones

    PubMed Central

    Lawrence, J Josh; Statland, Jeffrey M; Grinspan, Zachary M; McBain, Chris J

    2006-01-01

    Cholinergic signalling is critically involved in learning and memory processes in the hippocampus, but the postsynaptic impact of cholinergic modulation on morphologically defined subtypes of hippocampal interneurones remains unclear. We investigated the influence of muscarinic receptor (mAChR) activation on stratum oriens interneurones using whole-cell patch clamp recordings from hippocampal slices in vitro. Upon somatic depolarization, mAChR activation consistently enhanced firing frequency and produced large, sustained afterdepolarizations (ADPs) of stratum oriens–lacunosum moleculare (O-LM) interneurones. In contrast, stratum oriens cell types with axon arborization patterns different from O-LM cells not only lacked large muscarinic ADPs but also appeared to exhibit distinct responses to mAChR activation. The ADP in O-LM cells, mediated by M1/M3 receptors, was associated with inhibition of an M current, inhibition of a slow calcium-activated potassium current, and activation of a calcium-dependent non-selective cationic current (ICAT). An examination of ionic conductances generated by firing revealed that calcium entry through ICAT controls the emergence of the mAChR-mediated ADP. Our results indicate that cholinergic specializations are present within anatomically distinct subpopulations of hippocampal interneurones, suggesting that there may be organizing principles to cholinergic control of GABA release in the hippocampus. PMID:16322052

  13. Cell type-specific differences in β-glucan recognition and signalling in porcine innate immune cells.

    PubMed

    Baert, Kim; Sonck, Eva; Goddeeris, Bruno M; Devriendt, Bert; Cox, Eric

    2015-01-01

    β-glucans exert receptor-mediated immunomodulating activities, including oxidative burst activity and cytokine secretion. The role of the β-glucan receptors dectin-1 and complement receptor 3 (CR3) in the response of immune cells towards β-glucans is still unresolved. Dectin-1 is considered as the main β-glucan receptor in mice, while recent studies in man show that CR3 is more important in β-glucan-mediated responses. This incited us to elucidate which receptor contributes to the response of innate immune cells towards particulate β-glucans in pigs as the latter might serve as a better model for man. Our results show an important role of CR3 in β-glucan recognition, as blocking this receptor strongly reduced the phagocytosis of β-glucans and the β-glucan-induced ROS production by porcine neutrophils. Conversely, dectin-1 does not seem to play a major role in β-glucan recognition in neutrophils. However, recognition of β-glucans appeared cell type-specific as both dectin-1 and CR3 are involved in the β-glucan-mediated responses in pig macrophages. Moreover, CR3 signalling through focal adhesion kinase (FAK) was indispensable for β-glucan-mediated ROS production and cytokine production in neutrophils and macrophages, while the Syk-dependent pathway was only partly involved in these responses. We may conclude that CR3 plays a cardinal role in β-glucan signalling in porcine neutrophils, while macrophages use a more diverse receptor array to detect and respond towards β-glucans. Nonetheless, FAK acts as a master switch that regulates β-glucan-mediated responses in neutrophils as well as macrophages. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Intraindividual Temporal miRNA Variability in Serum, Plasma, and White Blood Cell Subpopulations.

    PubMed

    Ammerlaan, Wim; Betsou, Fay

    2016-10-01

    Blood microRNAs (miRNAs) are ideal biomarkers, and blood derivatives are often collected in the scope of miRNA research projects. However, knowledge of temporal variations of miRNAs in healthy individuals is lacking. In this study, miRNA variability was measured over a 1-year period in different blood derivatives, collected every 2-3 months from two healthy donors. There is a continuum of intraindividual temporal variability, with particularly stable (coefficient of variation [CV] <20%-30%) and particularly unstable (CV >100%-130%) miRNAs in serum, plasma, and specific white blood cell subpopulations. The temporal intraindividual variability of miRNAs should be taken into consideration in experimental design of biospecimen collections and validation of diagnostic biomarkers.

  15. So many pieces, one puzzle: cell type specification and visual circuitry in flies and mice

    PubMed Central

    Wernet, Mathias F.; Huberman, Andrew D.; Desplan, Claude

    2014-01-01

    The visual system is a powerful model for probing the development, connectivity, and function of neural circuits. Two genetically tractable species, mice and flies, are together providing a great deal of understanding of these processes. Current efforts focus on integrating knowledge gained from three cross-fostering fields of research: (1) understanding how the fates of different cell types are specified during development, (2) revealing the synaptic connections between identified cell types (“connectomics”) by high-resolution three-dimensional circuit anatomy, and (3) causal testing of how identified circuit elements contribute to visual perception and behavior. Here we discuss representative examples from fly and mouse models to illustrate the ongoing success of this tripartite strategy, focusing on the ways it is enhancing our understanding of visual processing and other sensory systems. PMID:25452270

  16. Isolation, Structure Elucidation, and (Bio)Synthesis of Haprolid, a Cell-Type-Specific Myxobacterial Cytotoxin.

    PubMed

    Steinmetz, Heinrich; Li, Jun; Fu, Chengzhang; Zaburannyi, Nestor; Kunze, Birgitte; Harmrolfs, Kirsten; Schmitt, Viktoria; Herrmann, Jennifer; Reichenbach, Hans; Höfle, Gerhard; Kalesse, Markus; Müller, Rolf

    2016-08-16

    Myxobacteria are well-established sources for novel natural products exhibiting intriguing bioactivities. We here report on haprolid (1) isolated from Byssovorax cruenta Har1. The compound exhibits an unprecedented macrolactone comprising four modified amino acids and a polyketide fragment. As configurational assignment proved difficult, a bioinformatic analysis of the biosynthetic gene cluster was chosen to predict the configuration of each stereocenter. In-depth analysis of the corresponding biosynthetic proteins established a hybrid polyketide synthase/nonribosomal peptide synthetase origin of haprolid and allowed for stereochemical assignments. A subsequent total synthesis yielded haprolid and corroborated all predictions made. Intriguingly, haprolid showed cytotoxicity against several cell lines in the nanomolar range whereas other cells were almost unaffected by treatment with the compound. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Cell-type-specific differentiation of chloroplasts in C4 plants.

    PubMed

    Majeran, Wojciech; van Wijk, Klaas J

    2009-02-01

    In leaves of C4 grasses such as maize, photosynthetic activities are partitioned between bundle-sheath and mesophyll cells, leading to increased photosynthetic yield, particularly under stress conditions. As we discuss here, recent comparative chloroplast proteome analyses in maize have shown specific adaptation to C4-cell-specific differentiation of the photosynthetic apparatus, primary and secondary metabolism and metabolite transporters, as well as the chloroplast protein homeostasis machinery. Furthermore, a novel bundle-sheath-enriched 1000-kDa NADPH dehydrogenase 'supercomplex' has been identified, and we discuss its possible role in inorganic carbon concentration. These breakthroughs provide new opportunities to further unravel C4 pathways and to increase crop productivity through metabolic engineering of C4 pathways into C3 plants, such as rice.

  18. Cell-Type-Specific Activity in Prefrontal Cortex during Goal-Directed Behavior.

    PubMed

    Pinto, Lucas; Dan, Yang

    2015-07-15

    The prefrontal cortex (PFC) plays a key role in controlling goal-directed behavior. Although a variety of task-related signals have been observed in the PFC, whether they are differentially encoded by various cell types remains unclear. Here we performed cellular-resolution microendoscopic Ca(2+) imaging from genetically defined cell types in the dorsomedial PFC of mice performing a PFC-dependent sensory discrimination task. We found that inhibitory interneurons of the same subtype were similar to each other, but different subtypes preferentially signaled different task-related events: somatostatin-positive neurons primarily signaled motor action (licking), vasoactive intestinal peptide-positive neurons responded strongly to action outcomes, whereas parvalbumin-positive neurons were less selective, responding to sensory cues, motor action, and trial outcomes. Compared to each interneuron subtype, pyramidal neurons showed much greater functional heterogeneity, and their responses varied across cortical layers. Such cell-type and laminar differences in neuronal functional properties may be crucial for local computation within the PFC microcircuit.

  19. Cell type-specific long-range connections of basal forebrain circuit

    PubMed Central

    Do, Johnny Phong; Xu, Min; Lee, Seung-Hee; Chang, Wei-Cheng; Zhang, Siyu; Chung, Shinjae; Yung, Tyler J; Fan, Jiang Lan; Miyamichi, Kazunari; Luo, Liqun; Dan, Yang

    2016-01-01

    The basal forebrain (BF) plays key roles in multiple brain functions, including sleep-wake regulation, attention, and learning/memory, but the long-range connections mediating these functions remain poorly characterized. Here we performed whole-brain mapping of both inputs and outputs of four BF cell types – cholinergic, glutamatergic, and parvalbumin-positive (PV+) and somatostatin-positive (SOM+) GABAergic neurons – in the mouse brain. Using rabies virus -mediated monosynaptic retrograde tracing to label the inputs and adeno-associated virus to trace axonal projections, we identified numerous brain areas connected to the BF. The inputs to different cell types were qualitatively similar, but the output projections showed marked differences. The connections to glutamatergic and SOM+ neurons were strongly reciprocal, while those to cholinergic and PV+ neurons were more unidirectional. These results reveal the long-range wiring diagram of the BF circuit with highly convergent inputs and divergent outputs and point to both functional commonality and specialization of different BF cell types. DOI: http://dx.doi.org/10.7554/eLife.13214.001 PMID:27642784

  20. Cell-Type Specific Determinants of NRAMP1 Expression in Professional Phagocytes

    PubMed Central

    Cellier, Mathieu F. M.

    2013-01-01

    The Natural resistance-associated macrophage protein 1 (Nramp1 or Solute carrier 11 member 1, Slc11a1) transports divalent metals across the membrane of late endosomes and lysosomes in professional phagocytes. Nramp1 represents an ancient eukaryotic cell-autonomous defense whereas the gene duplication that yielded Nramp1 and Nramp2 predated the origin of Sarcopterygians (lobe-finned fishes and tetrapods). SLC11A1 genetic polymorphisms associated with human resistance to tuberculosis consist of potential regulatory variants. Herein, current knowledge of the regulation of SLC11A1 gene expression is reviewed and comprehensive analysis of ENCODE data available for hematopoietic cell-types suggests a hypothesis for the regulation of SLC11A1 expression during myeloid development and phagocyte functional polarization. SLC11A1 is part of a 34.6 kb CTCF-insulated locus scattered with predicted regulatory elements: a 3' enhancer, a large 5' enhancer domain and four elements spread around the transcription start site (TSS), including several C/EBP and PU.1 sites. SLC11A1 locus ends appear mobilized by ETS-related factors early during myelopoiesis; activation of both 5' and 3' enhancers in myelo-monocytic cells correlate with transcription factor binding at the TSS. Characterizing the corresponding cis/trans determinants functionally will establish the mechanisms involved and possibly reveal genetic variation that impacts susceptibility to infectious or immune diseases. PMID:24832660

  1. Organization of human ACAT-2 gene and its cell-type-specific promoter activity.

    PubMed

    Song, B L; Qi, W; Yang, X Y; Chang, C C; Zhu, J Q; Chang, T Y; Li, B L

    2001-03-30

    Acyl-CoA:cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis. Two ACAT genes exist in mammals. We report here the genomic organization of human ACAT-2 gene and analysis of its promoter activity in various cell lines. The human ACAT-2 gene spans over 18 kb and contains 15 exons. Three transcription start sites and one poly(A) site are identified by the 5'/3'-RACE. In addition, the human ACAT-2 gene is linked to the insulin-like growth factor binding protein 6 (IGFBP-6) gene in a head-to-tail manner with a small intergenic region of about 1.2 kb. The 5'-flanking region of human ACAT-2 gene contains many potential cis-acting elements for multiple transcriptional regulatory factors but lacks TATA and CCAAT boxes. Using promoter-luciferase reporter assays, we demonstrate the transcriptional activity of ACAT-2 gene promoter is high in Caco-2 cells, especially after these cells become postconfluent and behave as intestinal enterocytes.

  2. Cell type specificity and mechanism of control of a gene may be reverted in different strains of Dictyostelium discoideum.

    PubMed

    Mangiarotti, G; Giorda, R

    2000-06-21

    Twelve genes which are expressed exclusively in pre-spore cells of Dictyostelium strain AX3 are expressed exclusively in pre-stalk cells of strain AX2. One gene has the opposite behavior: it is expressed in pre-stalk cells in AX3 and in pre-spore cells in AX2. The change in cell type specificity involves a change in the mechanism of control of gene expression. When they are expressed in pre-stalk cells, genes are controlled at the level of transcription, whilst in pre-spore cells, they are controlled at the level of mRNA stability. Genes expressed in pre-stalk cells in strain AX2, fused with an AX2 pre-spore specific promoter, become regulated at the level of mRNA stability. These findings indicate that at least a group of pre-stalk mRNAs possess the cis-destabilizing element typical of pre-spore mRNAs, though they are not destabilized in disaggregated cells. This is due to the fact that ribosomal protein S6, phosphorylation of which is responsible for controlling the stability of pre-spore mRNAs, is not dephosphorylated in disaggregated pre-stalk cells. These cells lack an S6 phosphatase activity which has been purified from disaggregated pre-spore cells.

  3. Cell-type specific inactivation of hippocampal CA1 disrupts location-dependent object recognition in the mouse

    PubMed Central

    Haettig, Jakob; Sun, Yanjun; Wood, Marcelo A.; Xu, Xiangmin

    2013-01-01

    The allatostatin receptor (AlstR)/ligand inactivation system enables potent regulation of neuronal circuit activity. To examine how different cell types participate in memory formation, we have used this system through Cre-directed, cell-type specific expression in mouse hippocampal CA1 in vivo and examined functional effects of inactivation of excitatory vs. inhibitory neurons on memory formation. We chose to use a hippocampus-dependent behavioral task involving location-dependent object recognition (LOR). The double transgenic mice, with the AlstRs selectively expressed in excitatory pyramidal neurons or inhibitory interneurons, were cannulated, targeting dorsal hippocampus to allow the infusion of the receptor ligand (the allatostatin [AL] peptide) in a time dependent manner. Compared to control animals, AL-infused animals showed no long-term memory for object location. While inactivation of excitatory or inhibitory neurons produced opposite effects on hippocampal circuit activity in vitro, the effects in vivo were similar. Both types of inactivation experiments resulted in mice exhibiting no long-term memory for object location. Together, these results demonstrate that the Cre-directed, AlstR-based system is a powerful tool for cell-type specific manipulations in a behaving animal and suggest that activity of either excitatory neurons or inhibitory interneurons is essential for proper long-term object location memory formation. PMID:23418393

  4. Discovery of Cell-Type-Specific and Disease-Related Enzymatic Activity Changes via Global Evaluation of Peptide Metabolism.

    PubMed

    Onagi, Jun; Komatsu, Toru; Ichihashi, Yuki; Kuriki, Yugo; Kamiya, Mako; Terai, Takuya; Ueno, Tasuku; Hanaoka, Kenjiro; Matsuzaki, Hiroyuki; Hata, Keisuke; Watanabe, Toshiaki; Nagano, Tetsuo; Urano, Yasuteru

    2017-03-08

    Cellular homeostasis is maintained by a complex network of reactions catalyzed by enormous numbers of enzymatic activities (the enzymome), which serve to determine the phenotypes of cells. Here, we focused on the enzymomics of proteases and peptidases because these enzymes are an important class of disease-related proteins. We describe a system that (A) simultaneously evaluates metabolic activities of peptides using a series of exogenous peptide substrates and (B) identifies the enzymes that metabolize the specified peptide substrate with high throughput. We confirmed that the developed system was able to discover cell-type-specific and disease-related exo- and endopeptidase activities and identify the responsible enzymes. For example, we found that the activity of the endopeptidase neurolysin is highly elevated in human colorectal tumor tissue samples. This simple but powerful enzymomics platform should be widely applicable to uncover cell-type-specific reactions and altered enzymatic functions with potential value as biomarkers or drug targets in various disease states and to investigate the mechanisms of the underlying pathologies.

  5. Flux analysis of cholesterol biosynthesis in vivo reveals multiple tissue and cell-type specific pathways

    PubMed Central

    Mitsche, Matthew A; McDonald, Jeffrey G; Hobbs, Helen H; Cohen, Jonathan C

    2015-01-01

    Two parallel pathways produce cholesterol: the Bloch and Kandutsch-Russell pathways. Here we used stable isotope labeling and isotopomer analysis to trace sterol flux through the two pathways in mice. Surprisingly, no tissue used the canonical K–R pathway. Rather, a hybrid pathway was identified that we call the modified K–R (MK–R) pathway. Proportional flux through the Bloch pathway varied from 8% in preputial gland to 97% in testes, and the tissue-specificity observed in vivo was retained in cultured cells. The distribution of sterol isotopomers in plasma mirrored that of liver. Sterol depletion in cultured cells increased flux through the Bloch pathway, whereas overexpression of 24-dehydrocholesterol reductase (DHCR24) enhanced usage of the MK–R pathway. Thus, relative use of the Bloch and MK–R pathways is highly variable, tissue-specific, flux dependent, and epigenetically fixed. Maintenance of two interdigitated pathways permits production of diverse bioactive sterols that can be regulated independently of cholesterol. DOI: http://dx.doi.org/10.7554/eLife.07999.001 PMID:26114596

  6. Dopaminergic neurons write and update memories with cell-type-specific rules

    PubMed Central

    Aso, Yoshinori; Rubin, Gerald M

    2016-01-01

    Associative learning is thought to involve parallel and distributed mechanisms of memory formation and storage. In Drosophila, the mushroom body (MB) is the major site of associative odor memory formation. Previously we described the anatomy of the adult MB and defined 20 types of dopaminergic neurons (DANs) that each innervate distinct MB compartments (Aso et al., 2014a, 2014b). Here we compare the properties of memories formed by optogenetic activation of individual DAN cell types. We found extensive differences in training requirements for memory formation, decay dynamics, storage capacity and flexibility to learn new associations. Even a single DAN cell type can either write or reduce an aversive memory, or write an appetitive memory, depending on when it is activated relative to odor delivery. Our results show that different learning rules are executed in seemingly parallel memory systems, providing multiple distinct circuit-based strategies to predict future events from past experiences. DOI: http://dx.doi.org/10.7554/eLife.16135.001 PMID:27441388

  7. ICK is essential for cell type-specific ciliogenesis and the regulation of ciliary transport.

    PubMed

    Chaya, Taro; Omori, Yoshihiro; Kuwahara, Ryusuke; Furukawa, Takahisa

    2014-06-02

    Cilia and flagella are formed and maintained by intraflagellar transport (IFT) and play important roles in sensing and moving across species. At the distal tip of the cilia/flagella, IFT complexes turn around to switch from anterograde to retrograde transport; however, the underlying regulatory mechanism is unclear. Here, we identified ICK localization at the tip of cilia as a regulator of ciliary transport. In ICK-deficient mice, we found ciliary defects in neuronal progenitor cells with Hedgehog signal defects. ICK-deficient cells formed cilia with mislocalized Hedgehog signaling components. Loss of ICK caused the accumulation of IFT-A, IFT-B, and BBSome components at the ciliary tips. In contrast, overexpression of ICK induced the strong accumulation of IFT-B, but not IFT-A or BBSome components at ciliary tips. In addition, ICK directly phosphorylated Kif3a, while inhibition of this Kif3a phosphorylation affected ciliary formation. Our results suggest that ICK is a Kif3a kinase and essential for proper ciliogenesis in development by regulating ciliary transport at the tip of cilia. © 2014 The Authors.

  8. Deep brain optical measurements of cell type-specific neural activity in behaving mice.

    PubMed

    Cui, Guohong; Jun, Sang Beom; Jin, Xin; Luo, Guoxiang; Pham, Michael D; Lovinger, David M; Vogel, Steven S; Costa, Rui M

    2014-01-01

    Recent advances in genetically encoded fluorescent sensors enable the monitoring of cellular events from genetically defined groups of neurons in vivo. In this protocol, we describe how to use a time-correlated single-photon counting (TCSPC)-based fiber optics system to measure the intensity, emission spectra and lifetime of fluorescent biosensors expressed in deep brain structures in freely moving mice. When combined with Cre-dependent selective expression of genetically encoded Ca(2+) indicators (GECIs), this system can be used to measure the average neural activity from a specific population of cells in mice performing complex behavioral tasks. As an example, we used viral expression of GCaMPs in striatal projection neurons (SPNs) and recorded the fluorescence changes associated with calcium spikes from mice performing a lever-pressing operant task. The whole procedure, consisting of virus injection, behavior training and optical recording, takes 3-4 weeks to complete. With minor adaptations, this protocol can also be applied to recording cellular events from other cell types in deep brain regions, such as dopaminergic neurons in the ventral tegmental area. The simultaneously recorded fluorescence signals and behavior events can be used to explore the relationship between the neural activity of specific brain circuits and behavior.

  9. Dynamic temporal and cell type-specific expression of Wnt signaling components in the developing midbrain

    SciTech Connect

    Rawal, Nina; Castelo-Branco, Goncalo; Sousa, Kyle M.; Kele, Julianna; Kobayashi, Kazuto; Okano, Hideyuki; Arenas, Ernest . E-mail: Ernest.Arenas@ki.se

    2006-05-15

    Wnt1 and -5a have been shown to modulate the proliferation and differentiation of midbrain dopaminergic (DA) neurons. However, it is not known whether other Wnts or which Frizzled (Fz) receptors are expressed in the developing midbrain. We found that 13 out of 19 Wnts, all 10 Fzs, and several intracellular Wnt signaling modulators, including Axin, FRAT, Naked, Par-1, and Ltap are developmentally regulated between embryonic days (E) 10.5 and 15.5. Next, we studied whether Fzs are differentially expressed in different cell types and examined neuronal-progenitor- or glial-enriched cultures and DA neurons isolated from TH-GFP reporter mice. We found that Fz8 is expressed at high levels in DA neurons at E11.5 and E13.5. Fz6 and -7 are the predominant transcripts in glial precursors, and Fz9, which is absent in DA neurons at E11.5, is the main receptor expressed in neuronal precursors. We therefore examined the function of Fz9 in DA cells and found that overexpression of Fz9 reduced Wnt5a- but not Wnt3a-induced hyperphosphorylation of Dishevelled. Thus, our results show that Fzs are developmentally regulated and differentially expressed in VM precursors, DA neurons, and glia. These findings suggest that Fz expression contributes to provide specificity to Wnt-mediated effects.

  10. Thalamocortical Innervation Pattern in Mouse Auditory and Visual Cortex: Laminar and Cell-Type Specificity.

    PubMed

    Ji, Xu-Ying; Zingg, Brian; Mesik, Lukas; Xiao, Zhongju; Zhang, Li I; Tao, Huizhong W

    2016-06-01

    Despite many previous studies, the functional innervation pattern of thalamic axons and their target specificity remains to be investigated thoroughly. Here, in primary auditory cortical slices, we examined thalamic innervation patterns for excitatory and different types of inhibitory neurons across laminae, by optogenetically stimulating axons from the medial geniculate body. We found that excitatory cells and parvalbumin (PV)-expressing inhibitory neurons across layer 2/3 (L2/3) to L6 are directly innervated by thalamic projections, with the strongest innervation occurring in L4. The innervation of PV neurons is stronger than that of excitatory neurons in the same layer, with a relatively constant ratio between their innervation strengths across layers. For somatostatin and vasoactive intestinal peptide inhibitory neurons, essentially only L4 neurons were innervated by thalamic axons and the innervation was much weaker compared with excitatory and PV cells. In addition, more than half of inhibitory neurons in L1 were innervated, relatively strongly, by thalamic axons. Similar innervation patterns were also observed in the primary visual cortex. Thus, thalamic information can be processed independently and differentially by different cortical layers, in addition to the generally thought hierarchical processing starting from L4. This parallel processing is likely shaped by feedforward inhibition from PV neurons in each individual lamina, and may extend the computation power of sensory cortices.

  11. Dissecting cell-type-specific roles of androgen receptor in prostate homeostasis and regeneration through lineage tracing.

    PubMed

    Xie, Qing; Liu, Yueli; Cai, Tao; Horton, Corrigan; Stefanson, Joshua; Wang, Zhu A

    2017-01-23

    Androgen signals through androgen receptor (AR) to influence prostate development and cancer. How stromal and epithelial AR regulate prostate homeostasis remains unclear. Using genetic lineage tracing, we systematically investigated the role of cell-autonomous AR in different prostate epithelial cell types. Here we show that AR is dispensable for basal cell maintenance, but is cell-autonomously required for the luminal differentiation of rare basal stem cells. In contrast, AR deletion in luminal cells alters cell morphology and induces transient over-proliferation, without affecting androgen-mediated luminal cell survival or regeneration. However, AR is selectively required for the maintenance of daughter cells produced by castration-resistant Nkx3.1-expressing luminal stem cells (CARNs). Notably, Pten loss can override AR-loss effects in both basal and luminal compartments to initiate tumours. Our data reveal distinct cell-type-specific roles of epithelial AR in orchestrating prostate homeostasis, and question the notion that epithelial AR serves as a tumour suppressor in early cancer initiation.

  12. Dissecting cell-type-specific roles of androgen receptor in prostate homeostasis and regeneration through lineage tracing

    PubMed Central

    Xie, Qing; Liu, Yueli; Cai, Tao; Horton, Corrigan; Stefanson, Joshua; Wang, Zhu A.

    2017-01-01

    Androgen signals through androgen receptor (AR) to influence prostate development and cancer. How stromal and epithelial AR regulate prostate homeostasis remains unclear. Using genetic lineage tracing, we systematically investigated the role of cell-autonomous AR in different prostate epithelial cell types. Here we show that AR is dispensable for basal cell maintenance, but is cell-autonomously required for the luminal differentiation of rare basal stem cells. In contrast, AR deletion in luminal cells alters cell morphology and induces transient over-proliferation, without affecting androgen-mediated luminal cell survival or regeneration. However, AR is selectively required for the maintenance of daughter cells produced by castration-resistant Nkx3.1-expressing luminal stem cells (CARNs). Notably, Pten loss can override AR-loss effects in both basal and luminal compartments to initiate tumours. Our data reveal distinct cell-type-specific roles of epithelial AR in orchestrating prostate homeostasis, and question the notion that epithelial AR serves as a tumour suppressor in early cancer initiation. PMID:28112153

  13. Cell-Type-Specific Sensorimotor Processing in Striatal Projection Neurons during Goal-Directed Behavior.

    PubMed

    Sippy, Tanya; Lapray, Damien; Crochet, Sylvain; Petersen, Carl C H

    2015-10-21

    Goal-directed sensorimotor transformation drives important aspects of mammalian behavior. The striatum is thought to play a key role in reward-based learning and action selection, receiving glutamatergic sensorimotor signals and dopaminergic reward signals. Here, we obtain whole-cell membrane potential recordings from the dorsolateral striatum of mice trained to lick a reward spout after a whisker deflection. Striatal projection neurons showed strong task-related modulation, with more depolarization and action potential firing on hit trials compared to misses. Direct pathway striatonigral neurons, but not indirect pathway striatopallidal neurons, exhibited a prominent early sensory response. Optogenetic stimulation of direct pathway striatonigral neurons, but not indirect pathway striatopallidal neurons, readily substituted for whisker stimulation evoking a licking response. Our data are consistent with direct pathway striatonigral neurons contributing a "go" signal for goal-directed sensorimotor transformation leading to action initiation. VIDEO ABSTRACT.

  14. A chemical proteomic atlas of brain serine hydrolases identifies cell type-specific pathways regulating neuroinflammation.

    PubMed

    Viader, Andreu; Ogasawara, Daisuke; Joslyn, Christopher M; Sanchez-Alavez, Manuel; Mori, Simone; Nguyen, William; Conti, Bruno; Cravatt, Benjamin F

    2016-01-18

    Metabolic specialization among major brain cell types is central to nervous system function and determined in large part by the cellular distribution of enzymes. Serine hydrolases are a diverse enzyme class that plays fundamental roles in CNS metabolism and signaling. Here, we perform an activity-based proteomic analysis of primary mouse neurons, astrocytes, and microglia to furnish a global portrait of the cellular anatomy of serine hydrolases in the brain. We uncover compelling evidence for the cellular compartmentalization of key chemical transmission pathways, including the functional segregation of endocannabinoid (eCB) biosynthetic enzymes diacylglycerol lipase-alpha (DAGLα) and -beta (DAGLβ) to neurons and microglia, respectively. Disruption of DAGLβ perturbed eCB-eicosanoid crosstalk specifically in microglia and suppressed neuroinflammatory events in vivo independently of broader effects on eCB content. Mapping the cellular distribution of metabolic enzymes thus identifies pathways for regulating specialized inflammatory responses in the brain while avoiding global alterations in CNS function.

  15. Cell type-specific loss of BDNF signaling mimics optogenetic control of cocaine reward.

    PubMed

    Lobo, Mary Kay; Covington, Herbert E; Chaudhury, Dipesh; Friedman, Allyson K; Sun, HaoSheng; Damez-Werno, Diane; Dietz, David M; Zaman, Samir; Koo, Ja Wook; Kennedy, Pamela J; Mouzon, Ezekiell; Mogri, Murtaza; Neve, Rachael L; Deisseroth, Karl; Han, Ming-Hu; Nestler, Eric J

    2010-10-15

    The nucleus accumbens is a key mediator of cocaine reward, but the distinct roles of the two subpopulations of nucleus accumbens projection neurons, those expressing dopamine D1 versus D2 receptors, are poorly understood. We show that deletion of TrkB, the brain-derived neurotrophic factor (BDNF) receptor, selectively from D1+ or D2+ neurons oppositely affects cocaine reward. Because loss of TrkB in D2+ neurons increases their neuronal excitability, we next used optogenetic tools to control selectively the firing rate of D1+ and D2+ nucleus accumbens neurons and studied consequent effects on cocaine reward. Activation of D2+ neurons, mimicking the loss of TrkB, suppresses cocaine reward, with opposite effects induced by activation of D1+ neurons. These results provide insight into the molecular control of D1+ and D2+ neuronal activity as well as the circuit-level contribution of these cell types to cocaine reward.

  16. Cell type-specific regulation of IL-10 expression in inflammation and disease

    PubMed Central

    Hedrich, Christian M.; Bream, Jay H.

    2010-01-01

    IL-10 plays an essential part in controlling inflammation and instructing adaptive immune responses. Consequently, dysregulation of IL-10 is linked with susceptibility to numerous infectious and autoimmune diseases in mouse models and in humans. It has become increasingly clear that appropriate temporal/spatial expression of IL-10 may be the key to how IL-10 contributes to the delicate balance between inflammation and immunoregulation. The mechanisms that govern the cell type- and receptor-specific induction of IL-10, however, remain unclear. This is due largely to the wide distribution of cellular sources that express IL-10 under diverse stimulation conditions and in a variety of tissue compartments. Further complicating the issue is the fact that human IL-10 expression patterns appear to be under genetic influence resulting in differential expression and disease susceptibility. In this review, we discuss the cellular sources of IL-10, their link to disease phenotypes and the molecular mechanisms implicated in IL-10 regulation. PMID:20087682

  17. Homeostasis or channelopathy? Acquired cell type-specific ion channel changes in temporal lobe epilepsy and their antiepileptic potential

    PubMed Central

    Wolfart, Jakob; Laker, Debora

    2015-01-01

    Neurons continuously adapt the expression and functionality of their ion channels. For example, exposed to chronic excitotoxicity, neurons homeostatically downscale their intrinsic excitability. In contrast, the “acquired channelopathy” hypothesis suggests that proepileptic channel characteristics develop during epilepsy. We review cell type-specific channel alterations under different epileptic conditions and discuss the potential of channels that undergo homeostatic adaptations, as targets for antiepileptic drugs (AEDs). Most of the relevant studies have been performed on temporal lobe epilepsy (TLE), a widespread AED-refractory, focal epilepsy. The TLE patients, who undergo epilepsy surgery, frequently display hippocampal sclerosis (HS), which is associated with degeneration of cornu ammonis subfield 1 pyramidal cells (CA1 PCs). Although the resected human tissue offers insights, controlled data largely stem from animal models simulating different aspects of TLE and other epilepsies. Most of the cell type-specific information is available for CA1 PCs and dentate gyrus granule cells (DG GCs). Between these two cell types, a dichotomy can be observed: while DG GCs acquire properties decreasing the intrinsic excitability (in TLE models and patients with HS), CA1 PCs develop channel characteristics increasing intrinsic excitability (in TLE models without HS only). However, thorough examination of data on these and other cell types reveals the coexistence of protective and permissive intrinsic plasticity within neurons. These mechanisms appear differentially regulated, depending on the cell type and seizure condition. Interestingly, the same channel molecules that are upregulated in DG GCs during HS-related TLE, appear as promising targets for future AEDs and gene therapies. Hence, GCs provide an example of homeostatic ion channel adaptation which can serve as a primer when designing novel anti-epileptic strategies. PMID:26124723

  18. Regulation of cancer metastasis by cell-free miRNAs

    PubMed Central

    Alečković, Maša; Kang, Yibin

    2014-01-01

    MicroRNAs (miRNAs) are integral molecules in the regulation of numerous physiological cellular processes that have emerged as critical players in cancer initiation and metastatic progression, both by promoting and suppressing metastasis. Recently, cell-free miRNAs shed from cancer cells into circulation have been reported in cancer patients, raising hope for development of novel biomarkers that can be routinely measured in easily accessible samples. In fact, establishing miRNA expression in the circulation likely has advantages over determination in primary tumor tissue, further augmenting the potential applications of miRNA detection in oncological practice. In addition, secretion of miRNAs impacting distant cell signaling or promoting the formation of a niche that sustains a distant tumor microenvironment allows for new treatment approaches to thwart cancer progression. PMID:25450578

  19. The epigenetic architecture at gene promoters determines cell type-specific LPS tolerance.

    PubMed

    Klein, Kerstin; Frank-Bertoncelj, Mojca; Karouzakis, Emmanuel; Gay, Renate E; Kolling, Christoph; Ciurea, Adrian; Bostanci, Nagihan; Belibasakis, Georgios N; Lin, Lih-Ling; Distler, Oliver; Gay, Steffen; Ospelt, Caroline

    2017-09-01

    Synovial fibroblasts (SF) drive inflammation and joint destruction in chronic arthritis. Here we show that SF possess a distinct type of LPS tolerance compared to macrophages and other types of fibroblasts. In SF and dermal fibroblasts, genes that were non-tolerizable after repeated LPS stimulation included pro-inflammatory cytokines, chemokines and matrix metalloproteinases, whereas anti-viral genes were tolerizable. In macrophages, all measured genes were tolerizable, whereas in gingival and foreskin fibroblasts these genes were non-tolerizable. Repeated stimulation of SF with LPS resulted in loss of activating histone marks only in promoters of tolerizable genes. The epigenetic landscape at promoters of tolerizable genes was similar in unstimulated SF and monocytes, whereas the basal configuration of histone marks profoundly differed in genes that were non-tolerizable in SF only. Our data suggest that the epigenetic configuration at gene promoters regulates cell-specific LPS-induced responses and primes SF to sustain their inflammatory response in chronic arthritis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. A chemical proteomic atlas of brain serine hydrolases identifies cell type-specific pathways regulating neuroinflammation

    PubMed Central

    Viader, Andreu; Ogasawara, Daisuke; Joslyn, Christopher M; Sanchez-Alavez, Manuel; Mori, Simone; Nguyen, William; Conti, Bruno; Cravatt, Benjamin F

    2016-01-01

    Metabolic specialization among major brain cell types is central to nervous system function and determined in large part by the cellular distribution of enzymes. Serine hydrolases are a diverse enzyme class that plays fundamental roles in CNS metabolism and signaling. Here, we perform an activity-based proteomic analysis of primary mouse neurons, astrocytes, and microglia to furnish a global portrait of the cellular anatomy of serine hydrolases in the brain. We uncover compelling evidence for the cellular compartmentalization of key chemical transmission pathways, including the functional segregation of endocannabinoid (eCB) biosynthetic enzymes diacylglycerol lipase-alpha (DAGLα) and –beta (DAGLβ) to neurons and microglia, respectively. Disruption of DAGLβ perturbed eCB-eicosanoid crosstalk specifically in microglia and suppressed neuroinflammatory events in vivo independently of broader effects on eCB content. Mapping the cellular distribution of metabolic enzymes thus identifies pathways for regulating specialized inflammatory responses in the brain while avoiding global alterations in CNS function. DOI: http://dx.doi.org/10.7554/eLife.12345.001 PMID:26779719

  1. Cell type-specific thalamic innervation in a column of rat vibrissal cortex.

    PubMed

    Meyer, Hanno S; Wimmer, Verena C; Hemberger, Mike; Bruno, Randy M; de Kock, Christiaan P J; Frick, Andreas; Sakmann, Bert; Helmstaedter, Moritz

    2010-10-01

    This is the concluding article in a series of 3 studies that investigate the anatomical determinants of thalamocortical (TC) input to excitatory neurons in a cortical column of rat primary somatosensory cortex (S1). We used viral synaptophysin-enhanced green fluorescent protein expression in thalamic neurons and reconstructions of biocytin-labeled cortical neurons in TC slices to quantify the number and distribution of boutons from the ventral posterior medial (VPM) and posteromedial (POm) nuclei potentially innervating dendritic arbors of excitatory neurons located in layers (L)2-6 of a cortical column in rat somatosensory cortex. We found that 1) all types of excitatory neurons potentially receive substantial TC input (90-580 boutons per neuron); 2) pyramidal neurons in L3-L6 receive dual TC input from both VPM and POm that is potentially of equal magnitude for thick-tufted L5 pyramidal neurons (ca. 300 boutons each from VPM and POm); 3) L3, L4, and L5 pyramidal neurons have multiple (2-4) subcellular TC innervation domains that match the dendritic compartments of pyramidal cells; and 4) a subtype of thick-tufted L5 pyramidal neurons has an additional VPM innervation domain in L4. The multiple subcellular TC innervation domains of L5 pyramidal neurons may partly explain their specific action potential patterns observed in vivo. We conclude that the substantial potential TC innervation of all excitatory neuron types in a cortical column constitutes an anatomical basis for the initial near-simultaneous representation of a sensory stimulus in different neuron types.

  2. Cell Type-Specific Sexual Dimorphism in Rat Pituitary Gene Expression During Maturation1

    PubMed Central

    Bjelobaba, Ivana; Janjic, Marija M.; Kucka, Marek; Stojilkovic, Stanko S.

    2015-01-01

    The most obvious functional differences between mammalian males and females are related to the control of reproductive physiology and include patterns of GnRH and gonadotropin release, the timing of puberty, sexual and social behavior, and the regulation of food intake and body weight. Using the rat as the best-studied mammalian model for maturation, we examined the expression of major anterior pituitary genes in five secretory cell types of developing males and females. Corticotrophs show comparable Pomc profiles in both sexes, with the highest expression occurring during the infantile period. Somatotrophs and lactotrophs also exhibit no difference in Gh1 and Prl profiles during embryonic to juvenile age but show the amplification of Prl expression in females and Gh1 expression in males during peripubertal and postpubertal ages. Gonadotrophs exhibit highly synchronized Lhb, Fshb, Cga, and Gnrhr expression in both sexes, but the peak of expression occurs during the infantile period in females and at the end of the juvenile period in males. Thyrotrophs also show different developmental Tshb profiles, which are synchronized with the expression of gonadotroph genes in males but not in females. These results indicate the lack of influence of sex on Pomc expression and the presence of two patterns of sexual dimorphism in the expression of other pituitary genes: a time shift in the peak expression during postnatal development, most likely reflecting the perinatal sex-specific brain differentiation, and modulation of the amplitude of expression during late development, which is secondary to the establishment of the hypothalamic-pituitary-gonadal and -thyroid axes. PMID:26063874

  3. MultiSite Gateway-Compatible Cell Type-Specific Gene-Inducible System for Plants1[OPEN

    PubMed Central

    Siligato, Riccardo; Wang, Xin; Yadav, Shri Ram; Lehesranta, Satu; Ma, Guojie; Ursache, Robertas; Sevilem, Iris; Zhang, Jing; Gorte, Maartje; Prasad, Kalika; Heidstra, Renze

    2016-01-01

    A powerful method to study gene function is expression or overexpression in an inducible, cell type-specific system followed by observation of consequent phenotypic changes and visualization of linked reporters in the target tissue. Multiple inducible gene overexpression systems have been developed for plants, but very few of these combine plant selection markers, control of expression domains, access to multiple promoters and protein fusion reporters, chemical induction, and high-throughput cloning capabilities. Here, we introduce a MultiSite Gateway-compatible inducible system for Arabidopsis (Arabidopsis thaliana) plants that provides the capability to generate such constructs in a single cloning step. The system is based on the tightly controlled, estrogen-inducible XVE system. We demonstrate that the transformants generated with this system exhibit the expected cell type-specific expression, similar to what is observed with constitutively expressed native promoters. With this new system, cloning of inducible constructs is no longer limited to a few special cases but can be used as a standard approach when gene function is studied. In addition, we present a set of entry clones consisting of histochemical and fluorescent reporter variants designed for gene and promoter expression studies. PMID:26644504

  4. MOBE-ChIP: a large-scale chromatin immunoprecipitation assay for cell type-specific studies.

    PubMed

    Lau, On Sun; Bergmann, Dominique C

    2015-10-01

    Cell type-specific transcriptional regulators play critical roles in the generation and maintenance of multicellularity. As they are often expressed at low levels, in vivo DNA-binding studies of these regulators by standard chromatin immunoprecipitation (ChIP) assays are technically challenging. We describe here an optimized ChIP protocol termed Maximized Objects for Better Enrichment (MOBE)-ChIP, which enhances the sensitivity of ChIP assays for detecting cell type-specific signals. The protocol, which is based on the disproportional increase of target signals over background at higher scales, uses substantially greater volume of starting materials than conventional ChIPs to achieve high signal enrichment. This technique can capture weak binding events that are ambiguous in standard ChIP assays, and is useful both in gene-specific and whole-genome analysis. This protocol has been optimized for Arabidopsis, but should be applicable to other model systems with minor modifications. The full procedure can be completed within 3 days. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  5. miRNAs in B-cell lymphoma: Molecular mechanisms and biomarker potential.

    PubMed

    Solé, Carla; Larrea, Erika; Di Pinto, Giovanni; Tellaetxe, Maitena; Lawrie, Charles Henderson

    2017-10-01

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate many human genes including those involved in normal B-cell development. When these miRNAs are aberrantly expressed in B-cells they play key pathogenetic roles in the development and maintenance of B-cell lymphomas and by association may serve as useful biomarkers. In this review, we provide an overview of the importance of miRNAs to B-cell lymphomagenesis, as well as considering their use as biomarkers, and their potential usefulness for the clinic. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Cell type-specific functions of period genes revealed by novel adipocyte and hepatocyte circadian clock models.

    PubMed

    Ramanathan, Chidambaram; Xu, Haiyan; Khan, Sanjoy K; Shen, Yang; Gitis, Paula J; Welsh, David K; Hogenesch, John B; Liu, Andrew C

    2014-04-01

    In animals, circadian rhythms in physiology and behavior result from coherent rhythmic interactions between clocks in the brain and those throughout the body. Despite the many tissue specific clocks, most understanding of the molecular core clock mechanism comes from studies of the suprachiasmatic nuclei (SCN) of the hypothalamus and a few other cell types. Here we report establishment and genetic characterization of three cell-autonomous mouse clock models: 3T3 fibroblasts, 3T3-L1 adipocytes, and MMH-D3 hepatocytes. Each model is genetically tractable and has an integrated luciferase reporter that allows for longitudinal luminescence recording of rhythmic clock gene expression using an inexpensive off-the-shelf microplate reader. To test these cellular models, we generated a library of short hairpin RNAs (shRNAs) against a panel of known clock genes and evaluated their impact on circadian rhythms. Knockdown of Bmal1, Clock, Cry1, and Cry2 each resulted in similar phenotypes in all three models, consistent with previous studies. However, we observed cell type-specific knockdown phenotypes for the Period and Rev-Erb families of clock genes. In particular, Per1 and Per2, which have strong behavioral effects in knockout mice, appear to play different roles in regulating period length and amplitude in these peripheral systems. Per3, which has relatively modest behavioral effects in knockout mice, substantially affects period length in the three cellular models and in dissociated SCN neurons. In summary, this study establishes new cell-autonomous clock models that are of particular relevance to metabolism and suitable for screening for clock modifiers, and reveals previously under-appreciated cell type-specific functions of clock genes.

  7. ON Bipolar Cells in Macaque Retina: Type-Specific Synaptic Connectivity with Special Reference to OFF Counterparts

    PubMed Central

    Tsukamoto, Yoshihiko; Omi, Naoko

    2016-01-01

    To date, 12 macaque bipolar cell types have been described. This list includes all morphology types first outlined by Polyak (1941) using the Golgi method in the primate retina and subsequently identified by other researchers using electron microscopy (EM) combined with the Golgi method, serial section transmission EM (SSTEM), and immunohistochemical imaging. We used SSTEM for the rod-dense perifoveal area of macaque retina, reconfirmed ON (cone) bipolar cells to be classified as invaginating midget bipolar (IMB), diffuse bipolar (DB)4, DB5, DB6, giant bipolar (GB), and blue bipolar (BB) types, and clarified their type-specific connectivity. DB4 cells made reciprocal synapses with a kind of ON-OFF lateral amacrine cell, similar to OFF DB2 cells. GB cells contacted rods and cones, similar to OFF DB3b cells. Retinal circuits formed by GB and DB3b cells are thought to substantiate the psychophysical finding of fast rod signals in mesopic vision. DB6 cell output synapses were directed to ON midget ganglion (MG) cells at 70% of ribbon contacts, similar to OFF DB1 cells that directed 60% of ribbon contacts to OFF MG cells. IMB cells contacted medium- or long-wavelength sensitive (M/L-) cones but not short-wavelength sensitive (S-) cones, while BB cells contacted S-cones but not M/L-cones. However, IMB and BB dendrites had similar morphological architectures, and a BB cell contacting a single S-cone resembled an IMB cell. Thus, both IMB and BB may be the ON bipolar counterparts of the OFF flat midget bipolar (FMB) type, likewise DB4 of DB2, DB5 of DB3a, DB6 of DB1, and GB of DB3b OFF bipolar type. The ON DB plus GB, and OFF DB cells predominantly contacted M/L-cones and their outputs were directed mainly to parasol ganglion (PG) cells but also moderately to MG cells. BB cells directed S-cone-driven outputs almost exclusively to small bistratified ganglion (SBG) cells. Some FMB cells predominantly contacted S-cones and their outputs were directed to OFF MG cells. Thus, two

  8. New miRNAs network in human mesenchymal stem cells derived from skin and amniotic fluid.

    PubMed

    Lazzarini, R; Sorgentoni, G; Caffarini, M; Sayeed, M A; Olivieri, F; Di Primio, R; Orciani, M

    2016-09-01

    Mesenchymal stem cells (MSCs), isolated from different adult sources, have great appeal for therapeutic applications due to their simple isolation, extensive expansion potential, and high differentiative potential.In our previous studies we isolated MSCs form amniotic fluid (AF-MSCs) and skin (S-MSCs) and characterized them according to their phenotype, pluripotency, and mRNA/microRNAs (miRNAs) profiling using Card A from Life Technologies.Here, we enlarge the profiling of AF-MCSs and S-MSCs to the more recently discovered miRNAs (Card B by Life Technologies) to identify the miRNAs putative target genes and the relative signaling pathways. Card B, in fact, contains miRNAs whose role and target are not yet elucidated.The expression of the analyzed miRNAs is changing between S-MSCs and AF-MSCs, indicating that these two types of MSCs show differences potentially related to their source. Interestingly, the pathways targeted by the miRNAS deriving from Card B are the same found during the analysis of miRNAs from Card A.This result confirms the key role played by WNT and TGF-β pathways in stem cell fate, underlining as other miRNAs partially ignored up to now deserve to be reconsidered. In addition, this analysis allows including Adherens junction pathways among the mechanisms finely regulated in stem cell behavior. © The Author(s) 2015.

  9. Transcriptional analysis of Volvox photoreceptors suggests the existence of different cell-type specific light-signaling pathways.

    PubMed

    Kianianmomeni, Arash; Hallmann, Armin

    2015-02-01

    Photosynthetic organisms, e.g., plants including green algae, use a sophisticated light-sensing system, composed of primary photoreceptors and additional downstream signaling components, to monitor changes in the ambient light environment towards adjust their growth and development. Although a variety of cellular processes, e.g., initiation of cleavage division and final cellular differentiation, have been shown to be light-regulated in the green alga Volvox carteri, little is known about the underlying light perception and signaling pathways. This multicellular alga possesses at least 12 photoreceptors, i.e., one phototropin (VcPhot), four cryptochromes (VcCRYa, VcCRYp, VcCRYd1, and VcCRYd2), and seven members of rhodopsin-like photoreceptors (VR1, VChR1, VChR2, VcHKR1, VcHKR2, VcHKR3, and VcHKR4), which display distinct light-dependent chemical processes based on their protein architectures and associated chromophores. Gene expression analyses could show that the transcript levels of some of the photoreceptor genes (e.g., VChR1 and VcHKR1) accumulate during division cleavages, while others (e.g., VcCRYa, VcCRYp, and VcPhot) accumulate during final cellular differentiation. However, the pattern of transcript accumulation changes when the alga switches to the sexual development. Eight photoreceptor genes, e.g., VcPhot, VcCRYp, and VcHKR1, are highly expressed in the somatic cells, while only the animal-type rhodopsin VR1 was found to be highly expressed in the reproductive cells/embryos during both asexual and sexual life cycles. Moreover, accumulation of VChR1 and VcCRYa transcripts is more sensitive to light and changes in response to more than one light quality. Obviously, different regulatory mechanisms underlying gene expression control transcript accumulation of photoreceptors not only during development, but also in a cell-type specific way and in response to various external signals such as light quality. The transcriptional patterns described in this study

  10. B-Cell and Monocyte Contribution to Systemic Lupus Erythematosus Identified by Cell-Type-Specific Differential Expression Analysis in RNA-Seq Data

    PubMed Central

    Dozmorov, Mikhail G.; Dominguez, Nicolas; Bean, Krista; Macwana, Susan R.; Roberts, Virginia; Glass, Edmund; James, Judith A.; Guthridge, Joel M.

    2015-01-01

    Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by complex interplay among immune cell types. SLE activity is experimentally assessed by several blood tests, including gene expression profiling of heterogeneous populations of cells in peripheral blood. To better understand the contribution of different cell types in SLE pathogenesis, we applied the two methods in cell-type-specific differential expression analysis, csSAM and DSection, to identify cell-type-specific gene expression differences in heterogeneous gene expression measures obtained using RNA-seq technology. We identified B-cell-, monocyte-, and neutrophil-specific gene expression differences. Immunoglobulin-coding gene expression was altered in B-cells, while a ribosomal signature was prominent in monocytes. On the contrary, genes differentially expressed in the heterogeneous mixture of cells did not show any functional enrichment. Our results identify antigen binding and structural constituents of ribosomes as functions altered by B-cell- and monocyte-specific gene expression differences, respectively. Finally, these results position both csSAM and DSection methods as viable techniques for cell-type-specific differential expression analysis, which may help uncover pathogenic, cell-type-specific processes in SLE. PMID:26512198

  11. Cell type-specific regulation of von Willebrand factor expression by the E4BP4 transcriptional repressor.

    PubMed

    Hough, Christine; Cuthbert, Carla D; Notley, Colleen; Brown, Christine; Hegadorn, Carol; Berber, Ergul; Lillicrap, David

    2005-02-15

    Mechanisms of tissue-restricted patterns of von Willebrand factor (VWF) expression involve activators and repressors that limit expression to endothelial cells and megakaryocytes. The relative transcriptional activity of the proximal VWF promoter was assessed in VWF-producing and -nonproducing cells, and promoter activity was highest in endothelial cells followed by megakaryocytes. Only basal VWF promoter activity was seen in nonendothelial cells. Here we identify a negative response element located at nucleotides (nts) +96/+105 and demonstrate, using chromatin immunoprecipitation (ChIP) analysis, that in vivo this sequence interacts with the E4BP4 transcriptional repressor. Differences in size and relative abundance of nuclear E4BP4 were observed. In HepG2 cells, low levels of larger forms of E4BP4 are present that directly interact with the negative response element. In VWF-expressing cells, high levels of smaller forms predominate with no evidence of direct DNA binding. However, in endothelial cells, mutation of the VWF E4BP4 binding motif not only restores but also further elevates VWF promoter activity, suggesting that E4BP4 may be part of a coordinated binding complex. These observations implicate this binding motif in repressing both activated and basal levels of VWF transcription by different cell type-specific mechanisms, and support the hypothesis that E4BP4 sequesters negative regulators of transcription, thereby enhancing activated gene expression.

  12. Optogenetic determination of the myocardial requirements for extrasystoles by cell type-specific targeting of ChannelRhodopsin-2

    PubMed Central

    Zaglia, Tania; Pianca, Nicola; Borile, Giulia; Da Broi, Francesca; Richter, Claudia; Campione, Marina; Lehnart, Stephan E.; Luther, Stefan; Corrado, Domenico; Miquerol, Lucile; Mongillo, Marco

    2015-01-01

    Extrasystoles lead to several consequences, ranging from uneventful palpitations to lethal ventricular arrhythmias, in the presence of pathologies, such as myocardial ischemia. The role of working versus conducting cardiomyocytes, as well as the tissue requirements (minimal cell number) for the generation of extrasystoles, and the properties leading ectopies to become arrhythmia triggers (topology), in the normal and diseased heart, have not been determined directly in vivo. Here, we used optogenetics in transgenic mice expressing ChannelRhodopsin-2 selectively in either cardiomyocytes or the conduction system to achieve cell type-specific, noninvasive control of heart activity with high spatial and temporal resolution. By combining measurement of optogenetic tissue activation in vivo and epicardial voltage mapping in Langendorff-perfused hearts, we demonstrated that focal ectopies require, in the normal mouse heart, the simultaneous depolarization of at least 1,300–1,800 working cardiomyocytes or 90–160 Purkinje fibers. The optogenetic assay identified specific areas in the heart that were highly susceptible to forming extrasystolic foci, and such properties were correlated to the local organization of the Purkinje fiber network, which was imaged in three dimensions using optical projection tomography. Interestingly, during the acute phase of myocardial ischemia, focal ectopies arising from this location, and including both Purkinje fibers and the surrounding working cardiomyocytes, have the highest propensity to trigger sustained arrhythmias. In conclusion, we used cell-specific optogenetics to determine with high spatial resolution and cell type specificity the requirements for the generation of extrasystoles and the factors causing ectopies to be arrhythmia triggers during myocardial ischemia. PMID:26204914

  13. Age-associated and cell-type-specific neurofibrillary pathology in transgenic mice expressing the human midsized neurofilament subunit.

    PubMed

    Vickers, J C; Morrison, J H; Friedrich, V L; Elder, G A; Perl, D P; Katz, R N; Lazzarini, R A

    1994-09-01

    Alterations in neurofilaments are a common occurrence in neurons of the human nervous system during aging and diseases associated with aging. Such pathologic changes may be attributed to species-specific properties of human neurofilaments as well as cell-type-specific regulation of this element of the cytoskeleton. The development of transgenic animals containing human neurofilament subunits offers an opportunity to study the effects of aging and other experimental conditions on the human-specific form of these proteins in a rodent model. The present study shows that mice from the transgenic line NF(M)27, which express the human midsized neurofilament subunit at low levels (2-25% of the endogenous NF-M), develop neurofilamentous accumulations in specific subgroups of neurons that are age dependent, affecting 78% of transgenic mice over 12 months of age. Similar accumulations do not occur in age-matched, wild-type littermates or in 3-month-old transgenic mice. In 12-month-old transgenic mice, somatic neurofilament accumulations resembling neurofibrillary tangles were present predominantly in layers III and V of the neocortex, as well as in select subpopulations of subcortical neurons. Intraperikaryal, spherical neurofilamentous accumulations were particularly abundant in cell bodies in layer II of the neocortex, and neurofilament-containing distentions of Purkinje cell proximal axons occurred in the cerebellum. These pathological accumulations contained mouse as well as human NF subunits, but could be distinguished by their content of phosphorylation-dependent NF epitopes. These cytoskeletal alterations closely resemble the cell-type-specific alterations in neurofilaments that occur during normal human aging and in diseases associated with aging, indicating that these transgenic animals may serve as models of some aspects of the pathologic features of human neurodegenerative diseases.

  14. Cell type-specific localization of Ephs pairing with ephrin-B2 in the rat postnatal pituitary gland.

    PubMed

    Yoshida, Saishu; Kato, Takako; Kanno, Naoko; Nishimura, Naoto; Nishihara, Hiroto; Horiguchi, Kotaro; Kato, Yukio

    2017-06-28

    Sox2-expressing stem/progenitor cells in the anterior lobe of the pituitary gland form two types of micro-environments (niches): the marginal cell layer and dense cell clusters in the parenchyma. In relation to the mechanism of regulation of niches, juxtacrine signaling via ephrin and its receptor Eph is known to play important roles in various niches. The ephrin and Eph families are divided into two subclasses to create ephrin/Eph signaling in co-operation with confined partners. Recently, we reported that ephrin-B2 localizes specifically to both pituitary niches. However, the Ephs interacting with ephrin-B2 in these pituitary niches have not yet been identified. Therefore, the present study aims to identify the Ephs interacting with ephrin-B2 and the cells that produce them in the rat pituitary gland. In situ hybridization and immunohistochemistry demonstrated cell type-specific localization of candidate interacting partners for ephrin-B2, including EphA4 in cells located in the posterior lobe, EphB1 in gonadotropes, EphB2 in corticotropes, EphB3 in stem/progenitor cells and EphB4 in endothelial cells in the adult pituitary gland. In particular, double-immunohistochemistry showed cis-interactions between EphB3 and ephrin-B2 in the apical cell membranes of stem/progenitor cell niches throughout life and trans-interactions between EphB2 produced by corticotropes and ephrin-B2 located in the basolateral cell membranes of stem/progenitor cells in the early postnatal pituitary gland. These data indicate that ephrin-B2 plays a role in pituitary stem/progenitor cell niches by selective interaction with EphB3 in cis and EphB2 in trans.

  15. Limited expression of nuclear pore membrane glycoprotein 210 in cell lines and tissues suggests cell-type specific nuclear pores in metazoans.

    PubMed

    Olsson, Magnus; Schéele, Susanne; Ekblom, Peter

    2004-01-15

    The nuclear pore complex (NPC) is the only known gateway for nucleocytoplasmic traffic. The nuclear pore membrane glycoprotein 210 (POM210/gp210) is considered to be important for the assembly and structure of pore complexes in metazoan cells. However, here we demonstrate cell-type specific expression of the gp210 protein during mouse organogenesis. As shown previously for its mRNA, distinct expression of the gp210 was seen in developing epithelia and some other cell types, whereas it was undetectable in nuclei of several other embryonic tissue compartments. In sharp contrast, monoclonal antibody 414 recognizing four non-membrane nucleoporins, stained the nuclear envelope of all cell types. In four cultured mouse cell lines, gp210 mRNA and protein were below detection levels, in contrast to some other nucleoporins tested. Distinct expression of gp210 mRNA and protein was seen in cultured mouse embryonic stem (ES) cells. These findings support the view of cell-type specific NPCs in metazoans and that the gp210 gene is regulated by cell-type specific control elements not shared by other nucleoporins. Although it cannot be excluded that very low expression levels of gp210 are sufficient to allow attachment of NPCs, a more likely alternative is that it has cell-type specific functions.

  16. Distinct AGO1 and AGO2 associated miRNA profiles in human cells and blood plasma

    PubMed Central

    Turchinovich, Andrey; Burwinkel, Barbara

    2012-01-01

    Studies of miRNA association with Argonaute (AGO) proteins in mammalian cells have indicated lack of bias toward particular AGO. However, to our knowledge, the use of quantitative methods for studying miRNA association with different AGOs has not been reported so far. In this work we compared the total miRNA content in AGO1 and AGO2 immunoprecipitates obtained from MCF7 adenocarcinoma cells using TaqMan Low Density miRNA Arrays and successfully verified selected miRNAs with qPCR. For most of the miRNA species AGO1 and AGO2 profiles were well correlated, however, some miRNAs demonstrated consistent biases toward one of the Argonautes. Furthermore, miRNAs which were predominantly AGO2-associated derived mostly from sense strands of the corresponding pre-miRNAs while the majority of AGO1 biased miRNAs originated from antisense strands of the pre-miRNAs. Additionally, we show that circulating miRNA in human blood plasma can be immunoprecipitated with both AGO1 and AGO2 antibody. However, unlike in cell lysates, AGO1 and AGO2 associated miRNA profiles in plasma did not correlate, indicating that many cell types contribute to circulating miRNA (given that expression of AGO proteins is tissue specific). Furthermore, AGO-specific miRNA profiles in blood cells differed significantly from miRNAs profiles in plasma indicating that most circulating miRNAs are likely to derive from non-blood cells. Since circulating miRNAs hold great promise as biomarkers for numerous cancers and other diseases, we hypothesize that AGO-specific miRNA profiles might add an additional dimension to circulating miRNA-based diagnostics. PMID:22858679

  17. miRNA profiling of B-cell subsets: specific miRNA profile for germinal center B cells with variation between centroblasts and centrocytes.

    PubMed

    Tan, Lu Ping; Wang, Miao; Robertus, Jan-Lukas; Schakel, Rikst Nynke; Gibcus, Johan H; Diepstra, Arjan; Harms, Geert; Peh, Suat-Cheng; Reijmers, Rogier M; Pals, Steven T; Kroesen, Bart-Jan; Kluin, Philip M; Poppema, Sibrand; van den Berg, Anke

    2009-06-01

    MicroRNAs (miRNAs) are an important class of small RNAs that regulate gene expression at the post-transcriptional level. It has become evident that miRNAs are involved in hematopoiesis, and that deregulation of miRNAs may give rise to hematopoietic malignancies. The aim of our study was to establish miRNA profiles of naïve, germinal center (GC) and memory B cells, and validate their expression patterns in normal lymphoid tissues. Quantitative (q) RT-PCR profiling revealed that several miRNAs were elevated in GC B cells, including miR-17-5p, miR-106a and miR-181b. One of the most abundant miRNAs in all three B-cell subsets analyzed was miR-150, with a more than 10-fold lower level in GC B cell as compared with the other two subsets. miRNA in situ hybridization (ISH) in tonsil tissue sections confirmed the findings from the profiling work. Interestingly, gradual decrease of miR-17-5p, miR-106a and miR-181b staining intensity from the dark to the light zone was observed in GC. A strong cytoplasmic staining of miR-150 was observed in a minority of the centroblasts in the dark zone of the GC. Inverse staining pattern of miR-150 against c-Myb and Survivin was observed in tonsil tissue sections, suggesting possible targeting of these genes by miR-150. In line with this, the experimental induction of miR-150 lead to reduced c-Myb, Survivin and Foxp1 expression levels in the Burkitt's lymphoma cell line, DG75. In conclusion, miRNA profiles of naïve, GC and memory B cells were established and validated by miRNA ISH. Within the GC cells, a marked difference was observed between the light and the dark zone.

  18. Stress and Cocaine Trigger Divergent and Cell Type-Specific Regulation of Synaptic Transmission at Single Spines in Nucleus Accumbens.

    PubMed

    Khibnik, Lena A; Beaumont, Michael; Doyle, Marie; Heshmati, Mitra; Slesinger, Paul A; Nestler, Eric J; Russo, Scott J

    2016-06-01

    Repeated exposure to cocaine or social stress leads to lasting structural and functional synaptic alterations in medium spiny neurons (MSNs) of nucleus accumbens (NAc). Although cocaine-induced and stress-induced structural changes in dendritic spines have been well documented, few studies have investigated functional consequences of cocaine and stress at the level of single spines. We exposed mice to chronic cocaine or chronic social defeat stress and used two-photon laser scanning microscopy with glutamate photo-uncaging and whole-cell recording to examine synaptic strength at individual spines on two distinct types of NAc MSNs in acute slices after 24 hours of cocaine withdrawal and after chronic social defeat stress. In animals treated with cocaine, average synaptic strength was reduced specifically at large mushroom spines of MSNs expressing dopamine receptor type 1 (D1-MSNs). In contrast, cocaine promoted a rightward shift in the distribution of synaptic weights toward larger synaptic responses in MSNs expressing dopamine receptor type 2 (D2-MSNs). After chronic social defeat stress, resilient animals displayed an upregulation of synaptic strength at large mushroom spines of D1-MSNs and a concomitant downregulation in D2-MSNs. Although susceptible mice did not exhibit a significant overall change in synaptic strength on D1-MSNs or D2-MSNs, we observed a slight leftward shift in cumulative distribution of large synaptic responses in both cell types. This study provides the first functional cell type-specific and spine type-specific comparison of synaptic strength at a single spine level between cocaine-induced and stress-induced neuroadaptations and demonstrates that psychoactive drugs and stress trigger divergent changes in synaptic function in NAc. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  19. Gamma-Retrovirus Integration Marks Cell Type-Specific Cancer Genes: A Novel Profiling Tool in Cancer Genomics.

    PubMed

    Gilroy, Kathryn L; Terry, Anne; Naseer, Asif; de Ridder, Jeroen; Allahyar, Amin; Wang, Weiwei; Carpenter, Eric; Mason, Andrew; Wong, Gane K-S; Cameron, Ewan R; Kilbey, Anna; Neil, James C

    2016-01-01

    Retroviruses have been foundational in cancer research since early studies identified proto-oncogenes as targets for insertional mutagenesis. Integration of murine gamma-retroviruses into the host genome favours promoters and enhancers and entails interaction of viral integrase with host BET/bromodomain factors. We report that this integration pattern is conserved in feline leukaemia virus (FeLV), a gamma-retrovirus that infects many human cell types. Analysis of FeLV insertion sites in the MCF-7 mammary carcinoma cell line revealed strong bias towards active chromatin marks with no evidence of significant post-integration growth selection. The most prominent FeLV integration targets had little overlap with the most abundantly expressed transcripts, but were strongly enriched for annotated cancer genes. A meta-analysis based on several gamma-retrovirus integration profiling (GRIP) studies in human cells (CD34+, K562, HepG2) revealed a similar cancer gene bias but also remarkable cell-type specificity, with prominent exceptions including a universal integration hotspot at the long non-coding RNA MALAT1. Comparison of GRIP targets with databases of super-enhancers from the same cell lines showed that these have only limited overlap and that GRIP provides unique insights into the upstream drivers of cell growth. These observations elucidate the oncogenic potency of the gamma-retroviruses and support the wider application of GRIP to identify the genes and growth regulatory circuits that drive distinct cancer types.

  20. Gamma-Retrovirus Integration Marks Cell Type-Specific Cancer Genes: A Novel Profiling Tool in Cancer Genomics

    PubMed Central

    Gilroy, Kathryn L.; Terry, Anne; Naseer, Asif; de Ridder, Jeroen; Wang, Weiwei; Carpenter, Eric; Mason, Andrew; Wong, Gane K-S.; Kilbey, Anna; Neil, James C.

    2016-01-01

    Retroviruses have been foundational in cancer research since early studies identified proto-oncogenes as targets for insertional mutagenesis. Integration of murine gamma-retroviruses into the host genome favours promoters and enhancers and entails interaction of viral integrase with host BET/bromodomain factors. We report that this integration pattern is conserved in feline leukaemia virus (FeLV), a gamma-retrovirus that infects many human cell types. Analysis of FeLV insertion sites in the MCF-7 mammary carcinoma cell line revealed strong bias towards active chromatin marks with no evidence of significant post-integration growth selection. The most prominent FeLV integration targets had little overlap with the most abundantly expressed transcripts, but were strongly enriched for annotated cancer genes. A meta-analysis based on several gamma-retrovirus integration profiling (GRIP) studies in human cells (CD34+, K562, HepG2) revealed a similar cancer gene bias but also remarkable cell-type specificity, with prominent exceptions including a universal integration hotspot at the long non-coding RNA MALAT1. Comparison of GRIP targets with databases of super-enhancers from the same cell lines showed that these have only limited overlap and that GRIP provides unique insights into the upstream drivers of cell growth. These observations elucidate the oncogenic potency of the gamma-retroviruses and support the wider application of GRIP to identify the genes and growth regulatory circuits that drive distinct cancer types. PMID:27097319

  1. Getting miRNA Therapeutics into the Target Cells for Neurodegenerative Diseases: A Mini-Review

    PubMed Central

    Wen, Ming Ming

    2016-01-01

    miRNAs play important roles in modulating gene expression in varying cellular processes and disease pathogenesis, including neurodegenerative diseases. Several miRNAs are expressed in the brain, control brain development and are identified as important biomarkers in the pathogenesis of motor—and neuro-cognitive diseases such as Alzheimer’s (AD), Huntington’s and Parkinson’s diseases (PD) and amyotrophic lateral sclerosis. These remarkable miRNAs could be used as diagnostic markers and therapeutic targeting potential for many stressful and untreatable progressive neurodegenerative diseases. To modulate these miRNA activities, there are currently two strategies involved; first one is to therapeutically restore the suppressed miRNA level by miRNA mimics (agonist), and the other one is to inhibit miRNA function by using anti-miR (antagonist) to repress overactive miRNA function. However, RNAi-based therapeutics often faces in vivo instability because naked nucleic acids are subject to enzyme degradation before reaching the target sites. Therefore, an effective, safe and stable bio-responsive delivery system is necessary to protect the nucleic acids from serum degradation and assist their entrance to the cells. Since neuronal cells are non-regenerating, to design engineered miRNAs to be delivered to the central nervous system (CNS) for long term gene expression and knockdown is representing an enormous challenge for scientists. This article provides an insight summary on some of the innovative strategies employed to deliver miRNA into target cells. These viral and non-viral carrier systems hold promise in RNA therapy delivery for neurodegenerative diseases. PMID:27920668

  2. miRNAs Signature in Head and Neck Squamous Cell Carcinoma Metastasis: A Literature Review

    PubMed Central

    Irani, Soussan

    2016-01-01

    Statement of the Problem Head and neck cancers include epithelial tumors arising in the oral cavity, pharynx, larynx, paranasal sinuses, and nasal cavity. Metastasis is a hallmark of cancer. MicroRNAs (miRNAs) are endogenous small non-coding RNAs involved in cell proliferation, development, differentiation and metastasis. It is believed that miRNA alterations correlate with initiation and progression of cancer cell proliferation or inhibition of tumorigenesis. Moreover, miRNAs have different roles in development, progression, and metastasis of head and neck squamous cell carcinoma (HNSCC). Altered expression of miRNAs could be novel molecular biomarkers for the definite diagnosis of cancer, metastatic site, cancer stage, and its progression. Purpose The purpose of this review was to provide a comprehensive literature review of the role of miRNAs in head and neck cancer metastasis. Search strategy A relevant English literature search in PubMed, ScienceDirect, and Google Scholar was performed. The keywords ‘miRNA’, ‘head and neck’, and ‘cancer’ were searched in title and abstract of publications; limited from 1990 to 2015. The inclusion criterion was the role of miRNAs in cancer metastasis. The exclusion criterion was the other functions of miRNAs in cancers. Out of 15221 articles, the full texts of 442 articles were retrieved and only 133 articles met the inclusion criteria. Conclusion Despite the advances in cancer treatment, the mortality rate of HNSCC is still high. The potential application of miRNAs for cancer therapy has been demonstrated in many studies; miRNAs function as either tumor suppressor or oncogene. The recognition of metastamir and their targets may lead to better understanding of HNSCC oncogenesis, and consequently, development of new therapeutic strategies which is a necessity in cancer treatment. Development of therapeutic agents based on miRNAs is a promising target. PMID:27284551

  3. Neuropilin-1 is a receptor for extracellular miRNA and AGO2/miRNA complexes and mediates the internalization of miRNAs that modulate cell function.

    PubMed

    Prud'homme, Gerald J; Glinka, Yelena; Lichner, Zsuzsanna; Yousef, George M

    2016-10-18

    Extracellular miRNAs are increasingly studied as markers for specific diseases. They are released in biological fluids in a remarkably stable form, and may play a role in intercellular communication. They are thought to be protected against degradation by either encapsulation within microparticles, or by binding to proteins (mostly AGO2). The particulate forms may be internalized by endocytosis or membrane fusion, but the protein-bound forms require a receptor mechanism for their uptake. A major question is whether there are natural cell-membrane receptors that capture and internalize protein-bound functional miRNAs. We examined neuropilin-1 (NRP1), in view of its properties as a receptor for many ligands, including growth factors such as vascular endothelial growth factor (VEGF), and efficiency at mediating ligand internalization. It is expressed by endothelial cells, many other normal cell types, and cancer cells. Here, we report that NRP1 binds miRNAs with high affinity, and promotes their entry into the cell. Furthermore, the internalized miRNAs remain functional, as they specifically regulate proliferation and migration of cancer cells, as well as tube formation by human endothelial cells. Anti-NRP1 antibodies or NRP1 siRNA knockdown block miRNA effects, further confirming NRP1-mediated uptake. VEGF does not compete with miRNAs for binding to NRP1. In addition, NRP1 binds extracellular AGO2 (carrying miRNA or not), and internalizes AGO2/miRNA complexes. Because miRNA bound to AGO2 appears to the most abundant form in body fluids, this may have important physiological and pathological effects.

  4. Neuropilin-1 is a receptor for extracellular miRNA and AGO2/miRNA complexes and mediates the internalization of miRNAs that modulate cell function

    PubMed Central

    Prud'homme, Gerald J.; Glinka, Yelena; Lichner, Zsuzsanna; Yousef, George M.

    2016-01-01

    Extracellular miRNAs are increasingly studied as markers for specific diseases. They are released in biological fluids in a remarkably stable form, and may play a role in intercellular communication. They are thought to be protected against degradation by either encapsulation within microparticles, or by binding to proteins (mostly AGO2). The particulate forms may be internalized by endocytosis or membrane fusion, but the protein-bound forms require a receptor mechanism for their uptake. A major question is whether there are natural cell-membrane receptors that capture and internalize protein-bound functional miRNAs. We examined neuropilin-1 (NRP1), in view of its properties as a receptor for many ligands, including growth factors such as vascular endothelial growth factor (VEGF), and efficiency at mediating ligand internalization. It is expressed by endothelial cells, many other normal cell types, and cancer cells. Here, we report that NRP1 binds miRNAs with high affinity, and promotes their entry into the cell. Furthermore, the internalized miRNAs remain functional, as they specifically regulate proliferation and migration of cancer cells, as well as tube formation by human endothelial cells. Anti-NRP1 antibodies or NRP1 siRNA knockdown block miRNA effects, further confirming NRP1-mediated uptake. VEGF does not compete with miRNAs for binding to NRP1. In addition, NRP1 binds extracellular AGO2 (carrying miRNA or not), and internalizes AGO2/miRNA complexes. Because miRNA bound to AGO2 appears to the most abundant form in body fluids, this may have important physiological and pathological effects. PMID:27486976

  5. Cell-type-specific and hypoxia-inducible expression of the human erythropoietin gene in transgenic mice

    SciTech Connect

    Semenza, G.L.; Nejfelt, M.K.; Gearhart, J.D.; Antonarakis, S.E. ); Koury, S.T. )

    1991-10-01

    Synthesis of erythropoietin, the primary humoral regulator or erythropoiesis, in liver and kidney is inducible by anemia or hypoxia. Analysis of human erythropoietin gene expression in transgenic mice revealed that sequences located 6-14 kilobases 5{prime} to the gene direct expression to the kidney, whereas sequences within the immediate 3{prime}-flanking region control hepatocyte-specific expression. Human erythropoietin transcription initiation sites were differentially utilized in liver and kidney. Inducible transgene expression was precisely targeted to peritubular interstitial cells in the renal cortex that synthesize endogenous mouse erythropoietin. These studies demonstrate that multiple erythropoietin gene regulatory elements control cello-type-specific expression and inducibility by a fundamental physiologic stimulus, hypoxia.

  6. Dual-Specificity Anti-sigma Factor Reinforces Control of Cell-Type Specific Gene Expression in Bacillus subtilis

    PubMed Central

    Serrano, Mónica; Gao, JinXin; Bota, João; Bate, Ashley R.; Meisner, Jeffrey; Eichenberger, Patrick; Moran, Charles P.; Henriques, Adriano O.

    2015-01-01

    Gene expression during spore development in Bacillus subtilis is controlled by cell type-specific RNA polymerase sigma factors. σFand σE control early stages of development in the forespore and the mother cell, respectively. When, at an intermediate stage in development, the mother cell engulfs the forespore, σF is replaced by σG and σE is replaced by σK. The anti-sigma factor CsfB is produced under the control of σF and binds to and inhibits the auto-regulatory σG, but not σF. A position in region 2.1, occupied by an asparagine in σG and by a glutamate in οF, is sufficient for CsfB discrimination of the two sigmas, and allows it to delay the early to late switch in forespore gene expression. We now show that following engulfment completion, csfB is switched on in the mother cell under the control of σK and that CsfB binds to and inhibits σE but not σK, possibly to facilitate the switch from early to late gene expression. We show that a position in region 2.3 occupied by a conserved asparagine in σE and by a conserved glutamate in σK suffices for discrimination by CsfB. We also show that CsfB prevents activation of σG in the mother cell and the premature σG-dependent activation of σK. Thus, CsfB establishes negative feedback loops that curtail the activity of σE and prevent the ectopic activation of σG in the mother cell. The capacity of CsfB to directly block σE activity may also explain how CsfB plays a role as one of the several mechanisms that prevent σE activation in the forespore. Thus the capacity of CsfB to differentiate between the highly similar σF/σG and σE/σK pairs allows it to rinforce the cell-type specificity of these sigma factors and the transition from early to late development in B. subtilis, and possibly in all sporeformers that encode a CsfB orthologue. PMID:25835496

  7. Improving sensitivity of linear regression-based cell type-specific differential expression deconvolution with per-gene vs. global significance threshold.

    PubMed

    Glass, Edmund R; Dozmorov, Mikhail G

    2016-10-06

    The goal of many human disease-oriented studies is to detect molecular mechanisms different between healthy controls and patients. Yet, commonly used gene expression measurements from blood samples suffer from variability of cell composition. This variability hinders the detection of differentially expressed genes and is often ignored. Combined with cell counts, heterogeneous gene expression may provide deeper insights into the gene expression differences on the cell type-specific level. Published computational methods use linear regression to estimate cell type-specific differential expression, and a global cutoff to judge significance, such as False Discovery Rate (FDR). Yet, they do not consider many artifacts hidden in high-dimensional gene expression data that may negatively affect linear regression. In this paper we quantify the parameter space affecting the performance of linear regression (sensitivity of cell type-specific differential expression detection) on a per-gene basis. We evaluated the effect of sample sizes, cell type-specific proportion variability, and mean squared error on sensitivity of cell type-specific differential expression detection using linear regression. Each parameter affected variability of cell type-specific expression estimates and, subsequently, the sensitivity of differential expression detection. We provide the R package, LRCDE, which performs linear regression-based cell type-specific differential expression (deconvolution) detection on a gene-by-gene basis. Accounting for variability around cell type-specific gene expression estimates, it computes per-gene t-statistics of differential detection, p-values, t-statistic-based sensitivity, group-specific mean squared error, and several gene-specific diagnostic metrics. The sensitivity of linear regression-based cell type-specific differential expression detection differed for each gene as a function of mean squared error, per group sample sizes, and variability of the proportions

  8. Role of miRNAs in CD4 T cell plasticity during inflammation and tolerance

    PubMed Central

    Sethi, Apoorva; Kulkarni, Neeraja; Sonar, Sandip; Lal, Girdhari

    2013-01-01

    Gene expression is tightly regulated in a tuneable, cell-specific and time-dependent manner. Recent advancement in epigenetics and non-coding RNA (ncRNA) revolutionized the concept of gene regulation. In order to regulate the transcription, ncRNA can promptly response to the extracellular signals as compared to transcription factors present in the cells. microRNAs (miRNAs) are ncRNA (~22 bp) encoded in the genome, and present as intergenic or oriented antisense to neighboring genes. The strategic location of miRNA in coding genes helps in the coupled regulation of its expression with host genes. miRNA together with complex machinery called RNA-induced silencing complex (RISC) interacts with target mRNA and degrade the mRNA or inhibits the translation. CD4 T cells play an important role in the generation and maintenance of inflammation and tolerance. Cytokines and chemokines present in the inflamed microenvironment controls the differentiation and function of various subsets of CD4 T cells [Th1, Th2, Th17, and regulatory CD4 T cells (Tregs)]. Recent studies suggest that miRNAs play an important role in the development and function of all subsets of CD4 T cells. In current review, we focused on how various miRNAs are regulated by cell's extrinsic and intrinsic signaling, and how miRNAs affect the transdifferentiation of subsets of CD4 T cell and controls their plasticity during inflammation and tolerance. PMID:23386861

  9. Cell-Type-Specific Profiling of Gene Expression and Chromatin Binding without Cell Isolation: Assaying RNA Pol II Occupancy in Neural Stem Cells

    PubMed Central

    Southall, Tony D.; Gold, Katrina S.; Egger, Boris; Davidson, Catherine M.; Caygill, Elizabeth E.; Marshall, Owen J.; Brand, Andrea H.

    2013-01-01

    Summary Cell-type-specific transcriptional profiling often requires the isolation of specific cell types from complex tissues. We have developed “TaDa,” a technique that enables cell-specific profiling without cell isolation. TaDa permits genome-wide profiling of DNA- or chromatin-binding proteins without cell sorting, fixation, or affinity purification. The method is simple, sensitive, highly reproducible, and transferable to any model system. We show that TaDa can be used to identify transcribed genes in a cell-type-specific manner with considerable temporal precision, enabling the identification of differential gene expression between neuroblasts and the neuroepithelial cells from which they derive. We profile the genome-wide binding of RNA polymerase II in these adjacent, clonally related stem cells within intact Drosophila brains. Our data reveal expression of specific metabolic genes in neuroepithelial cells, but not in neuroblasts, and highlight gene regulatory networks that may pattern neural stem cell fates. PMID:23792147

  10. Cell-type-specific recruitment of amygdala interneurons to hippocampal theta rhythm and noxious stimuli in vivo.

    PubMed

    Bienvenu, Thomas C M; Busti, Daniela; Magill, Peter J; Ferraguti, Francesco; Capogna, Marco

    2012-06-21

    Neuronal synchrony in the basolateral amygdala (BLA) is critical for emotional behavior. Coordinated theta-frequency oscillations between the BLA and the hippocampus and precisely timed integration of salient sensory stimuli in the BLA are involved in fear conditioning. We characterized GABAergic interneuron types of the BLA and determined their contribution to shaping these network activities. Using in vivo recordings in rats combined with the anatomical identification of neurons, we found that the firing of BLA interneurons associated with network activities was cell type specific. The firing of calbindin-positive interneurons targeting dendrites was precisely theta-modulated, but other cell types were heterogeneously modulated, including parvalbumin-positive basket cells. Salient sensory stimuli selectively triggered axo-axonic cells firing and inhibited firing of a disctinct projecting interneuron type. Thus, GABA is released onto BLA principal neurons in a time-, domain-, and sensory-specific manner. These specific synaptic actions likely cooperate to promote amygdalo-hippocampal synchrony involved in emotional memory formation.

  11. Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling

    PubMed Central

    Steiner, Florian A.; Talbert, Paul B.; Kasinathan, Sivakanthan; Deal, Roger B.; Henikoff, Steven

    2012-01-01

    An understanding of developmental processes requires knowledge of transcriptional and epigenetic landscapes at the level of tissues and ultimately individual cells. However, obtaining tissue- or cell-type-specific expression and chromatin profiles for animals has been challenging. Here we describe a method for purifying nuclei from specific cell types of animal models that allows simultaneous determination of both expression and chromatin profiles. The method is based on in vivo biotin-labeling of the nuclear envelope and subsequent affinity purification of nuclei. We describe the use of the method to isolate nuclei from muscle of adult Caenorhabditis elegans and from mesoderm of Drosophila melanogaster embryos. As a case study, we determined expression and nucleosome occupancy profiles for affinity-purified nuclei from C. elegans muscle. We identified hundreds of genes that are specifically expressed in muscle tissues and found that these genes are depleted of nucleosomes at promoters and gene bodies in muscle relative to other tissues. This method should be universally applicable to all model systems that allow transgenesis and will make it possible to determine epigenetic and expression profiles of different tissues and cell types. PMID:22219512

  12. Epigenetic Regulation of miRNAs and Breast Cancer Stem Cells

    PubMed Central

    Duru, Nadire; Gernapudi, Ramkishore; Eades, Gabriel; Eckert, Richard; Zhou, Qun

    2015-01-01

    MicroRNAs have emerged as important targets of chemopreventive strategies in breast cancer. We have found that miRNAs are dysregulated at an early stage in breast cancer, in non-malignant Ductal Carcinoma In Situ. Many dietary chemoprevention agents can act by epigenetically activating miRNA-signaling pathways involved in tumor cell proliferation and invasive progression. In addition, many miRNAs activated via chemopreventive strategies target cancer stem cell signaling and prevent tumor progression or relapse. Specifically, we have found that miRNAs regulate DCIS stem cells, which may play important roles in breast cancer progression to invasive disease. We have shown that chemopreventive agents can directly inhibit DCIS stem cells and block tumor formation in vivo, via activation of tumor suppressor miRNAs. PMID:26052481

  13. Differential miRNA expressions in peripheral blood mononuclear cells for diagnosis of lung cancer.

    PubMed

    Ma, Jie; Lin, Yanli; Zhan, Min; Mann, Dean L; Stass, Sanford A; Jiang, Feng

    2015-10-01

    Tremendous efforts have been made to develop cancer biomarkers by detecting circulating extracellular miRNAs directly released from tumors. Yet, none of the cell-free biomarkers has been accepted to be used for early detection of non-small cell lung cancer (NSCLC). Peripheral blood mononucleated cells (PBMCs) act as the first line of defense against malignancy in immune system, their dysfunction may occur as an early event in cancer immunogenicity or immune evasion. We proposed to investigate whether analysis of miRNA expressions of PBMCs has diagnostic value for NSCLC. We first used a microarray to analyze PBMCs of 16 stage I NSCLC patients and 16 cancer-free smokers, and identified seven PBMC miRNAs with a significantly altered expression level in NSCLC patients. In a training set of 84 NSCLC patients and 69 cancer-free smokers, a panel of two miRNAs (miRs-19b-3p and -29b-3p) were developed from the seven PBMC miRNAs, producing 72.62% sensitivity and 82.61% specificity in identifying NSCLC. Furthermore, the miRNAs could identify squamous cell lung carcinoma (SCC), a major type of NSCLC, with 80.00% sensitivity and 89.86% specificity. The expression levels of the miRNAs were independent of disease stage. In a testing set of 56 NSCLC patients and 46 controls, the performance of the biomarkers was reproducibly confirmed. The study presents the first in-depth analysis of PBMC miRNA profile of NSCLC patients. The assessment of PBMC miRNAs may provide a new diagnostic approach for the early detection of NSCLC.

  14. miRNA expression profile during osteogenic differentiation of human adipose-derived stem cells.

    PubMed

    Zhang, Zi-ji; Zhang, Hao; Kang, Yan; Sheng, Pu-yi; Ma, Yuan-chen; Yang, Zi-bo; Zhang, Zhi-qi; Fu, Ming; He, Ai-shan; Liao, Wei-ming

    2012-03-01

    Human adipose-derived stem cells (hADSC) are capable of differentiating into an osteogenic lineage. It is believed that microRNAs (miRNAs) play important roles in regulating this osteogenic differentiation of human adipose-derived cells, although its molecular mechanism remains unclear. We investigated the miRNA expression profile during osteogenic differentiation of hADSCs, and assessed the roles of involved miRNAs during the osteogenic differentiation. We obtained and cultured human adipose-derived stems cells from donors who underwent elective liposuction or other abdominal surgery at our institution. miRNA expression profiles pre- and post-osteogenic induction were obtained using microarray essay, and differently expressed miRNAs were verified using quantitative real-time polymerase chain reaction (qRT-PCR). The expression of osteogenic proteins was detected using an enzyme-linked immunosorbent assay. Putative targets of the miRNAs were predicted using online software MiRanda, TargetScan, and miRBase. Eight miRNAs were found differently expressed pre- and post-osteogenic induction, among which four miRNAs (miR-17, miR-20a, miR-20b, and miR-106a) were up-regulated and four miRNAs (miR-31, miR-125a-5p, miR-125b, and miR-193a) were down-regulated. qRT-PCR analysis further confirmed the results. Predicted target genes of the differentially expressed miRNAs based on the overlap from three public prediction algorithms: MiRanda, TargetScan, and miRBase Target have the known functions of regulating stem cell osteogenic differentiation, self-renewal, signal transduction, and cell cycle control. We identified a group of miRNAs that may play important roles in regulating hADSC cell differentiation toward an osteoblast lineage. Further study of these miRNAs may elucidate the mechanism of hADSC differentiation into adipose tissue, and thus provide basis for tissue engineering. © 2011 Wiley Periodicals, Inc.

  15. Immuno-Navigator, a batch-corrected coexpression database, reveals cell type-specific gene networks in the immune system

    PubMed Central

    Vandenbon, Alexis; Dinh, Viet H.; Mikami, Norihisa; Kitagawa, Yohko; Teraguchi, Shunsuke; Ohkura, Naganari; Sakaguchi, Shimon

    2016-01-01

    High-throughput gene expression data are one of the primary resources for exploring complex intracellular dynamics in modern biology. The integration of large amounts of public data may allow us to examine general dynamical relationships between regulators and target genes. However, obstacles for such analyses are study-specific biases or batch effects in the original data. Here we present Immuno-Navigator, a batch-corrected gene expression and coexpression database for 24 cell types of the mouse immune system. We systematically removed batch effects from the underlying gene expression data and showed that this removal considerably improved the consistency between inferred correlations and prior knowledge. The data revealed widespread cell type-specific correlation of expression. Integrated analysis tools allow users to use this correlation of expression for the generation of hypotheses about biological networks and candidate regulators in specific cell types. We show several applications of Immuno-Navigator as examples. In one application we successfully predicted known regulators of importance in naturally occurring Treg cells from their expression correlation with a set of Treg-specific genes. For one high-scoring gene, integrin β8 (Itgb8), we confirmed an association between Itgb8 expression in forkhead box P3 (Foxp3)-positive T cells and Treg-specific epigenetic remodeling. Our results also suggest that the regulation of Treg-specific genes within Treg cells is relatively independent of Foxp3 expression, supporting recent results pointing to a Foxp3-independent component in the development of Treg cells. PMID:27078110

  16. OFF bipolar cells in macaque retina: type-specific connectivity in the outer and inner synaptic layers

    PubMed Central

    Tsukamoto, Yoshihiko; Omi, Naoko

    2015-01-01

    OFF bipolar cells in the macaque retina were recently classified into five types: flat midget bipolar (FMB) and diffuse bipolar (DB) 1, 2, 3a, and 3b. We examined all parallel pathways from cone photoreceptors via OFF bipolar cells to parasol and midget ganglion cells by serial section transmission electron microscopy. Basal contacts of OFF bipolar cells to cone pedicles were previously categorized as triad-associated (TA) and non-TA (NTA). The latter was further divided into two groups located in the middle and marginal areas of the pedicle at the present eccentricity of 15°. We then mapped the distributions of all three basal contacts of the five OFF bipolar cell types in the same area of cone pedicles. TA contacts were more numerous than NTA contacts in FMB (93%), DB1 (67%), and DB3a (81%) cells, but less in DB2 (30%) and DB3b (21%) cells. Cluster analysis of these contact parameters reconfirmed five distinct OFF bipolar cell types and showed these positional configurations of basal synapses to be cell type-specific. This architecture is thought to provide a spatial framework for the interstitial diffusion and local uptake of the neurotransmitter (glutamate) that spills over from ribbon synapses. All five OFF bipolar cell types formed ribbon-synaptic contacts to both parasol and midget ganglion cells. DB2 and 3a, DB1 and 3b, and FMB predominantly, moderately, and negligibly contacted parasol ganglion cells, respectively. FMB almost exclusively contacted midget ganglion cells, to which DB1 provided dominant output (58%), and DB2, 3a, and 3b provided between 3% and 10% of their output. Consequently, the cone signal sampling routes of a midget ganglion cell consisted of two substructures: the narrow (mainly 2-3 cones) FMB pathway and the wide (mainly 10 cones) DB pathway, where connection strength was four-fold greater in the FMB than DB pathway. The narrow and strong FMB pathway may confer the highest spatial resolution and sporadically may include blue cone

  17. Mapping cell type-specific transcriptional enhancers using high affinity, lineage-specific Ep300 bioChIP-seq

    PubMed Central

    Zhou, Pingzhu; Gu, Fei; Zhang, Lina; Akerberg, Brynn N; Ma, Qing; Li, Kai; He, Aibin; Lin, Zhiqiang; Stevens, Sean M; Zhou, Bin; Pu, William T

    2017-01-01

    Understanding the mechanisms that regulate cell type-specific transcriptional programs requires developing a lexicon of their genomic regulatory elements. We developed a lineage-selective method to map transcriptional enhancers, regulatory genomic regions that activate transcription, in mice. Since most tissue-specific enhancers are bound by the transcriptional co-activator Ep300, we used Cre-directed, lineage-specific Ep300 biotinylation and pulldown on immobilized streptavidin followed by next generation sequencing of co-precipitated DNA to identify lineage-specific enhancers. By driving this system with lineage-specific Cre transgenes, we mapped enhancers active in embryonic endothelial cells/blood or skeletal muscle. Analysis of these enhancers identified new transcription factor heterodimer motifs that likely regulate transcription in these lineages. Furthermore, we identified candidate enhancers that regulate adult heart- or lung- specific endothelial cell specialization. Our strategy for tissue-specific protein biotinylation opens new avenues for studying lineage-specific protein-DNA and protein-protein interactions. DOI: http://dx.doi.org/10.7554/eLife.22039.001 PMID:28121289

  18. Balancing intestinal and systemic inflammation through cell type-specific expression of the aryl hydrocarbon receptor repressor

    PubMed Central

    Brandstätter, Olga; Schanz, Oliver; Vorac, Julia; König, Jessica; Mori, Tetsushi; Maruyama, Toru; Korkowski, Markus; Haarmann-Stemmann, Thomas; von Smolinski, Dorthe; Schultze, Joachim L.; Abel, Josef; Esser, Charlotte; Takeyama, Haruko; Weighardt, Heike; Förster, Irmgard

    2016-01-01

    As a sensor of polyaromatic chemicals the aryl hydrocarbon receptor (AhR) exerts an important role in immune regulation besides its requirement for xenobiotic metabolism. Transcriptional activation of AhR target genes is counterregulated by the AhR repressor (AhRR) but the exact function of the AhRR in vivo is currently unknown. We here show that the AhRR is predominantly expressed in immune cells of the skin and intestine, different from other AhR target genes. Whereas AhRR antagonizes the anti-inflammatory function of the AhR in the context of systemic endotoxin shock, AhR and AhRR act in concert to dampen intestinal inflammation. Specifically, AhRR contributes to the maintenance of colonic intraepithelial lymphocytes and prevents excessive IL-1β production and Th17/Tc17 differentiation. In contrast, the AhRR enhances IFN-γ-production by effector T cells in the inflamed gut. Our findings highlight the physiologic importance of cell-type specific balancing of AhR/AhRR expression in response to microbial, nutritional and other environmental stimuli. PMID:27184933

  19. Peroxisome proliferator-activated receptor subtype- and cell-type-specific activation of genomic target genes upon adenoviral transgene delivery.

    PubMed

    Nielsen, Ronni; Grøntved, Lars; Stunnenberg, Hendrik G; Mandrup, Susanne

    2006-08-01

    Investigations of the molecular events involved in activation of genomic target genes by peroxisome proliferator-activated receptors (PPARs) have been hampered by the inability to establish a clean on/off state of the receptor in living cells. Here we show that the combination of adenoviral delivery and chromatin immunoprecipitation (ChIP) is ideal for dissecting these mechanisms. Adenoviral delivery of PPARs leads to a rapid and synchronous expression of the PPAR subtypes, establishment of transcriptional active complexes at genomic loci, and immediate activation of even silent target genes. We demonstrate that PPARgamma2 possesses considerable ligand-dependent as well as independent transactivation potential and that agonists increase the occupancy of PPARgamma2/retinoid X receptor at PPAR response elements. Intriguingly, by direct comparison of the PPARs (alpha, gamma, and beta/delta), we show that the subtypes have very different abilities to gain access to target sites and that in general the genomic occupancy correlates with the ability to activate the corresponding target gene. In addition, the specificity and potency of activation by PPAR subtypes are highly dependent on the cell type. Thus, PPAR subtype-specific activation of genomic target genes involves an intricate interplay between the properties of the subtype- and cell-type-specific settings at the individual target loci.

  20. Peroxisome Proliferator-Activated Receptor Subtype- and Cell-Type-Specific Activation of Genomic Target Genes upon Adenoviral Transgene Delivery

    PubMed Central

    Nielsen, Ronni; Grøntved, Lars; Stunnenberg, Hendrik G.; Mandrup, Susanne

    2006-01-01

    Investigations of the molecular events involved in activation of genomic target genes by peroxisome proliferator-activated receptors (PPARs) have been hampered by the inability to establish a clean on/off state of the receptor in living cells. Here we show that the combination of adenoviral delivery and chromatin immunoprecipitation (ChIP) is ideal for dissecting these mechanisms. Adenoviral delivery of PPARs leads to a rapid and synchronous expression of the PPAR subtypes, establishment of transcriptional active complexes at genomic loci, and immediate activation of even silent target genes. We demonstrate that PPARγ2 possesses considerable ligand-dependent as well as independent transactivation potential and that agonists increase the occupancy of PPARγ2/retinoid X receptor at PPAR response elements. Intriguingly, by direct comparison of the PPARs (α, γ, and β/δ), we show that the subtypes have very different abilities to gain access to target sites and that in general the genomic occupancy correlates with the ability to activate the corresponding target gene. In addition, the specificity and potency of activation by PPAR subtypes are highly dependent on the cell type. Thus, PPAR subtype-specific activation of genomic target genes involves an intricate interplay between the properties of the subtype- and cell-type-specific settings at the individual target loci. PMID:16847324

  1. Shoot Na+ exclusion and increased salinity tolerance engineered by cell type-specific alteration of Na+ transport in Arabidopsis.

    PubMed

    Møller, Inge S; Gilliham, Matthew; Jha, Deepa; Mayo, Gwenda M; Roy, Stuart J; Coates, Juliet C; Haseloff, Jim; Tester, Mark

    2009-07-01

    Soil salinity affects large areas of cultivated land, causing significant reductions in crop yield globally. The Na+ toxicity of many crop plants is correlated with overaccumulation of Na+ in the shoot. We have previously suggested that the engineering of Na+ exclusion from the shoot could be achieved through an alteration of plasma membrane Na+ transport processes in the root, if these alterations were cell type specific. Here, it is shown that expression of the Na+ transporter HKT1;1 in the mature root stele of Arabidopsis thaliana decreases Na+ accumulation in the shoot by 37 to 64%. The expression of HKT1;1 specifically in the mature root stele is achieved using an enhancer trap expression system for specific and strong overexpression. The effect in the shoot is caused by the increased influx, mediated by HKT1;1, of Na+ into stelar root cells, which is demonstrated in planta and leads to a reduction of root-to-shoot transfer of Na+. Plants with reduced shoot Na+ also have increased salinity tolerance. By contrast, plants constitutively expressing HKT1;1 driven by the cauliflower mosaic virus 35S promoter accumulated high shoot Na+ and grew poorly. Our results demonstrate that the modification of a specific Na+ transport process in specific cell types can reduce shoot Na+ accumulation, an important component of salinity tolerance of many higher plants.

  2. Developmentally Programmed 3′ CpG Island Methylation Confers Tissue- and Cell-Type-Specific Transcriptional Activation

    PubMed Central

    Yu, Da-Hai; Ware, Carol; Waterland, Robert A.; Zhang, Jiexin; Chen, Miao-Hsueh; Gadkari, Manasi; Kunde-Ramamoorthy, Govindarajan; Nosavanh, Lagina M.

    2013-01-01

    During development, a small but significant number of CpG islands (CGIs) become methylated. The timing of developmentally programmed CGI methylation and associated mechanisms of transcriptional regulation during cellular differentiation, however, remain poorly characterized. Here, we used genome-wide DNA methylation microarrays to identify epigenetic changes during human embryonic stem cell (hESC) differentiation. We discovered a group of CGIs associated with developmental genes that gain methylation after hESCs differentiate. Conversely, erasure of methylation was observed at the identified CGIs during subsequent reprogramming to induced pluripotent stem cells (iPSCs), further supporting a functional role for the CGI methylation. Both global gene expression profiling and quantitative reverse transcription-PCR (RT-PCR) validation indicated opposing effects of CGI methylation in transcriptional regulation during differentiation, with promoter CGI methylation repressing and 3′ CGI methylation activating transcription. By studying diverse human tissues and mouse models, we further confirmed that developmentally programmed 3′ CGI methylation confers tissue- and cell-type-specific gene activation in vivo. Importantly, luciferase reporter assays provided evidence that 3′ CGI methylation regulates transcriptional activation via a CTCF-dependent enhancer-blocking mechanism. These findings expand the classic view of mammalian CGI methylation as a mechanism for transcriptional silencing and indicate a functional role for 3′ CGI methylation in developmental gene regulation. PMID:23459939

  3. Implementing the LIM code: the structural basis for cell type-specific assembly of LIM-homeodomain complexes

    SciTech Connect

    Bhati, Mugdha; Lee, Christopher; Nancarrow, Amy L.; Lee, Mihwa; Craig, Vanessa J.; Bach, Ingolf; Guss, J. Mitchell; Mackay, Joel P.; Matthews, Jacqueline M.

    2008-09-03

    LIM-homeodomain (LIM-HD) transcription factors form a combinatorial 'LIM code' that contributes to the specification of cell types. In the ventral spinal cord, the binary LIM homeobox protein 3 (Lhx3)/LIM domain-binding protein 1 (Ldb1) complex specifies the formation of V2 interneurons. The additional expression of islet-1 (Isl1) in adjacent cells instead specifies the formation of motor neurons through assembly of a ternary complex in which Isl1 contacts both Lhx3 and Ldb1, displacing Lhx3 as the binding partner of Ldb1. However, little is known about how this molecular switch occurs. Here, we have identified the 30-residue Lhx3-binding domain on Isl1 (Isl1{sub LBD}). Although the LIM interaction domain of Ldb1 (Ldb1{sub LID}) and Isl1{sub LBD} share low levels of sequence homology, X-ray and NMR structures reveal that they bind Lhx3 in an identical manner, that is, Isl1{sub LBD} mimics Ldb1{sub LID}. These data provide a structural basis for the formation of cell type-specific protein-protein interactions in which unstructured linear motifs with diverse sequences compete to bind protein partners. The resulting alternate protein complexes can target different genes to regulate key biological events.

  4. Involvement of miRNAs and Cell-Secreted Vesicles in Mammalian Ovarian Antral Follicle Development.

    PubMed

    da Silveira, Juliano C; de Andrade, Gabriella M; Nogueira, Marcelo F G; Meirelles, Flávio V; Perecin, Felipe

    2015-12-01

    Ovarian follicular development is a controlled series of events culminating with an ovulatory or atretic follicle. MicroRNAs (miRNAs) are small noncoding RNAs involved in translational regulation of genes in different developmental processes. Deletion of Dicer in mice ovaries demonstrated the importance of miRNAs in reproduction, which led to infertility. The miRNAs were thought to act only within host cells; however, these molecules are also present in cell-secreted vesicles. These vesicles are present in body fluids such as milk, serum, and ovarian follicular fluid. Vesicles are secreted in extracellular fluids and travel from donor to target cells, mediating transfer of bioactive material. Herein we discuss the role of hormonal-regulated miRNAs within different ovarian follicular cells as well as cell-secreted vesicles participation in mammalian ovarian follicular fluid. Furthermore, we discuss the possibility of miRNAs transference mediated by cell-secreted vesicles present in ovarian follicular fluid, increasing the versatility of miRNA functions during antral follicle development. © The Author(s) 2015.

  5. MiRNA182 regulates percentage of myeloid and erythroid cells in chronic myeloid leukemia.

    PubMed

    Arya, Deepak; Sachithanandan, Sasikala P; Ross, Cecil; Palakodeti, Dasaradhi; Li, Shang; Krishna, Sudhir

    2017-01-12

    The deregulation of lineage control programs is often associated with the progression of haematological malignancies. The molecular regulators of lineage choices in the context of tyrosine kinase inhibitor (TKI) resistance remain poorly understood in chronic myeloid leukemia (CML). To find a potential molecular regulator contributing to lineage distribution and TKI resistance, we undertook an RNA-sequencing approach for identifying microRNAs (miRNAs). Following an unbiased screen, elevated miRNA182-5p levels were detected in Bcr-Abl-inhibited K562 cells (CML blast crisis cell line) and in a panel of CML patients. Earlier, miRNA182-5p upregulation was reported in several solid tumours and haematological malignancies. We undertook a strategy involving transient modulation and CRISPR/Cas9 (clustered regularly interspersed short palindromic repeats)-mediated knockout of the MIR182 locus in CML cells. The lineage contribution was assessed by methylcellulose colony formation assay. The transient modulation of miRNA182-5p revealed a biased phenotype. Strikingly, Δ182 cells (homozygous deletion of MIR182 locus) produced a marked shift in lineage distribution. The phenotype was rescued by ectopic expression of miRNA182-5p in Δ182 cells. A bioinformatic analysis and Hes1 modulation data suggested that Hes1 could be a putative target of miRNA182-5p. A reciprocal relationship between miRNA182-5p and Hes1 was seen in the context of TK inhibition. In conclusion, we reveal a key role for miRNA182-5p in restricting the myeloid development of leukemic cells. We propose that the Δ182 cell line will be valuable in designing experiments for next-generation pharmacological interventions.

  6. MicroRNA-494 plays a role in fiber type-specific skeletal myogenesis in human induced pluripotent stem cells.

    PubMed

    Iwasaki, Hirotaka; Imamura, Takeshi; Morino, Katsutaro; Shimosato, Takashi; Tawa, Masashi; Ugi, Satoshi; Sakurai, Hidetoshi; Maegawa, Hiroshi; Okamura, Tomio

    Mitochondrial oxidative capacity in skeletal muscle is known to decrease in diabetic patients, and sarcopenia is a risk factor for diabetes, particularly in elderly people. We previously revealed that microRNA (miR)-494 inhibits mitochondrial biogenesis during myogenic differentiation in murine C2C12 cells and others reported that exercise regulates miR-494 levels in obese sedentary individuals with increased risk of type 2 diabetes. In this study, to investigate the therapeutic potential of miR-494, we first investigated the role of miR-494 during human skeletal myogenesis. Using human induced pluripotent stem (hiPS) cells stably transfected with the Tet/ON-myogenic differentiation 1(MYOD1) gene (MyoD-hiPS cells), we found that miR-494 expression transiently increased and was downregulated after myogenic induction. In miR-494 transfected MyoD-hiPS cells, the level of high oxidative fiber (type IIa) marker proteins specifically decreased, while no change in the total number of cells was observed. In contrast, the expression of both type I and type IIx markers was unaffected by miR-494 overexpression. Furthermore, miR-494 overexpression suppressed basal oxygen consumption rate concomitant with the inhibition of myotube formation and without significant effects on the mitochondrial content. These results suggest that miR-494 plays a novel role in the fiber type-specific skeletal myogenesis in MyoD-hiPS cells, distinct from murine C2C12 myogenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. In Vivo Senescence in the Sbds-Deficient Murine Pancreas: Cell-Type Specific Consequences of Translation Insufficiency.

    PubMed

    Tourlakis, Marina E; Zhang, Siyi; Ball, Heather L; Gandhi, Rikesh; Liu, Hongrui; Zhong, Jian; Yuan, Julie S; Guidos, Cynthia J; Durie, Peter R; Rommens, Johanna M

    2015-06-01

    Genetic models of ribosome dysfunction show selective organ failure, highlighting a gap in our understanding of cell-type specific responses to translation insufficiency. Translation defects underlie a growing list of inherited and acquired cancer-predisposition syndromes referred to as ribosomopathies. We sought to identify molecular mechanisms underlying organ failure in a recessive ribosomopathy, with particular emphasis on the pancreas, an organ with a high and reiterative requirement for protein synthesis. Biallelic loss of function mutations in SBDS are associated with the ribosomopathy Shwachman-Diamond syndrome, which is typified by pancreatic dysfunction, bone marrow failure, skeletal abnormalities and neurological phenotypes. Targeted disruption of Sbds in the murine pancreas resulted in p53 stabilization early in the postnatal period, specifically in acinar cells. Decreased Myc expression was observed and atrophy of the adult SDS pancreas could be explained by the senescence of acinar cells, characterized by induction of Tgfβ, p15(Ink4b) and components of the senescence-associated secretory program. This is the first report of senescence, a tumour suppression mechanism, in association with SDS or in response to a ribosomopathy. Genetic ablation of p53 largely resolved digestive enzyme synthesis and acinar compartment hypoplasia, but resulted in decreased cell size, a hallmark of decreased translation capacity. Moreover, p53 ablation resulted in expression of acinar dedifferentiation markers and extensive apoptosis. Our findings indicate a protective role for p53 and senescence in response to Sbds ablation in the pancreas. In contrast to the pancreas, the Tgfβ molecular signature was not detected in fetal bone marrow, liver or brain of mouse models with constitutive Sbds ablation. Nevertheless, as observed with the adult pancreas phenotype, disease phenotypes of embryonic tissues, including marked neuronal cell death due to apoptosis, were determined to

  8. Cell Type-Specific Replication of Simian Virus 40 Conferred by Hormone Response Elements in the Late Promoter

    PubMed Central

    Farrell, Michael L.; Mertz, Janet E.

    2002-01-01

    The late genes of SV40 are not expressed at significant levels until after the onset of viral DNA replication. We previously identified two hormone response elements (HREs) in the late promoter that contribute to this delay. Mutants defective in these HREs overexpress late RNA at early, but not late, times after transfection of CV-1PD cells. Overexpression of nuclear receptors (NRs) that recognize these HREs leads to repression of the late promoter in a sequence-specific and titratable manner, resulting in a delay in late gene expression. These observations led to a model in which the late promoter is repressed at early times after infection by NRs, with this repression being relieved by titration of these repressors through simian virus 40 (SV40) genome replication to high copy number. Here, we tested this model in the context of the viral life cycle. SV40 genomes containing mutations in either or both HREs that significantly reduce NR binding without altering the coding of any proteins were constructed. Competition for replication between mutant and wild-type viruses in low-multiplicity coinfections indicated that the +1 HRE offered a significant selective advantage to the virus within a few cycles of infection in African green monkey kidney cell lines CV-1, CV-1P, TC-7, MA-134, and Vero but not in CV-1PD′ cells. Interestingly, the +55 HRE offered a selective disadvantage in MA-134 cells but had no effect in CV-1, CV-1P, TC-7, Vero, and CV-1PD′ cells. Thus, we conclude that these HREs are biologically important to the virus, but in a cell type-specific manner. PMID:12050389

  9. In Vivo Senescence in the Sbds-Deficient Murine Pancreas: Cell-Type Specific Consequences of Translation Insufficiency

    PubMed Central

    Tourlakis, Marina E.; Zhang, Siyi; Ball, Heather L.; Gandhi, Rikesh; Liu, Hongrui; Zhong, Jian; Yuan, Julie S.; Guidos, Cynthia J.; Durie, Peter R.; Rommens, Johanna M.

    2015-01-01

    Genetic models of ribosome dysfunction show selective organ failure, highlighting a gap in our understanding of cell-type specific responses to translation insufficiency. Translation defects underlie a growing list of inherited and acquired cancer-predisposition syndromes referred to as ribosomopathies. We sought to identify molecular mechanisms underlying organ failure in a recessive ribosomopathy, with particular emphasis on the pancreas, an organ with a high and reiterative requirement for protein synthesis. Biallelic loss of function mutations in SBDS are associated with the ribosomopathy Shwachman-Diamond syndrome, which is typified by pancreatic dysfunction, bone marrow failure, skeletal abnormalities and neurological phenotypes. Targeted disruption of Sbds in the murine pancreas resulted in p53 stabilization early in the postnatal period, specifically in acinar cells. Decreased Myc expression was observed and atrophy of the adult SDS pancreas could be explained by the senescence of acinar cells, characterized by induction of Tgfβ, p15Ink4b and components of the senescence-associated secretory program. This is the first report of senescence, a tumour suppression mechanism, in association with SDS or in response to a ribosomopathy. Genetic ablation of p53 largely resolved digestive enzyme synthesis and acinar compartment hypoplasia, but resulted in decreased cell size, a hallmark of decreased translation capacity. Moreover, p53 ablation resulted in expression of acinar dedifferentiation markers and extensive apoptosis. Our findings indicate a protective role for p53 and senescence in response to Sbds ablation in the pancreas. In contrast to the pancreas, the Tgfβ molecular signature was not detected in fetal bone marrow, liver or brain of mouse models with constitutive Sbds ablation. Nevertheless, as observed with the adult pancreas phenotype, disease phenotypes of embryonic tissues, including marked neuronal cell death due to apoptosis, were determined to

  10. Knockdown of the fat mass and obesity gene disrupts cellular energy balance in a cell-type specific manner.

    PubMed

    Pitman, Ryan T; Fong, Jason T; Billman, Penny; Puri, Neelu

    2012-01-01

    Recent studies suggest that FTO variants strongly correlate with obesity and mainly influence energy intake with little effect on the basal metabolic rate. We suggest that FTO influences eating behavior by modulating intracellular energy levels and downstream signaling mechanisms which control energy intake and metabolism. Since FTO plays a particularly important role in adipocytes and in hypothalamic neurons, SH-SY5Y neuronal cells and 3T3-L1 adipocytes were used to understand how siRNA mediated knockdown of FTO expression alters cellular energy homeostasis. Cellular energy status was evaluated by measuring ATP levels using a luminescence assay and uptake of fluorescent glucose. FTO siRNA in SH-SY5Y cells mediated mRNA knockdown (-82%), increased ATP concentrations by up to 46% (P = 0.013) compared to controls, and decreased phosphorylation of AMPk and Akt in SH-SY5Y by -52% and -46% respectively as seen by immunoblotting. In contrast, FTO siRNA in 3T3-L1 cells decreased ATP concentration by -93% (p<0.0005), and increased AMPk and Akt phosphorylation by 204% and 70%, respectively suggesting that FTO mediates control of energy levels in a cell-type specific manner. Furthermore, glucose uptake was decreased in both SH-SY5Y (-51% p = 0.015) and 3T3-L1 cells (-30%, p = 0.0002). We also show that FTO knockdown decreases NPY mRNA expression in SH-SY5Y cells (-21%) through upregulation of pSTAT3 (118%). These results provide important evidence that FTO-variant linked obesity may be associated with altered metabolic functions through activation of downstream metabolic mediators including AMPk.

  11. Identification of miRNAs Involved in Reprogramming Acinar Cells into Insulin Producing Cells

    PubMed Central

    Teichenne, Joan; Morró, Meritxell; Casellas, Alba; Jimenez, Veronica; Tellez, Noelia; Leger, Adrien; Bosch, Fatima; Ayuso, Eduard

    2015-01-01

    Reprogramming acinar cells into insulin producing cells using adenoviral (Ad)-mediated delivery of Pdx1, Ngn3 and MafA (PNM) is an innovative approach for the treatment of diabetes. Here, we aimed to investigate the molecular mechanisms involved in this process and in particular, the role of microRNAs. To this end, we performed a comparative study of acinar-to-β cell reprogramming efficiency in the rat acinar cell line AR42J and its subclone B13 after transduction with Ad-PNM. B13 cells were more efficiently reprogrammed than AR42J cells, which was demonstrated by a strong activation of β cell markers (Ins1, Ins2, IAPP, NeuroD1 and Pax4). miRNome panels were used to analyze differentially expressed miRNAs in acinar cells under four experimental conditions (i) non-transduced AR42J cells, (ii) non-transduced B13 cells, (iii) B13 cells transduced with Ad-GFP vectors and (iv) B13 cells transduced with Ad-PNM vectors. A total of 59 miRNAs were found to be differentially expressed between non-transduced AR42J and B13 cells. Specifically, the miR-200 family was completely repressed in B13 cells, suggesting that these cells exist in a less differentiated state than AR42J cells and as a consequence they present a greater plasticity. Adenoviral transduction per se induced dedifferentiation of acinar cells and 11 miRNAs were putatively involved in this process, whereas 8 miRNAs were found to be associated with PNM expression. Of note, Ad-PNM reprogrammed B13 cells presented the same levels of miR-137-3p, miR-135a-5p, miR-204-5p and miR-210-3p of those detected in islets, highlighting their role in the process. In conclusion, this study led to the identification of miRNAs that might be of compelling importance to improve acinar-to-β cell conversion for the future treatment of diabetes. PMID:26690959

  12. Identification of miRNAs Involved in Reprogramming Acinar Cells into Insulin Producing Cells.

    PubMed

    Teichenne, Joan; Morró, Meritxell; Casellas, Alba; Jimenez, Veronica; Tellez, Noelia; Leger, Adrien; Bosch, Fatima; Ayuso, Eduard

    2015-01-01

    Reprogramming acinar cells into insulin producing cells using adenoviral (Ad)-mediated delivery of Pdx1, Ngn3 and MafA (PNM) is an innovative approach for the treatment of diabetes. Here, we aimed to investigate the molecular mechanisms involved in this process and in particular, the role of microRNAs. To this end, we performed a comparative study of acinar-to-β cell reprogramming efficiency in the rat acinar cell line AR42J and its subclone B13 after transduction with Ad-PNM. B13 cells were more efficiently reprogrammed than AR42J cells, which was demonstrated by a strong activation of β cell markers (Ins1, Ins2, IAPP, NeuroD1 and Pax4). miRNome panels were used to analyze differentially expressed miRNAs in acinar cells under four experimental conditions (i) non-transduced AR42J cells, (ii) non-transduced B13 cells, (iii) B13 cells transduced with Ad-GFP vectors and (iv) B13 cells transduced with Ad-PNM vectors. A total of 59 miRNAs were found to be differentially expressed between non-transduced AR42J and B13 cells. Specifically, the miR-200 family was completely repressed in B13 cells, suggesting that these cells exist in a less differentiated state than AR42J cells and as a consequence they present a greater plasticity. Adenoviral transduction per se induced dedifferentiation of acinar cells and 11 miRNAs were putatively involved in this process, whereas 8 miRNAs were found to be associated with PNM expression. Of note, Ad-PNM reprogrammed B13 cells presented the same levels of miR-137-3p, miR-135a-5p, miR-204-5p and miR-210-3p of those detected in islets, highlighting their role in the process. In conclusion, this study led to the identification of miRNAs that might be of compelling importance to improve acinar-to-β cell conversion for the future treatment of diabetes.

  13. In silico screening of alleged miRNAs associated with cell competition: an emerging cellular event in cancer.

    PubMed

    Patel, Manish; Antala, Bhavesh; Shrivastava, Neeta

    2015-12-01

    Cell competition is identified as a crucial phenomenon for cancer and organ development. There is a possibility that microRNAs (miRNAs) may play an important role in the regulation of expression of genes involved in cell competition. In silico screening of miRNAs is an effort to abridge, economize and expedite the experimental approaches to identification of potential miRNAs involved in cell competition, as no study has reported involvement of miRNAs in cell competition to date. In this study, we used multiple screening steps as follows: (i) selection of cell competition related genes of Drosophila through a literature survey; (ii) homology study of selected cell competition related genes; (iii) identification of miRNAs that target conserved cell competition-related genes through prediction tools; (iv) sequence conservation analysis of identified miRNAs with human genome; (v) identification of conserved cell competition miRNAs using their expression profiles and exploration of roles of their homologous human miRNAs. This study led to the identification of nine potential cell competition miRNAs in the Drosophila genome. Importantly, eighteen human homologs of these nine potential Drosophila miRNAs are well reported for their involvement in different types of cancers. This confirms their probable involvement in cell competition as well, because cell competition is well justified for its involvement in cancer initiation and maintenance.

  14. Role of miRNAs in muscle stem cell biology: proliferation, differentiation and death.

    PubMed

    Crippa, Stefania; Cassano, Marco; Sampaolesi, Maurilio

    2012-01-01

    miRNAs are small non-coding RNAs that regulate post-transcriptionally gene expression by degradation or translational repression of specific target mRNAs. In the 90s, lin-4 and let-7 were firstly identified as small regulatory RNAs able to control C. elegans larval development, by specifically targeting the 3'UTR of lin-14 and lin-28, respectively. These findings have introduced a novel and wide layer of complexity in the regulation of mRNA and protein expression. Lin-4 and let-7 are now considered the founding members of an abundant class of small fine-tuned RNAs, called microRNAs (miRNAs), in viruses, green algae, plants, flies, worms, and in mammals. In humans, the estimated number of genes encoding for miRNAs is as high as 1000 and around 30% of the protein-coding genes are post-transcriptionally controlled by miRNAs. This article reviews the role of miRNAs in regulating several biological responses in muscle cells, ranging from proliferation, differentiation and adaptation to stress cues. Cardiac and skeletal muscles are powerful examples to summarize the activity of miRNAs in cell fate specification, lineage differentiation and metabolic pathways. Indeed, specific miRNAs control the number of proliferating muscle progenitors to guarantee the proper formation of the heart and muscle fibers and to assure the self-renewal of muscle progenitors during adult tissue regeneration. On the other side, several other miRNAs promote the differentiation of muscle progenitors into skeletal myofibers or into cardiomyocytes, where metabolic activity, survival and remodeling process in response to stress, injury and chronic diseases are also fine-tuned by miRNAs.

  15. Regulation of serum response factor by miRNA-200 and miRNA-9 modulates oligodendrocyte progenitor cell differentiation

    PubMed Central

    Buller, Benjamin; Chopp, Michael; Ueno, Yuji; Zhang, Li; Zhang, Rui Lan; Morris, Daniel; Zhang, Yi; Zhang, Zheng Gang

    2012-01-01

    Serum response factor (SRF) is a transcription factor that transactivates actin associated genes, and has been implicated in oligodendrocyte (OL) differentiation. To date, it has not been investigated in cerebral ischemia. We investigated the dynamics of SRF expression after stroke in vivo and the role of SRF in oligodendrocyte differentiation in vitro. Using immunohistochemistry, we found that SRF was upregulated in OLs and OL precursor cells (OPCs) after stroke. Moreover, upregulation of SRF was concurrent with downregulation of the microRNAs (miRNAs) miR-9 and the miR-200 family in the ischemic white matter region, the corpus callosum. Inhibition of SRF activation by CCG-1423, a specific inhibitor of SRF function, blocked OPCs from differentiating into OLs. Over-expression of miR-9 and miR-200 in cultured OPCs suppressed SRF expression and inhibited OPC differentiation. Moreover, co-expression of miR-9 and miR-200 attenuated activity of a luciferase reporter assay containing the Srf 3′ untranslated region (UTR). Collectively, this study is the first to show that stroke upregulates SRF expression in OPCs and OLs, and that SRF levels are mediated by miRNAs and regulate OPC differentiation. PMID:22907787

  16. Pluripotent and Multipotent Stem Cells Display Distinct Hypoxic miRNA Expression Profiles

    PubMed Central

    Agrawal, Rahul; Dale, Tina P.; Al-Zubaidi, Mohammed A.; Benny Malgulwar, Prit; Forsyth, Nicholas R.; Kulshreshtha, Ritu

    2016-01-01

    MicroRNAs are reported to have a crucial role in the regulation of self-renewal and differentiation of stem cells. Hypoxia has been identified as a key biophysical element of the stem cell culture milieu however, the link between hypoxia and miRNA expression in stem cells remains poorly understood. We therefore explored miRNA expression in hypoxic human embryonic and mesenchymal stem cells (hESCs and hMSCs). A total of 50 and 76 miRNAs were differentially regulated by hypoxia (2% O2) in hESCs and hMSCs, respectively, with a negligible overlap of only three miRNAs. We found coordinate regulation of precursor and mature miRNAs under hypoxia suggesting their regulation mainly at transcriptional level. Hypoxia response elements were located upstream of 97% of upregulated hypoxia regulated miRNAs (HRMs) suggesting hypoxia-inducible-factor (HIF) driven transcription. HIF binding to the candidate cis-elements of specific miRNAs under hypoxia was confirmed by Chromatin immunoprecipitation coupled with qPCR. Role analysis of a subset of upregulated HRMs identified linkage to reported inhibition of differentiation while a downregulated subset of HRMs had a putative role in the promotion of differentiation. MiRNA-target prediction correlation with published hypoxic hESC and hMSC gene expression profiles revealed HRM target genes enriched in the cytokine:cytokine receptor, HIF signalling and pathways in cancer. Overall, our study reveals, novel and distinct hypoxia-driven miRNA signatures in hESCs and hMSCs with the potential for application in optimised culture and differentiation models for both therapeutic application and improved understanding of stem cell biology. PMID:27783707

  17. Roles of miRNAs in microcystin-LR-induced Sertoli cell toxicity

    SciTech Connect

    Zhou, Yuan; Wang, Hui; Wang, Cong; Qiu, Xuefeng; Benson, Mikael; Yin, Xiaoqin; Xiang, Zou; Li, Dongmei; and others

    2015-08-15

    Microcystin (MC)-LR, a cyclic heptapeptide, is a potent reproductive system toxin. To understand the molecular mechanisms of MC-induced reproductive system cytotoxicity, we evaluated global changes of miRNA and mRNA expression in mouse Sertoli cells following MC-LR treatment. Our results revealed that the exposure to MC-LR resulted in an altered miRNA expression profile that might be responsible for the modulation of mRNA expression. Bio-functional analysis indicated that the altered genes were involved in specific cellular processes, including cell death and proliferation. Target gene analysis suggested that junction injury in Sertoli cells exposed to MC-LR might be mediated by miRNAs through the regulation of the Sertoli cell-Sertoli cell pathway. Collectively, these findings may enhance our understanding on the modes of action of MC-LR on mouse Sertoli cells as well as the molecular mechanisms underlying the toxicity of MC-LR on the male reproductive system. - Highlights: • miRNAs were altered in Sertoli cells exposed to MC-LR. • Alerted genes were involved in different cell functions including the cell morphology. • MC-LR adversely affected Sertoli cell junction formation through the regulating miRNAs.

  18. Measurement of precursor miRNA in exosomes from human ESC-derived mesenchymal stem cells.

    PubMed

    Chen, Tian Sheng; Lim, Sai Kiang

    2013-01-01

    Mesenchymal stem cells (MSCs) derived from human embryonic stem cells (ESCs) have been shown to secrete exosomes that are cardioprotective against myocardial ischemia reperfusion injury in a mouse model. To elucidate this cardioprotective mechanism, we have characterized the protein, nucleic acid, and lipid composition of MSC exosomes. Here we describe the isolation and analysis of RNA in MSC exosome. We have previously reported that RNAs in MSC exosome are primarily small RNA molecules of <300 nt and they include many miRNAs. Many of these miRNAs are in the precursor form suggesting that pre-miRNAs, and not mature miRNAs are preferentially loaded into exosomes. The protocols described here include assays to ascertain the presence of pre-miRNAs, profiling of miRNA and pre-miRNA, and quantitative estimation of mature and pre-miRNA.

  19. Multiplexed miRNA Fluorescence In Situ Hybridization for Formalin-Fixed Paraffin-Embedded Tissues

    PubMed Central

    Renwick, Neil; Cekan, Pavol; Bognanni, Claudia; Tuschl, Thomas

    2015-01-01

    Multiplexed miRNA fluorescence in situ hybridization (miRNA FISH) is an advanced method for visualizing differentially expressed miRNAs, together with other reference RNAs, in archival tissues. Some miRNAs are excellent disease biomarkers due to their abundance and cell-type specificity. However, these short RNA molecules are difficult to visualize due to loss by diffusion, probe mishybridization, and signal detection and signal amplification issues. Here, we describe a reliable and adjustable method for visualizing and normalizing miRNA signals in formalin-fixed paraffin-embedded (FFPE) tissue sections. PMID:25218385

  20. Multiplexed miRNA fluorescence in situ hybridization for formalin-fixed paraffin-embedded tissues.

    PubMed

    Renwick, Neil; Cekan, Pavol; Bognanni, Claudia; Tuschl, Thomas

    2014-01-01

    Multiplexed miRNA fluorescence in situ hybridization (miRNA FISH) is an advanced method for visualizing differentially expressed miRNAs, together with other reference RNAs, in archival tissues. Some miRNAs are excellent disease biomarkers due to their abundance and cell-type specificity. However, these short RNA molecules are difficult to visualize due to loss by diffusion, probe mishybridization, and signal detection and signal amplification issues. Here, we describe a reliable and adjustable method for visualizing and normalizing miRNA signals in formalin-fixed paraffin-embedded (FFPE) tissue sections.

  1. Improved Salinity Tolerance of Rice Through Cell Type-Specific Expression of AtHKT1;1

    PubMed Central

    Plett, Darren; Safwat, Gehan; Gilliham, Matthew; Skrumsager Møller, Inge; Roy, Stuart; Shirley, Neil; Jacobs, Andrew; Johnson, Alexander; Tester, Mark

    2010-01-01

    Previously, cell type-specific expression of AtHKT1;1, a sodium transporter, improved sodium (Na+) exclusion and salinity tolerance in Arabidopsis. In the current work, AtHKT1;1, was expressed specifically in the root cortical and epidermal cells of an Arabidopsis GAL4-GFP enhancer trap line. These transgenic plants were found to have significantly improved Na+ exclusion under conditions of salinity stress. The feasibility of a similar biotechnological approach in crop plants was explored using a GAL4-GFP enhancer trap rice line to drive expression of AtHKT1;1 specifically in the root cortex. Compared with the background GAL4-GFP line, the rice plants expressing AtHKT1;1 had a higher fresh weight under salinity stress, which was related to a lower concentration of Na+ in the shoots. The root-to-shoot transport of 22Na+ was also decreased and was correlated with an upregulation of OsHKT1;5, the native transporter responsible for Na+ retrieval from the transpiration stream. Interestingly, in the transgenic Arabidopsis plants overexpressing AtHKT1;1 in the cortex and epidermis, the native AtHKT1;1 gene responsible for Na+ retrieval from the transpiration stream, was also upregulated. Extra Na+ retrieved from the xylem was stored in the outer root cells and was correlated with a significant increase in expression of the vacuolar pyrophosphatases (in Arabidopsis and rice) the activity of which would be necessary to move the additional stored Na+ into the vacuoles of these cells. This work presents an important step in the development of abiotic stress tolerance in crop plants via targeted changes in mineral transport. PMID:20838445

  2. Cell-type-specific expression of STAT transcription factors in tissue samples from patients with lymphocytic thyroiditis.

    PubMed

    Staab, Julia; Barth, Peter J; Meyer, Thomas

    2012-09-01

    Expression of cytokine-regulated signal transducer and activator of transcription (STAT) proteins was histochemically assessed in patients diagnosed as having Hashimoto's disease or focal lymphocytic thyroiditis (n = 10). All surgical specimens showed histological features of lymphocytic thyroiditis, including a diffuse infiltration with mononuclear cells and an incomplete loss of thyroid follicles, resulting in the destruction of glandular tissue architecture. Immunohistochemical analysis demonstrated differential expression patterns of the various members of the STAT transcription factors examined, indicating that each member of this conserved protein family has its distinct functions in the development of the disease. Using an antibody that specifically recognized the phosphorylated tyrosine residue in position 701, we detected activated STAT1 dimers in numerous germinal macrophages and infiltrating lymphocytes as well as in oncocytes. In contrast, STAT3 expression was restricted to epithelial cells and showed a clear colocalization with the antiapoptotic protein Bcl-2. Moreover, expression of phospho-STAT3 was associated with low levels of stromal fibrosis, suggesting that STAT3 serves as a protective factor in the remodeling of the inflamed thyroid gland. Phospho-STAT5 immunoreactivity was detected in numerous infiltrating cells of hematopoietic origin and, additionally, in hyperplastic follicular epithelia. This tissue distribution demonstrated that activated STAT5 molecules participate in both lymphocytopoiesis and possibly also in the buildup of regenerating thyroid follicles. Taken together, the cell-type-specific expression patterns of STAT proteins in human lymphocytic thyroiditis reflect their distinct and partially antagonistic roles in orchestrating the balance between degenerating and regenerating processes within a changing cytokine environment.

  3. Hippo signaling regulates Microprocessor and links cell density-dependent miRNA biogenesis to cancer

    PubMed Central

    Mori, Masaki; Triboulet, Robinson; Mohseni, Morvarid; Schlegelmilch, Karin; Shrestha, Kriti; Camargo, Fernando D.; Gregory, Richard I.

    2014-01-01

    SUMMARY Global downregulation of microRNAs (miRNAs) is commonly observed in human cancers and can have a causative role in tumorigenesis. The mechanisms responsible for this phenomenon remain poorly understood. Here we show that YAP, the downstream target of the tumor-suppressive Hippo signaling pathway regulates miRNA biogenesis in a cell density-dependent manner. At low cell density, nuclear YAP binds and sequesters p72 (DDX17), a regulatory component of the miRNA processing machinery. At high cell density, Hippo-mediated cytoplasmic retention of YAP facilitates p72 association with Microprocessor and binding to a specific sequence motif in pri-miRNAs. Inactivation of the Hippo pathway or expression of constitutively active YAP causes widespread miRNA suppression in cells and tumors and a corresponding post-transcriptional induction of MYC expression. Thus, the Hippo pathway links contact-inhibition regulation to miRNA biogenesis and may be responsible for the widespread miRNA repression observed in cancer. PMID:24581491

  4. Cell-Type-Specific Modulation of Sensory Responses in Olfactory Bulb Circuits by Serotonergic Projections from the Raphe Nuclei

    PubMed Central

    Brunert, Daniela; Tsuno, Yusuke; Rothermel, Markus; Shipley, Michael T.

    2016-01-01

    Serotonergic neurons in the brainstem raphe nuclei densely innervate the olfactory bulb (OB), where they can modulate the initial representation and processing of olfactory information. Serotonergic modulation of sensory responses among defined OB cell types is poorly characterized in vivo. Here, we used cell-type-specific expression of optical reporters to visualize how raphe stimulation alters sensory responses in two classes of GABAergic neurons of the mouse OB glomerular layer, periglomerular (PG) and short axon (SA) cells, as well as mitral/tufted (MT) cells carrying OB output to piriform cortex. In PG and SA cells, brief (1–4 s) raphe stimulation elicited a large increase in the magnitude of responses linked to inhalation of ambient air, as well as modest increases in the magnitude of odorant-evoked responses. Near-identical effects were observed when the optical reporter of glutamatergic transmission iGluSnFR was expressed in PG and SA cells, suggesting enhanced excitatory input to these neurons. In contrast, in MT cells imaged from the dorsal OB, raphe stimulation elicited a strong increase in resting GCaMP fluorescence with only a slight enhancement of inhalation-linked responses to odorant. Finally, optogenetically stimulating raphe serotonergic afferents in the OB had heterogeneous effects on presumptive MT cells recorded extracellularly, with an overall modest increase in resting and odorant-evoked responses during serotonergic afferent stimulation. These results suggest that serotonergic afferents from raphe dynamically modulate olfactory processing through distinct effects on multiple OB targets, and may alter the degree to which OB output is shaped by inhibition during behavior. SIGNIFICANCE STATEMENT Modulation of the circuits that process sensory information can profoundly impact how information about the external world is represented and perceived. This study investigates how the serotonergic system modulates the initial processing of olfactory

  5. Cell-Type-Specific Modulation of Sensory Responses in Olfactory Bulb Circuits by Serotonergic Projections from the Raphe Nuclei.

    PubMed

    Brunert, Daniela; Tsuno, Yusuke; Rothermel, Markus; Shipley, Michael T; Wachowiak, Matt

    2016-06-22

    Serotonergic neurons in the brainstem raphe nuclei densely innervate the olfactory bulb (OB), where they can modulate the initial representation and processing of olfactory information. Serotonergic modulation of sensory responses among defined OB cell types is poorly characterized in vivo Here, we used cell-type-specific expression of optical reporters to visualize how raphe stimulation alters sensory responses in two classes of GABAergic neurons of the mouse OB glomerular layer, periglomerular (PG) and short axon (SA) cells, as well as mitral/tufted (MT) cells carrying OB output to piriform cortex. In PG and SA cells, brief (1-4 s) raphe stimulation elicited a large increase in the magnitude of responses linked to inhalation of ambient air, as well as modest increases in the magnitude of odorant-evoked responses. Near-identical effects were observed when the optical reporter of glutamatergic transmission iGluSnFR was expressed in PG and SA cells, suggesting enhanced excitatory input to these neurons. In contrast, in MT cells imaged from the dorsal OB, raphe stimulation elicited a strong increase in resting GCaMP fluorescence with only a slight enhancement of inhalation-linked responses to odorant. Finally, optogenetically stimulating raphe serotonergic afferents in the OB had heterogeneous effects on presumptive MT cells recorded extracellularly, with an overall modest increase in resting and odorant-evoked responses during serotonergic afferent stimulation. These results suggest that serotonergic afferents from raphe dynamically modulate olfactory processing through distinct effects on multiple OB targets, and may alter the degree to which OB output is shaped by inhibition during behavior. Modulation of the circuits that process sensory information can profoundly impact how information about the external world is represented and perceived. This study investigates how the serotonergic system modulates the initial processing of olfactory information by the

  6. Layer- and cell-type-specific subthreshold and suprathreshold effects of long-term monocular deprivation in rat visual cortex.

    PubMed

    Medini, Paolo

    2011-11-23

    Connectivity and dendritic properties are determinants of plasticity that are layer and cell-type specific in the neocortex. However, the impact of experience-dependent plasticity at the level of synaptic inputs and spike outputs remains unclear along vertical cortical microcircuits. Here I compared subthreshold and suprathreshold sensitivity to prolonged monocular deprivation (MD) in rat binocular visual cortex in layer 4 and layer 2/3 pyramids (4Ps and 2/3Ps) and in thick-tufted and nontufted layer 5 pyramids (5TPs and 5NPs), which innervate different extracortical targets. In normal rats, 5TPs and 2/3Ps are the most binocular in terms of synaptic inputs, and 5NPs are the least. Spike responses of all 5TPs were highly binocular, whereas those of 2/3Ps were dominated by either the contralateral or ipsilateral eye. MD dramatically shifted the ocular preference of 2/3Ps and 4Ps, mostly by depressing deprived-eye inputs. Plasticity was profoundly different in layer 5. The subthreshold ocular preference shift was sevenfold smaller in 5TPs because of smaller depression of deprived inputs combined with a generalized loss of responsiveness, and was undetectable in 5NPs. Despite their modest ocular dominance change, spike responses of 5TPs consistently lost their typically high binocularity during MD. The comparison of MD effects on 2/3Ps and 5TPs, the main affected output cells of vertical microcircuits, indicated that subthreshold plasticity is not uniquely determined by the initial degree of input binocularity. The data raise the question of whether 5TPs are driven solely by 2/3Ps during MD. The different suprathreshold plasticity of the two cell populations could underlie distinct functional deficits in amblyopia.

  7. The Modulatory Effects of the Polymorphisms in GLA 5'-Untranslated Region Upon Gene Expression Are Cell-Type Specific.

    PubMed

    Ferreira, Susana; Reguenga, Carlos; Oliveira, João Paulo

    2015-01-01

    Lysosomal α-galactosidase A (αGal) is the enzyme deficient in Fabry disease (FD). The 5'-untranslated region (5'UTR) of the αGal gene (GLA) shows a remarkable degree of variation with three common single nucleotide polymorphisms at nucleotide positions c.-30G>A, c.-12G>A and c.-10C>T. We have recently identified in young Portuguese stroke patients a fourth polymorphism, at c.-44C>T, co-segregating in cis with the c.-12A allele. In vivo, the c.-30A allele is associated with higher enzyme activity in plasma, whereas c.-10T is associated with moderately decreased enzyme activity in leucocytes. Limited data suggest that c.-44T might be associated with increased plasma αGal activity. We have used a luciferase reporter system to experimentally assess the relative modulatory effects on gene expression of the different GLA 5'UTR polymorphisms, as compared to the wild-type sequence, in four different human cell lines. Group-wise, the relative luciferase expression patterns of the various GLA variant isoforms differed significantly in all four cell lines, as evaluated by non-parametric statistics, and were cell-type specific. Some of the post hoc pairwise statistical comparisons were also significant, but the observed effects of the GLA 5'UTR polymorphisms upon the luciferase transcriptional activity in vitro did not consistently replicate the in vivo observations.These data suggest that the GLA 5'UTR polymorphisms are possible modulators of the αGal expression. Further studies are needed to elucidate the biological and clinical implications of these observations, particularly to clarify the effect of these polymorphisms in individuals carrying GLA variants associated with high residual enzyme activity, with no or mild FD clinical phenotypes.

  8. Adolescent maturation of inhibitory inputs onto cingulate cortex neurons is cell-type specific and TrkB dependent

    PubMed Central

    Vandenberg, Angela; Piekarski, David J.; Caporale, Natalia; Munoz-Cuevas, Francisco Javier; Wilbrecht, Linda

    2015-01-01

    The maturation of inhibitory circuits during adolescence may be tied to the onset of mental health disorders such as schizophrenia. Neurotrophin signaling likely plays a critical role in supporting inhibitory circuit development and is also implicated in psychiatric disease. Within the neocortex, subcircuits may mature at different times and show differential sensitivity to neurotrophin signaling. We measured miniature inhibitory and excitatory postsynaptic currents (mIPSCs and mEPSCs) in Layer 5 cell-types in the mouse anterior cingulate (Cg) across the periadolescent period. We differentiated cell-types mainly by Thy1 YFP transgene expression and also retrobead injection labeling in the contralateral Cg and ipsilateral pons. We found that YFP− neurons and commissural projecting neurons had lower frequency of mIPSCs than neighboring YFP+ neurons or pons projecting neurons in juvenile mice (P21–25). YFP− neurons and to a lesser extent commissural projecting neurons also showed a significant increase in mIPSC amplitude during the periadolescent period (P21–25 vs. P40–50), which was not seen in YFP+ neurons or pons projecting neurons. Systemic disruption of tyrosine kinase receptor B (TrkB) signaling during P23–50 in TrkBF616A mice blocked developmental changes in mIPSC amplitude, without affecting miniature excitatory post synaptic currents (mEPSCs). Our data suggest that the maturation of inhibitory inputs onto Layer 5 pyramidal neurons is cell-type specific. These data may inform our understanding of adolescent brain development across species and aid in identifying candidate subcircuits that may show greater vulnerability in mental illness. PMID:25762898

  9. Identification of sequence elements that confer cell-type-specific control of MF alpha 1 expression in Saccharomyces cerevisiae.

    PubMed Central

    Inokuchi, K; Nakayama, A; Hishinuma, F

    1987-01-01

    The MF alpha 1 gene of Saccharomyces cerevisiae, a major structural gene for mating pheromone alpha factor, is an alpha-specific gene whose expression is regulated by the mating-type locus. To study the role of sequences upstream of MF alpha 1 in its expression and regulation, we generated two sets of promoter deletions: upstream deletions and internal deletions. By analyzing these deletions, we have identified a TATA box and two closely related, tandemly arranged upstream activation sites as necessary elements for MF alpha 1 expression. Two upstream activation sites were located ca. 300 and 250 base pairs upstream of the MF alpha 1 transcription start points, which were also determined in this study. Each site contained a homologous 22-base-pair sequence, and both sites were required for maximum transcription level. The distance between the upstream activation sites and the transcription start points could be altered without causing loss of transcription efficiency, and the sites were active in either orientation with respect to the coding region. These elements conferred cell type-specific expression on a heterologous promoter. Analysis with host mating-type locus mutants indicates that these sequences are the sites through which the MAT alpha 1 product exerts its action to activate the MF alpha 1 gene. Homologous sequences with these elements were found in other alpha-specific genes, MF alpha 2 and STE3, and may mediate activation of this set of genes by MAT alpha 1. Images PMID:2959859

  10. Post-ischaemic long-term synaptic potentiation in the striatum: a putative mechanism for cell type-specific vulnerability.

    PubMed

    Calabresi, Paolo; Saulle, Emilia; Centonze, Diego; Pisani, Antonio; Marfia, Girolama A; Bernardi, Giorgio

    2002-04-01

    In the present in vitro study of rat brain, we report that transient oxygen and glucose deprivation (in vitro ischaemia) induced a post-ischaemic long-term synaptic potentiation (i-LTP) at corticostriatal synapses. We compared the physiological and pharmacological characteristics of this pathological form of synaptic plasticity with those of LTP induced by tetanic stimulation of corticostriatal fibres (t-LTP), which is thought to represent a cellular substrate of learning and memory. Activation of N-methyl-D-aspartate (NMDA) receptors was required for the induction of both forms of synaptic plasticity. The intraneuronal injection of the calcium chelator BAPTA [bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate] and inhibitors of the mitogen-activated protein kinase pathway blocked both forms of synaptic plasticity. However, while t-LTP showed input specificity, i-LTP occurred also at synaptic pathways inactive during the ischaemic period. In addition, scopolamine, a muscarinic receptor antagonist, prevented the induction of t-LTP but not of i-LTP, indicating that endogenous acetylcholine is required for physiological but not for pathological synaptic potentiation. Finally, we found that striatal cholinergic interneurones, which are resistant to in vivo ischaemia, do not express i-LTP while they express t-LTP. We suggest that i-LTP represents a pathological form of synaptic plasticity that may account for the cell type-specific vulnerability observed in striatal spiny neurones following ischaemia and energy deprivation.

  11. Cell-type-specific modulation of targets and distractors by dopamine D1 receptors in primate prefrontal cortex

    PubMed Central

    Jacob, Simon N.; Stalter, Maximilian; Nieder, Andreas

    2016-01-01

    The prefrontal cortex (PFC) is crucial for maintaining relevant information in working memory and resisting interference. PFC neurons are strongly regulated by dopamine, but it is unknown whether dopamine receptors are involved in protecting target memories from distracting stimuli. We investigated the prefrontal circuit dynamics and dopaminergic modulation of targets and distractors in monkeys trained to ignore interfering stimuli in a delayed-match-to-numerosity task. We found that dopamine D1 receptors (D1Rs) modulate the recovery of task-relevant information following a distracting stimulus. The direction of modulation is cell-type-specific: in putative pyramidal neurons, D1R inhibition enhances and D1R stimulation attenuates coding of the target stimulus after the interference, while the opposite pattern is observed in putative interneurons. Our results suggest that dopaminergic neuromodulation of PFC circuits regulates mental representations of behaviourally relevant stimuli that compete with task-irrelevant input and could play a central role for cognitive functioning in health and disease. PMID:27807366

  12. Human Milk Cells Contain Numerous miRNAs that May Change with Milk Removal and Regulate Multiple Physiological Processes.

    PubMed

    Alsaweed, Mohammed; Lai, Ching Tat; Hartmann, Peter E; Geddes, Donna T; Kakulas, Foteini

    2016-06-17

    Human milk (HM) is a complex biofluid conferring nutritional, protective and developmental components for optimal infant growth. Amongst these are maternal cells, which change in response to feeding and were recently shown to be a rich source of miRNAs. We used next generation sequencing to characterize the cellular miRNA profile of HM collected before and after feeding. HM cells conserved higher miRNA content than the lipid and skim HM fractions or other body fluids, in accordance with previous studies. In total, 1467 known mature and 1996 novel miRNAs were identified, with 89 high-confidence novel miRNAs. HM cell content was higher post-feeding (p < 0.05), and was positively associated with total miRNA content (p = 0.014) and species number (p < 0.001). This coincided with upregulation of 29 known and 2 novel miRNAs, and downregulation of 4 known and 1 novel miRNAs post-feeding, but no statistically significant change in expression was found for the remaining miRNAs. These findings suggest that feeding may influence the miRNA content of HM cells. The most highly and differentially expressed miRNAs were key regulators of milk components, with potential diagnostic value in lactation performance. They are also involved in the control of body fluid balance, thirst, appetite, immune response, and development, implicating their functional significance for the infant.

  13. Human Milk Cells Contain Numerous miRNAs that May Change with Milk Removal and Regulate Multiple Physiological Processes

    PubMed Central

    Alsaweed, Mohammed; Lai, Ching Tat; Hartmann, Peter E.; Geddes, Donna T.; Kakulas, Foteini

    2016-01-01

    Human milk (HM) is a complex biofluid conferring nutritional, protective and developmental components for optimal infant growth. Amongst these are maternal cells, which change in response to feeding and were recently shown to be a rich source of miRNAs. We used next generation sequencing to characterize the cellular miRNA profile of HM collected before and after feeding. HM cells conserved higher miRNA content than the lipid and skim HM fractions or other body fluids, in accordance with previous studies. In total, 1467 known mature and 1996 novel miRNAs were identified, with 89 high-confidence novel miRNAs. HM cell content was higher post-feeding (p < 0.05), and was positively associated with total miRNA content (p = 0.014) and species number (p < 0.001). This coincided with upregulation of 29 known and 2 novel miRNAs, and downregulation of 4 known and 1 novel miRNAs post-feeding, but no statistically significant change in expression was found for the remaining miRNAs. These findings suggest that feeding may influence the miRNA content of HM cells. The most highly and differentially expressed miRNAs were key regulators of milk components, with potential diagnostic value in lactation performance. They are also involved in the control of body fluid balance, thirst, appetite, immune response, and development, implicating their functional significance for the infant. PMID:27322254

  14. Cell type-specific filamin complex regulation by a novel class of HECT ubiquitin ligase is required for normal cell motility and patterning

    PubMed Central

    Blagg, Simone L.; Battom, Suzanne E.; Annesley, Sarah J.; Keller, Thomas; Parkinson, Katie; Wu, Jasmine M. F.; Fisher, Paul R.; Thompson, Christopher R. L.

    2011-01-01

    Differential cell motility, which plays a key role in many developmental processes, is perhaps most evident in examples of pattern formation in which the different cell types arise intermingled before sorting out into discrete tissues. This is thought to require heterogeneities in responsiveness to differentiation-inducing signals that result in the activation of cell type-specific genes and ‘salt and pepper’ patterning. How differential gene expression results in cell sorting is poorly defined. Here we describe a novel gene (hfnA) that provides the first mechanistic link between cell signalling, differential gene expression and cell type-specific sorting in Dictyostelium. HfnA defines a novel group of evolutionarily conserved HECT ubiquitin ligases with an N-terminal filamin domain (HFNs). HfnA expression is induced by the stalk differentiation-inducing factor DIF-1 and is restricted to a subset of prestalk cells (pstO). hfnA− pstO cells differentiate but their sorting out is delayed. Genetic interactions suggest that this is due to misregulation of filamin complex activity. Overexpression of filamin complex members phenocopies the hfnA− pstO cell sorting defect, whereas disruption of filamin complex function in a wild-type background results in pstO cells sorting more strongly. Filamin disruption in an hfnA− background rescues pstO cell localisation. hfnA− cells exhibit altered slug phototaxis phenotypes consistent with filamin complex hyperactivity. We propose that HfnA regulates filamin complex activity and cell type-specific motility through the breakdown of filamin complexes. These findings provide a novel mechanism for filamin regulation and demonstrate that filamin is a crucial mechanistic link between responses to differentiation signals and cell movement in patterning based on ‘salt and pepper’ differentiation and sorting out. PMID:21389049

  15. Fiber Type-Specific Satellite Cell Content in Cyclists Following Heavy Training with Carbohydrate and Carbohydrate-Protein Supplementation

    PubMed Central

    McKenzie, Alec I.; D'Lugos, Andrew C.; Saunders, Michael J.; Gworek, Keith D.; Luden, Nicholas D.

    2016-01-01

    The central purpose of this study was to evaluate the fiber type-specific satellite cell and myonuclear responses of endurance-trained cyclists to a block of intensified training, when supplementing with carbohydrate (CHO) vs. carbohydrate-protein (PRO). In a crossover design, endurance-trained cyclists (n = 8) performed two consecutive training periods, once supplementing with CHO (de facto “control” condition) and the other with PRO. Each training period consisted of 10 days of intensified cycle training (ICT–120% increase in average training duration) followed by 10 days of recovery (RVT–reduced volume training; 33% volume reduction vs. normal training). Skeletal muscle biopsies were obtained from the vastus lateralis before and after ICT and again following RVT. Immunofluorescent microscopy was used to quantify SCs (Pax7+), myonuclei (DAPI+), and myosin heavy chain I (MyHC I). Data are expressed as percent change ± 90% confidence limits. The 10-day block of ICTCHO increased MyHC I SC content (35 ± 28%) and myonuclear density (16 ± 6%), which remained elevated following RVTCHO (SC = 69 ± 50% vs. PRE; Nuclei = 17 ± 15% vs. PRE). MyHC II SC and myonuclei were not different following ICTCHO, but were higher following RVTCHO (SC = +33 ± 31% vs. PRE; Nuclei = 15 ± 14% vs. PRE), indicating a delayed response compared to MyHC I fibers. The MyHC I SC pool increased following ICTPRO (37 ± 37%), but without a concomitant increase in myonuclei. There were no changes in MyHC II SC or myonuclei following ICTPRO. Collectively, these trained endurance cyclists possessed a relatively large pool of SCs that facilitated rapid (MyHC I) and delayed (MyHC II) satellite cell proliferation and myonuclear accretion under carbohydrate conditions. The current findings strengthen the growing body of evidence demonstrating alterations in satellite cell number in the absence of hypertrophy. Satellite cell pool expansion is typically viewed as an advantageous response to

  16. Fiber Type-Specific Satellite Cell Content in Cyclists Following Heavy Training with Carbohydrate and Carbohydrate-Protein Supplementation.

    PubMed

    McKenzie, Alec I; D'Lugos, Andrew C; Saunders, Michael J; Gworek, Keith D; Luden, Nicholas D

    2016-01-01

    The central purpose of this study was to evaluate the fiber type-specific satellite cell and myonuclear responses of endurance-trained cyclists to a block of intensified training, when supplementing with carbohydrate (CHO) vs. carbohydrate-protein (PRO). In a crossover design, endurance-trained cyclists (n = 8) performed two consecutive training periods, once supplementing with CHO (de facto "control" condition) and the other with PRO. Each training period consisted of 10 days of intensified cycle training (ICT-120% increase in average training duration) followed by 10 days of recovery (RVT-reduced volume training; 33% volume reduction vs. normal training). Skeletal muscle biopsies were obtained from the vastus lateralis before and after ICT and again following RVT. Immunofluorescent microscopy was used to quantify SCs (Pax7+), myonuclei (DAPI+), and myosin heavy chain I (MyHC I). Data are expressed as percent change ± 90% confidence limits. The 10-day block of ICTCHO increased MyHC I SC content (35 ± 28%) and myonuclear density (16 ± 6%), which remained elevated following RVTCHO (SC = 69 ± 50% vs. PRE; Nuclei = 17 ± 15% vs. PRE). MyHC II SC and myonuclei were not different following ICTCHO, but were higher following RVTCHO (SC = +33 ± 31% vs. PRE; Nuclei = 15 ± 14% vs. PRE), indicating a delayed response compared to MyHC I fibers. The MyHC I SC pool increased following ICTPRO (37 ± 37%), but without a concomitant increase in myonuclei. There were no changes in MyHC II SC or myonuclei following ICTPRO. Collectively, these trained endurance cyclists possessed a relatively large pool of SCs that facilitated rapid (MyHC I) and delayed (MyHC II) satellite cell proliferation and myonuclear accretion under carbohydrate conditions. The current findings strengthen the growing body of evidence demonstrating alterations in satellite cell number in the absence of hypertrophy. Satellite cell pool expansion is typically viewed as an advantageous response to exercise

  17. Cell-Type Specific Channelopathies in the Prefrontal Cortex of the fmr1-/y Mouse Model of Fragile X Syndrome.

    PubMed

    Kalmbach, Brian E; Johnston, Daniel; Brager, Darrin H

    2015-01-01

    Fragile X syndrome (FXS) is caused by transcriptional silencing of the fmr1 gene resulting in the loss of fragile X mental retardation protein (FMRP) expression. FXS patients display several behavioral phenotypes associated with prefrontal cortex (PFC) dysfunction. Voltage-gated ion channels, some of which are regulated by FMRP, heavily influence PFC neuron function. Although there is evidence for brain region-specific alterations to the function a single type of ion channel in FXS, it is unclear whether subtypes of principal neurons within a brain region are affected uniformly. We tested for alterations to ion channels critical in regulating neural excitability in two subtypes of prefrontal L5 pyramidal neurons. Using somatic and dendritic patch-clamp recordings, we provide evidence that the functional expression of h-channels (Ih) is down-regulated, whereas A-type K(+) channel function is up-regulated in pyramidal tract-projecting (PT) neurons in the fmr1-/y mouse PFC. This is the opposite pattern of results from published findings from hippocampus where Ih is up-regulated and A-type K(+) channel function is down-regulated. Additionally, we find that somatic Kv1-mediated current is down-regulated, resulting in increased excitability of fmr1-/y PT neurons. Importantly, these h- and K(+) channel differences do not extend to neighboring intratelencephalic-projecting neurons. Thus, the absence of FMRP has divergent effects on the function of individual types of ion channels not only between brain regions, but also variable effects across cell types within the same brain region. Given the importance of ion channels in regulating neural circuits, these results suggest cell-type-specific phenotypes for the disease.

  18. Dendritic cell type-specific HIV-1 activation in effector T cells: implications for latent HIV-1 reservoir establishment.

    PubMed

    van der Sluis, Renée M; van Capel, Toni M M; Speijer, Dave; Sanders, Rogier W; Berkhout, Ben; de Jong, Esther C; Jeeninga, Rienk E; van Montfort, Thijs

    2015-06-01

    Latent HIV type I (HIV-1) infections can frequently occur in short-lived proliferating effector T lymphocytes. These latently infected cells could revert into resting T lymphocytes and thereby contribute to the establishment of the long-lived viral reservoir. Monocyte-derived dendritic cells can revert latency in effector T cells in vitro. Here we investigated the latency activation properties of tissue-specific immune cells, including a large panel of dendritic cell subsets, to explore in which body compartments effector T cells are most likely to maintain latent HIV-1 provirus and thus potentially contribute to the long-lived reservoir. Our results demonstrate that blood or genital tract dendritic cells do not activate latent provirus in effector T cells, whereas gut or lymphoid dendritic cells induce virus production from latently infected effector T cells in our in-vitro model for latency. Toll-like receptor 3-induced interferon production by myeloid dendritic cells abolished the dendritic cells' ability to induce viral gene expression. In this study, we show that HIV-1 provirus residing in effector T cells is activated from latency by tissue-specific dendritic cell subsets and other immune cells with remarkably different efficiencies.Our new assay system points to an important, neglected aspect of HIV-1 research: the ability of other immune cells, especially dendritic cells, to differentially affect latency establishment as well as virus reactivation.

  19. Altered miRNA expression in aniline-mediated cell cycle progression in rat spleen.

    PubMed

    Wang, Gangduo; Wang, Jianling; Khan, M Firoze

    2017-09-01

    Aniline exposure is associated with toxicity to the spleen, however, early molecular events in aniline-induced cell cycle progression in the spleen remain unknown. MicroRNAs (miRNAs) have been implicated in tumor development by modulating key cell cycle regulators and controlling cell proliferation. This study was, therefore, undertaken on the expression of miRNAs, regulation of cyclins and cyclin-dependent kinases (CDKs) in an experimental condition that precedes a tumorigenic response. Male SD rats were treated with aniline (1 mmol/kg/day by gavage) for 7 days, and expression of miRNAs, cyclins and CDKs in rat spleens were analyzed. Microarray and/or qPCR analyses showed that aniline exposure led to significantly decreased miRNA expression of let-7a, miR-24, miR-34c, miR-100, miR-125b, and greatly increased miR-181a. The aberrant expression of miRNAs was associated with significantly increased protein expression of cyclins A, B1, D3 and E. Furthermore, remarkably enhanced expression of CDKs like CDK1, CDK2, CDK4, CDK6, especially p-CDK1 and p-CDK2 as well as alternations in the expression of pRB, p27, and CDC25A in the spleens of aniline-treated rats was also observed. The data suggest that aniline exposure leads to aberrant expression of miRNAs in the spleen which could be important in the regulation of cell cycle proteins. Our findings, thus, provide new insight into the role of miRNAs in cell cycle progression, which may contribute to aniline-induced tumorigenic response in the spleen.

  20. Mating type-specific cell-cell recognition of Saccharomyces cerevisiae: cell wall attachment and active sites of a- and alpha-agglutinin.

    PubMed Central

    Cappellaro, C; Baldermann, C; Rachel, R; Tanner, W

    1994-01-01

    Mating type-specific agglutination of Saccharomyces cerevisiae a and alpha cells depends on the heterophilic interaction of two cell surface glycoproteins, the gene products of AG alpha 1 and AGA2. Evidence is presented with immunogold labelling that the alpha-agglutinin is part of the outer fimbrial cell wall coat. The a-agglutinin is bound via two S-S bridges (Cys7 and Cys50) to a cell wall component, most probably the gene product of AGA1. His273 of alpha-agglutinin has previously been shown to be essential for a- and alpha-agglutinin interaction and a model based on two opposing ion-pairs had been proposed. By site-directed mutagenesis this possibility has now been excluded. With the help of various peptides, either chemically synthesized, obtained by proteolysis of intact glycosylated a-agglutinin or prepared from a fusion protein expressed in Escherichia coli, the biologically active region of a-agglutinin was located at the C-terminus of the molecule. A peptide consisting of the C-terminal 10 amino acids (GSPIN-TQYVF) was active in nanomolar concentrations. Saccharide moieties, therefore, are not essential for the mating type-specific cell-cell interaction; glycosylated peptides are, however, four to five times more active than non-glycosylated ones. Comparisons of the recognition sequences of the S. cerevisiae agglutinins with that of the Dictyostelium contact site A glycoprotein (gp80), as well as with those of the various families of cell adhesion molecules of higher eucaryotes, have been made and are discussed. Images PMID:7957044

  1. A cell-type-specific defect in border cell formation in the Acacia mangium root cap developing an extraordinary sheath of sloughed-off cells

    PubMed Central

    Endo, Izuki; Tange, Takeshi; Osawa, Hiroki

    2011-01-01

    Background and Aims Root caps release border cells, which play central roles in microbe interaction and root protection against soil stresses. However, the number and connectivity of border cells differ widely among plant species. Better understanding of key border-cell phenotype across species will help define the total function of border cells and associated genes. Methods The spatio-temporal detachment of border cells in the leguminous tree Acacia mangium was investigated by using light and fluorescent microscopy with fluorescein diacetate, and their number and structural connectivity compared with that in soybean (Glycine max). Key Results Border-like cells with a sheet structure peeled bilaterally from the lateral root cap of A. mangium. Hydroponic root elongation partially facilitated acropetal peeling of border-like cells, which accumulate as a sheath that covers the 0- to 4-mm tip within 1 week. Although root elongation under friction caused basipetal peeling, lateral root caps were minimally trimmed as compared with hydroponic roots. In the meantime, A. mangium columella caps simultaneously released single border cells with a number similar to those in soybean. Conclusions These results suggest that cell type-specific inhibitory factors induce a distinct defective phenotype in single border-cell formation in A. mangium lateral root caps. PMID:21712296

  2. A cell-type-specific defect in border cell formation in the Acacia mangium root cap developing an extraordinary sheath of sloughed-off cells.

    PubMed

    Endo, Izuki; Tange, Takeshi; Osawa, Hiroki

    2011-08-01

    Root caps release border cells, which play central roles in microbe interaction and root protection against soil stresses. However, the number and connectivity of border cells differ widely among plant species. Better understanding of key border-cell phenotype across species will help define the total function of border cells and associated genes. The spatio-temporal detachment of border cells in the leguminous tree Acacia mangium was investigated by using light and fluorescent microscopy with fluorescein diacetate, and their number and structural connectivity compared with that in soybean (Glycine max). Border-like cells with a sheet structure peeled bilaterally from the lateral root cap of A. mangium. Hydroponic root elongation partially facilitated acropetal peeling of border-like cells, which accumulate as a sheath that covers the 0- to 4-mm tip within 1 week. Although root elongation under friction caused basipetal peeling, lateral root caps were minimally trimmed as compared with hydroponic roots. In the meantime, A. mangium columella caps simultaneously released single border cells with a number similar to those in soybean. These results suggest that cell type-specific inhibitory factors induce a distinct defective phenotype in single border-cell formation in A. mangium lateral root caps.

  3. Role of apoptosis-related miRNAs in resveratrol-induced breast cancer cell death.

    PubMed

    Venkatadri, R; Muni, T; Iyer, A K V; Yakisich, J S; Azad, N

    2016-02-18

    Breast cancer is the most frequently diagnosed cancer in women, and one of the leading causes of cancer-related deaths worldwide. Recent evidences indicate that dietary agents such as resveratrol may inhibit cancer progression through modulation of microRNAs (miRNAs). We demonstrate that resveratrol regulates apoptotic and cell cycle machinery in breast cancer cells by modulating key tumor-suppressive miRNAs including miR-125b-5p, miR-200c-3p, miR-409-3p, miR-122-5p and miR-542-3p. Resveratrol-mediated miRNA modulation regulates key anti-apoptotic and cell cycle proteins including Bcl-2, X-linked inhibitor of apoptosis protein and CDKs, which are critical for its activity. Modulating miRNAs with mimics or inhibitors further validated a key role for miR-542-3p in MCF-7 and miR-122-5p in MDA-MB-231 breast cancer cell death in response to resveratrol. In conclusion, this study reveals novel miRNAs modulated by resveratrol that have a key role in breast cancer cell death.

  4. High Intensity Training May Reverse the Fiber Type Specific Decline in Myogenic Stem Cells in Multiple Sclerosis Patients

    PubMed Central

    Farup, Jean; Dalgas, Ulrik; Keytsman, Charly; Eijnde, Bert O.; Wens, Inez

    2016-01-01

    Multiple sclerosis (MS) is associated with loss of skeletal muscle mass and function. The myogenic stem cells (satellite cells—SCs) are instrumental to accretion of myonuclei, but remain to be investigated in MS. The present study aimed to compare the SC and myonuclei content between MS patients (n = 23) and age matched healthy controls (HC, n = 18). Furthermore, the effects of 12 weeks of high intensity training on SC and myonuclei content were explored in MS. Muscle biopsies were obtained from m. Vastus Lateralis at baseline (MS and HC) and following 12 weeks of training (MS only). Frozen biopsies were sectioned followed by immunohistochemical analysis for fiber type specific SCs (Pax7+), myonuclei (MN) and central nuclei content and fiber cross-sectional area (fCSA) was quantified using ATPase histochemistry. At baseline the SCs per fiber was lower in type II compared to type I fibers in both MS (119%, p < 0.01) and HC (69%, p < 0.05), whereas the SCs per fCSA was lower in type II fibers compared to type I only in MS (72%, p < 0.05). No differences were observed in MN or central nuclei between MS and HC. Following training the type II fiber SCs per fiber and per fCSA in MS patients increased by 165% (p < 0.05) and 135% (p < 0.05), respectively. Furthermore, the type II fiber MN content tended (p = 0.06) to be increased by 35% following training. In conclusion, the SC content is lower in type II compared to type I fibers in both MS and HC. Furthermore, high intensity training was observed to selectively increase the SC and myonuclei content in type II fibers in MS patients. PMID:27303309

  5. Cell type-specific and common characteristics of exosomes derived from mouse cell lines: Yield, physicochemical properties, and pharmacokinetics.

    PubMed

    Charoenviriyakul, Chonlada; Takahashi, Yuki; Morishita, Masaki; Matsumoto, Akihiro; Nishikawa, Makiya; Takakura, Yoshinobu

    2017-01-01

    Exosomes are small membrane vesicles secreted from cells and are expected to be used as drug delivery systems. Important characteristics of exosomes, such as yield, physicochemical properties, and pharmacokinetics, may be different among different cell types. However, there is limited information about the effect of cell type on these characteristics. In the present study, we evaluated these characteristics of exosomes derived from five different types of mouse cell lines: B16BL6 murine melanoma cells, C2C12 murine myoblast cells, NIH3T3 murine fibroblasts cells, MAEC murine aortic endothelial cells, and RAW264.7 murine macrophage-like cells. Exosomes were collected using a differential ultracentrifugation method. The exosomes collected from all the cell types were negatively charged globular vesicles with a diameter of approximately 100nm. C2C12 and RAW264.7 cells produced more exosomes than the other types of cells. The exosomes were labeled with a fusion protein of Gaussia luciferase and lactadherin to evaluate their pharmacokinetics. After intravenous injection into mice, all the exosomes rapidly disappeared from the systemic circulation and mainly distributed to the liver. In conclusion, the exosome yield was significantly different among the cell types, and all the exosomes evaluated in this study showed comparable physicochemical and pharmacokinetic properties.

  6. Cell-type specific photoreceptors and light signaling pathways in the multicellular green alga volvox carteri and their potential role in cellular differentiation

    PubMed Central

    Kianianmomeni, Arash

    2015-01-01

    The formation of multicellular organisms requires genetically predefined signaling pathways in various cell types. Besides differences in size, energy balance and life time, cell types should be enable to modulate appropriate developmental and adaptive responses in ever-changing surrounding environment. One of the most important environmental cues is light which regulates a variety of physiological and cellular processes. During evolution, diverse light-sensitive proteins, so-called photoreceptors, and corresponding signaling pathways have evolved, in almost all kingdoms of life, to monitor light continuously and adjust their growth and development accordingly. However, considering the fact that different cell types should be enable to trigger distinct light signaling pathways according to their needs, cell-type specific light signaling pathways are required to guarantee cell type-matched modulation of cellular and developmental processes in response to different light signals. The multicellular green alga Volvox carteri, which has only 2 cell types with clear division of labor, possesses cell-type specific photoreceptors and light signaling pathways which allow differential regulation of genes involved in various cellular and metabolic pathways in response to environmental light. The existence of cell-type specific light signaling pathways in muticellular organism like Volvox reflects an early development of cell-type specific signaling mechanisms during evolution to ensure maintenance of differentiation. PMID:25874475

  7. Cell-type specific photoreceptors and light signaling pathways in the multicellular green alga Volvox carteri and their potential role in cellular differentiation.

    PubMed

    Kianianmomeni, Arash

    2015-01-01

    The formation of multicellular organisms requires genetically predefined signaling pathways in various cell types. Besides differences in size, energy balance and life time, cell types should be enable to modulate appropriate developmental and adaptive responses in ever-changing surrounding environment. One of the most important environmental cues is light which regulates a variety of physiological and cellular processes. During evolution, diverse light-sensitive proteins, so-called photoreceptors, and corresponding signaling pathways have evolved, in almost all kingdoms of life, to monitor light continuously and adjust their growth and development accordingly. However, considering the fact that different cell types should be enable to trigger distinct light signaling pathways according to their needs, cell-type specific light signaling pathways are required to guarantee cell type-matched modulation of cellular and developmental processes in response to different light signals. The multicellular green alga Volvox carteri, which has only 2 cell types with clear division of labor, possesses cell-type specific photoreceptors and light signaling pathways which allow differential regulation of genes involved in various cellular and metabolic pathways in response to environmental light. The existence of cell-type specific light signaling pathways in multicellular organism like Volvox reflects an early development of cell-type specific signaling mechanisms during evolution to ensure maintenance of differentiation.

  8. Fractionation of human spermatogenic cells using STA-PUT gravity sedimentation and their miRNA profiling.

    PubMed

    Liu, Yun; Niu, Minghui; Yao, Chencheng; Hai, Yanan; Yuan, Qingqing; Liu, Yang; Guo, Ying; Li, Zheng; He, Zuping

    2015-01-30

    Human spermatogenic cells have not yet been isolated, and notably, their global miRNA profiles remain unknown. Here we have effectively isolated human spermatogonia, pachytene spermatocytes and round spermatids using STA-PUT velocity sedimentation. RT-PCR, immunocytochemistry and meiosis spread assays revealed that the purities of isolated human spermatogonia, pachytene spermatocytes, and round spermatids were 90%, and the viability of these isolated cells was over 98%. MiRNA microarrays showed distinct global miRNA profiles among human spermatogonia, pachytene spermatocytes, and round spermatids. Thirty-two miRNAs were significantly up-regulated whereas 78 miRNAs were down-regulated between human spermatogonia and pachytene spermatocytes, suggesting that these miRNAs are involved in the meiosis and mitosis, respectively. In total, 144 miRNAs were significantly up-regulated while 29 miRNAs were down-regulated between pachytene spermatocytes and round spermatids, reflecting potential roles of these miRNAs in mediating spermiogenesis. A number of novel binding targets of miRNAs were further identified using various softwares and verified by real-time PCR. Our ability of isolating human spermatogonia, pachytene spermatocytes and round spermatids and unveiling their distinct global miRNA signatures and novel targets could provide novel small RNA regulatory mechanisms mediating three phases of human spermatogenesis and offer new targets for the treatment of male infertility.

  9. Fractionation of human spermatogenic cells using STA-PUT gravity sedimentation and their miRNA profiling

    PubMed Central

    Liu, Yun; Niu, Minghui; Yao, Chencheng; Hai, Yanan; Yuan, Qingqing; Liu, Yang; Guo, Ying; Li, Zheng; He, Zuping

    2015-01-01

    Human spermatogenic cells have not yet been isolated, and notably, their global miRNA profiles remain unknown. Here we have effectively isolated human spermatogonia, pachytene spermatocytes and round spermatids using STA-PUT velocity sedimentation. RT-PCR, immunocytochemistry and meiosis spread assays revealed that the purities of isolated human spermatogonia, pachytene spermatocytes, and round spermatids were 90%, and the viability of these isolated cells was over 98%. MiRNA microarrays showed distinct global miRNA profiles among human spermatogonia, pachytene spermatocytes, and round spermatids. Thirty-two miRNAs were significantly up-regulated whereas 78 miRNAs were down-regulated between human spermatogonia and pachytene spermatocytes, suggesting that these miRNAs are involved in the meiosis and mitosis, respectively. In total, 144 miRNAs were significantly up-regulated while 29 miRNAs were down-regulated between pachytene spermatocytes and round spermatids, reflecting potential roles of these miRNAs in mediating spermiogenesis. A number of novel binding targets of miRNAs were further identified using various softwares and verified by real-time PCR. Our ability of isolating human spermatogonia, pachytene spermatocytes and round spermatids and unveiling their distinct global miRNA signatures and novel targets could provide novel small RNA regulatory mechanisms mediating three phases of human spermatogenesis and offer new targets for the treatment of male infertility. PMID:25634318

  10. Double-stranded microRNA mimics can induce length- and passenger strand-dependent effects in a cell type-specific manner.

    PubMed

    Goldgraben, Mae A; Russell, Roslin; Rueda, Oscar M; Caldas, Carlos; Git, Anna

    2016-02-01

    MicroRNAs are short (17-26) noncoding RNAs driving or modulating physiological and pathological cellular events. Overexpression of miR-155 is pathogenic in B-cell malignancy but was also reported in a number of solid tumors-in particular, in breast cancer, where its role remains unclear and often contradictory. Using representative cell line models, we sought to determine whether the discrepant miR-155 effects in breast cancer could be explained by the heterogeneity of the disease. The growth of six breast cancer cell lines transfected with several miRNA mimics was analyzed. We found MCF-7 cell growth to be inhibited by miR-155 and miR-145 mimics, both 23-nt long, but not by a number of shorter mimics, including a universal commercial negative control. Microarray and Western blot analyses revealed induction of apoptosis, associated with interferon-β after activation of the double-stranded RNA sensor pathway. 3' Trimming of the miRNA mimics to 21 nt substantially reduced their growth-inhibitory potency. Mutating the canonical seed of the miR-155 mimic had no effect on the induced inhibition, which was abolished by mutating the miRNA seed of the artificial passenger strand. A panel of breast cancer cell lines showed a wide range of sensitivities to 23-mer mimics, broadly consistent with the sensitivity of the cell lines to Poly (I:C). We demonstrate two sources for nonspecific in vitro effects by miRNA mimics: duplex length and the artificial passenger strand. We highlight the danger of a universal 21-mer negative control and the importance of using matched seed mutants for reliable interpretation of phenotypes.

  11. Molecular beacon-enabled purification of living cells by targeting cell type-specific mRNAs.

    PubMed

    Wile, Brian M; Ban, Kiwon; Yoon, Young-Sup; Bao, Gang

    2014-10-01

    Molecular beacons (MBs) are dual-labeled oligonucleotides that fluoresce only in the presence of complementary mRNA. The use of MBs to target specific mRNAs allows sorting of specific cells from a mixed cell population. In contrast to existing approaches that are limited by available surface markers or selectable metabolic characteristics, the MB-based method enables the isolation of a wide variety of cells. For example, the ability to purify specific cell types derived from pluripotent stem cells (PSCs) is important for basic research and therapeutics. In addition to providing a general protocol for MB design, validation and nucleofection into cells, we describe how to isolate a specific cell population from differentiating PSCs. By using this protocol, we have successfully isolated cardiomyocytes differentiated from mouse or human PSCs (hPSCs) with ∼ 97% purity, as confirmed by electrophysiology and immunocytochemistry. After designing MBs, their ordering and validation requires 2 weeks, and the isolation process requires 3 h.

  12. STI571 SENSITIZES BREAST CANCER CELLS TO 5-FLUOROURACIL, CISPLATIN AND CAMPTOTHECIN IN A CELL TYPE-SPECIFIC MANNER

    PubMed Central

    Sims, Jonathan T.; Ganguly, Sourik; Fiore, Leann S.; Holler, Chris J.; Park, Eun-Sil; Plattner, Rina

    2009-01-01

    Previously, we demonstrated that Abl kinases are highly active in invasive breast cancer cell lines, and contribute to survival in response to nutrient deprivation, invasion and proliferation. To determine whether an Abl kinase inhibitor, STI571 (Gleevec; imatinib mesylate) sensitizes breast cancer cells to chemotherapeutic agents, we treated three breast cancer cell lines (BT-549, MDA-MB-231, and MDA-MB-468) that have active Abl kinases, with STI571 in combination with several conventional chemotherapeutic drugs frequently used to treat breast cancer, and assessed the effect on cell viability, proliferation, and apoptosis. We found that STI571 had synergistic effects with cisplatin in BT-549 and to some extent in MDA-MB-468 cells, STI571 synergized with camptothecin using an alternate dosing regimen in MDA-MB-231 cells, and STI571 synergistically sensitized MDA-MB-468 cells to paclitaxel and to high doses of 5-fluorouracil. Significantly, STI571 increased the ability of cisplatin to inhibit constitutive activation of PI3K/Akt, synergized with camptothecin to increase the stability of IκB in MDA-MB-231 cells, and in MDA-MB-468 cells, camptothecin and 5-fluorouracil inhibited STI571-dependent activation of STAT3. In other cell line/drug combinations, STI571 had additive or antagonistic effects, indicating that the ability of STI571 to sensitize breast cancer cells to chemotherapeutic agents is cell type-dependent. Significantly, unlike cisplatin, paclitaxel, and camptothecin, mechloroethamine was strongly antagonistic to STI571, and the effect was not cell line-dependent. Taken together, these data indicate that the cellular milieu governs the response of breast cancer cells to STI571/chemotherapeutic combination regimens, which suggests that treatment with these combinations requires individualization. PMID:19427998

  13. Acute kidney injury: a paradigm for miRNA regulation of the cell cycle.

    PubMed

    Khalid, Usman; Bowen, Timothy; Fraser, Donald J; Jenkins, Robert H

    2014-08-01

    miRNAs are small, endogenous, post-transcriptional regulators of gene expression. AKI (acute kidney injury) of various aetiologies, including trauma, sepsis and IRI (ischaemia/reperfusion injury) in the context of kidney transplantation, or drug toxicity, has a high morbidity and mortality rate and presents a significant burden to health services worldwide. AKI primarily affects the renal cortex, in particular PTCs (proximal tubular epithelial cells). Current research demonstrates causality between G2/M cell cycle arrest of PTCs and AKI. Recent findings from our laboratory and others presented in this review implicate miRNA regulation of the cell cycle in the pathology of AKI.

  14. Inference of gene regulation via miRNAs during ES cell differentiation using MiRaGE method.

    PubMed

    Yoshizawa, Masato; Taguchi, Y-H; Yasuda, Jun

    2011-01-01

    MicroRNA (miRNA) is a critical regulator of cell growth, differentiation, and development. To identify important miRNAs in a biological process, many bioinformatical tools have been developed. We have developed MiRaGE (MiRNA Ranking by Gene Expression) method to infer the regulation of gene expression by miRNAs from changes of gene expression profiles. The method does not require precedent array normalization. We applied the method to elucidate possibly important miRNAs during embryonic stem (ES) cell differentiation to neuronal cells and we infer that certain miRNAs, including miR-200 family, miR-429, miR-302 family, and miR-17-92 cluster members may be important to the maintenance of undifferentiated status in ES cells.

  15. Inference of Gene Regulation via miRNAs During ES Cell Differentiation Using MiRaGE Method

    PubMed Central

    Yoshizawa, Masato; Taguchi, Y-h.; Yasuda, Jun

    2011-01-01

    MicroRNA (miRNA) is a critical regulator of cell growth, differentiation, and development. To identify important miRNAs in a biological process, many bioinformatical tools have been developed. We have developed MiRaGE (MiRNA Ranking by Gene Expression) method to infer the regulation of gene expression by miRNAs from changes of gene expression profiles. The method does not require precedent array normalization. We applied the method to elucidate possibly important miRNAs during embryonic stem (ES) cell differentiation to neuronal cells and we infer that certain miRNAs, including miR-200 family, miR-429, miR-302 family, and miR-17-92 cluster members may be important to the maintenance of undifferentiated status in ES cells. PMID:22272132

  16. Epidrug mediated re-expression of miRNA targeting the HMGA transcripts in pituitary cells.

    PubMed

    Kitchen, Mark O; Yacqub-Usman, Kiren; Emes, Richard D; Richardson, Alan; Clayton, Richard N; Farrell, William E

    2015-10-01

    Transgenic mice overexpressing the high mobility group A (HMGA) genes, Hmga1 or Hmga2 develop pituitary tumours and their overexpression is also a frequent finding in human pituitary adenomas. In some cases, increased expression of HMGA2 but not that of HMGA1 is consequent to genetic perturbations. However, recent studies show that down-regulation of microRNA (miRNA), that contemporaneously target the HMGA1 and HMGA2 transcripts, are associated with their overexpression. In a cohort of primary pituitary adenoma we determine the impact of epigenetic modifications on the expression of HMGA-targeting miRNA. For these miRNAs, chromatin immunoprecipitations showed that transcript down-regulation is correlated with histone tail modifications associated with condensed silenced genes. The functional impact of epigenetic modification on miRNA expression was determined in the rodent pituitary cell line, GH3. In these cells, histone tail, miRNA-associated, modifications were similar to those apparent in human adenoma and likely account for their repression. Indeed, challenge of GH3 cells with the epidrugs, zebularine and TSA, led to enrichment of the histone modification, H3K9Ac, associated with active genes, and depletion of the modification, H3K27me3, associated with silent genes and re-expression of HMGA-targeting miRNA. Moreover, epidrugs challenges were also associated with a concomitant decrease in hmga1 transcript and protein levels and concurrent increase in bmp-4 expression. These findings show that the inverse relationship between HMGA expression and targeting miRNA is reversible through epidrug interventions. In addition to showing a mechanistic link between epigenetic modifications and miRNA expression these findings underscore their potential as therapeutic targets in this and other diseases.

  17. MiRNAs in β-Cell Development, Identity, and Disease

    PubMed Central

    Martinez-Sanchez, Aida; Rutter, Guy A.; Latreille, Mathieu

    2017-01-01

    Pancreatic β-cells regulate glucose metabolism by secreting insulin, which in turn stimulates the utilization or storage of the sugar by peripheral tissues. Insulin insufficiency and a prolonged period of insulin resistance are usually the core components of type 2 diabetes (T2D). Although, decreased insulin levels in T2D have long been attributed to a decrease in β-cell function and/or mass, this model has recently been refined with the recognition that a loss of β-cell “identity” and dedifferentiation also contribute to the decline in insulin production. MicroRNAs (miRNAs) are key regulatory molecules that display tissue-specific expression patterns and maintain the differentiated state of somatic cells. During the past few years, great strides have been made in understanding how miRNA circuits impact β-cell identity. Here, we review current knowledge on the role of miRNAs in regulating the acquisition of the β-cell fate during development and in maintaining mature β-cell identity and function during stress situations such as obesity, pregnancy, aging, or diabetes. We also discuss how miRNA function could be harnessed to improve our ability to generate β-cells for replacement therapy for T2D. PMID:28123396

  18. Cell-type-specific profiling of protein-DNA interactions without cell isolation using targeted DamID with next-generation sequencing.

    PubMed

    Marshall, Owen J; Southall, Tony D; Cheetham, Seth W; Brand, Andrea H

    2016-09-01

    This protocol is an extension to: Nat. Protoc. 2, 1467-1478 (2007); doi:10.1038/nprot.2007.148; published online 7 June 2007The ability to profile transcription and chromatin binding in a cell-type-specific manner is a powerful aid to understanding cell-fate specification and cellular function in multicellular organisms. We recently developed targeted DamID (TaDa) to enable genome-wide, cell-type-specific profiling of DNA- and chromatin-binding proteins in vivo without cell isolation. As a protocol extension, this article describes substantial modifications to an existing protocol, and it offers additional applications. TaDa builds upon DamID, a technique for detecting genome-wide DNA-binding profiles of proteins, by coupling it with the GAL4 system in Drosophila to enable both temporal and spatial resolution. TaDa ensures that Dam-fusion proteins are expressed at very low levels, thus avoiding toxicity and potential artifacts from overexpression. The modifications to the core DamID technique presented here also increase the speed of sample processing and throughput, and adapt the method to next-generation sequencing technology. TaDa is robust, reproducible and highly sensitive. Compared with other methods for cell-type-specific profiling, the technique requires no cell-sorting, cross-linking or antisera, and binding profiles can be generated from as few as 10,000 total induced cells. By profiling the genome-wide binding of RNA polymerase II (Pol II), TaDa can also identify transcribed genes in a cell-type-specific manner. Here we describe a detailed protocol for carrying out TaDa experiments and preparing the material for next-generation sequencing. Although we developed TaDa in Drosophila, it should be easily adapted to other organisms with an inducible expression system. Once transgenic animals are obtained, the entire experimental procedure-from collecting tissue samples to generating sequencing libraries-can be accomplished within 5 d.

  19. MiRNA-133b promotes the proliferation of human Sertoli cells through targeting GLI3

    PubMed Central

    Yao, Chencheng; Sun, Min; Yuan, Qingqing; Niu, Minghui; Chen, Zheng; Hou, Jingmei; Wang, Hong; Wen, Liping; Liu, Yun; Li, Zheng; He, Zuping

    2016-01-01

    Sertoli cells play critical roles in regulating spermatogenesis and they can be reprogrammed to the cells of other lineages, highlighting that they have significant applications in reproductive and regenerative medicine. The fate determinations of Sertoli cells are regulated precisely by epigenetic factors. However, the expression, roles, and targets of microRNA (miRNA) in human Sertoli cells remain unknown. Here we have for the first time revealed that 174 miRNAs were distinctly expressed in human Sertoli cells between Sertoli-cell-only syndrome (SCOS) patients and obstructive azoospermia (OA) patients with normal spermatogenesis using miRNA microarrays and real time PCR, suggesting that these miRNAs may be associated with the pathogenesis of SCOS. MiR-133b is upregulated in Sertoli cells of SCOS patients compared to OA patients. Proliferation assays with miRNA mimics and inhibitors showed that miR-133b enhanced the proliferation of human Sertoli cells. Moreover, we demonstrated that GLI3 was a direct target of miR-133b and the expression of Cyclin B1 and Cyclin D1 was enhanced by miR-133b mimics but decreased by its inhibitors. Gene silencing of GLI3 using RNA inference stimulated the growth of human Sertoli cells. Collectively, miR-133b promoted the proliferation of human Sertoli cells by targeting GLI3. This study thus sheds novel insights into epigenetic regulation of human Sertoli cells and the etiology of azoospermia and offers new targets for treating male infertility PMID:26755652

  20. Regulation of miRNAs Affects Radiobiological Response of Lung Cancer Stem Cells

    PubMed Central

    Xu, Yan-mei; Liao, Xing-yun; Chen, Xie-wan; Li, De-zhi; Sun, Jian-guo; Liao, Rong-xia

    2015-01-01

    Radiotherapy (RT) is a key therapeutic strategy for lung cancer, the most common cause of cancer-related deaths worldwide, but radioresistance often occurs and leads to failure of RT. It is therefore important to clarify the mechanism underlying radioresistance in lung cancer. Cancer stem cells (CSCs) are considered the fundamental reason for radioresistance. MicroRNAs (miRNAs) have been regarded as important regulatory molecules of CSCs, carcinogenesis, and treatment response of cancers. It is crucial to clarify how regulation of miRNAs affects repair of DNA damage, redistribution, repopulation, reoxygenation, and radiosensitivity (5R) of lung cancer stem cells (LCSCs). A thorough understanding of the regulation of miRNAs affecting 5R of LCSCs has potential impact on identifying novel targets and thus may improve the efficacy of lung cancer radiotherapy. PMID:25815339

  1. miRNAs modified by dietary lipids in Caco-2 cells. A microarray screening

    PubMed Central

    Daimiel, Lidia; Ordovás, Jose Mª.; Dávalos, Alberto

    2015-01-01

    We performed a screening of miRNAs regulated by dietary lipids in a cellular model of enterocytes, Caco-2 cells. Our aim was to describe new lipid-modified miRNAs with an implication in lipid homeostasis and cardiovascular disease [1], [2]. For that purpose, we treated differentiated Caco-2 cells with micelles containing the assayed lipids (cholesterol, conjugated linoleic acid and docosahexaenoic acid) and the screening of miRNAs was carried out by microarray using the μParaflo®Microfluidic Biochip Technology of LC Sciences (Huston, TX, USA). Experimental design, microarray description and raw data have been made available in the GEO database with the reference number of GSE59153. Here we described in detail the experimental design and methods used to obtain the relative expression data. PMID:26484250

  2. Monitoring cell physiology by expression profiles and discovering cell type-specific genes by compiled expression profiles

    SciTech Connect

    Okubo, Kousaku; Itoh, Kouichi; Fukushima, Atsushi; Yoshii, Junji; Matsubara, Kenichi

    1995-11-20

    A gene expression profile is the list showing the expressed gene species and the abundance of their transcripts in a given cell or tissue. This list is made by constructing 3{prime}-directed cDNA libraries consisting of only the 3{prime}-termini of mRNA and sequencing randomly selected clones from such libraries: genes are identified by the sequences, and the composition of mRNA, which reflects gene activities, is measured from the frequency of appearance of the gene transcripts. For practical reasons, the number of sequenced clones has been limited to approximately 1000 per library at present, but the resulting profile covers almost all highly or moderately expressed genes, along with many less active genes. We constructed expression profiles from the HL60 human promyelocytic cell line and two of its derivatives, granulocytoids induced by DMSO and monocytoids induced by TPA. In HL60, a significant fraction of the abundantly expressed genes was for protein synthesis. Upon induction, these genes were partially or totally silenced; transcripts for proteins that characterize the granulocytes and monocyte-macrophages became abundant. By compiling and comparing different expression profiles, genes can be categorized into those expressed in diverse cell types and those active only in limited cell types. Although at present, the number of expression profiles that can be compiled is limited and this categorization is applicable only to abundantly expressed genes, 13 novel genes that may represent granulocyte- or monocyte-specific functions have been discovered. 37 refs., 1 fig., 2 tabs.

  3. Delta-9-tetrahydrocannabinol accumulation, metabolism and cell-type-specific adverse effects in aggregating brain cell cultures.

    PubMed

    Monnet-Tschudi, Florianne; Hazekamp, Arno; Perret, Nicolas; Zurich, Marie-Gabrielle; Mangin, Patrice; Giroud, Christian; Honegger, Paul

    2008-04-01

    Despite the widespread use of Cannabis as recreational drug or as medicine, little is known about its toxicity. The accumulation, metabolism and toxicity of THC were analyzed 10 days after a single treatment, and after repeated exposures during 10 days. Mixed-cell aggregate cultures of fetal rat telencephalon were used as in vitro model, as well as aggregates enriched either in neurons or in glial cells. It was found that THC accumulated preferentially in neurons, and that glia-neuron interactions decreased THC accumulation. The quantification of 11-OH-THC and of THC-COOH showed that brain aggregates were capable of THC metabolism. No cell-type difference was found for the metabolite 11-OH-THC, whereas the THC-COOH content was higher in mixed-cell cultures. No cell death was found at THC concentrations of 2 microM in single treatment and of 1 microM and 2 microM in repeated treatments. Neurons, and particularly GABAergic neurons, were most sensitive to THC. Only the GABAergic marker was affected after the single treatment, whereas the GABAergic, cholinergic and astrocytic markers were decreased after the repeated treatments. JWH 015, a CB2 receptor agonist, showed effects similar to THC, whereas ACEA, a CB1 receptor agonist, had no effect. The expression of the cytokine IL-6 was upregulated 48 h after the single treatment with 5 microM of THC or JWH 015, whereas the expression of TNF-alpha remained unchanged. These results suggest that the adverse effects of THC were related either to THC accumulation or to cannabinoid receptor activation and associated with IL-6 upregulation.

  4. Delta-9-tetrahydrocannabinol accumulation, metabolism and cell-type-specific adverse effects in aggregating brain cell cultures

    SciTech Connect

    Monnet-Tschudi, Florianne Hazekamp, Arno; Perret, Nicolas; Zurich, Marie-Gabrielle; Mangin, Patrice; Giroud, Christian; Honegger, Paul

    2008-04-01

    Despite the widespread use of Cannabis as recreational drug or as medicine, little is known about its toxicity. The accumulation, metabolism and toxicity of THC were analyzed 10 days after a single treatment, and after repeated exposures during 10 days. Mixed-cell aggregate cultures of fetal rat telencephalon were used as in vitro model, as well as aggregates enriched either in neurons or in glial cells. It was found that THC accumulated preferentially in neurons, and that glia-neuron interactions decreased THC accumulation. The quantification of 11-OH-THC and of THC-COOH showed that brain aggregates were capable of THC metabolism. No cell-type difference was found for the metabolite 11-OH-THC, whereas the THC-COOH content was higher in mixed-cell cultures. No cell death was found at THC concentrations of 2 {mu}M in single treatment and of 1 {mu}M and 2 {mu}M in repeated treatments. Neurons, and particularly GABAergic neurons, were most sensitive to THC. Only the GABAergic marker was affected after the single treatment, whereas the GABAergic, cholinergic and astrocytic markers were decreased after the repeated treatments. JWH 015, a CB2 receptor agonist, showed effects similar to THC, whereas ACEA, a CB1 receptor agonist, had no effect. The expression of the cytokine IL-6 was upregulated 48 h after the single treatment with 5 {mu}M of THC or JWH 015, whereas the expression of TNF-{alpha} remained unchanged. These results suggest that the adverse effects of THC were related either to THC accumulation or to cannabinoid receptor activation and associated with IL-6 upregulation.

  5. Regulation of PP2Cm expression by miRNA-204/211 and miRNA-22 in mouse and human cells

    PubMed Central

    Pan, Bang-fen; Gao, Chen; Ren, Shu-xun; Wang, Yi-bin; Sun, Hai-peng; Zhou, Mei-yi

    2015-01-01

    Aim: The mitochondrial targeted 2C-type serine/threonine protein phosphatase (PP2Cm) is encoded by the gene PPM1K and is highly conserved among vertebrates. PP2Cm plays a critical role in branched-chain amino acid catabolism and regulates cell survival. Its expression is dynamically regulated by the nutrient environment and pathological stresses. However, little is known about the molecular mechanism underlying the regulation of PPM1K gene expression. In this study, we aimed to reveal how PPM1K expression is affected by miRNA-mediated post-transcriptional regulation. Methods: Computational analysis based on conserved miRNA binding motifs was applied to predict the candidate miRNAs that potentially affect PPM1K expression. Dual-luciferase reporter assay was performed to verify the miRNAs' binding sites in the PPM1K gene and their influence on PPM1K 3′UTR activity. We further over-expressed the mimics of these miRNAs in human and mouse cells to examine whether miRNAs affected the mRNA level of PPM1K. Results: Computational analysis identified numerous miRNAs potentially targeting PPM1K. Luciferase reporter assays demonstrated that the 3′UTR of PPM1K gene contained the recognition sites of miR-204 and miR-211. Overexpression of these miRNAs in human and mouse cells diminished the 3′UTR activity and the endogenous mRNA level of PPM1K. However, the miR-22 binding site was found only in human and not mouse PPM1K 3′UTR. Accordingly, PPM1K 3′UTR activity was suppressed by miR-22 overexpression in human but not mouse cells. Conclusion: These data suggest that different miRNAs contribute to the regulation of PP2Cm expression in a species-specific manner. miR-204 and miR-211 are efficient in both mouse and human cells, while miR-22 regulates PP2Cm expression only in human cells. PMID:26592513

  6. A high-throughput screen identifies miRNA inhibitors regulating lung cancer cell survival and response to paclitaxel

    PubMed Central

    Du, Liqin; Borkowski, Robert; Zhao, Zhenze; Ma, Xiuye; Yu, Xiaojie; Xie, Xian-Jin; Pertsemlidis, Alexander

    2013-01-01

    microRNAs (miRNAs) are small RNAs endogenously expressed in multiple organisms that regulate gene expression largely by decreasing levels of target messenger RNAs (mRNAs). Over the past few years, numerous studies have demonstrated critical roles for miRNAs in the pathogenesis of many cancers, including lung cancer. Cellular miRNA levels can be easily manipulated, showing the promise of developing miRNA-targeted oligos as next-generation therapeutic agents. In a comprehensive effort to identify novel miRNA-based therapeutic agents for lung cancer treatment, we combined a high-throughput screening platform with a library of chemically synthesized miRNA inhibitors to systematically identify miRNA inhibitors that reduce lung cancer cell survival and those that sensitize cells to paclitaxel. By screening three lung cancer cell lines with different genetic backgrounds, we identified miRNA inhibitors that potentially have a universal cytotoxic effect on lung cancer cells and miRNA inhibitors that sensitize cells to paclitaxel treatment, suggesting the potential of developing these miRNA inhibitors as therapeutic agents for lung cancer. We then focused on characterizing the inhibitors of three miRNAs (miR-133a/b, miR-361-3p, and miR-346) that have the most potent effect on cell survival. We demonstrated that two of the miRNA inhibitors (miR-133a/b and miR-361-3p) decrease cell survival by activating caspase-3/7-dependent apoptotic pathways and inducing cell cycle arrest in S phase. Future studies are certainly needed to define the mechanisms by which the identified miRNA inhibitors regulate cell survival and drug response, and to explore the potential of translating the current findings into clinical applications. PMID:24157646

  7. Characterisation of CD4 T cells in healthy and diseased koalas (Phascolarctos cinereus) using cell-type-specific monoclonal antibodies.

    PubMed

    Mangar, Chandan; Armitage, Charles W; Timms, Peter; Corcoran, Lynn M; Beagley, Kenneth W

    2016-07-01

    The koala (Phascolarctos cinereus) is an arboreal herbivorous marsupial that is an Australian icon. Koalas in many parts of Australia are under multiple threats including habitat destruction, dog attacks, vehicular accidents, and infectious diseases such as Chlamydia spp. and the koala retrovirus (KoRV), which may contribute to the incidence of lymphoma and leukaemia in this species. Due to a lack of koala-specific immune reagents and assays there is currently no way to adequately analyse the immune response in healthy, diseased or vaccinated animals. This paper reports the production and characterisation of the first anti-koala CD4 monoclonal antibody (mAb). The koala CD4 gene was identified and used to develop recombinant proteins for mAb production. Fluorochrome-conjugated anti-CD4 mAb was used to measure the levels of CD4(+) lymphocytes collected from koala spleens (41.1%, range 20-45.1%) lymph nodes (36.3%, range 19-55.9%) and peripheral blood (23.8%, range 17.3-35%) by flow cytometry. Biotin-conjugated anti-CD4 mAb was used for western blot to determine an approximate size of 52 kDa for the koala CD4 molecule and used in immunohistochemistry to identify CD4(+) cells in the paracortical region and germinal centres of spleen and lymph nodes. Using the anti-CD4 mab we showed that CD4 cells from vaccinated, but not control, koalas proliferated following in vitro stimulation with UV-inactivated Chlamydia pecorum and recombinant chlamydial antigens. Since CD4(+) T cells have been shown to play a pivotal role in clearing chlamydial infection in both human and mouse infections, using this novel antibody will help determine the role CD4(+) T cells play in protection against chlamydial infection in koalas and also enhance our knowledge of how KoRV affects the koala immune system.

  8. Integrative miRNA and Gene Expression Profiling Analysis of Human Quiescent Hepatic Stellate Cells.

    PubMed

    Coll, Mar; El Taghdouini, Adil; Perea, Luis; Mannaerts, Inge; Vila-Casadesús, Maria; Blaya, Delia; Rodrigo-Torres, Daniel; Affò, Silvia; Morales-Ibanez, Oriol; Graupera, Isabel; Lozano, Juan José; Najimi, Mustapha; Sokal, Etienne; Lambrecht, Joeri; Ginès, Pere; van Grunsven, Leo A; Sancho-Bru, Pau

    2015-06-22

    Unveiling the regulatory pathways maintaining hepatic stellate cells (HSC) in a quiescent (q) phenotype is essential to develop new therapeutic strategies to treat fibrogenic diseases. To uncover the miRNA-mRNA regulatory interactions in qHSCs, HSCs were FACS-sorted from healthy livers and activated HSCs (aHSCs) were generated in vitro. MiRNA Taqman array analysis showed HSCs expressed a low number of miRNAs (n = 259), from which 47 were down-regulated and 212 up-regulated upon activation. Computational integration of miRNA and gene expression profiles revealed that 66% of qHSC-associated miRNAs correlated with more than 6 altered target mRNAs (17,28 ± 10,7 targets/miRNA) whereas aHSC-associated miRNAs had an average of 1,49 targeted genes. Interestingly, interaction networks generated by miRNA-targeted genes in qHSCs were associated with key HSC activation processes. Next, selected miRNAs were validated in healthy and cirrhotic human livers and miR-192 was chosen for functional analysis. Down-regulation of miR-192 in HSCs was found to be an early event during fibrosis progression in mouse models of liver injury. Moreover, mimic assays for miR-192 in HSCs revealed its role in HSC activation, proliferation and migration. Together, these results uncover the importance of miRNAs in the maintenance of the qHSC phenotype and form the basis for understanding the regulatory networks in HSCs.

  9. Integrative miRNA and Gene Expression Profiling Analysis of Human Quiescent Hepatic Stellate Cells

    PubMed Central

    Coll, Mar; Taghdouini, Adil El; Perea, Luis; Mannaerts, Inge; Vila-Casadesús, Maria; Blaya, Delia; Rodrigo-Torres, Daniel; Affò, Silvia; Morales-Ibanez, Oriol; Graupera, Isabel; Lozano, Juan José; Najimi, Mustapha; Sokal, Etienne; Lambrecht, Joeri; Ginès, Pere; van Grunsven, Leo A.; Sancho-Bru, Pau

    2015-01-01

    Unveiling the regulatory pathways maintaining hepatic stellate cells (HSC) in a quiescent (q) phenotype is essential to develop new therapeutic strategies to treat fibrogenic diseases. To uncover the miRNA-mRNA regulatory interactions in qHSCs, HSCs were FACS-sorted from healthy livers and activated HSCs (aHSCs) were generated in vitro. MiRNA Taqman array analysis showed HSCs expressed a low number of miRNAs (n = 259), from which 47 were down-regulated and 212 up-regulated upon activation. Computational integration of miRNA and gene expression profiles revealed that 66% of qHSC-associated miRNAs correlated with more than 6 altered target mRNAs (17,28 ± 10,7 targets/miRNA) whereas aHSC-associated miRNAs had an average of 1,49 targeted genes. Interestingly, interaction networks generated by miRNA-targeted genes in qHSCs were associated with key HSC activation processes. Next, selected miRNAs were validated in healthy and cirrhotic human livers and miR-192 was chosen for functional analysis. Down-regulation of miR-192 in HSCs was found to be an early event during fibrosis progression in mouse models of liver injury. Moreover, mimic assays for miR-192 in HSCs revealed its role in HSC activation, proliferation and migration. Together, these results uncover the importance of miRNAs in the maintenance of the qHSC phenotype and form the basis for understanding the regulatory networks in HSCs. PMID:26096707

  10. Transient Gene and miRNA Expression Profile Changes of Confluent Human Fibroblast Cells in Space

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Lu, Tao; Wong, Michael; Feiveson, Alan; Stodieck, Louis; Karouia, Fathi; Wang, Xiaoyu; Wu, Honglu

    2015-01-01

    Microgravity or an altered gravity environment from the static 1 gravitational constant has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies conducted in space or using simulated microgravity on the ground have focused on the growth or differentiation of the cells. Whether non-dividing cultured cells will sense the presence of microgravity in space has not been specifically addressed. In an experiment conducted on the International Space Station, confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 days for investigations of gene and miRNA (microRNA) expression profile changes in these cells. A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the structural framework for tissues, and plays a critical role in wound healing and other functions. Results of the experiment showed that on Day 3, both the flown and ground cells were still proliferating slowly even though they were confluent, as measured by the expression of the protein Ki-67 positive cells, and the cells in space grew slightly faster. Gene and miRNA expression data indicated activation of NF(sub kappa)B (nuclear factor kappa-light-chain-enhancer of activated B cells) and other growth related pathways involving HGF and VEGF in the flown cells. On Day 14 when the cells were mostly non-dividing, the gene and miRNA expression profiles between the flight and ground samples were indistinguishable. Comparison of gene and miRNA expressions in the Day 3 samples in respect to Day 14 revealed that most of the changes observed on Day 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeleton changes by immunohistochemistry staining of the cells with antibodies for alpha-tubulin showed no difference between the flight and ground samples. Results of our study suggest that in true non-dividing human fibroblast cells, microgravity in

  11. miRNA Signature of Mouse Helper T Cell Hyper-Proliferation

    PubMed Central

    Sommers, Connie L.; Rouquette-Jazdanian, Alexandre K.; Robles, Ana I.; Kortum, Robert L.; Merrill, Robert K.; Li, Wenmei; Nath, Nandan; Wohlfert, Elizabeth; Sixt, Katherine M.; Belkaid, Yasmine; Samelson, Lawrence E.

    2013-01-01

    Helper T cells from a mutant mouse model, LAT Y136F, hyper-proliferate and cause a severe lymphoproliferative disease that kills the mice by six months of age. LAT Y136F mice carry a tyrosine to phenylalanine mutation in the Linker for Activation of T cells (LAT) gene. This mutation leads to a number of changes in T cells that result in altered cytokine production including increased IL-4 production, increased proliferation, and decreased apoptosis. Hyper-proliferation of the mutant T cells contributes to lymphadenopathy, splenomegaly, and multi-organ T cell infiltration. miRNAs are short non-coding RNAs that regulate expression of cohorts of genes. This study investigates which miRNAs are expressed in LAT Y136F T cells and compares these to miRNAs expressed in wild type T cells that are undergoing proliferation in two other settings. The first setting is homeostatic proliferation, which was modeled by adoptive transfer of wild type T cells into T cell-deficient mice. The second setting is proliferation in response to infection, which was modeled by infection of wild type mice with the nematode H. polygyrus. By comparing miRNA expression in these three proliferative states (LAT Y136F hyper-proliferation, homeostatic proliferation and proliferation in response to H. polygyrus infection) to expression in wild type naïve CD4+ T cells, we found miRNAs that were highly regulated in all three proliferative states (miR-21 and miR-146a) and some that were more specific to individual settings of proliferation such as those more specific for LAT Y136F lymphoproliferative disease (miR-669f, miR-155 and miR-466a/b). Future experiments that modulate levels of the miRNAs identified in this study may reveal the roles of these miRNAs in T cell proliferation and/or lymphoproliferative disease. PMID:23825558

  12. Developmental and cell type-specific expression of thyroid hormone transporters in the mouse brain and in primary brain cells.

    PubMed

    Braun, Doreen; Kinne, Anita; Bräuer, Anja U; Sapin, Remy; Klein, Marc O; Köhrle, Josef; Wirth, Eva K; Schweizer, Ulrich

    2011-03-01

    Cellular thyroid hormone uptake and efflux are mediated by transmembrane transport proteins. One of these, monocarboxylate transporter 8 (MCT8) is mutated in Allan-Herndon-Dudley syndrome, a severe mental retardation associated with abnormal thyroid hormone constellations. Since mice deficient in Mct8 exhibit a milder neurological phenotype than patients, we hypothesized that alternative thyroid hormone transporters may compensate in murine brain cells for the lack of Mct8. Using qPCR, Western Blot, and immunocytochemistry, we investigated the expression of three different thyroid hormone transporters, i.e., Mct8 and L-type amino acid transporters Lat1 and Lat2, in mouse brain. All three thyroid hormone transporters are expressed from corticogenesis and peak around birth. Primary cultures of neurons and astrocytes express Mct8, Lat1, and Lat2. Microglia specifically expresses Mct10 and Slco4a1 in addition to high levels of Lat2 mRNA and protein. As in vivo, a brain microvascular endothelial cell line expressed Mct8 and Lat1. 158N, an oligodendroglial cell line expressed Mct8 protein, consistent with delayed myelination in MCT8-deficient patients. Functional T(3)- and T(4)-transport assays into primary astrocytes showed K(M) values of 4.2 and 3.7 μM for T(3) and T(4). Pharmacological inhibition of L-type amino acid transporters by BCH and genetic inactivation of Lat2 reduced astrocytic T(3) uptake to the same extent. BSP, a broad spectrum inhibitor, including Mct8, reduced T(3) uptake further suggesting the cooperative activity of several T(3) transporters in astrocytes.

  13. Dose-dependent and cell type-specific cell death and proliferation following in vitro exposure to radial extracorporeal shock waves

    PubMed Central

    Hochstrasser, Tanja; Frank, Hans-Georg; Schmitz, Christoph

    2016-01-01

    Radial extracorporeal shock wave (rESW) therapy is widely used in musculoskeletal disorders and wound repair. However, the mechanisms of action are still largely unknown. The current study compared the effects of rESWs on two cell types. Human fetal foreskin fibroblasts (HFFF2) and human placental choriocarcinoma cell line JEG-3 were exposed to 0, 100, 200, 500 or 5000 rESWs generated with a Swiss DolorClast device (2.5 bar, 1 Hz). FACS analysis immediately after rESW exposure showed that initially, rESWs rather induced mechanical cell destruction than regulated or programmed cell death. Cell damage was nearly negated by reducing cavitation. Furthermore, cell viability decreased progressively with higher numbers of rESWs. Exposure to rESWs had no impact on growth potential of JEG-3 cells, but dose-dependently increased growth potential of HFFF2 cells. Cultivation of cells that were initially exposed to sham-rESWs in conditioned media increased the growth potential of HFFF2 cells, nevertheless, an even stronger effect was achieved by direct exposure to rESWs. Additionally, cell cycle distribution analysis demonstrated a shift in proportion from G0/G1 to G2/M phase in HFFF2 cells, but not in JEG-3 cells. These data demonstrate that rESWs leads to initial and subsequent dose-dependent and cell type-specific effects in vitro. PMID:27477873

  14. The cell cycle- and insulin-signaling-inhibiting miRNA expression pattern of very small embryonic-like stem cells contributes to their quiescent state.

    PubMed

    Maj, Magdalena; Schneider, Gabriela; Ratajczak, Janina; Suszynska, Malwina; Kucia, Magda; Ratajczak, Mariusz Z

    2015-08-01

    Murine Oct4(+), very small embryonic-like stem cells (VSELs), are a quiescent stem cell population that requires a supportive co-culture layer to proliferate and/or to differentiate in vitro. Gene expression studies have revealed that the quiescence of these cells is due to changes in expression of parentally imprinted genes, including genes involved in cell cycle regulation and insulin and insulin-like growth factor signaling (IIS). To investigate the role of microRNAs (miRNAs) in VSEL quiescence, we performed miRNA studies in highly purified VSELs and observed a unique miRNA expression pattern in these cells. Specifically, we observed significant differences in the expression of certain miRNA species (relative to a reference cell population), including (i) miRNA-25_1 and miRNA-19 b, whose downregulation has the effect of upregulating cell cycle checkpoint genes and (ii) miRNA-675-3 p and miRNA-675-5 p, miRNA-292-5 p, miRNA-184, and miRNA-125 b, whose upregulation attenuates IIS. These observations are important for understanding the biology of these cells and for developing efficient ex vivo expansion strategies for VSELs isolated from adult tissues.

  15. Identification of miRNAs differentially expressed in human epilepsy with or without granule cell pathology.

    PubMed

    Zucchini, Silvia; Marucci, Gianluca; Paradiso, Beatrice; Lanza, Giovanni; Roncon, Paolo; Cifelli, Pierangelo; Ferracin, Manuela; Giulioni, Marco; Michelucci, Roberto; Rubboli, Guido; Simonato, Michele

    2014-01-01

    The microRNAs (miRNAs) are small size non-coding RNAs that regulate expression of target mRNAs at post-transcriptional level. miRNAs differentially expressed under pathological conditions may help identifying mechanisms underlying the disease and may represent biomarkers with prognostic value. However, this kind of studies are difficult in the brain because of the cellular heterogeneity of the tissue and of the limited access to fresh tissue. Here, we focused on a pathology affecting specific cells in a subpopulation of epileptic brains (hippocampal granule cells), an approach that bypasses the above problems. All patients underwent surgery for intractable temporal lobe epilepsy and had hippocampal sclerosis associated with no granule cell pathology in half of the cases and with type-2 granule cell pathology (granule cell layer dispersion or bilamination) in the other half. The expression of more than 1000 miRNAs was examined in the laser-microdissected dentate granule cell layer. Twelve miRNAs were differentially expressed in the two groups. One of these, miR487a, was confirmed to be expressed at highly differential levels in an extended cohort of patients, using RT-qPCR. Bioinformatics searches and RT-qPCR verification identified ANTXR1 as a possible target of miR487a. ANTXR1 may be directly implicated in granule cell dispersion because it is an adhesion molecule that favors cell spreading. Thus, miR487a could be the first identified element of a miRNA signature that may be useful for prognostic evaluation of post-surgical epilepsy and may drive mechanistic studies leading to the identification of therapeutic targets.

  16. Identification of miRNAs Differentially Expressed in Human Epilepsy with or without Granule Cell Pathology

    PubMed Central

    Paradiso, Beatrice; Lanza, Giovanni; Roncon, Paolo; Cifelli, Pierangelo; Ferracin, Manuela; Giulioni, Marco; Michelucci, Roberto; Simonato, Michele

    2014-01-01

    The microRNAs (miRNAs) are small size non-coding RNAs that regulate expression of target mRNAs at post-transcriptional level. miRNAs differentially expressed under pathological conditions may help identifying mechanisms underlying the disease and may represent biomarkers with prognostic value. However, this kind of studies are difficult in the brain because of the cellular heterogeneity of the tissue and of the limited access to fresh tissue. Here, we focused on a pathology affecting specific cells in a subpopulation of epileptic brains (hippocampal granule cells), an approach that bypasses the above problems. All patients underwent surgery for intractable temporal lobe epilepsy and had hippocampal sclerosis associated with no granule cell pathology in half of the cases and with type-2 granule cell pathology (granule cell layer dispersion or bilamination) in the other half. The expression of more than 1000 miRNAs was examined in the laser-microdissected dentate granule cell layer. Twelve miRNAs were differentially expressed in the two groups. One of these, miR487a, was confirmed to be expressed at highly differential levels in an extended cohort of patients, using RT-qPCR. Bioinformatics searches and RT-qPCR verification identified ANTXR1 as a possible target of miR487a. ANTXR1 may be directly implicated in granule cell dispersion because it is an adhesion molecule that favors cell spreading. Thus, miR487a could be the first identified element of a miRNA signature that may be useful for prognostic evaluation of post-surgical epilepsy and may drive mechanistic studies leading to the identification of therapeutic targets. PMID:25148080

  17. Potential Inhibitory Influence of miRNA 210 on Regulatory T Cells during Epicutaneous Chemical Sensitization

    PubMed Central

    Long, Carrie Mae; Lukomska, Ewa; Marshall, Nikki B.; Nayak, Ajay; Anderson, Stacey E.

    2016-01-01

    Toluene diisocyanate (TDI) is a potent low molecular weight chemical sensitizer and a leading cause of chemical-induced occupational asthma. The regulatory potential of microRNAs (miRNAs) has been recognized in a variety of disease states, including allergic disease; however, the roles of miRNAs in chemical sensitization are largely unknown. In a previous work, increased expression of multiple miRNAs during TDI sensitization was observed and several putative mRNA targets identified for these miRNAs were directly related to regulatory T-cell (Treg) differentiation and function including Foxp3 and Runx3. In this work, we show that miR-210 expression is increased in the mouse draining lymph node (dLN) and Treg subsets following dermal TDI sensitization. Alterations in dLN mRNA and protein expression of Treg related genes/putative miR-210 targets (foxp3, runx3, ctla4, and cd25) were observed at multiple time points following TDI exposure and in ex vivo systems. A Treg suppression assay, including a miR-210 mimic, was utilized to investigate the suppressive ability of Tregs. Cells derived from TDI sensitized mice treated with miR-210 mimic had less expression of miR-210 compared to the acetone control suggesting other factors, such as additional miRNAs, might be involved in the regulation of the functional capabilities of these cells. These novel findings indicate that miR-210 may have an inhibitory role in Treg function during TDI sensitization. Because the functional roles of miRNAs have not been previously elucidated in a model of chemical sensitization, these data contribute to the understanding of the potential immunologic mechanisms of chemical induced allergic disease. PMID:28035981

  18. Integrative Modeling of eQTLs and Cis-Regulatory Elements Suggests Mechanisms Underlying Cell Type Specificity of eQTLs

    PubMed Central

    Brown, Christopher D.; Mangravite, Lara M.; Engelhardt, Barbara E.

    2013-01-01

    Genetic variants in cis-regulatory elements or trans-acting regulators frequently influence the quantity and spatiotemporal distribution of gene transcription. Recent interest in expression quantitative trait locus (eQTL) mapping has paralleled the adoption of genome-wide association studies (GWAS) for the analysis of complex traits and disease in humans. Under the hypothesis that many GWAS associations tag non-coding SNPs with small effects, and that these SNPs exert phenotypic control by modifying gene expression, it has become common to interpret GWAS associations using eQTL data. To fully exploit the mechanistic interpretability of eQTL-GWAS comparisons, an improved understanding of the genetic architecture and causal mechanisms of cell type specificity of eQTLs is required. We address this need by performing an eQTL analysis in three parts: first we identified eQTLs from eleven studies on seven cell types; then we integrated eQTL data with cis-regulatory element (CRE) data from the ENCODE project; finally we built a set of classifiers to predict the cell type specificity of eQTLs. The cell type specificity of eQTLs is associated with eQTL SNP overlap with hundreds of cell type specific CRE classes, including enhancer, promoter, and repressive chromatin marks, regions of open chromatin, and many classes of DNA binding proteins. These associations provide insight into the molecular mechanisms generating the cell type specificity of eQTLs and the mode of regulation of corresponding eQTLs. Using a random forest classifier with cell specific CRE-SNP overlap as features, we demonstrate the feasibility of predicting the cell type specificity of eQTLs. We then demonstrate that CREs from a trait-associated cell type can be used to annotate GWAS associations in the absence of eQTL data for that cell type. We anticipate that such integrative, predictive modeling of cell specificity will improve our ability to understand the mechanistic basis of human complex phenotypic

  19. Dissecting miRNA gene repression on single cell level with an advanced fluorescent reporter system

    PubMed Central

    Lemus-Diaz, Nicolas; Böker, Kai O.; Rodriguez-Polo, Ignacio; Mitter, Michael; Preis, Jasmin; Arlt, Maximilian; Gruber, Jens

    2017-01-01

    Despite major advances on miRNA profiling and target predictions, functional readouts for endogenous miRNAs are limited and frequently lead to contradicting conclusions. Numerous approaches including functional high-throughput and miRISC complex evaluations suggest that the functional miRNAome differs from the predictions based on quantitative sRNA profiling. To resolve the apparent contradiction of expression versus function, we generated and applied a fluorescence reporter gene assay enabling single cell analysis. This approach integrates and adapts a mathematical model for miRNA-driven gene repression. This model predicts three distinct miRNA-groups with unique repression activities (low, mid and high) governed not just by expression levels but also by miRNA/target-binding capability. Here, we demonstrate the feasibility of the system by applying controlled concentrations of synthetic siRNAs and in parallel, altering target-binding capability on corresponding reporter-constructs. Furthermore, we compared miRNA-profiles with the modeled predictions of 29 individual candidates. We demonstrate that expression levels only partially reflect the miRNA function, fitting to the model-projected groups of different activities. Furthermore, we demonstrate that subcellular localization of miRNAs impacts functionality. Our results imply that miRNA profiling alone cannot define their repression activity. The gene regulatory function is a dynamic and complex process beyond a minimalistic conception of “highly expressed equals high repression”. PMID:28338079

  20. MiRNA expression profiling in human gliomas: upregulated miR-363 increases cell survival and proliferation.

    PubMed

    Conti, Alfredo; Romeo, Sara G; Cama, Annamaria; La Torre, Domenico; Barresi, Valeria; Pezzino, Gaetana; Tomasello, Chiara; Cardali, Salvatore; Angileri, Filippo F; Polito, Francesca; Ferlazzo, Guido; Di Giorgio, Rosamaria; Germanò, Antonino; Aguennouz, M'hammed

    2016-10-01

    The role of microRNAs (miRNAs) in glioma biology is increasingly recognized. To investigate the regulatory mechanisms governing the malignant signature of gliomas with different grades of malignancy, we analyzed miRNA expression profiles in human grade I-IV tumor samples and primary glioma cell cultures. Multiplex real-time PCR was used to profile miRNA expression in a set of World Health Organization (WHO) grade I (pilocytic astrocytoma), II (diffuse fibrillary astrocytoma), and IV (glioblastoma multiforme) astrocytic tumors and primary glioma cell cultures. Primary glioma cell cultures were used to evaluate the effect of transfection of specific miRNAs and miRNA inhibitors. miRNA microarray showed that a set of miRNAs was consistently upregulated in all glioma samples. miR-363 was upregulated in all tumor specimens and cell lines, and its expression correlated with tumor grading. The transfection of glioma cells with the specific inhibitor of miR-363 increased the expression level of tumor suppressor growth-associated protein 43 (GAP-43). Transfection of miR-363 induced cell survival, while inhibition of miR-363 significantly reduced glioma cell viability. Furthermore, miRNA-363 inhibition induced the downregulation of AKT, cyclin-D1, matrix metalloproteinase (MMP)-2, MMP-9, and Bcl-2 and upregulation of caspase 3. Together, these data suggest that the upregulation of miR-363 may play a role in malignant glioma signature.

  1. Cell type-specific long-term plasticity at glutamatergic synapses onto hippocampal interneurons expressing either parvalbumin or CB1 cannabinoid receptor.

    PubMed

    Nissen, Wiebke; Szabo, Andras; Somogyi, Jozsef; Somogyi, Peter; Lamsa, Karri P

    2010-01-27

    Different GABAergic interneuron types have specific roles in hippocampal function, and anatomical as well as physiological features vary greatly between interneuron classes. Long-term plasticity of interneurons has mostly been studied in unidentified GABAergic cells and is known to be very heterogeneous. Here we tested whether cell type-specific plasticity properties in distinct GABAergic interneuron types might underlie this heterogeneity. We show that long-term potentiation (LTP) and depression (LTD), two common forms of synaptic plasticity, are expressed in a highly cell type-specific manner at glutamatergic synapses onto hippocampal GABAergic neurons. Both LTP and LTD are generated in interneurons expressing parvalbumin (PV+), whereas interneurons with similar axon distributions but expressing cannabinoid receptor-1 show no lasting plasticity in response to the same protocol. In addition, LTP or LTD occurs in PV+ interneurons with different efferent target domains. Perisomatic-targeting PV+ basket and axo-axonic interneurons express LTP, whereas glutamatergic synapses onto PV+ bistratified cells display LTD. Both LTP and LTD are pathway specific, independent of NMDA receptors, and occur at synapses with calcium-permeable (CP) AMPA receptors. Plasticity in interneurons with CP-AMPA receptors strongly modulates disynaptic GABAergic transmission onto CA1 pyramidal cells. We propose that long-term plasticity adjusts the synaptic strength between pyramidal cells and interneurons in a cell type-specific manner and, in the defined CA1 interneurons, shifts the spatial pattern of inhibitory weight from pyramidal cell dendrites to the perisomatic region.

  2. The development and function of dendritic cell populations and their regulation by miRNAs.

    PubMed

    Zhou, Haibo; Wu, Li

    2017-03-31

    Dendritic cells (DCs) are important immune cells linking innate and adaptive immune responses. DCs encounter various self and non-self antigens present in the environment and induce different types of antigen specific adaptive immune responses. DCs can be classified into lymphoid tissue-resident DCs, migratory DCs, non-lymphoid resident DCs, and monocyte derived DCs (moDCs). Recent work has also established that DCs consist of developmentally and functionally distinct subsets that differentially regulate T lymphocyte function. The development of different DC subsets has been found to be regulated by a network of different cytokines and transcriptional factors. Moreover, the response of DC is tightly regulated to maintain the homeostasis of immune system. MicroRNAs (miRNAs) are an important class of cellular regulators that modulate gene expression and thereby influence cell fate and function. In the immune system, miRNAs act at checkpoints during hematopoietic development and cell subset differentiation, they modulate effector cell function, and are implicated in the maintenance of homeostasis. DCs are also regulated by miRNAs. In the past decade, much progress has been made to understand the role of miRNAs in regulating the development and function of DCs. In this review, we summarize the origin and distribution of different mouse DC subsets in both lymphoid and non-lymphoid tissues. The DC subsets identified in human are also described. Recent progress on the function of miRNAs in the development and activation of DCs and their functional relevance to autoimmune diseases are discussed.

  3. Inference of Target Gene Regulation via miRNAs during Cell Senescence by Using the MiRaGE Server

    PubMed Central

    Taguchi, Y-h.

    2012-01-01

    miRNAs have recently been shown to play a key role in cell senescence, by downregulating target genes. Thus, inference of those miRNAs that critically downregulate target genes is important. However, inference of target gene regulation by miRNAs is difficult and is often achieved simply by investigating significant upregulation during cell senescence. Here, we inferred the regulation of target genes by miRNAs, using the recently developed MiRaGE server, together with the change in miRNA expression during fibroblast IMR90 cell senescence. We revealed that the simultaneous consideration of 2 criteria, the up(down)regulation and the down(up) regulatiion of target genes, yields more feasible miRNA, i.e., those that are most frequently reported to be down/upregulated and/or to possess biological backgrounds that induce cell senescence. Thus, when analyzing miRNAs that critically contribute to cell senescence, it is important to consider the level of target gene regulation, simultaneously with the change in miRNA expression. PMID:23185711

  4. Inference of Target Gene Regulation via miRNAs during Cell Senescence by Using the MiRaGE Server.

    PubMed

    Taguchi, Y-H

    2012-08-01

    miRNAs have recently been shown to play a key role in cell senescence, by downregulating target genes. Thus, inference of those miRNAs that critically downregulate target genes is important. However, inference of target gene regulation by miRNAs is difficult and is often achieved simply by investigating significant upregulation during cell senescence. Here, we inferred the regulation of target genes by miRNAs, using the recently developed MiRaGE server, together with the change in miRNA expression during fibroblast IMR90 cell senescence. We revealed that the simultaneous consideration of 2 criteria, the up(down)regulation and the down(up) regulatiion of target genes, yields more feasible miRNA, i.e., those that are most frequently reported to be down/upregulated and/or to possess biological backgrounds that induce cell senescence. Thus, when analyzing miRNAs that critically contribute to cell senescence, it is important to consider the level of target gene regulation, simultaneously with the change in miRNA expression.

  5. MicroRNA (miRNA) expression is regulated by butyrate induced epigenetic modulation of gene expression in bovine cells

    USDA-ARS?s Scientific Manuscript database

    We present evidence that butyrate induced histone acetylation regulates miRNA expression. MicroRNA expression microarray profiling revealed that 35 miRNA transcripts are significantly (p <0.05) differentially expressed after cells were treated with 10 mM butyrate. Among them, 11 transcripts are dif...

  6. AmpA protein functions by different mechanisms to influence early cell type specification and to modulate cell adhesion and actin polymerization in Dictyostelium discoideum.

    PubMed

    Cost, Hoa N; Noratel, Elizabeth F; Blumberg, Daphne D

    2013-01-01

    The Dictyostelium discoideum ampA gene encodes a multifunctional regulator protein that modulates cell-cell and cell-substrate adhesions and actin polymerization during growth and is necessary for correct cell type specification and patterning during development. Insertional inactivation of the ampA gene results in defects that define two distinct roles for the ampA gene during development. AmpA is necessary in a non-cell autonomous manner to prevent premature expression of a prespore gene marker. It is also necessary in a cell autonomous manner for the anterior like cells, which express the ampA gene, to migrate to the upper cup during culmination. It is also necessary to prevent excessive cell-cell agglutination when cells are developed in a submerged suspension culture. Here, we demonstrate that a supernatant source of AmpA protein, added extracellularly, can prevent the premature mis-expression of the prespore marker. Synthetic oligopeptides are used to identify the domain of the AmpA protein that is important for preventing cells from mis-expressing the prespore gene. We further demonstrate that a factor capable of inducing additional cells to express the prespore gene marker accumulates extracellularly in the absence of AmpA protein. While the secreted AmpA acts extracellularly to suppress prespore gene expression, the effects of AmpA on cell agglutination and on actin polymerization in growing cells are not due to an extracellular role of secreted AmpA protein. Rather, these effects appear to reflect a distinct cell autonomous role of the ampA gene. Finally, we show that secretion of AmpA protein is brought about by elevating the levels of expression of ampA so that the protein accumulates to an excessive level.

  7. Hepatitis A virus-encoded miRNAs attenuate the accumulation of viral genomic RNAs in infected cells.

    PubMed

    Shi, Jiandong; Sun, Jing; Wu, Meini; Hu, Ningzhu; Hu, Yunzhang

    2016-06-01

    The establishment of persistent infection with hepatitis A virus (HAV) is the common result of most HAV/cell culture systems. Previous observations show that the synthesis of viral RNAs is reduced during infection. However, the underlying mechanism is poorly understood. We characterized three HAV-encoded miRNAs in our previous study. In this study, we aim to investigate the impact of these miRNAs on the accumulation of viral RNAs. The results indicated that the synthesis of viral genomic RNAs was dramatically reduced (more than 75 % reduction, P < 0.05) when transfected with one or two viral miRNA mimics. Conversely, they were significantly increased (more than 3.3-fold addition, P < 0.05) when transfected with one or two viral miRNA inhibitors. The luciferase reporter assay of miRNA targets showed that viral miRNAs were fully complementary to specific sites of the viral plus or minus strand RNA and strongly inhibited their expressions. Further data showed that the relative abundance of viral genomic RNA fragments that contain miRNA targets was also dramatically reduced (more than 80 % reduction, P < 0.05) when viral miRNAs were overexpressed with miRNA mimics. In contrast, they were significantly increased (approximately 2-fold addition, P < 0.05) when viral miRNAs were inhibited with miRNA inhibitors. In conclusion, these data suggest a possible mechanism for the reduction of viral RNA synthesis during HAV infection. Thus, we propose that it is likely that RNA virus-derived miRNA could serve as a self-mediated feedback regulator during infection.

  8. Comparative profiling of miRNA expression of lung adenocarcinoma cells in two-dimensional and three-dimensional cultures

    PubMed Central

    Li, Cui; Nguyen, Hong T.; Zhuang, Yan; Lin, Zhen; Flemington, Erik K.; Zhuo, Ying; Kantrow, Stephen P.; Morris, Gilbert F.; Sullivan, Deborah E.; Shan, Bin

    2012-01-01

    Three-dimensional organotypic culture using reconstituted basement membrane matrix (rBM 3-D) is an invaluable tool to characterize morphogenesis of epithelial cells and to elucidate the tumor-modulating actions of extracellular matrix. microRNAs (miRNA) are a novel class of tumor modulating genes. A substantial amount of investigation of miRNAs in cancer is carried out using monolayer 2-D culture on plastic substratum, which lacks a consideration of the matrix-mediated regulation of miRNAs. In the current study we compared the expression of miRNAs in rBM 3-D and 2-D cultures of two lung adenocarcinoma cell lines. Our findings revealed a profound difference in miRNA profiles between 2-D and rBM 3-D cultures of lung adenocarcinoma cells. The rBM 3-D culture-specific miRNA profile was highlighted with higher expression of the tumor suppressive miRNAs (i.e., miR-200 family) and lower expression of the oncogenic miRNAs (i.e., miR-17~92 cluster and miR-21) than that of 2-D culture. Moreover, the expression pattern of miR-17, miR-21, and miR-200a in rBM 3-D culture correlated with the expression of their targets and acinar morphogenesis, a differentiation behavior of lung epithelial cells in rBM 3-D culture. Over-expression of miR-21 suppressed its target PTEN and disrupted acinar morphogenesis. In summary, we provide the first miRNA profile of lung adenocarcinoma cells in rBM 3-D culture with respect to acinar morphogenesis. These results indicate that rBM 3-D culture is essential to a comprehensive understanding of the miRNA biology in lung epithelial cells pertinent to lung adenocarcinoma. PMID:23036707

  9. The effect of antisense inhibitor of miRNA 106b∼25 on the proliferation, invasion, migration, and apoptosis of gastric cancer cell.

    PubMed

    Zhang, Rupeng; Li, Fangxuan; Wang, Weijia; Wang, Xuejun; Li, Shixia; Liu, Juntian

    2016-08-01

    Accumulating data has demonstrated that miRNA 106b∼25, which are composed of the highly conserved miRNA 106b, miRNA 93, and miRNA 25, play carcinogenic roles in cancers. We investigated the expression of miRNA 106b∼25 in gastric cancer cells (SGC 7901, MGC 803, BGC 823) and normal gastric epithelial cell then inhibited miRNA 106b∼25 expression via transiently transfecting their antisense inhibitor. After miRNA 106b∼25 cluster was inhibited, MTT, Scratch test, Transwell invasion test, and flow cytometry were applied to investigate the proliferation, invasion, migration, cell cycle, and apoptosis of gastric cancer cell. The expression of miRNA 106b, miRNA 93, and miRNA 25 in gastric cancer cells SGC 7901, MGC 803, and BGC 823 was significantly higher than in gastric epithelial cell GES-1. The most significant suppression of miRNA 106b∼25 expressions can be detected in MGC 803 cell after transiently transfecting their antisense inhibitors. So, MGC 803 cell was selected as our research object. After inhibiting miRNA 106b and miRNA 93 respectively and combined, the proliferation, migration, and invasion of gastric cancer cell MGC 803 were significantly suppressed. The most significant suppression was observed in combined inhibiting group. After miRNA 106b∼25 cluster was inhibited respectively or combined, more gastric cancer cells were arrested in the G0G1 phase. However, there was no statistical difference in comparing with control groups. While the percentages of apoptotic cells increased after miRNA 106b∼25 cluster was inhibited, the statistical difference was detected only in combined inhibiting group. Inhibiting miRNA 106b∼25 cluster via transfecting antisense inhibitor can influence biological behavior of gastric cancer cell.

  10. The urologic epithelial stem cell database (UESC) – a web tool for cell type-specific gene expression and immunohistochemistry images of the prostate and bladder

    PubMed Central

    Pascal, Laura E; Deutsch, Eric W; Campbell, David S; Korb, Martin; True, Lawrence D; Liu, Alvin Y

    2007-01-01

    Background Public databases are crucial for analysis of high-dimensional gene and protein expression data. The Urologic Epithelial Stem Cells (UESC) database is a public database that contains gene and protein information for the major cell types of the prostate, prostate cancer cell lines, and a cancer cell type isolated from a primary tumor. Similarly, such information is available for urinary bladder cell types. Description Two major data types were archived in the database, protein abundance localization data from immunohistochemistry images, and transcript abundance data principally from DNA microarray analysis. Data results were organized in modules that were made to operate independently but built upon a core functionality. Gene array data and immunostaining images for human and mouse prostate and bladder were made available for interrogation. Data analysis capabilities include: (1) CD (cluster designation) cell surface protein data. For each cluster designation molecule, a data summary allows easy retrieval of images (at multiple magnifications). (2) Microarray data. Single gene or batch search can be initiated with Affymetrix Probeset ID, Gene Name, or Accession Number together with options of coalescing probesets and/or replicates. Conclusion Databases are invaluable for biomedical research, and their utility depends on data quality and user friendliness. UESC provides for database queries and tools to examine cell type-specific gene expression (normal vs. cancer), whereas most other databases contain only whole tissue expression datasets. The UESC database provides a valuable tool in the analysis of differential gene expression in prostate cancer genes in cancer progression. PMID:18072977

  11. Influence of gestational diabetes mellitus on human umbilical vein endothelial cell miRNA.

    PubMed

    Tryggestad, Jeanie B; Vishwanath, Anu; Jiang, Shaoning; Mallappa, Ashwini; Teague, April M; Takahashi, Yusuke; Thompson, David M; Chernausek, Steven D

    2016-11-01

    We aimed to identify miRNAs whose expression levels in fetal tissues are altered by exposure to a diabetic milieu and elucidate the impact on target protein expression. Gestational diabetes mellitus (GDM) affects both immediate and future disease risk in the offspring. We hypothesized that GDM alters miRNA expression in human umbilical vein endothelial cells (HUVECs) that may influence metabolic processes. A cross-sectional design compared differences in miRNA expression in HUVECs and target protein abundance in placentae between infants of women with GDM (IGDM) and infants born to normoglycaemic controls. miRNAs were identified using microarray profiling and literature review and validated by quantitative PCR (qPCR). In vitro transfection studies explored the impact of the miRNA on target protein expression. Expression of seven miRNA species, miR-30c-5p, miR-452-5p, miR-126-3p, miR-130b-3p, miR-148a-3p, miR-let-7a-5p and miR-let-7g-5p, was higher in the HUVECs of IGDM. Abundance of the catalytic subunit of AMP-activated protein kinase α1 (AMPKα1) was decreased in the HUVECs and BeWo cells (transformed trophoblast cell line) transfected with miR-130b and miR-148a mimics. AMPKα1 expression was also decreased in placental tissues of IGDM. The expression of several miRNAs were altered by in utero exposure to DM in infants of women whose dysglycaemia was very well controlled by current standards. Decreased expression of AMPKα1 as a result of increased levels of miR-130b and miR-148a may potentially explain the decrease in fat oxidation we reported in infants at 1 month of age and, if persistent, may predispose offspring to future metabolic disease. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  12. Benzene-Induced Aberrant miRNA Expression Profile in Hematopoietic Progenitor Cells in C57BL/6 Mice.

    PubMed

    Wei, Haiyan; Zhang, Juan; Tan, Kehong; Sun, Rongli; Yin, Lihong; Pu, Yuepu

    2015-11-12

    Benzene is a common environmental pollutant that causes hematological alterations. MicroRNAs (miRNAs) may play a role in benzene-induced hematotoxicity. In this study, C57BL/6 mice showed significant hematotoxicity after exposure to 150 mg/kg benzene for 4 weeks. Benzene exposure decreased not only the number of cells in peripheral blood but also hematopoietic progenitor cells in the bone marrow. Meanwhile, RNA from Lin(-) cells sorted from the bone marrow was applied to aberrant miRNA expression profile using Illumina sequencing. We found that 5 miRNAs were overexpressed and 45 miRNAs were downregulated in the benzene exposure group. Sequencing results were confirmed through qRT-PCR. Furthermore, we also identified five miRNAs which significantly altered in Lin(-)c-Kit⁺ cells obtained from benzene-exposed mice, including mmu-miR-34a-5p; mmu-miR-342-3p; mmu-miR-100-5p; mmu-miR-181a-5p; and mmu-miR-196b-5p. In summary, we successfully established a classical animal model to induce significant hematotoxicity by benzene injection. Benzene exposure may cause severe hematotoxicity not only to blood cells in peripheral circulation but also to hematopoietic cells in bone marrow. Benzene exposure also alters miRNA expression in hematopoietic progenitor cells. This study suggests that benzene induces alteration in hematopoiesis and hematopoiesis-associated miRNAs.

  13. Benzene-Induced Aberrant miRNA Expression Profile in Hematopoietic Progenitor Cells in C57BL/6 Mice

    PubMed Central

    Wei, Haiyan; Zhang, Juan; Tan, Kehong; Sun, Rongli; Yin, Lihong; Pu, Yuepu

    2015-01-01

    Benzene is a common environmental pollutant that causes hematological alterations. MicroRNAs (miRNAs) may play a role in benzene-induced hematotoxicity. In this study, C57BL/6 mice showed significant hematotoxicity after exposure to 150 mg/kg benzene for 4 weeks. Benzene exposure decreased not only the number of cells in peripheral blood but also hematopoietic progenitor cells in the bone marrow. Meanwhile, RNA from Lin− cells sorted from the bone marrow was applied to aberrant miRNA expression profile using Illumina sequencing. We found that 5 miRNAs were overexpressed and 45 miRNAs were downregulated in the benzene exposure group. Sequencing results were confirmed through qRT-PCR. Furthermore, we also identified five miRNAs which significantly altered in Lin−c-Kit+ cells obtained from benzene-exposed mice, including mmu-miR-34a-5p; mmu-miR-342-3p; mmu-miR-100-5p; mmu-miR-181a-5p; and mmu-miR-196b-5p. In summary, we successfully established a classical animal model to induce significant hematotoxicity by benzene injection. Benzene exposure may cause severe hematotoxicity not only to blood cells in peripheral circulation but also to hematopoietic cells in bone marrow. Benzene exposure also alters miRNA expression in hematopoietic progenitor cells. This study suggests that benzene induces alteration in hematopoiesis and hematopoiesis-associated miRNAs. PMID:26569237

  14. Systematic identification and annotation of human methylation marks based on bisulfite sequencing methylomes reveals distinct roles of cell type-specific hypomethylation in the regulation of cell identity genes.

    PubMed

    Liu, Hongbo; Liu, Xiaojuan; Zhang, Shumei; Lv, Jie; Li, Song; Shang, Shipeng; Jia, Shanshan; Wei, Yanjun; Wang, Fang; Su, Jianzhong; Wu, Qiong; Zhang, Yan

    2016-01-08

    DNA methylation is a key epigenetic mark that is critical for gene regulation in multicellular eukaryotes. Although various human cell types may have the same genome, these cells have different methylomes. The systematic identification and characterization of methylation marks across cell types are crucial to understand the complex regulatory network for cell fate determination. In this study, we proposed an entropy-based framework termed SMART to integrate the whole genome bisulfite sequencing methylomes across 42 human tissues/cells and identified 757 887 genome segments. Nearly 75% of the segments showed uniform methylation across all cell types. From the remaining 25% of the segments, we identified cell type-specific hypo/hypermethylation marks that were specifically hypo/hypermethylated in a minority of cell types using a statistical approach and presented an atlas of the human methylation marks. Further analysis revealed that the cell type-specific hypomethylation marks were enriched through H3K27ac and transcription factor binding sites in cell type-specific manner. In particular, we observed that the cell type-specific hypomethylation marks are associated with the cell type-specific super-enhancers that drive the expression of cell identity genes. This framework provides a complementary, functional annotation of the human genome and helps to elucidate the critical features and functions of cell type-specific hypomethylation.

  15. Discovery and characterization of miRNA during cellular senescence in bone marrow-derived human mesenchymal stem cells.

    PubMed

    Yoo, Jung Ki; Kim, Chang-Hyun; Jung, Ho Yong; Lee, Dong Ryul; Kim, Jin Kyeoung

    2014-10-01

    Cellular senescence is an irreversible cell cycle arrest in which specific mRNAs and miRNAs are involved in senescence progression. miRNAs interact with specific mRNAs to regulate various cellular mechanisms, including metabolism, proliferation, apoptosis, senescence and differentiation. In this study, we identify and characterize miRNAs during cellular senescence in mesenchymal stem cells (MSCs). Using previously reported miRNAs, expression profiling of 23 miRNAs was performed using real-time PCR analysis. Among these miRNAs, 19 miRNAs showed upregulated expression patterns in senescent MSCs compared with young MSCs, and 5 miRNAs were downregulated. These miRNAs have not been previously identified as being related to cellular senescence but seem to be related. miR-103-2*, miR-140-5p and miR-330-5p are highly upregulated, while miR-29b and miR-199b-5p are significantly downregulated in senescent MSCs. We identify unique functions of 5 miRNAs and predict putative target genes of 5 miRNAs using our previous report. Among them, miR-199b-5p directly suppressed LAMC1 expression, as shown in a luciferase assay. miR-199b-5p significantly regulates translational activity but does not control post-transcriptional activity. Likewise, miR-199b-5p modulates LAMC networks, which demonstrates the resulting phenomenon during cellular senescence, namely, that miR-199b-5p indirectly regulates cellular senescence in MSCs.

  16. Fluorescent metal nanoshell probe to detect single miRNA in lung cancer cell.

    PubMed

    Zhang, Jian; Fu, Yi; Mei, Yuping; Jiang, Feng; Lakowicz, Joseph R

    2010-06-01

    In this study, fluorescent metal nanoshells were synthesized as a molecular imaging agent to detect single microRNA (miRNA) molecules in the cells positive to lung cancer. These metal nanoshells were composed of silica spheres with encapsulated Ru(bpy)(3)(2+) complexes as cores and thin silver layers as shells. Compared with the silica spheres in the absence of metal, the metal nanoshells displayed an enhanced emission intensity, shortened lifetime, and extended photostability. The single-stranded probe oligonucleotides were covalently bound on the metal nanoshells to hybridize with the target miRNA-486 molecules in the cells. It was shown that with stronger emission intensity and longer lifetime, the conjugated metal nanoshells were isolated distinctly from the cellular autofluorescence on the cell images. These emission spots on the cell images were counted accurately and analyzed with a pool of cells representing the miRNA-486 expression levels in the cells. The results may reflect a genomic signal change and provide a reference to lung cancer early diagnosis as well as other diseases.

  17. Parecoxib inhibits glioblastoma cell proliferation, migration and invasion by upregulating miRNA-29c

    PubMed Central

    Li, Lin-Yong; Xiao, Jie; Liu, Qiang

    2017-01-01

    ABSTRACT Glioblastoma (GBM) is one of the most lethal brain cancers worldwide, and there is an urgent need for development of novel therapeutic approaches. Parecoxib is a well-known cyclooxygenase-2 (COX-2) inhibitor, and had already been developed for postoperative analgesia with high efficacy and low adverse reaction. A recent study has suggested that parecoxib potently enhances immunotherapeutic efficacy of GBM, but its effects on GBM growth, migration and invasion have not previously been studied. In the present study, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and BrdU (5-bromo-2-deoxyuridine) incorporation assays were used to evaluate the cell proliferation of GBM cells. Wound-healing and transwell assays were preformed to analyze GBM cell migration and invasion, respectively. The results suggested that parecoxib inhibits cell proliferation, migration and invasion of GBM cells in a dose-dependent manner. RT-qPCR (real-time quantitative PCR) analysis demonstrated that miRNA-29c can be significantly induced by parecoxib. Furthermore, our data suggests that a miRNA-29c inhibitor can significantly attenuate parecoxib's effect on proliferation, migration and invasion of GBM. In conclusion, the present study suggests that parecoxib inhibits GBM cell proliferation, migration and invasion by upregulating miRNA-29c. PMID:27895048

  18. Cell type-specific adaptation of cellular and nuclear volume in micro-engineered 3D environments.

    PubMed

    Greiner, Alexandra M; Klein, Franziska; Gudzenko, Tetyana; Richter, Benjamin; Striebel, Thomas; Wundari, Bayu G; Autenrieth, Tatjana J; Wegener, Martin; Franz, Clemens M; Bastmeyer, Martin

    2015-11-01

    Bio-functionalized three-dimensional (3D) structures fabricated by direct laser writing (DLW) are structurally and mechanically well-defined and ideal for systematically investigating the influence of three-dimensionality and substrate stiffness on cell behavior. Here, we show that different fibroblast-like and epithelial cell lines maintain normal proliferation rates and form functional cell-matrix contacts in DLW-fabricated 3D scaffolds of different mechanics and geometry. Furthermore, the molecular composition of cell-matrix contacts forming in these 3D micro-environments and under conventional 2D culture conditions is identical, based on the analysis of several marker proteins (paxillin, phospho-paxillin, phospho-focal adhesion kinase, vinculin, β1-integrin). However, fibroblast-like and epithelial cells differ markedly in the way they adapt their total cell and nuclear volumes in 3D environments. While fibroblast-like cell lines display significantly increased cell and nuclear volumes in 3D substrates compared to 2D substrates, epithelial cells retain similar cell and nuclear volumes in 2D and 3D environments. Despite differential cell volume regulation between fibroblasts and epithelial cells in 3D environments, the nucleus-to-cell (N/C) volume ratios remain constant for all cell types and culture conditions. Thus, changes in cell and nuclear volume during the transition from 2D to 3D environments are strongly cell type-dependent, but independent of scaffold stiffness, while cells maintain the N/C ratio regardless of culture conditions.

  19. Cell type specific applicability of 5-ethynyl-2'-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry.

    PubMed

    Diermeier-Daucher, Simone; Clarke, Scott T; Hill, Dani; Vollmann-Zwerenz, Arabel; Bradford, Jolene A; Brockhoff, Gero

    2009-06-01

    Using the nucleoside analogue EdU (5-ethynyl-2'-deoxyuridine) for thymidine substitution instead of BrdU (5-bromo-2'-deoxyuridine) in cell proliferation assays has recently been proposed. However, the effect of EdU on cell viability, DNA synthesis, and cell cycle progression and consequently its usability for dynamic cell proliferation analysis in vitro has not been explored. We compared the effect of EdU and BrdU incorporation into SK-BR-3 and BT474 breast cancer cells and the impact on cell cycle kinetics, cell viability, and DNA damage. We found that EdU can be used not only for pulse but also for continuous cell labeling and henceforth in high resolution EdU/Hoechst quenching assays. BrdU and EdU proliferation assays based on click chemistry revealed comparable results. However, cell viability of SK-BR-3 breast cancer cells was highly affected by long term exposure to EdU. Both SK-BR-3 as well as BT474 cells show cell cycle arrests upon long term EdU treatment whereas only SK-BR-3 cells were driven into necrotic cell death by long term exposure to EdU. In contrast BT474 cells appeared essentially unharmed by EdU treatment in terms of viability. Consequently using EdU enables highly sensitive and quantitative detection of proliferating cells and facilitates even continuous cell cycle assessment. Nevertheless, potential cellular susceptibility needs to be individually evaluated.

  20. miRNA-149* promotes cell proliferation and suppresses apoptosis by mediating JunB in T-cell acute lymphoblastic leukemia.

    PubMed

    Fan, Sheng-Jin; Li, Hui-Bo; Cui, Gang; Kong, Xiao-Lin; Sun, Li-Li; Zhao, Yan-Qiu; Li, Ying-Hua; Zhou, Jin

    2016-02-01

    MicroRNA-149* (miRNA-149*) functions as an oncogenic regulator in human melanoma. However, the effect of miRNA-149* on T-cell acute lymphoblastic leukemia (T-ALL) is unclear. Here we aimed to analyze the effects of miRNA-149* on in vitro T-ALL cells and to uncover the target for miRNA-149* in these cells. The miRNA-149* level was determined in multiple cell lines and bone marrow cells derived from patients with T-ALL, B acute lymphoblastic leukemia (B-ALL), acute myelocytic leukemia (AML), and healthy donors. We found that miRNA-149* was highly expressed in T-ALL cell lines and T-ALL patients' bone marrow samples. JunB was identified as a direct target of miR-149*. miRNA-149* mimics downregulated JunB levels in Molt-4 and Jurkat cells, while miRNA-149* inhibitors dramatically upregulated JunB expression in these cells. miRNA-149* mimics promoted proliferation, decreased the proportion of cells in G1 phase, and reduced cell apoptosis in T-ALL cells, while miRNA-149* inhibitors prevented these effects. miRNA-149* mimics downregulated p21 and upregulated cyclinD1, 4EBP1, and p70s6k in Molt-4 and Jurkat cells. Again, inhibitors prevented these effects. Our findings demonstrate that miRNA-149* may serve as an oncogenic regulator in T-ALL by negatively regulating JunB.

  1. Cell type-specific anti-cancer properties of valproic acid: independent effects on HDAC activity and Erk1/2 phosphorylation

    PubMed Central

    2010-01-01

    Background The anti-epileptic drug valproic acid (VPA) has attracted attention as an anti-cancer agent. Methods The present study investigated effects of VPA exposure on histone deacetylase (HDAC) inhibition, cell growth, cell speed, and the degree of Erk1/2 phosphorylation in 10 cell lines (BT4C, BT4Cn, U87MG, N2a, PC12-E2, CSML0, CSML100, HeLa, L929, Swiss 3T3). Results VPA induced significant histone deacetylase (HDAC) inhibition in most of the cell lines, but the degree of inhibition was highly cell type-specific. Moreover, cell growth, motility and the degree of Erk1/2 phosphorylation were inhibited, activated, or unaffected by VPA in a cell type-specific manner. Importantly, no relationship was found between the effects of VPA on HDAC inhibition and changes in the degree of Erk1/2 phosphorylation, cell growth, or motility. In contrast, VPA-induced modulation of the MAPK pathway downstream of Ras but upstream of MEK (i.e., at the level of Raf) was important for changes in cell speed. Conclusions These results suggest that VPA can modulate the degree of Erk1/2 phosphorylation in a manner unrelated to HDAC inhibition and emphasize that changes in the degree of Erk1/2 phosphorylation are also important for the anti-cancer properties of VPA. PMID:20663132

  2. Rickettsia rickettsii infection of human macrovascular and microvascular endothelial cells reveals activation of both common and cell type-specific host response mechanisms.

    PubMed

    Rydkina, Elena; Turpin, Loel C; Sahni, Sanjeev K

    2010-06-01

    Although inflammation and altered barrier functions of the vasculature, due predominantly to the infection of endothelial cell lining of small and medium-sized blood vessels, represent salient pathological features of human rickettsioses, the interactions between pathogenic rickettsiae and microvascular endothelial cells remain poorly understood. We have investigated the activation of nuclear transcription factor-kappa B (NF-kappaB) and p38 mitogen-activated protein (MAP) kinase, expression of heme oxygenase 1 (HO-1) and cyclooxygenase 2 (COX-2), and secretion of chemokines and prostaglandins after Rickettsia rickettsii infection of human cerebral, dermal, and pulmonary microvascular endothelial cells in comparison with pulmonary artery cells of macrovascular origin. NF-kappaB and p38 kinase activation and increased HO-1 mRNA expression were clearly evident in all cell types, along with relatively similar susceptibility to R. rickettsii infection in vitro but considerable variations in the intensities/kinetics of the aforementioned host responses. As expected, the overall activation profiles of macrovascular endothelial cells derived from human pulmonary artery and umbilical vein were nearly identical. Interestingly, cerebral endothelial cells displayed a marked refractoriness in chemokine production and secretion, while all other cell types secreted various levels of interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) in response to infection. A unique feature of all microvascular endothelial cells was the lack of induced COX-2 expression and resultant inability to secrete prostaglandin E(2) after R. rickettsii infection. Comparative evaluation thus yields the first experimental evidence for the activation of both common and unique cell type-specific host response mechanisms in macrovascular and microvascular endothelial cells infected with R. rickettsii, a prototypical species known to cause Rocky Mountain spotted fever in humans.

  3. 1α,25(OH)2D3 differentially regulates miRNA expression in human bladder cancer cells

    PubMed Central

    Ma, Yingyu; Hu, Qiang; Luo, Wei; Pratt, Rachel N.; Glenn, Sean T.; Liu, Song; Trump, Donald L.; Johnson, Candace S.

    2014-01-01

    Bladder cancer is the fourth most commonly diagnosed cancer in men and eighth leading cause of cancer-related death in the US. Epidemiological and experimental studies strongly suggest a role for 1α,25(OH)2D3 in cancer prevention and treatment. The antitumor activities of 1α,25(OH)2D3 are mediated by the induction of cell cycle arrest, apoptosis, differentiation and the inhibition of angiogenesis and metastasis. MiRNAs play important regulatory roles in cancer development and progression. However, the role of 1α,25(OH)2D3 in the regulation of miRNA expression and the potential impact in bladder cancer has not been investigated. Therefore, we studied 1α,25(OH)2D3-regulated miRNA expression profiles in human bladder cancer cell line 253J and the highly tumorigenic and metastatic derivative line 253J-BV by miRNA qPCR panels. 253 J and 253J-BV cells express endogenous vitamin D receptor (VDR) which can be further induced by 1α,25(OH)2D3. VDR target gene 24-hydroxylase was induced by 1α,25(OH)2D3 in both cell lines, indicating functional 1α,25(OH)2D3 signaling. The miRNA qPCR panel assay results showed that 253J and 253J-BV cells have distinct miRNA expression profiles. Further, 1α,25(OH)2D3 differentially regulated miRNA expression profiles in 253J and 253 J-BV cells in a dynamic manner. Pathway analysis of the miRNA target genes revealed distinct patterns of contribution to the molecular functions and biological processes in the two cell lines. In conclusion, 1α,25(OH)2D3 differentially regulates the expression of miRNAs, which may contribute to distinct biological functions, in human bladder 253J and 253J-BV cells. PMID:25263658

  4. Epigenetic Changes Induced by Air Toxics: Formaldehyde Exposure Alters miRNA Expression Profiles in Human Lung Cells

    PubMed Central

    Rager, Julia E.; Smeester, Lisa; Jaspers, Ilona; Sexton, Kenneth G.; Fry, Rebecca C.

    2011-01-01

    Background Exposure to formaldehyde, a known air toxic, is associated with cancer and lung disease. Despite the adverse health effects of formaldehyde, the mechanisms underlying formaldehyde-induced disease remain largely unknown. Research has uncovered microRNAs (miRNAs) as key posttranscriptional regulators of gene expression that may influence cellular disease state. Although studies have compared different miRNA expression patterns between diseased and healthy tissue, this is the first study to examine perturbations in global miRNA levels resulting from formaldehyde exposure. Objectives We investigated whether cellular miRNA expression profiles are modified by formaldehyde exposure to test the hypothesis that formaldehyde exposure disrupts miRNA expression levels within lung cells, representing a novel epigenetic mechanism through which formaldehyde may induce disease. Methods Human lung epithelial cells were grown at air–liquid interface and exposed to gaseous formaldehyde at 1 ppm for 4 hr. Small RNAs and protein were collected and analyzed for miRNA expression using microarray analysis and for interleukin (IL-8) protein levels by enzyme-linked immunosorbent assay (ELISA). Results Gaseous formaldehyde exposure altered the miRNA expression profiles in human lung cells. Specifically, 89 miRNAs were significantly down-regulated in formaldehyde-exposed samples versus controls. Functional and molecular network analysis of the predicted miRNA transcript targets revealed that formaldehyde exposure potentially alters signaling pathways associated with cancer, inflammatory response, and endocrine system regulation. IL-8 release increased in cells exposed to formaldehyde, and results were confirmed by real-time polymerase chain reaction. Conclusions Formaldehyde alters miRNA patterns that regulate gene expression, potentially leading to the initiation of a variety of diseases. PMID:21147603

  5. Epigenetic/Genetic Mismatch: Using Transdifferentiation as a Potential Cancer Therapy to Exploit the Cell Type Specificity of Cancer.

    PubMed

    Mendelsohn, Andrew R; Lei, Jennifer L; Chatterjee, Devasis

    2015-01-01

    Every cell type capable of proliferation can be malignantly transformed. However, there appears to be no naturally occurring universal set of genetic mutations capable of converting every cell type to a malignant state. Any specific cell type is generally resistant to transformation by the cancer mutations accumulated by cells of different lineages, presumably due to epigenetic differences. Evidence for this idea derives from experiments in which the developmental fates of cancer cells are altered to reduce malignancy. Reprogramming cancer cells to more primitive developmental states using pluripotency factors (IPS) or somatic nuclear transfer suppresses the malignant phenotype, as does subsequent directed differentiation to mature cells of lineages distinct from the originating cell. Direct transdifferentiation to an alternative cell fate also reduces tumorigenicity. In contrast, after reprogramming, cells induced to redifferentiate toward the original tumor cell type are tumorigenic. In these types of experiments an epigenetic/genetic mismatch often results in suppression of malignancy or cell death. Elucidating the specific transcription and cell signaling network incompatibilities will identify new targets for cancer therapy. Moreover, novel strategies to induce an incompatible transdifferentiated state, in which expression of thousands of genes are altered, will prove useful in controlling malignancies that otherwise easily evolve resistance to single target-based therapeutics. Engineering small molecules, genetic vectors, cytokines, growth factors, targeted extracellular vesicles, and cell fusion will help realize transdifferentiation-based therapeutics for cancer.

  6. Mammalian non-CG methylations are conserved and cell-type specific and may have been involved in the evolution of transposon elements

    PubMed Central

    Guo, Weilong; Zhang, Michael Q.; Wu, Hong

    2016-01-01

    Although non-CG methylations are abundant in several mammalian cell types, their biological significance is sparsely characterized. We gathered 51 human and mouse DNA methylomes from brain neurons, embryonic stem cells and induced pluripotent stem cells, primordial germ cells and oocytes. We utilized an unbiased sub-motif prediction method and reported CW as the representative non-CG methylation context, which is distinct from CC methylation in terms of sequence context and genomic distribution. A two-dimensional comparison of non-CG methylations across cell types and species was performed. Unambiguous studies of sequence preferences and genomic region enrichment showed that CW methylation is cell-type specific and is also conserved between humans and mice. In brain neurons, it was found that active long interspersed nuclear element-1 (LINE-1) lacked CW methylations but not CG methylations. Coincidentally, both human Alu and mouse B1 elements preferred high CW methylations at specific loci during their respective evolutionary development. Last, the strand-specific distributions of CW methylations in introns and long interspersed nuclear elements are also cell-type specific and conserved. In summary, our results illustrate that CW methylations are highly conserved among species, are dynamically regulated in each cell type, and are potentially involved in the evolution of transposon elements. PMID:27573482

  7. Mammalian non-CG methylations are conserved and cell-type specific and may have been involved in the evolution of transposon elements.

    PubMed

    Guo, Weilong; Zhang, Michael Q; Wu, Hong

    2016-08-30

    Although non-CG methylations are abundant in several mammalian cell types, their biological significance is sparsely characterized. We gathered 51 human and mouse DNA methylomes from brain neurons, embryonic stem cells and induced pluripotent stem cells, primordial germ cells and oocytes. We utilized an unbiased sub-motif prediction method and reported CW as the representative non-CG methylation context, which is distinct from CC methylation in terms of sequence context and genomic distribution. A two-dimensional comparison of non-CG methylations across cell types and species was performed. Unambiguous studies of sequence preferences and genomic region enrichment showed that CW methylation is cell-type specific and is also conserved between humans and mice. In brain neurons, it was found that active long interspersed nuclear element-1 (LINE-1) lacked CW methylations but not CG methylations. Coincidentally, both human Alu and mouse B1 elements preferred high CW methylations at specific loci during their respective evolutionary development. Last, the strand-specific distributions of CW methylations in introns and long interspersed nuclear elements are also cell-type specific and conserved. In summary, our results illustrate that CW methylations are highly conserved among species, are dynamically regulated in each cell type, and are potentially involved in the evolution of transposon elements.

  8. Peroxisome proliferator-activated receptor gamma-mediated differentiation: a mutation in colon cancer cells reveals divergent and cell type-specific mechanisms.

    PubMed

    Gupta, Rajnish A; Sarraf, Pasha; Mueller, Elisabetta; Brockman, Jeffrey A; Prusakiewicz, Jeffery J; Eng, Charis; Willson, Timothy M; DuBois, Raymond N

    2003-06-20

    Activation of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits cell growth and induces differentiation in both adipocyte and epithelial cell lineages, although it is unclear whether this occurs through common or cell-type specific mechanisms. We have identified four human colon cancer cell lines that do no undergo growth inhibition or induce markers of differentiation after exposure to PPARgamma agonists. Sequence analysis of the PPARgamma gene revealed that all four cell lines contain a previously unidentified point mutation in the ninth alpha-helix of the ligand binding domain at codon 422 (K422Q). The mutant receptor did not exhibit any defects in DNA binding or retinoid X receptor heterodimerization and was transcriptionally active in an artificial reporter assay. However, only retroviral transduction of the wild-type (WT), but not mutant, receptor could restore PPARgamma ligand-induced growth inhibition and differentiation in resistant colon cancer cell lines. In contrast, there was no difference in the ability of fibroblast cells expressing WT or K422Q mutant receptor to undergo growth inhibition, express adipocyte differentiation markers, or uptake lipid after treatment with a PPARgamma agonist. Finally, analysis of direct PPARgamma target genes in colon cancer cells expressing the WT or K422Q mutant allele suggests that the mutation may disrupt the ability of PPARgamma to repress the basal expression of a subset of genes in the absence of exogenous ligand. Collectively, these data argue that codon 422 may be a part of a co-factor(s) interaction domain necessary for PPARgamma to induce terminal differentiation in epithelial, but not adipocyte, cell lineages and argues that the receptor induces growth inhibition and differentiation via cell lineage-specific mechanisms.

  9. HPV-type-specific response of cervical cancer cells to cisplatin after silencing replication licensing factor MCM4.

    PubMed

    Das, Mitali; Prasad, Shyam Babu; Yadav, Suresh Singh; Modi, Arusha; Singh, Sunita; Pradhan, Satyajit; Narayan, Gopeshwar

    2015-12-01

    Minichoromosome maintenance (MCM) proteins play key role in cell cycle progression by licensing DNA replication only once per cell cycle. These proteins are found to be overexpressed in cervical cancer cells. In this study, we depleted MCM4, one of the MCM 2-7 complex components by RNA interference (RNAi) in four cervical cancer cell lines. The four cell lines were selected on the basis of their human papillomavirus (HPV) infection: HPV16-positive SiHa, HPV18-positive ME-180, HPV16- and HPV18-positive CaSki, and HPV-negative C-33A. The MCM4-deficient cells irrespective of their HPV status grow for several generations and maintain regular cell cycle. We did not find any evidence of augmented response to a short-term (48 h) cisplatin treatment in these MCM4-deficient cells. However, MCM4-/HPV16+ SiHa cells cannot withstand a prolonged treatment (up to 5 days) of even a sublethal dosage of cisplatin. They show increased chromosomal instability compared to their control counterparts. On the other hand, MCM4-deficient CaSki cells (both HPV16+ and 18+) remain resistant to a prolonged exposure to cisplatin. Our study indicates that cervical cancer cells may be using excess MCMs as a backup for replicative stress; however, its regulatory mechanism is dependent on the HPV status of the cells.

  10. Genome-wide miRNA response to anacardic acid in breast cancer cells.

    PubMed

    Schultz, David J; Muluhngwi, Penn; Alizadeh-Rad, Negin; Green, Madelyn A; Rouchka, Eric C; Waigel, Sabine J; Klinge, Carolyn M

    2017-01-01

    MicroRNAs are biomarkers and potential therapeutic targets for breast cancer. Anacardic acid (AnAc) is a dietary phenolic lipid that inhibits both MCF-7 estrogen receptor α (ERα) positive and MDA-MB-231 triple negative breast cancer (TNBC) cell proliferation with IC50s of 13.5 and 35 μM, respectively. To identify potential mediators of AnAc action in breast cancer, we profiled the genome-wide microRNA transcriptome (microRNAome) in these two cell lines altered by the AnAc 24:1n5 congener. Whole genome expression profiling (RNA-seq) and subsequent network analysis in MetaCore Gene Ontology (GO) algorithm was used to characterize the biological pathways altered by AnAc. In MCF-7 cells, 69 AnAc-responsive miRNAs were identified, e.g., increased let-7a and reduced miR-584. Fewer, i.e., 37 AnAc-responsive miRNAs were identified in MDA-MB-231 cells, e.g., decreased miR-23b and increased miR-1257. Only two miRNAs were increased by AnAc in both cell lines: miR-612 and miR-20b; however, opposite miRNA arm preference was noted: miR-20b-3p and miR-20b-5p were upregulated in MCF-7 and MDA-MB-231, respectively. miR-20b-5p target EFNB2 transcript levels were reduced by AnAc in MDA-MB-231 cells. AnAc reduced miR-378g that targets VIM (vimentin) and VIM mRNA transcript expression was increased in AnAc-treated MCF-7 cells, suggesting a reciprocal relationship. The top three enriched GO terms for AnAc-treated MCF-7 cells were B cell receptor signaling pathway and ribosomal large subunit biogenesis and S-adenosylmethionine metabolic process for AnAc-treated MDA-MB-231 cells. The pathways modulated by these AnAc-regulated miRNAs suggest that key nodal molecules, e.g., Cyclin D1, MYC, c-FOS, PPARγ, and SIN3, are targets of AnAc activity.

  11. MiRNA-30a-mediated autophagy inhibition sensitizes renal cell carcinoma cells to sorafenib.

    PubMed

    Zheng, Bing; Zhu, Hua; Gu, Donghua; Pan, Xiaodong; Qian, Lin; Xue, Boxin; Yang, Dongrong; Zhou, Jundong; Shan, Yuxi

    2015-04-03

    Chemotherapy-induced autophagy activation often contributes to cancer resistance. MiRNA-30a (miR-30a) is a potent inhibitor of autophagy by downregulating Beclin-1. In this study, we characterized the role of miR-30a in sorafenib-induced activity in renal cell carcinoma (RCC) cells. We found that expression of miR-30a was significantly downregulated in several human RCC tissues and in RCC cell lines. Accordingly, its targeted gene Beclin-1 was upregulated. Sorafenib activated autophagy in RCC cells (786-0 and A489 lines), evidenced by p62 degradation, Beclin-1/autophagy protein 5 (ATG-5) upregulation and light chain (LC)3B-I/-II conversion. Exogenously expressing miR-30a in 786-0 or A489 cells inhibited Beclin-1 expression and enhanced sorafenib-induced cytotoxicity. In contrast, knockdown of miR-30a by introducing antagomiR-30a increased Beclin-1 expression, and inhibited sorafenib-induced cytotoxicity against RCC cells. Autophagy inhibitors, including chloroquine, 3-methyaldenine or Bafliomycin A1, enhanced sorafenib activity, causing substantial cell apoptosis. Meanwhile, knockdown of Beclin-1 or ATG-5 by targeted siRNAs also increased sorafenib-induced cytotoxicity in above RCC cells. These findings indicate that dysregulation of miR-30a in RCC may interfere with the effectiveness of sorafenib-mediated apoptosis by an autophagy-dependent pathway, thus representing a novel potential therapeutic target for RCC.

  12. A cell type-specific view on the translation of mRNAs from ROS-responsive genes upon paraquat treatment of Arabidopsis thaliana leaves.

    PubMed

    Benina, Maria; Ribeiro, Dimas Mendes; Gechev, Tsanko S; Mueller-Roeber, Bernd; Schippers, Jos H M

    2015-02-01

    Oxidative stress causes dramatic changes in the expression levels of many genes. The formation of a functional protein through successful mRNA translation is central to a coordinated cellular response. To what extent the response towards reactive oxygen species (ROS) is regulated at the translational level is poorly understood. Here we analysed leaf- and tissue-specific translatomes using a set of transgenic Arabidopsis thaliana lines expressing a FLAG-tagged ribosomal protein to immunopurify polysome-bound mRNAs before and after oxidative stress. We determined transcript levels of 171 ROS-responsive genes upon paraquat treatment, which causes formation of superoxide radicals, at the whole-organ level. Furthermore, the translation of mRNAs was determined for five cell types: mesophyll, bundle sheath, phloem companion, epidermal and guard cells. Mesophyll and bundle sheath cells showed the strongest response to paraquat treatment. Interestingly, several ROS-responsive transcription factors displayed cell type-specific translation patterns, while others were translated in all cell types. In part, cell type-specific translation could be explained by the length of the 5'-untranslated region (5'-UTR) and the presence of upstream open reading frames (uORFs). Our analysis reveals insights into the translational regulation of ROS-responsive genes, which is important to understanding cell-specific responses and functions during oxidative stress.

  13. Anorganic bovine bone (Bio-Oss) regulates miRNA of osteoblast-like cells.

    PubMed

    Palmieri, Annalisa; Pezzetti, Furio; Brunelli, Giorgio; Martinelli, Marcella; Lo Muzio, Lorenzo; Scarano, Antonio; Scapoli, Luca; Arlotti, Marzia; Guerzoni, Laura; Carinci, Francesco

    2010-02-01

    Bio-Oss (Geistlich) is composed of an organic bovine bone and has been widely used in several bone regeneration procedures during oral surgery. However, how this biomaterial enhances osteoblast activity to promote bone formation is not completely understood. MicroRNAs (miRNAs) represent a class of small, functional, noncoding RNAs of 19 to 23 nucleotides that regulate the transcription of messenger RNAs (mRNAs) in proteins. In this study, the miRNA microarray technique was used to investigate translation regulation in an osteoblast-like cell line (MG63) exposed to Bio-Oss. Nine up-regulated miRNAs (mir-423, mir-492, mir-191, mir-23a, mir-377, mir-494, mir-214, mir-193b, mir-320) and 4 down-regulated miRNAs (mir-27a, mir-24, mir-188, let-7c) were identified. Because each miRNA regulates 100 mRNAs, only mRNAs related to bone formation were analyzed. The vast majority of detected mRNAs are down-regulated, including some homeobox genes (genes that regulate the morphogenesis of an entire segment of the body), such as noggin and EN1. An indirect positive effect was demonstrated on bone morphogenetic protein-4. To the authors' knowledge, the data reported here are the first on translation regulation in osteoblasts exposed to Bio-Oss. This study may be relevant in better understanding the molecular mechanism of bone regeneration and used as a potential tool for analyzing the combined use of cytokines.

  14. Cell type-specific and activity-dependent dynamics of action potential-evoked Ca2+ signals in dendrites of hippocampal inhibitory interneurons

    PubMed Central

    Evstratova, Alesya; Chamberland, Simon; Topolnik, Lisa

    2011-01-01

    Abstract In most central neurons, action potentials (APs), generated in the initial axon segment, propagate back into dendrites and trigger considerable Ca2+ entry via activation of voltage-sensitive calcium channels (VSCCs). Despite the similarity in its underlying mechanisms, however, AP-evoked dendritic Ca2+ signalling often demonstrates a cell type-specific profile that is determined by the neuron dendritic properties. Using two-photon Ca2+ imaging in combination with patch-clamp whole-cell recordings, we found that in distinct types of hippocampal inhibitory interneurons Ca2+ transients evoked by backpropagating APs not only were shaped by the interneuron-specific properties of dendritic Ca2+ handling but also involved specific Ca2+ mechanisms that were regulated dynamically by distinct activity patterns. In dendrites of regularly spiking basket cells, AP-evoked Ca2+ rises were of large amplitude and fast kinetics; however, they decreased with membrane hyperpolarization or following high-frequency firing episodes. In contrast, AP-evoked Ca2+ elevations in dendrites of Schaffer collateral-associated cells exhibited significantly smaller amplitude and slower kinetics, but increased with membrane hyperpolarization. These cell type-specific properties of AP-evoked dendritic Ca2+ signalling were determined by distinct endogenous buffer capacities of the interneurons examined and by specific types of VSCCs recruited by APs during different patterns of activity. Furthermore, AP-evoked Ca2+ transients summated efficiently during theta-like bursting and were associated with the induction of long-term potentiation at inhibitory synapses onto both types of interneurons. Therefore, the cell type-specific profile of AP-evoked dendritic Ca2+ signalling is shaped in an activity-dependent manner, such that the same pattern of hippocampal activity can be differentially translated into dendritic Ca2+ signals in different cell types. However, Cell type-specific differences in Ca

  15. The Role of miRNAs in the Regulation of Pancreatic Cancer Stem Cells

    PubMed Central

    Bimonte, Sabrina; Barbieri, Antonio; Leongito, Maddalena; Palma, Giuseppe; del Vecchio, Vitale; Falco, Michela; Palaia, Raffaele; Albino, Vittorio; Piccirillo, Mauro; Amore, Alfonso; Petrillo, Antonella; Granata, Vincenza; Izzo, Francesco

    2016-01-01

    Pancreatic ductal adenocarcinoma is currently one of the deadliest cancers with low overall survival rate. This disease leads to an aggressive local invasion and early metastases and is poorly responsive to treatment with chemotherapy or chemoradiotherapy. Several studies have shown that pancreatic cancer stem cells (PCSCs) play different roles in the regulation of drug resistance and recurrence in pancreatic cancer. MicroRNA (miRNA), a class of newly emerging small noncoding RNAs, is involved in the modulation of several biological activities ranging from invasion to metastases development, as well as drug resistance of pancreatic cancer. In this review, we synthesize the latest findings on the role of miRNAs in regulating different biological properties of pancreatic cancer stem cells. PMID:27006664

  16. The phosphorylated C-terminus of cAR1 plays a role in cell-type-specific gene expression and STATa tyrosine phosphorylation.

    PubMed

    Briscoe, C; Moniakis, J; Kim, J Y; Brown, J M; Hereld, D; Devreotes, P N; Firtel, R A

    2001-05-01

    cAMP receptors mediate some signaling pathways via coupled heterotrimeric G proteins, while others are G-protein-independent. This latter class includes the activation of the transcription factors GBF and STATa. Within the cellular mounds formed by aggregation of Dictyostelium, micromolar levels of cAMP activate GBF function, thereby inducing the transcription of postaggregative genes and initiating multicellular differentiation. Activation of STATa, a regulator of culmination and ecmB expression, results from cAMP receptor-dependent tyrosine phosphorylation and nuclear localization, also in mound-stage cells. During mound development, the cAMP receptor cAR1 is in a low-affinity state and is phosphorylated on multiple serine residues in its C-terminus. This paper addresses possible roles of cAMP receptor phosphorylation in the cAMP-mediated stimulation of GBF activity, STATa tyrosine phosphorylation, and cell-type-specific gene expression. To accomplish this, we have expressed cAR1 mutants in a strain in which the endogenous cAMP receptors that mediate postaggregative gene expression in vivo are deleted. We then examined the ability of these cells to undergo morphogenesis and induce postaggregative and cell-type-specific gene expression and STATa tyrosine phosphorylation. Analysis of cAR1 mutants in which the C-terminal tail is deleted or the ligand-mediated phosphorylation sites are mutated suggests that the cAR1 C-terminus is not essential for GBF-mediated postaggregative gene expression or STATa tyrosine phosphorylation, but may play a role in regulating cell-type-specific gene expression and morphogenesis. A mutant receptor, in which the C-terminal tail is constitutively phosphorylated, exhibits constitutive activation of STATa tyrosine phosphorylation in pulsed cells in suspension and a significantly impaired ability to induce cell-type-specific gene expression. The constitutively phosphorylated receptor also exerts a partial dominant negative effect on

  17. Differential expression analysis of miRNA in peripheral blood mononuclear cells of patients with non-segmental vitiligo.

    PubMed

    Wang, Yi; Wang, Keyu; Liang, Jianhua; Yang, Hong; Dang, Ningning; Yang, Xi; Kong, Yi

    2015-02-01

    Vitiligo is a common depigmentary skin disease that may follow a pattern of multifactorial inheritance. The essential factors of its immunopathogenesis is thought to be the selective destruction of melanocytes. As a new class of microregulators of gene expression, miRNA have been reported to play vital roles in autoimmune diseases, metabolic diseases and cancer. This study sought to characterize the different miRNA expression pattern in the peripheral blood mononuclear cells (PBMC) of patients with non-segmental vitiligo (NSV) and healthy individuals and to examine their direct responses to thymosin α1 (Tα1) treatment. The miRNA expression profile in the PBMC of patients with NSV was analyzed using Exiqon's miRCURY LNA microRNA Array. The differentially expressed miRNA were validated by real-time quantitative polymerase chain reaction. We found that the expression levels of miR-224-3p and miR-4712-3p were upregulated, and miR-3940-5p was downregulated in the PBMC. The common clinical immune modulator Tα1 changed the miRNA expression profile. Our analysis showed that differentially expressed miRNA were associated with the mechanism of immune imbalance of vitiligo and that Tα1 could play an important role in changing the expression of these miRNA in the PBMC of patients with NSV. This study provided further evidence that miRNA may serve as novel drug targets for vitiligo therapeutic evaluation.

  18. Cell type specific repression of the varicella zoster virus immediate early gene 62 promoter by the cellular Oct-2 transcription factor.

    PubMed

    Patel, Y; Gough, G; Coffin, R S; Thomas, S; Cohen, J I; Latchman, D S

    1998-05-11

    The cellular transcription factor Oct-2.1 has previously been shown to repress the transactivation of the varicella zoster virus (VZV) immediate early gene promoter by viral transactivators but not to inhibit its basal activity. In the case of the related virus herpes simplex virus (HSV), the effect of Oct-2 on the IE promoters has been shown to be cell type specific and to differ between the different alternatively spliced forms of Oct-2. Here we show that as well as Oct-2.1, the Oct-2.4 and 2.5 isoforms which are expressed in neuronal cells can inhibit transactivation of the VZV immediate early promoter regardless of the cell type used. In contrast, all the isoforms of Oct-2 can inhibit basal activity of the VZV promoter in neuronal cells but not in other cell types indicating that this effect is cell type specific. These effects are discussed in terms of the differential regulation of latent infections with HSV or VZV in dorsal root ganglia.

  19. Microarray Gene Expression Analysis to Evaluate Cell Type Specific Expression of Targets Relevant for Immunotherapy of Hematological Malignancies.

    PubMed

    Pont, M J; Honders, M W; Kremer, A N; van Kooten, C; Out, C; Hiemstra, P S; de Boer, H C; Jager, M J; Schmelzer, E; Vries, R G; Al Hinai, A S; Kroes, W G; Monajemi, R; Goeman, J J; Böhringer, S; Marijt, W A F; Falkenburg, J H F; Griffioen, M

    2016-01-01

    Cellular immunotherapy has proven to be effective in the treatment of hematological cancers by donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation and more recently by targeted therapy with chimeric antigen or T-cell receptor-engineered T cells. However, dependent on the tissue distribution of the antigens that are targeted, anti-tumor responses can be accompanied by undesired side effects. Therefore, detailed tissue distribution analysis is essential to estimate potential efficacy and toxicity of candidate targets for immunotherapy of hematological malignancies. We performed microarray gene expression analysis of hematological malignancies of different origins, healthy hematopoietic cells and various non-hematopoietic cell types from organs that are often targeted in detrimental immune responses after allogeneic stem cell transplantation leading to graft-versus-host disease. Non-hematopoietic cells were also cultured in the presence of IFN-γ to analyze gene expression under inflammatory circumstances. Gene expression was investigated by Illumina HT12.0 microarrays and quality control analysis was performed to confirm the cell-type origin and exclude contamination of non-hematopoietic cell samples with peripheral blood cells. Microarray data were validated by quantitative RT-PCR showing strong correlations between both platforms. Detailed gene expression profiles were generated for various minor histocompatibility antigens and B-cell surface antigens to illustrate the value of the microarray dataset to estimate efficacy and toxicity of candidate targets for immunotherapy. In conclusion, our microarray database provides a relevant platform to analyze and select candidate antigens with hematopoietic (lineage)-restricted expression as potential targets for immunotherapy of hematological cancers.

  20. MiRNA profile associated with replicative senescence, extended cell culture, and ectopic telomerase expression in human foreskin fibroblasts.

    PubMed

    Bonifacio, Laura N; Jarstfer, Michael B

    2010-09-01

    Senescence is a highly regulated process that limits cellular replication by enforcing a G1 arrest in response to various stimuli. Replicative senescence occurs in response to telomeric DNA erosion, and telomerase expression can offset replicative senescence leading to immortalization of many human cells. Limited data exists regarding changes of microRNA (miRNA) expression during senescence in human cells and no reports correlate telomerase expression with regulation of senescence-related miRNAs. We used miRNA microarrays to provide a detailed account of miRNA profiles for early passage and senescent human foreskin (BJ) fibroblasts as well as early and late passage immortalized fibroblasts (BJ-hTERT) that stably express the human telomerase reverse transcriptase subunit hTERT. Selected miRNAs that were differentially expressed in senescence were assayed for expression in quiescent cells to identify miRNAs that are specifically associated with senescence-associated growth arrest. From this group of senescence-associated miRNAs, we confirmed the ability of miR-143 to induce growth arrest after ectopic expression in young fibroblasts. Remarkably, miR-143 failed to induce growth arrest in BJ-hTERT cells. Importantly, the comparison of late passage immortalized fibroblasts to senescent wild type fibroblasts reveals that miR-146a, a miRNA with a validated role in regulating the senescence associated secretory pathway, is also regulated during extended cell culture independently of senescence. The discovery that miRNA expression is impacted by expression of ectopic hTERT as well as extended passaging in immortalized fibroblasts contributes to a comprehensive understanding of the connections between telomerase expression, senescence and processes of cellular aging.

  1. The pan-deacetylase inhibitor panobinostat suppresses the expression of oncogenic miRNAs in hepatocellular carcinoma cell lines.

    PubMed

    Henrici, Alexander; Montalbano, Roberta; Neureiter, Daniel; Krause, Michael; Stiewe, Thorsten; Slater, Emily Prentice; Quint, Karl; Ocker, Matthias; Di Fazio, Pietro

    2015-08-01

    Deacetylase inhibitors (DACi) are a new class of drugs with a broad spectrum of mechanisms that favor their application in cancer therapy. Currently, the exact mechanisms and cellular effects of DACi have not been fully elucidated. In addition to their effects on histone acetylation, DACi can interfere with gene expression via miRNA pathways. Treatment with panobinostat (LBH589), a novel potent DACi, led to the highly aberrant modulation of several miRNAs in hepatocellular carcinoma (HCC) cell lines as shown by miRNA array analysis. Among them, hsa-miR-19a, hsa-miR-19b1 and the corresponding precursors were down-regulated by panobinostat in TP53(-/-) Hep3B and TP53(+/+) HepG2 cell lines; hsa-miR30a-5p mature form only was suppressed in both HCC cell lines, as confirmed by further RT-qPCR analysis. In HCC cell lines, panobinostat caused the upregulation of the predicted miRNA targets APAF1 and Beclin1 protein levels. Transfection with oligonucleotides mimicking these miRNAs led to an increase in the viability rate of both cell lines as analyzed by impedance-based real-time cell analysis. In addition, transfecting miRNA mimicking oligonucleotides resulted in the decrease of APAF1, Beclin1 and PAK6 at the protein level, proving the regulating influence of the investigated miRNAs on gene final products. The overexpression of the above mentioned oncomiRs in Hep3B and HepG2 cell lines leads to cell proliferation and downregulation of cell death associated proteins. In our model, panobinostat exerts its anti-cancer effect by suppressing these miRNAs and restoring the expression of their corresponding tumor suppressor targets. © 2013 Wiley Periodicals, Inc.

  2. Tuft Cell Regulation of miRNAs in Pancreatic Cancer

    DTIC Science & Technology

    2014-12-01

    Stanton L Young Blvd , WP1345 Oklahoma City, OK 73104 9. SPONSORING I MONITORING AGENCY NAME(S) A ND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S) U.S...Sureban SM, May R, Lightfoot SA, Hoskins AB, Lerner M, Brackett DJ, et al. DCAMKL-1 regulates epithelial-mesenchymal transition in human pancreatic cells

  3. Identifying Cell Type-Specific Transcription Factors by Integrating ChIP-seq and eQTL Data-Application to Monocyte Gene Regulation.

    PubMed

    Choudhury, Mudra; Ramsey, Stephen A

    2016-01-01

    We describe a novel computational approach to identify transcription factors (TFs) that are candidate regulators in a human cell type of interest. Our approach involves integrating cell type-specific expression quantitative trait locus (eQTL) data and TF data from chromatin immunoprecipitation-to-tag-sequencing (ChIP-seq) experiments in cell lines. To test the method, we used eQTL data from human monocytes in order to screen for TFs. Using a list of known monocyte-regulating TFs, we tested the hypothesis that the binding sites of cell type-specific TF regulators would be concentrated in the vicinity of monocyte eQTLs. For each of 397 ChIP-seq data sets, we obtained an enrichment ratio for the number of ChIP-seq peaks that are located within monocyte eQTLs. We ranked ChIP-seq data sets according to their statistical significances for eQTL overlap, and from this ranking, we observed that monocyte-regulating TFs are more highly ranked than would be expected by chance. We identified 27 TFs that had significant monocyte enrichment scores and mapped them into a protein interaction network. Our analysis uncovered two novel candidate monocyte-regulating TFs, BCLAF1 and SIN3A. Our approach is an efficient method to identify candidate TFs that can be used for any cell/tissue type for which eQTL data are available.

  4. Identifying Cell Type-Specific Transcription Factors by Integrating ChIP-seq and eQTL Data–Application to Monocyte Gene Regulation

    PubMed Central

    Choudhury, Mudra; Ramsey, Stephen A.

    2016-01-01

    We describe a novel computational approach to identify transcription factors (TFs) that are candidate regulators in a human cell type of interest. Our approach involves integrating cell type-specific expression quantitative trait locus (eQTL) data and TF data from chromatin immunoprecipitation-to-tag-sequencing (ChIP-seq) experiments in cell lines. To test the method, we used eQTL data from human monocytes in order to screen for TFs. Using a list of known monocyte-regulating TFs, we tested the hypothesis that the binding sites of cell type-specific TF regulators would be concentrated in the vicinity of monocyte eQTLs. For each of 397 ChIP-seq data sets, we obtained an enrichment ratio for the number of ChIP-seq peaks that are located within monocyte eQTLs. We ranked ChIP-seq data sets according to their statistical significances for eQTL overlap, and from this ranking, we observed that monocyte-regulating TFs are more highly ranked than would be expected by chance. We identified 27 TFs that had significant monocyte enrichment scores and mapped them into a protein interaction network. Our analysis uncovered two novel candidate monocyte-regulating TFs, BCLAF1 and SIN3A. Our approach is an efficient method to identify candidate TFs that can be used for any cell/tissue type for which eQTL data are available. PMID:28008225

  5. Cell-type-specific expression of murine multifunctional galectin-3 and its association with follicular atresia/luteolysis in contrast to pro-apoptotic galectins-1 and -7.

    PubMed

    Lohr, Michaela; Kaltner, Herbert; Lensch, Martin; André, Sabine; Sinowatz, Fred; Gabius, Hans-Joachim

    2008-09-01

    Galectin-3 is a multifunctional protein with modular design. A distinct expression profile was determined in various murine organs when set into relation to homodimeric galectins-1 and -7. Fittingly, the signature of putative transcription-factor-binding sites in the promoter region of the galectin-3 gene affords a toolbox for a complex combinatorial regulation, distinct from the respective sequence stretches in galectins-1 and -7. A striking example for cell-type specificity was the ovary, where these two lectins were confined to the surface epithelium. Immunohistochemically, galectin-3 was found in macrophages of the cortical interstitium between developing follicles and medullary interstitium, matching the distribution of the F4/80 antigen. With respect to atresia and luteolysis strong signals in granulosa cells of atretic preantral but not antral follicles and increasing positivity in corpora lutea upon regression coincided with DNA fragmentation. Labeled galectin-3 revealed lactose-inhibitable binding to granulosa cells. Also, slender processes of vital granulosa cells which extended into the zona pellucida were positive. This study demonstrates cell-type specificity and cycle-associated regulation for galectin-3 with increased presence in atretic preantral follicles and in late stages of luteolysis.

  6. Histone deacetylase inhibitors modulate miRNA and mRNA expression, block metaphase, and induce apoptosis in inflammatory breast cancer cells.

    PubMed

    Chatterjee, Namita; Wang, Wei-Lin Winnie; Conklin, Tucker; Chittur, Sridar; Tenniswood, Martin

    2013-07-01

    To develop new therapies for inflammatory breast cancer (IBC) we have compared the effects of two hydroxamic acid-based histone deacetylase (HDAC) inhibitors, CG-1521 and Trichostatin A (TSA) on the biology of two IBC cell lines: SUM149PT and SUM190PT. CG-1521 and TSA induce dose (0-10 µM) and time-dependent (0-96 h) increases in the proportion of cells undergoing cell cycle arrest and apoptosis in the presence or absence of 17β-estradiol. In SUM 149PT cells, both CG-1521 and TSA increase the levels of acetylated α-tubulin; however the morphological effects are different: CG-1521 blocks mitotic spindle formation and prevents abscission during cytokinesis while TSA results in an increase in cell size. In SUM190PT cells CG-1521 does not cause an increase in acetylated-α-tubulin and even though TSA significantly increases the levels of acetylated tubulin, neither inhibitor alters the morphology of the cells. Microarray analysis demonstrates that CG-1521 modulates the expression of 876 mRNAs and 63 miRNAs in SUM149PT cells, and 1227 mRNAs and 35 miRNAs in SUM190PT cells. Only 9% of the genes are commonly modulated in both cell lines, suggesting that CG-1521 and TSA target different biological processes in the two cell lines most likely though the inhibition of different HDACs in these cell lines. Gene ontology (GO) analysis reveals that CG-1521 affects the expression of mRNAs that encode proteins associated with the spindle assembly checkpoint, chromosome segregation, and microtubule-based processes in both cell lines and has cell-type specific effects on lipid biosynthesis, response to DNA damage, and cell death.

  7. NCAM1 is the Target of miRNA-572: Validation in the Human Oligodendroglial Cell Line.

    PubMed

    Mancuso, Roberta; Agostini, Simone; Marventano, Ivana; Hernis, Ambra; Saresella, Marina; Clerici, Mario

    2017-03-22

    The neural cell adhesion molecule 1 (NCAM1) is a fundamental protein in cell-cell interaction and in cellular developmental processes, and its dysregulation is involved in a number of diseases including multiple sclerosis. Studies in rats suggest that the modulation of NCAM1 expression is regulated by miRNA-572, but no data are available confirming such interaction in the human system. We analyzed whether this is the case using a human oligodendroglial cell line (MO3.13). MO3.13 cells were transfected with miRNA-572 mimic and inhibitor separately; NCAM1 mRNA and protein expression levels were analyzed at different time points after transfection. Results indicated that NCAM1 expression is increased after transfection with miRNA-572 inhibitor, whereas it is decreased after transfection with the mimic (p < 0.005). The interaction between NCAM1 and miRNA-572 was subsequently confirmed in a Vero cell line that does not express NCAM1, by luciferase assay after transfection with NCAM1. These results confirm that miRNA-572 regulates NCAM1 and for the first time demonstrate that this interaction regulates NCAM1 expression in human cells. Data herein also support the hypothesis that miRNA-572 is involved in diseases associated with NCAM1 deregulation, suggesting its possible use as a biomarker in these diseases.

  8. Bivalent histone modifications in stem cells poise miRNA loci for CpG island hypermethylation in human cancer.

    PubMed

    Iliou, Maria S; Lujambio, Amaia; Portela, Anna; Brüstle, Oliver; Koch, Philipp; Andersson-Vincent, Per Henrik; Sundström, Erik; Hovatta, Outi; Esteller, Manel

    2011-11-01

    It has been proposed that the existence of stem cell epigenetic patterns confer a greater likelihood of CpG island hypermethylation on tumor suppressor-coding genes in cancer. The suggested mechanism is based on the Polycomb-mediated methylation of K27 of histone H3 and the recruitment of DNA methyltransferases on the promoters of tumor suppressor genes in cancer cells, when those genes are preferentially pre-marked in embryonic stem cells (ESCs) with bivalent chromatin domains. On the other hand, miRNAs appear to be dysregulated in cancer, with many studies reporting silencing of miRNA genes due to aberrant hypermethylation of their promoter regions. We wondered whether a pre-existing histone modification profile in stem cells might also contribute to the DNA methylation-associated silencing of miRNA genes in cancer. To address this, we examined a group of tumor suppressor miRNA genes previously reported to become hypermethylated and inactivated specifically in cancer cells. We analyzed the epigenetic events that take place along their promoters in human embryonic stem cells and in transformed cells. Our results suggest that there is a positive correlation between the existence of bivalent chromatin domains on miRNA promoters in ESCs and the hypermethylation of those genes in cancer, leading us to conclude that this epigenetic mark could be a mechanism that prepares miRNA promoters for further DNA hypermethylation in human tumors.

  9. The regulation of miRNA-211 expression and its role in melanoma cell invasiveness.

    PubMed

    Mazar, Joseph; DeYoung, Katherine; Khaitan, Divya; Meister, Edward; Almodovar, Alvin; Goydos, James; Ray, Animesh; Perera, Ranjan J

    2010-11-01

    The immediate molecular mechanisms behind invasive melanoma are poorly understood. Recent studies implicate microRNAs (miRNAs) as important agents in melanoma and other cancers. To investigate the role of miRNAs in melanoma, we subjected human melanoma cell lines to miRNA expression profiling, and report a range of variations in several miRNAs. Specifically, compared with expression levels in melanocytes, levels of miR-211 were consistently reduced in all eight non-pigmented melanoma cell lines we examined; they were also reduced in 21 out of 30 distinct melanoma samples from patients, classified as primary in situ, regional metastatic, distant metastatic, and nodal metastatic. The levels of several predicted target mRNAs of miR-211 were reduced in melanoma cell lines that ectopically expressed miR-211. In vivo target cleavage assays confirmed one such target mRNA encoded by KCNMA1. Mutating the miR-211 binding site seed sequences at the KCNMA1 3'-UTR abolished target cleavage. KCNMA1 mRNA and protein expression levels varied inversely with miR-211 levels. Two different melanoma cell lines ectopically expressing miR-211 exhibited significant growth inhibition and reduced invasiveness compared with the respective parental melanoma cell lines. An shRNA against KCNMA1 mRNA also demonstrated similar effects on melanoma cells. miR-211 is encoded within the sixth intron of TRPM1, a candidate suppressor of melanoma metastasis. The transcription factor MITF, important for melanocyte development and function, is needed for high TRPM1 expression. MITF is also needed for miR-211 expression, suggesting that the tumor-suppressor activities of MITF and/or TRPM1 may at least partially be due to miR-211's negative post transcriptional effects on the KCNMA1 transcript. Given previous reports of high KCNMA1 levels in metastasizing melanoma, prostate cancer and glioma, our findings that miR-211 is a direct posttranscriptional regulator of KCNMA1 expression as well as the dependence

  10. Control of the innate epithelial antimicrobial response is cell-type specific and dependent on relevant microenvironmental stimuli

    PubMed Central

    Schauber, Jürgen; Dorschner, Robert A; Yamasaki, Kenshi; Brouha, Brook; Gallo, Richard L

    2006-01-01

    Immune defence against microbes depends in part on the production of antimicrobial peptides, a process that occurs in a variety of cell types but is incompletely understood. In this study, the mechanisms responsible for the induction of cathelicidin and β-defensin antimicrobial peptides were found to be independent and specific to the cell type and stimulus. Vitamin D3 induced cathelicidin expression in keratinocytes and monocytes but not in colonic epithelial cells. Conversely, butyrate induced cathelicidin in colonic epithelia but not in keratinocytes or monocytes. Distinct factors induced β-defensin expression. In all cell types, vitamin D3 activated the cathelicidin promoter and was dependent on a functional vitamin D responsive element. However, in colonic epithelia butyrate induced cathelicidin expression without increasing promoter activity and vitamin D3 activated the cathelicidin promoter without a subsequent increase in transcript accumulation. Induction of cathelicidin transcript correlated with increased processed mature peptide and enhanced antimicrobial activity against Staphylococcus aureus. However, induction of β-defensin-2 expression did not alter the innate antimicrobial capacity of cells in culture. These data suggest that antimicrobial peptide expression is regulated in a tissue-specific manner at transcriptional, post-transcriptional and post-translational levels. Furthermore, these data show for the first time that innate antimicrobial activity can be triggered independently of the release of other pro-inflammatory molecules, and suggest strategies for augmenting innate immune defence without increasing inflammation. PMID:16895558

  11. Cell Type-Specific and Inducible PTEN Gene Silencing by a Tetracycline Transcriptional Activator-Regulated Short Hairpin RNA.

    PubMed

    Wang, Shan; Wang, Ting; Wang, Tao; Jia, Lintao

    2015-11-01

    Inducible and reversible gene silencing in desired types of cells is instrumental for deciphering gene functions using cultured cells or in vivo models. However, efficient conditional gene knockdown systems remain to be established. Here, we report the generation of an inducible expression system for short hairpin RNA (shRNA) targeted to PTEN, a well-documented dual-specificity phosphatase involved in tumor suppression and ontogenesis. Upon induction by doxycycline (DOX), the reverse tetracycline transcriptional activator (rtTA) switched on the concomitant expression of GFP and a miR-30 precursor, the subsequent processing of which released the embedded PTEN-targeted shRNA. The efficacy and reversibility of PTEN knockdown by this construct was validated in normal and neoplastic cells, in which PTEN deficiency resulted in accelerated cell proliferation, suppressed apoptosis, and increased invasiveness. Transgenic mice harboring the conditional shRNA-expression cassette were obtained; GFP expression and concurrent PTEN silencing were observed upon ectopic expression of rtTA and induction with Dox. Therefore, this study provides novel tools for the precise dissection of PTEN functions and the generation of PTEN loss of function models in specific subsets of cells during carcinogenesis and ontogenesis.

  12. Cell-type specific circuit connectivity of hippocampal CA1 revealed through Cre-dependent rabies tracing

    PubMed Central

    Sun, Yanjun; Nguyen, Amanda; Nguyen, Joseph; Le, Luc; Saur, Dieter; Choi, Jiwon; Callaway, Edward M.; Xu, Xiangmin

    2014-01-01

    Summary We applied a new Cre-dependent, genetically modified rabies-based tracing system to map direct synaptic connections to CA1 excitatory and inhibitory neuron types in mouse hippocampus. We found common inputs to excitatory and inhibitory CA1 neurons from CA3, CA2, entorhinal cortex and the medial septum (MS), and unexpectedly also from the subiculum. Excitatory CA1 neurons receive inputs from both cholinergic and GABAergic MS neurons while inhibitory CA1 neurons receive a great majority of input from GABAergic MS neurons; both cell types also receive weaker input from glutamatergic MS neurons. Comparisons of inputs to CA1 PV+ interneurons versus SOM+ interneurons showed similar strengths of input from the subiculum, but PV+ interneurons receive much stronger input than SOM+ neurons from CA3, entorhinal cortex and MS. Differential input from CA3 to specific CA1 cell types was also demonstrated functionally using laser scanning photostimulation and whole cell recordings. PMID:24656815

  13. Cell type-specific modulation of lipid mediator's formation in murine adipose tissue by omega-3 fatty acids.

    PubMed

    Kuda, Ondrej; Rombaldova, Martina; Janovska, Petra; Flachs, Pavel; Kopecky, Jan

    2016-01-15

    Mutual interactions between adipocytes and immune cells in white adipose tissue (WAT) are involved in modulation of lipid metabolism in the tissue and also in response to omega-3 polyunsaturated fatty acids (PUFA), which counteract adverse effects of obesity. This complex interplay depends in part on in situ formed anti- as well as pro-inflammatory lipid mediators, but cell types engaged in the synthesis of the specific mediators need to be better characterized. We used tissue fractionation and metabolipidomic analysis to identify cells producing lipid mediators in epididymal WAT of mice fed for 5 weeks obesogenic high-fat diet (lipid content 35% wt/wt), which was supplemented or not by omega-3 PUFA (4.3 mg eicosapentaenoic acid and 14.7 mg docosahexaenoic acid per g of diet). Our results demonstrate selective increase in levels of anti-inflammatory lipid mediators in WAT in response to omega-3, reflecting either their association with adipocytes (endocannabinoid-related N-docosahexaenoylethanolamine) or with stromal vascular cells (pro-resolving lipid mediator protectin D1). In parallel, tissue levels of obesity-associated pro-inflammatory endocannabinoids were suppressed. Moreover, we show that adipose tissue macrophages (ATMs), which could be isolated using magnetic force from the stromal vascular fraction, are not the major producers of protectin D1 and that omega-3 PUFA lowered lipid load in ATMs while promoting their less-inflammatory phenotype. Taken together, these results further document specific roles of various cell types in WAT in control of WAT inflammation and metabolism and they suggest that also other cells but ATMs are engaged in production of pro-resolving lipid mediators in response to omega-3 PUFA.

  14. BDNF Promotes Axon Branching of Retinal Ganglion Cells via miRNA-132 and p250GAP

    PubMed Central

    Marler, Katharine J.; Suetterlin, Philipp; Dopplapudi, Asha; Rubikaite, Aine; Adnan, Jihad; Maiorano, Nicola A.; Lowe, Andrew S.; Thompson, Ian D.; Pathania, Manav; Bordey, Angelique; Fulga, Tudor; Van Vactor, David L.; Hindges, Robert

    2014-01-01

    A crucial step in the development of the vertebrate visual system is the branching of retinal ganglion cell (RGC) axons within their target, the superior colliculus/tectum. A major player in this process is the neurotrophin brain-derived neurotrophic factor (BDNF). However, the molecular basis for the signaling pathways mediating BDNF action is less well understood. As BDNF exerts some of its functions by controlling the expression of microRNAs (miRNAs), we investigated whether miRNAs are also involved in BDNF-mediated retinal axon branching. Here, we demonstrate that the expression pattern of miRNA-132 in the retina is consistent with its involvement in this process, and that BDNF induces the upregulation of miRNA-132 in retinal cultures. Furthermore, in vitro gain-of-function and loss-of-function approaches in retinal cultures reveal that miRNA-132 mediates axon branching downstream of BDNF. A known target of miRNA-132 is the Rho family GTPase-activating protein, p250GAP. We find that p250GAP is expressed in RGC axons and mediates the effects of miRNA-132 in BDNF-induced branching. BDNF treatment or overexpression of miRNA-132 leads to a reduction in p250GAP protein levels in retinal cultures, whereas the overexpression of p250GAP abolishes BDNF-induced branching. Finally, we used a loss-of-function approach to show that miRNA-132 affects the maturation of RGC termination zones in the mouse superior colliculus in vivo, while their topographic targeting remains intact. Together, our data indicate that BDNF promotes RGC axon branching during retinocollicular/tectal map formation via upregulation of miRNA-132, which in turn downregulates p250GAP. PMID:24431455

  15. BDNF promotes axon branching of retinal ganglion cells via miRNA-132 and p250GAP.

    PubMed

    Marler, Katharine J; Suetterlin, Philipp; Dopplapudi, Asha; Rubikaite, Aine; Adnan, Jihad; Maiorano, Nicola A; Lowe, Andrew S; Thompson, Ian D; Pathania, Manav; Bordey, Angelique; Fulga, Tudor; Van Vactor, David L; Hindges, Robert; Drescher, Uwe

    2014-01-15

    A crucial step in the development of the vertebrate visual system is the branching of retinal ganglion cell (RGC) axons within their target, the superior colliculus/tectum. A major player in this process is the neurotrophin brain-derived neurotrophic factor (BDNF). However, the molecular basis for the signaling pathways mediating BDNF action is less well understood. As BDNF exerts some of its functions by controlling the expression of microRNAs (miRNAs), we investigated whether miRNAs are also involved in BDNF-mediated retinal axon branching. Here, we demonstrate that the expression pattern of miRNA-132 in the retina is consistent with its involvement in this process, and that BDNF induces the upregulation of miRNA-132 in retinal cultures. Furthermore, in vitro gain-of-function and loss-of-function approaches in retinal cultures reveal that miRNA-132 mediates axon branching downstream of BDNF. A known target of miRNA-132 is the Rho family GTPase-activating protein, p250GAP. We find that p250GAP is expressed in RGC axons and mediates the effects of miRNA-132 in BDNF-induced branching. BDNF treatment or overexpression of miRNA-132 leads to a reduction in p250GAP protein levels in retinal cultures, whereas the overexpression of p250GAP abolishes BDNF-induced branching. Finally, we used a loss-of-function approach to show that miRNA-132 affects the maturation of RGC termination zones in the mouse superior colliculus in vivo, while their topographic targeting remains intact. Together, our data indicate that BDNF promotes RGC axon branching during retinocollicular/tectal map formation via upregulation of miRNA-132, which in turn downregulates p250GAP.

  16. Transcription of mouse Sp2 yields alternatively spliced and sub-genomic mRNAs in a tissue- and cell type-specific fashion

    PubMed Central

    Yin, Haifeng; Nichols, Teresa D.; Horowitz, Jonathan M.

    2010-01-01

    The Sp-family of transcription factors is comprised by nine members, Sp1-9, that share a highly-conserved DNA-binding domain. Sp2 is a poorly characterized member of this transcription factor family that is widely expressed in murine and human cell lines yet exhibits little DNA-binding or trans-activation activity in these settings. As a prelude to the generation of a “knock-out” mouse strain, we isolated a mouse Sp2 cDNA and performed a detailed analysis of Sp2 transcription in embryonic and adult mouse tissues. We report that (1) the 5′ untranslated region of Sp2 is subject to alternative splicing, (2) Sp2 transcription is regulated by at least two promoters that differ in their cell-type specificity, (3) one Sp2 promoter is highly active in nine mammalian cell lines and strains and is regulated by at least five discrete stimulatory and inhibitory elements, (4) a variety of sub-genomic messages are synthesized from the Sp2 locus in a tissue- and cell type-specific fashion and these transcripts have the capacity to encode a novel partial-Sp2 protein, and (5) RNA in situ hybridization assays indicate that Sp2 is widely expressed during mouse embryogenesis, particularly in the embryonic brain, and robust Sp2 expression occurs in neurogenic regions of the post-natal and adult brain. PMID:20353838

  17. Identification of common and cell type specific LXXLL motif EcR cofactors using a bioinformatics refined candidate RNAi screen in Drosophila melanogaster cell lines

    PubMed Central

    2011-01-01

    Background During Drosophila development, titers of the steroid ecdysone trigger and maintain temporal and tissue specific biological transitions. Decades of evidence reveal that the ecdysone response is both unique to specific tissues and distinct among developmental timepoints. To achieve this diversity in response, the several isoforms of the Ecdysone Receptor, which transduce the hormone signal to the genome level, are believed to interact with tissue specific cofactors. To date, little is known about the identity of these cofactor interactions; therefore, we conducted a bioinformatics informed, RNAi luciferase reporter screen against a subset of putative candidate cofactors identified through an in silico proteome screen. Candidates were chosen based on criteria obtained from bioinformatic consensus of known nuclear receptor cofactors and homologs, including amino acid sequence motif content and context. Results The bioinformatics pre-screen of the Drosophila melanogaster proteome was successful in identifying an enriched putative candidate gene cohort. Over 80% of the genes tested yielded a positive hit in our reporter screen. We have identified both cell type specific and common cofactors which appear to be necessary for proper ecdysone induced gene regulation. We have determined that certain cofactors act as co-repressors to reduce target gene expression, while others act as co-activators to increase target gene expression. Interestingly, we find that a few of the cofactors shared among cell types have a reversible roles to function as co-repressors in certain cell types while in other cell types they serve as co-activators. Lastly, these proteins are highly conserved, with higher order organism homologs also harboring the LXXLL steroid receptor interaction domains, suggesting a highly conserved mode of steroid cell target specificity. Conclusions In conclusion, we submit these cofactors as novel components of the ecdysone signaling pathway in order to

  18. miRNA and TMPRSS2-ERG do not mind their own business in prostate cancer cells.

    PubMed

    Fayyaz, Sundas; Farooqi, Ammad Ahmad

    2013-05-01

    Oncogenic fusion proteins belong to an important class that disrupts gene expression networks in a cell. Astonishingly, fusion-positive prostate cancer cells enable the multi-gene regulatory capability of miRNAs to remodel the signal transduction landscape, enhancing or antagonizing the transmission of information to downstream effectors. Accumulating evidence substantiates the fact that miRNAs translate into dose-dependent responsiveness of cells to signaling regulators in transmembrane protease serine 2:ETS-related gene (TMPRSS2-ERG)-positive cells. Wide ranging signaling proteins are the targets for the degree of quantitative fluctuations imposed by miRNAs. miRNA signatures are aberrantly expressed in fusion-positive cancer cells, suggesting that they have a cumulative effect on tumor aggressiveness. It seems attractive to note that TMPRSS2:ERG fusion has a stronger effect as tumors positive for the oncogenic TMPRSS2:ERG have dysregulated oncomirs and tumor suppressor miRNA signature. It is undeniable that a comprehensive analysis of the prostate cancer microRNAome is necessary to uncover novel microRNAs and pathways associated with prostate cancer. Moreover, the identification and validation of miRNA signature in TMPRSS2-ERG-positive prostate cancer cells may help to identify novel molecular targets and pathways for personalized therapy.

  19. Cell Type-Specific Differences in Spike Timing and Spike Shape in the Rat Parasubiculum and Superficial Medial Entorhinal Cortex.

    PubMed

    Ebbesen, Christian Laut; Reifenstein, Eric Torsten; Tang, Qiusong; Burgalossi, Andrea; Ray, Saikat; Schreiber, Susanne; Kempter, Richard; Brecht, Michael

    2016-07-26

    The medial entorhinal cortex (MEC) and the adjacent parasubiculum are known for their elaborate spatial discharges (grid cells, border cells, etc.) and the precessing of spikes relative to the local field potential. We know little, however, about how spatio-temporal firing patterns map onto cell types. We find that cell type is a major determinant of spatio-temporal discharge properties. Parasubicular neurons and MEC layer 2 (L2) pyramids have shorter spikes, discharge spikes in bursts, and are theta-modulated (rhythmic, locking, skipping), but spikes phase-precess only weakly. MEC L2 stellates and layer 3 (L3) neurons have longer spikes, do not discharge in bursts, and are weakly theta-modulated (non-rhythmic, weakly locking, rarely skipping), but spikes steeply phase-precess. The similarities between MEC L3 neurons and MEC L2 stellates on one hand and parasubicular neurons and MEC L2 pyramids on the other hand suggest two distinct streams of temporal coding in the parahippocampal cortex.

  20. A roadmap of cell-type specific gene expression during sequential stages of the arbuscular mycorrhiza symbiosis

    PubMed Central

    2013-01-01

    Background About 80% of today’s land plants are able to establish an arbuscular mycorrhizal (AM) symbiosis with Glomeromycota fungi to improve their access to nutrients and water in the soil. On the molecular level, the development of AM symbioses is only partly understood, due to the asynchronous development of the microsymbionts in the host roots. Although many genes specifically activated during fungal colonization have been identified, genome-wide information on the exact place and time point of their activation remains limited. Results In this study, we relied on a combination of laser-microdissection and the use of Medicago GeneChips to perform a genome-wide analysis of transcription patterns in defined cell-types of Medicago truncatula roots mycorrhized with Glomus intraradices. To cover major stages of AM development, we harvested cells at 5-6 and at 21 days post inoculation (dpi). Early developmental stages of the AM symbiosis were analysed by monitoring gene expression in appressorial and non-appressorial areas from roots harbouring infection units at 5-6 dpi. Here, the use of laser-microdissection for the first time enabled the targeted harvest of those sites, where fungal hyphae first penetrate the root. Circumventing contamination with developing arbuscules, we were able to specifically detect gene expression related to early infection events. To cover the late stages of AM formation, we studied arbusculated cells, cortical cells colonized by intraradical hyphae, and epidermal cells from mature mycorrhizal roots at 21 dpi. Taken together, the cell-specific expression patterns of 18014 genes were revealed, including 1392 genes whose transcription was influenced by mycorrhizal colonization at different stages, namely the pre-contact phase, the infection of roots via fungal appressoria, the subsequent colonization of the cortex by fungal hyphae, and finally the formation of arbuscules. Our cellular expression patterns identified distinct groups of AM

  1. A roadmap of cell-type specific gene expression during sequential stages of the arbuscular mycorrhiza symbiosis.

    PubMed

    Hogekamp, Claudia; Küster, Helge

    2013-05-07

    About 80% of today's land plants are able to establish an arbuscular mycorrhizal (AM) symbiosis with Glomeromycota fungi to improve their access to nutrients and water in the soil. On the molecular level, the development of AM symbioses is only partly understood, due to the asynchronous development of the microsymbionts in the host roots. Although many genes specifically activated during fungal colonization have been identified, genome-wide information on the exact place and time point of their activation remains limited. In this study, we relied on a combination of laser-microdissection and the use of Medicago GeneChips to perform a genome-wide analysis of transcription patterns in defined cell-types of Medicago truncatula roots mycorrhized with Glomus intraradices. To cover major stages of AM development, we harvested cells at 5-6 and at 21 days post inoculation (dpi). Early developmental stages of the AM symbiosis were analysed by monitoring gene expression in appressorial and non-appressorial areas from roots harbouring infection units at 5-6 dpi. Here, the use of laser-microdissection for the first time enabled the targeted harvest of those sites, where fungal hyphae first penetrate the root. Circumventing contamination with developing arbuscules, we were able to specifically detect gene expression related to early infection events. To cover the late stages of AM formation, we studied arbusculated cells, cortical cells colonized by intraradical hyphae, and epidermal cells from mature mycorrhizal roots at 21 dpi. Taken together, the cell-specific expression patterns of 18014 genes were revealed, including 1392 genes whose transcription was influenced by mycorrhizal colonization at different stages, namely the pre-contact phase, the infection of roots via fungal appressoria, the subsequent colonization of the cortex by fungal hyphae, and finally the formation of arbuscules. Our cellular expression patterns identified distinct groups of AM-activated genes

  2. miRNA-155 modulates the malignant biological characteristics of NK/T-cell lymphoma cells by targeting FOXO3a gene.

    PubMed

    Ji, Wei-guo; Zhang, Xu-dong; Sun, Xiang-dong; Wang, Xiang-qi; Chang, Bao-ping; Zhang, Ming-zhi

    2014-12-01

    This study investigated the effects of miRNA-155 on malignant biological characteristics of NK/T-cell lymphoma cell lines and the possible mechanism. The expression of miRNA-155 was detected in lymphoma cell lines from different sources (SNK-6, YTS, Jurkat and DOHH2) by real-time PCR. Lentiviral vectors (pLL3.7) that could overexpress or downexpress miRNA-155 were constructed. Recombinant lentiviral particles were prepared and purified, and their titers determined. The expression of miRNA-155 in the infected SNK-6 cells and the cell proliferation were detected by PCR and CCK-8, respectively. Flow cytometry was used to determine the apoptosis of infected SNK-6 cells. The target of miRNA155 was predicted from Targetscan website. The effect of miRNA155 on FOXO3a expression was examined by Western blotting. The results showed that among the human NK/T-cell lymphoma cell lines SNK-6, YTS, Jurkat and DOHH2, the expression of miRNA-155 was highest in SNK-6. The infection efficiency of the recombinant lentivirus in SNK-6 was more than 70% at multiplicity of infection (MOI) of 100. The expression of miRNA-155 was significantly increased in SNK-6 cells infected by lentivirus vectors with high expression of miRNA-155 (4 times higher than the control group), and profoundly decreased in those infected with lentiviruses with low expression of miRNA-155. The proliferation of letivirus-infected SNK-6 cells was decreased as the expression of miRNA-155 reduced. The apoptosis rate was increased with the reduction in the expression of miRNA-155. FOXO3a was found to be a possible target of miRNA155, as suggested by Targetscan website. Western blotting showed that the expression of FOXO3a was significantly elevated in SNK-6 cells with miRNA-155 inhibition. It was concluded that reduction in miRNA-155 expression can inhibit the proliferation of SNK-6 lymphoma cells and promote their apoptosis, which may be associated with regulation of FOXO3a gene.

  3. Circulating osteocyte-derived exosomes contain miRNAs which are enriched in exosomes from MLO-Y4 cells

    PubMed Central

    Sato, Mari; Suzuki, Tomohide; Kawano, Mitsuoki; Tamura, Masato

    2017-01-01

    Signaling molecules produced by osteocytes have been proposed to serve as soluble factors that contribute to bone remodeling, as well as to homeostasis of other organs. However, to the best of our knowledge, there are currently no studies investigating the role of osteocyte-secreted exosomes. In the present study, ablation of osteocytes in mice [osteocyte-less (OL)] was used to examine the microRNA (miRNA) levels of plasma-circulating exosomes. In order to investigate the function of osteocyte-secreted exosomes, exosomes derived from MLO-Y4 cells were extracted and their miRNA expression levels were examined using miRNA array analysis and deep sequencing. Comparison of miRNA expression levels between plasma exosomes from OL mouse plasma and MLO-Y4-derived exosomes revealed that decreases in the number of miRNAs from exosomes circulating in the OL mouse plasma may be caused by a decrease in secretion of exosomes from osteocytes. These results suggest that osteocytes secrete exosomes containing characterized miRNAs and then circulate in the blood, and may thus transfer their components, including miRNAs, to recipient cells where they function as signaling molecules in other organs and/or tissues to regulate biological responses. PMID:28357077

  4. Cell-type-specific circuit connectivity of hippocampal CA1 revealed through Cre-dependent rabies tracing.

    PubMed

    Sun, Yanjun; Nguyen, Amanda Q; Nguyen, Joseph P; Le, Luc; Saur, Dieter; Choi, Jiwon; Callaway, Edward M; Xu, Xiangmin

    2014-04-10

    We developed and applied a Cre-dependent, genetically modified rabies-based tracing system to map direct synaptic connections to specific CA1 neuron types in the mouse hippocampus. We found common inputs to excitatory and inhibitory CA1 neurons from CA3, CA2, the entorhinal cortex (EC), the medial septum (MS), and, unexpectedly, the subiculum. Excitatory CA1 neurons receive inputs from both cholinergic and GABAergic MS neurons, whereas inhibitory neurons receive a great majority of inputs from GABAergic MS neurons. Both cell types also receive weaker input from glutamatergic MS neurons. Comparisons of inputs to CA1 PV+ interneurons versus SOM+ interneurons showed similar strengths of input from the subiculum, but PV+ interneurons received much stronger input than SOM+ neurons from CA3, the EC, and the MS. Thus, rabies tracing identifies hippocampal circuit connections and maps how the different input sources to CA1 are distributed with different strengths on each of its constituent cell types.

  5. Chen 10-marker miRNA signature for non-small cell lung cancer — EDRN Public Portal

    Cancer.gov

    A panel of 10 serum miRNAs has been identified that were found to have significantly different expression levels in non-small cell lung cancer (NSCLC) serum samples compared with the control serum samples. This panel of miRNAs was able to distinguish NSCLC cases from controls with high sensitivity and specificity. The ten miRNAs are: miR-20a, miR-24, miR-25, miR-145, miR-152, miR-199a-5p, miR-221, miR-222, miR-223, miR-320.

  6. Cell type-specific post-transcriptional regulation of production of the potent antiangiogenic and proatherogenic protein thrombospondin-1 by high glucose.

    PubMed

    Bhattacharyya, Sanghamitra; Marinic, Tina E; Krukovets, Irene; Hoppe, George; Stenina, Olga I

    2008-02-29

    Hyperglycemia is an independent risk factor for development of vascular diabetic complications. Vascular dysfunction in diabetics manifests in a tissue-specific manner; macrovasculature is affected by atherosclerotic lesions, and microvascular complications are described as "aberrant angiogenesis": in the same patient angiogenesis is increased in some tissues (e.g. retinal neovascularization) and decreased in others (e.g. in skin). Molecular cell- and tissue-specific mechanisms regulating the response of vasculature to hyperglycemia remain unclear. Thrombospondin-1 (TSP-1), a potent antiangiogenic and proatherogenic protein, has been implicated in the development of several vascular diabetic complications (atherosclerosis, nephropathy, and cardiomyopathy). This study examines cell type-specific regulation of production of thrombospondin-1 by high glucose. We previously reported the increased expression of TSP-1 in the large arteries of diabetic animals. mRNA and protein levels were up-regulated in response to high glucose. Unlike in macrovascular cells, TSP-1 protein levels are dramatically decreased in response to high glucose in microvascular endothelial cells and retinal pigment epithelial cells (RPE). This down-regulation is post-transcriptional; mRNA levels are increased. In situ mRNA hybridization and immunohistochemistry revealed that the level of mRNA is up-regulated in RPE of diabetic rats, whereas the protein level is decreased. This cell type-specific posttranscriptional suppression of TSP-1 production in response to high glucose in microvascular endothelial cells and RPE is controlled by untranslated regions of TSP-1 mRNA that regulate coupling of TSP-1 mRNA to polysomes and its translation. The cell-specific regulation of TSP-1 suggests a potential mechanism for the aberrant angiogenesis in diabetics and TSP-1 involvement in development of various vascular diabetic complications.

  7. Opening up the blackbox: an interpretable deep neural network-based classifier for cell-type specific enhancer predictions.

    PubMed

    Kim, Seong Gon; Theera-Ampornpunt, Nawanol; Fang, Chih-Hao; Harwani, Mrudul; Grama, Ananth; Chaterji, Somali

    2016-08-01

    Gene expression is mediated by specialized cis-regulatory modules (CRMs), the most prominent of which are called enhancers. Early experiments indicated that enhancers located far from the gene promoters are often responsible for mediating gene transcription. Knowing their properties, regulatory activity, and genomic targets is crucial to the functional understanding of cellular events, ranging from cellular homeostasis to differentiation. Recent genome-wide investigation of epigenomic marks has indicated that enhancer elements could be enriched for certain epigenomic marks, such as, combinatorial patterns of histone modifications. Our efforts in this paper are motivated by these recent advances in epigenomic profiling methods, which have uncovered enhancer-associated chromatin features in different cell types and organisms. Specifically, in this paper, we use recent state-of-the-art Deep Learning methods and develop a deep neural network (DNN)-based architecture, called EP-DNN, to predict the presence and types of enhancers in the human genome. It uses as features, the expression levels of the histone modifications at the peaks of the functional sites as well as in its adjacent regions. We apply EP-DNN to four different cell types: H1, IMR90, HepG2, and HeLa S3. We train EP-DNN using p300 binding sites as enhancers, and TSS and random non-DHS sites as non-enhancers. We perform EP-DNN predictions to quantify the validation rate for different levels of confidence in the predictions and also perform comparisons against two state-of-the-art computational models for enhancer predictions, DEEP-ENCODE and RFECS. We find that EP-DNN has superior accuracy and takes less time to make predictions. Next, we develop methods to make EP-DNN interpretable by computing the importance of each input feature in the classification task. This analysis indicates that the important histone modifications were distinct for different cell types, with some overlaps, e.g., H3K27ac was

  8. LaeA Control of Velvet Family Regulatory Proteins for Light-Dependent Development and Fungal Cell-Type Specificity

    PubMed Central

    Valerius, Oliver; Park, Hee Soo; Irniger, Stefan; Gerke, Jennifer; Ni, Min; Han, Kap-Hoon; Yu, Jae-Hyuk; Braus, Gerhard H.

    2010-01-01

    VeA is the founding member of the velvet superfamily of fungal regulatory proteins. This protein is involved in light response and coordinates sexual reproduction and secondary metabolism in Aspergillus nidulans. In the dark, VeA bridges VelB and LaeA to form the VelB-VeA-LaeA (velvet) complex. The VeA-like protein VelB is another developmental regulator, and LaeA has been known as global regulator of secondary metabolism. In this study, we show that VelB forms a second light-regulated developmental complex together with VosA, another member of the velvet family, which represses asexual development. LaeA plays a key role, not only in secondary metabolism, but also in directing formation of the VelB-VosA and VelB-VeA-LaeA complexes. LaeA controls VeA modification and protein levels and possesses additional developmental functions. The laeA null mutant results in constitutive sexual differentiation, indicating that LaeA plays a pivotal role in inhibiting sexual development in response to light. Moreover, the absence of LaeA results in the formation of significantly smaller fruiting bodies. This is due to the lack of a specific globose cell type (Hülle cells), which nurse the young fruiting body during development. This suggests that LaeA controls Hülle cells. In summary, LaeA plays a dynamic role in fungal morphological and chemical development, and it controls expression, interactions, and modification of the velvet regulators. PMID:21152013

  9. Integrated analysis of miRNA and mRNA expression in childhood medulloblastoma compared with neural stem cells.

    PubMed

    Genovesi, Laura A; Carter, Kim W; Gottardo, Nicholas G; Giles, Keith M; Dallas, Peter B

    2011-01-01

    Medulloblastoma (MB) is the most common malignant brain tumor in children and a leading cause of cancer-related mortality and morbidity. Several molecular sub-types of MB have been identified, suggesting they may arise from distinct cells of origin. Data from animal models indicate that some MB sub-types arise from multipotent cerebellar neural stem cells (NSCs). Hence, microRNA (miRNA) expression profiles of primary MB samples were compared to CD133+ NSCs, aiming to identify deregulated miRNAs involved in MB pathogenesis. Expression profiling of 662 miRNAs in primary MB specimens, MB cell lines, and human CD133+ NSCs and CD133- neural progenitor cells was performed by qRT-PCR. Clustering analysis identified two distinct sub-types of MB primary specimens, reminiscent of sub-types obtained from their mRNA profiles. 21 significantly up-regulated and 12 significantly down-regulated miRNAs were identified in MB primary specimens relative to CD133+ NSCs (p<0.01). The majority of up-regulated miRNAs mapped to chromosomal regions 14q32 and 17q. Integration of the predicted targets of deregulated miRNAs with mRNA expression data from the same specimens revealed enrichment of pathways regulating neuronal migration, nervous system development and cell proliferation. Transient over-expression of a down-regulated miRNA, miR-935, resulted in significant down-regulation of three of the seven predicted miR-935 target genes at the mRNA level in a MB cell line, confirming the validity of this approach. This study represents the first integrated analysis of MB miRNA and mRNA expression profiles and is the first to compare MB miRNA expression profiles to those of CD133+ NSCs. We identified several differentially expressed miRNAs that potentially target networks of genes and signaling pathways that may be involved in the transformation of normal NSCs to brain tumor stem cells. Based on this integrative approach, our data provide an important platform for future investigations aimed at

  10. A 350 bp region of the proximal promoter of Rds drives cell-type specific gene expression

    PubMed Central

    Cai, Xue; Conley, Shannon M.; Cheng, Tong; Al-Ubaidi, Muayyad R.; Naash, Muna I.

    2010-01-01

    RDS (retinal degeneration slow) is a photoreceptor-specific tetraspanin protein required for the biogenesis and maintenance of rod and cone outer segments. Mutations in the Rds gene are associated with multiple forms of rod- and cone-dominant retinal degeneration. To gain more insight into the mechanisms underlying the regulation of this gene the identification of regulatory sequences within the promoter of Rds was undertaken. A 3.5kb fragment of the 5′ flanking region of the mouse Rds gene was isolated and binding sites for Crx, Otx2, Nr2e3, RXR family members, Mef2C, Esrrb, NF1, AP1, and SP1 in addition to several E-boxes, GC-boxes and GAGA-boxes were identified. Crx binding sequences were conserved in all mammalian species examined. Truncation expression analysis of the Rds promoter region in Y-79 retinoblastoma cells showed maximal activity in the 350bp proximal promoter region. We also show that inclusion of more distal fragments reduced promoter activity to the basal level, and that the promoter activities are cell-type and direction specific. Co-transfection with Nrl increased promoter activity, suggesting that this gene positively regulates Rds expression. Based on these findings, a relatively small fragment of the Rds promoter may be useful in future gene transfer studies to drive gene expression in photoreceptors. PMID:20447394

  11. Developmental expression of COE across the Metazoa supports a conserved role in neuronal cell-type specification and mesodermal development.

    PubMed

    Jackson, Daniel J; Meyer, Néva P; Seaver, Elaine; Pang, Kevin; McDougall, Carmel; Moy, Vanessa N; Gordon, Kacy; Degnan, Bernard M; Martindale, Mark Q; Burke, Robert D; Peterson, Kevin J

    2010-12-01

    The transcription factor COE (collier/olfactory-1/early B cell factor) is an unusual basic helix-loop-helix transcription factor as it lacks a basic domain and is maintained as a single copy gene in the genomes of all currently analysed non-vertebrate Metazoan genomes. Given the unique features of the COE gene, its proposed ancestral role in the specification of chemosensory neurons and the wealth of functional data from vertebrates and Drosophila, the evolutionary history of the COE gene can be readily investigated. We have examined the ways in which COE expression has diversified among the Metazoa by analysing its expression from representatives of four disparate invertebrate phyla: Ctenophora (Mnemiopsis leidyi); Mollusca (Haliotis asinina); Annelida (Capitella teleta and Chaetopterus) and Echinodermata (Strongylocentrotus purpuratus). In addition, we have studied COE function with knockdown experiments in S. purpuratus, which indicate that COE is likely to be involved in repressing serotonergic cell fate in the apical ganglion of dipleurula larvae. These analyses suggest that COE has played an important role in the evolution of ectodermally derived tissues (likely primarily nervous tissues) and mesodermally derived tissues. Our results provide a broad evolutionary foundation from which further studies aimed at the functional characterisation and evolution of COE can be investigated.

  12. Cell-Type-Specific Repression by Methyl-CpG-Binding Protein 2 Is Biased toward Long Genes

    PubMed Central

    Hempel, Chris M.; Okaty, Benjamin W.; Arnson, Hannah A.; Kato, Saori; Dani, Vardhan S.

    2014-01-01

    Mutations in methyl-CpG-binding protein 2 (MeCP2) cause Rett syndrome and related autism spectrum disorders (Amir et al., 1999). MeCP2 is believed to be required for proper regulation of brain gene expression, but prior microarray studies in Mecp2 knock-out mice using brain tissue homogenates have revealed only subtle changes in gene expression (Tudor et al., 2002; Nuber et al., 2005; Jordan et al., 2007; Chahrour et al., 2008). Here, by profiling discrete subtypes of neurons we uncovered more dramatic effects of MeCP2 on gene expression, overcoming the “dilution problem” associated with assaying homogenates of complex tissues. The results reveal misregulation of genes involved in neuronal connectivity and communication. Importantly, genes upregulated following loss of MeCP2 are biased toward longer genes but this is not true for downregulated genes, suggesting MeCP2 may selectively repress long genes. Because genes involved in neuronal connectivity and communication, such as cell adhesion and cell–cell signaling genes, are enriched among longer genes, their misregulation following loss of MeCP2 suggests a possible etiology for altered circuit function in Rett syndrome. PMID:25232122

  13. Developmental expression of COE across the Metazoa supports a conserved role in neuronal cell-type specification and mesodermal development

    PubMed Central

    Meyer, Néva P.; Seaver, Elaine; Pang, Kevin; McDougall, Carmel; Moy, Vanessa N.; Gordon, Kacy; Degnan, Bernard M.; Martindale, Mark Q.; Burke, Robert D.; Peterson, Kevin J.

    2010-01-01

    The transcription factor COE (collier/olfactory-1/early B cell factor) is an unusual basic helix–loop–helix transcription factor as it lacks a basic domain and is maintained as a single copy gene in the genomes of all currently analysed non-vertebrate Metazoan genomes. Given the unique features of the COE gene, its proposed ancestral role in the specification of chemosensory neurons and the wealth of functional data from vertebrates and Drosophila, the evolutionary history of the COE gene can be readily investigated. We have examined the ways in which COE expression has diversified among the Metazoa by analysing its expression from representatives of four disparate invertebrate phyla: Ctenophora (Mnemiopsis leidyi); Mollusca (Haliotis asinina); Annelida (Capitella teleta and Chaetopterus) and Echinodermata (Strongylocentrotus purpuratus). In addition, we have studied COE function with knockdown experiments in S. purpuratus, which indicate that COE is likely to be involved in repressing serotonergic cell fate in the apical ganglion of dipleurula larvae. These analyses suggest that COE has played an important role in the evolution of ectodermally derived tissues (likely primarily nervous tissues) and mesodermally derived tissues. Our results provide a broad evolutionary foundation from which further studies aimed at the functional characterisation and evolution of COE can be investigated. Electronic supplementary material The online version of this article (doi:10.1007/s00427-010-0343-3) contains supplementary material, which is available to authorized users. PMID:21069538

  14. The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction

    PubMed Central

    Ren, Jianwei; Yao, Peng; Wang, Xiaowei; Wang, Zhe; Zhang, Qunye

    2016-01-01

    Background MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs. Methods and Results Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye) could firmly bind to the surface of adherent cells (Hela) and suspended cells (K562) even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein) to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it. Conclusions These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results. PMID

  15. Generation of cell type-specific monoclonal antibodies for the planarian and optimization of sample processing for immunolabeling.

    PubMed

    Forsthoefel, David J; Waters, Forrest A; Newmark, Phillip A

    2014-12-21

    Efforts to elucidate the cellular and molecular mechanisms of regeneration have required the application of methods to detect specific cell types and tissues in a growing cohort of experimental animal models. For example, in the planarian Schmidtea mediterranea, substantial improvements to nucleic acid hybridization and electron microscopy protocols have facilitated the visualization of regenerative events at the cellular level. By contrast, immunological resources have been slower to emerge. Specifically, the repertoire of antibodies recognizing planarian antigens remains limited, and a more systematic approach is needed to evaluate the effects of processing steps required during sample preparation for immunolabeling. To address these issues and to facilitate studies of planarian digestive system regeneration, we conducted a monoclonal antibody (mAb) screen using phagocytic intestinal cells purified from the digestive tracts of living planarians as immunogens. This approach yielded ten antibodies that recognized intestinal epitopes, as well as markers for the central nervous system, musculature, secretory cells, and epidermis. In order to improve signal intensity and reduce non-specific background for a subset of mAbs, we evaluated the effects of fixation and other steps during sample processing. We found that fixative choice, treatments to remove mucus and bleach pigment, as well as methods for tissue permeabilization and antigen retrieval profoundly influenced labeling by individual antibodies. These experiments led to the development of a step-by-step workflow for determining optimal specimen preparation for labeling whole planarians as well as unbleached histological sections. We generated a collection of monoclonal antibodies recognizing the planarian intestine and other tissues; these antibodies will facilitate studies of planarian tissue morphogenesis. We also developed a protocol for optimizing specimen processing that will accelerate future efforts to

  16. The Stage- and Cell Type-Specific Localization of Fragile X Mental Retardation Protein in Rat Ovaries.

    PubMed

    Takahashi, Noriyuki; Tarumi, Wataru; Itoh, Masanori T; Ishizuka, Bunpei

    2015-12-01

    Premutations of the fragile X mental retardation 1 (FMR1) gene are associated with increased risk of primary ovarian insufficiency. Here we examined the localization of the Fmr1 gene protein product, fragile X mental retardation protein (FMRP), in rat ovaries at different stages, including fetus, neonate, and old age. In ovaries dissected from 19 days postcoitum embryos, the germ cells were divided into 2 types: one with decondensed chromatin in the nucleus was FMRP positive in the cytoplasm, but the other with strongly condensed chromatin in the nucleus was FMRP negative in the cytoplasm. The FMRP was predominantly localized to the cytoplasm of oocytes in growing ovarian follicles. Levels of FMRP in oocytes from elderly (9 or 14 months of age) ovaries were lower than in those from younger ovaries. These results suggest that FMRP is associated with the activation of oogenesis and oocyte function. Especially, FMRP is likely to be implicated in germline development during oogenesis.

  17. A novel BAT3 sequence generated by alternative RNA splicing of exon 11B displays cell type-specific expression and impacts on subcellular localization.

    PubMed

    Kämper, Nadine; Kessler, Jörg; Temme, Sebastian; Wegscheid, Claudia; Winkler, Johannes; Koch, Norbert

    2012-01-01

    The human lymphocyte antigen (HLA) encoded BAT3/BAG6 recently attracted interest as a regulator of protein targeting and degradation, a function that could be exerted in the cytosol and in the nucleus. The BAT3 gene was described to consist of 25 exons. Diversity of transcripts can be generated by alternative RNA splicing, which may control subcellular distribution of BAT3. By cDNA sequencing we identified a novel alternatively spliced sequence of the BAT3 gene located between exons 11 and 12, which was designated as exon 11B. Using PCR and colony hybridization we identified six cDNA variants, which were produced by RNA splicing of BAT3 exons 5, 11B and 24. In four examined cell types the content of BAT3 splice variants was examined. Most of the cDNA clones from monocyte-derived dendritic cells contain exon 11B, whereas this sequence was almost absent in the B lymphoma Raji. Exon 5 was detected in most and exon 24 in approximately half of the cDNA clones. The subcellular distribution of endogenous BAT3 largely correlates with a cell type specific splicing pattern. In cells transfected with BAT3 variants, full-length and Δ24 BAT3 displayed nearly exclusive nuclear staining, whereas variants deleted of exon 11B showed substantial cytosolic expression. We show here that BAT3 is mainly expressed in the cytosol of Raji cells, while other cell types displayed both cytosolic and nuclear staining. Export of BAT3 from the nucleus to the cytosol is inhibited by treatment with leptomycin B, indicating that the Crm1 pathway is involved. Nuclear expression of BAT3 containing exon 11B suggests that this sequence plays a role for nuclear retention of the protein. Cell type-specific subcellular expression of BAT3 suggests distinct functions in the cytosol and in the nucleus. Differential expression of BAT3 variants may reconcile the multiple roles described for BAT3.

  18. Cell Type Specific Spatial and Functional Coupling Between Mammalian Brain Kv2.1 K+ Channels and Ryanodine Receptors

    PubMed Central

    Mandikian, Danielle; Bocksteins, Elke; Parajuli, Laxmi Kumar; Bishop, Hannah I.; Cerda, Oscar; Shigemoto, Ryuichi; Trimmer, James S.

    2014-01-01

    The Kv2.1 voltage-gated K+ channel is widely expressed throughout mammalian brain where it contributes to dynamic activity-dependent regulation of intrinsic neuronal excitability. Here we show that somatic plasma membrane Kv2.1 clusters are juxtaposed to clusters of intracellular ryanodine receptor (RyR) Ca2+-release channels in mouse brain neurons, most prominently in medium spiny neurons (MSNs) of the striatum. Electron microscopy-immunogold labeling shows that in MSNs, plasma membrane Kv2.1 clusters are adjacent to subsurface cisternae, placing Kv2.1 in close proximity to sites of RyR-mediated Ca2+ release. Immunofluorescence labeling in transgenic mice expressing GFP in specific MSN populations reveals the most prominent juxtaposed Kv2.1-RyR clusters in indirect pathway MSNs. Kv2.1 in both direct and indirect pathway MSNs exhibits markedly lower levels of labeling with phosphospecific antibodies directed against the S453, S563, and S603 phosphorylation site compared to levels observed in neocortical neurons, although labeling for Kv2.1 phosphorylation at S563 was significantly lower in indirect pathway MSNs compared to those in the direct pathway. Finally, acute stimulation of RyRs in heterologous cells causes a rapid hyperpolarizing shift in the voltage-dependence of activation of Kv2.1, typical of Ca2+/calcineurin-dependent Kv2.1 dephosphorylation. Together, these studies reveal that striatal MSNs are distinct in their expression of clustered Kv2.1 at plasma membrane sites juxtaposed to intracellular RyRs, as well as in Kv2.1 phosphorylation state. Differences in Kv2.1 expression and phosphorylation between MSNs in direct and indirect pathways provide a cell- and circuit-specific mechanism for coupling intracellular Ca2+ release to phosphorylation-dependent regulation of Kv2.1 to dynamically impact intrinsic excitability. PMID:24962901

  19. Fibronectin signals through integrin α5β1 to regulate cardiovascular development in a cell type-specific manner.

    PubMed

    Chen, Dongying; Wang, Xia; Liang, Dong; Gordon, Julie; Mittal, Ashok; Manley, Nancy; Degenhardt, Karl; Astrof, Sophie

    2015-11-15

    Fibronectin (Fn1) is an evolutionarily conserved extracellular matrix glycoprotein essential for embryonic development. Global deletion of Fn1 leads to mid-gestation lethality from cardiovascular defects. However, severe morphogenetic defects that occur early in embryogenesis in these embryos precluded assigning a direct role for Fn1 in cardiovascular development. We noticed that Fn1 is expressed in strikingly non-uniform patterns during mouse embryogenesis, and that its expression is particularly enriched in the pharyngeal region corresponding with the pharyngeal arches 3, 4, and 6. This region bears a special importance for the developing cardiovascular system, and we hypothesized that the localized enrichment of Fn1 in the pharyngeal region may be essential for cardiovascular morphogenesis. To test this hypothesis, we ablated Fn1 using the Isl1(Cre) knock-in strain of mice. Deletion of Fn1 using the Isl1(Cre) strain resulted in defective formation of the 4th pharyngeal arch arteries (PAAs), aberrant development of the cardiac outflow tract (OFT), and ventricular septum defects. To determine the cell types responding to Fn1 signaling during cardiovascular development, we deleted a major Fn1 receptor, integrin α5 using the Isl1(Cre) strain, and observed the same spectrum of abnormalities seen in the Fn1 conditional mutants. Additional conditional mutagenesis studies designed to ablate integrin α5 in distinct cell types within the Isl1(+) tissues and their derivatives, suggested that the expression of integrin α5 in the pharyngeal arch mesoderm, endothelium, surface ectoderm and the neural crest were not required for PAA formation. Our studies suggest that an (as yet unknown) integrin α5-dependent signal extrinsic to the pharyngeal endothelium mediates the formation of the 4th PAAs.

  20. High-throughput mRNA and miRNA profiling of epithelial-mesenchymal transition in MDCK cells.

    PubMed

    Shukla, Priyank; Vogl, Claus; Wallner, Barbara; Rigler, Doris; Müller, Mathias; Macho-Maschler, Sabine

    2015-11-16

    Epithelial-mesenchymal transition (EMT) is an important process in embryonic development, especially during gastrulation and organ formation. Furthermore EMT is widely observed in pathological conditions, e.g., fibrosis, tumor progression and metastasis. Madin-Darby Canine Kidney (MDCK) cells are widely used for studies of EMT and epithelial plasticity. MDCK cells show an epithelial phenotype, while oncogenic Ras-transformed MDCK (MDCK-Ras) cells undergo EMT and show a mesenchymal phenotype. RNA-Seq and miRNA-Seq analyses were performed on MDCK and MDCK-Ras cells. Data were validated by qRT-PCR. Gene signature analyses were carried out to identify pathways and gene ontology terms. For selected miRNAs target prediction was performed. With RNA-Seq, mRNAs of approximately half of the genes known for dog were detected. These were screened for differential regulation during Ras-induced EMT. We went further and performed gene signature analyses and found Gene Ontology (GO) terms and pathways important for epithelial polarity and implicated in EMT. Among the identified pathways, TGFβ1 emerged as a central signaling factor in many EMT related pathways and biological processes. With miRNA-Seq, approximately half of the known canine miRNAs were found expressed in MDCK and MDCK-Ras cells. Furthermore, among differentially expressed miRNAs, miRNAs that are known to be important regulators of EMT were detected and new candidates were predicted. New dog miRNAs were discovered after aligning our reads to that of other species in miRBase. Importantly, we could identify 25 completely novel miRNAs with a stable hairpin structure. Two of these novel miRNAs were differentially expressed. We validated the two novel miRNAs with the highest read counts by RT-qPCR. Target prediction of a particular novel miRNA highly expressed in mesenchymal MDCK-Ras cells revealed that it targets components of epithelial cell junctional complexes. Combining target prediction for the most upregulated miRNAs

  1. MMAD: microarray microdissection with analysis of differences is a computational tool for deconvoluting cell type-specific contributions from tissue samples.

    PubMed

    Liebner, David A; Huang, Kun; Parvin, Jeffrey D

    2014-03-01

    One of the significant obstacles in the development of clinically relevant microarray-derived biomarkers and classifiers is tissue heterogeneity. Physical cell separation techniques, such as cell sorting and laser-capture microdissection, can enrich samples for cell types of interest, but are costly, labor intensive and can limit investigation of important interactions between different cell types. We developed a new computational approach, called microarray microdissection with analysis of differences (MMAD), which performs microdissection in silico. Notably, MMAD (i) allows for simultaneous estimation of cell fractions and gene expression profiles of contributing cell types, (ii) adjusts for microarray normalization bias, (iii) uses the corrected Akaike information criterion during model optimization to minimize overfitting and (iv) provides mechanisms for comparing gene expression and cell fractions between samples in different classes. Computational microdissection of simulated and experimental tissue mixture datasets showed tight correlations between predicted and measured gene expression of pure tissues as well as tight correlations between reported and estimated cell fraction for each of the individual cell types. In simulation studies, MMAD showed superior ability to detect differentially expressed genes in mixed tissue samples when compared with standard metrics, including both significance analysis of microarrays and cell type-specific significance analysis of microarrays. We have developed a new computational tool called MMAD, which is capable of performing robust tissue microdissection in silico, and which can improve the detection of differentially expressed genes. MMAD software as implemented in MATLAB is publically available for download at http://sourceforge.net/projects/mmad/.

  2. Two-Photon Intravital Fluorescence Lifetime Imaging of the Kidney Reveals Cell-Type Specific Metabolic Signatures.

    PubMed

    Hato, Takashi; Winfree, Seth; Day, Richard; Sandoval, Ruben M; Molitoris, Bruce A; Yoder, Mervin C; Wiggins, Roger C; Zheng, Yi; Dunn, Kenneth W; Dagher, Pierre C

    2017-03-01

    In the live animal, tissue autofluorescence arises from a number of biologically important metabolites, such as the reduced form of nicotinamide adenine dinucleotide. Because autofluorescence changes with metabolic state, it can be harnessed as a label-free imaging tool with which to study metabolism in vivo Here, we used the combination of intravital two-photon microscopy and frequency-domain fluorescence lifetime imaging microscopy (FLIM) to map cell-specific metabolic signatures in the kidneys of live animals. The FLIM images are analyzed using the phasor approach, which requires no prior knowledge of metabolite species and can provide unbiased metabolic fingerprints for each pixel of the lifetime image. Intravital FLIM revealed the metabolic signatures of S1 and S2 proximal tubules to be distinct and resolvable at the subcellular level. Notably, S1 and distal tubules exhibited similar metabolic profiles despite apparent differences in morphology and autofluorescence emission with traditional two-photon microscopy. Time-lapse imaging revealed dynamic changes in the metabolic profiles of the interstitium, urinary lumen, and glomerulus-areas that are not resolved by traditional intensity-based two-photon microscopy. Finally, using a model of endotoxemia, we present examples of the way in which intravital FLIM can be applied to study kidney diseases and metabolism. In conclusion, intravital FLIM of intrinsic metabolites is a bias-free approach with which to characterize and monitor metabolism in vivo, and offers the unique opportunity to uncover dynamic metabolic changes in living animals with subcellular resolution.

  3. Behavioral-state modulation of inhibition is context-dependent and cell type specific in mouse visual cortex

    PubMed Central

    Pakan, Janelle MP; Lowe, Scott C; Dylda, Evelyn; Keemink, Sander W; Currie, Stephen P; Coutts, Christopher A; Rochefort, Nathalie L

    2016-01-01

    Cortical responses to sensory stimuli are modulated by behavioral state. In the primary visual cortex (V1), visual responses of pyramidal neurons increase during locomotion. This response gain was suggested to be mediated through inhibitory neurons, resulting in the disinhibition of pyramidal neurons. Using in vivo two-photon calcium imaging in layers 2/3 and 4 in mouse V1, we reveal that locomotion increases the activity of vasoactive intestinal peptide (VIP), somatostatin (SST) and parvalbumin (PV)-positive interneurons during visual stimulation, challenging the disinhibition model. In darkness, while most VIP and PV neurons remained locomotion responsive, SST and excitatory neurons were largely non-responsive. Context-dependent locomotion responses were found in each cell type, with the highest proportion among SST neurons. These findings establish that modulation of neuronal activity by locomotion is context-dependent and contest the generality of a disinhibitory circuit for gain control of sensory responses by behavioral state. DOI: http://dx.doi.org/10.7554/eLife.14985.001 PMID:27552056

  4. Cell-type-specific long-range looping interactions identify distant regulatory elements of the CFTR gene

    PubMed Central

    Gheldof, Nele; Smith, Emily M.; Tabuchi, Tomoko M.; Koch, Christoph M.; Dunham, Ian; Stamatoyannopoulos, John A.; Dekker, Job

    2010-01-01

    Identification of regulatory elements and their target genes is complicated by the fact that regulatory elements can act over large genomic distances. Identification of long-range acting elements is particularly important in the case of disease genes as mutations in these elements can result in human disease. It is becoming increasingly clear that long-range control of gene expression is facilitated by chromatin looping interactions. These interactions can be detected by chromosome conformation capture (3C). Here, we employed 3C as a discovery tool for identification of long-range regulatory elements that control the cystic fibrosis transmembrane conductance regulator gene, CFTR. We identified four elements in a 460-kb region around the locus that loop specifically to the CFTR promoter exclusively in CFTR expressing cells. The elements are located 20 and 80 kb upstream; and 109 and 203 kb downstream of the CFTR promoter. These elements contain DNase I hypersensitive sites and histone modification patterns characteristic of enhancers. The elements also interact with each other and the latter two activate the CFTR promoter synergistically in reporter assays. Our results reveal novel long-range acting elements that control expression of CFTR and suggest that 3C-based approaches can be used for discovery of novel regulatory elements. PMID:20360044

  5. Mitochondrial Abnormality Associates with Type-Specific Neuronal Loss and Cell Morphology Changes in the Pedunculopontine Nucleus in Parkinson Disease

    PubMed Central

    Pienaar, Ilse S.; Elson, Joanna L.; Racca, Claudia; Nelson, Glyn; Turnbull, Douglass M.; Morris, Christopher M.

    2014-01-01

    Cholinergic neuronal loss in the pedunculopontine nucleus (PPN) associates with abnormal functions, including certain motor and nonmotor symptoms. This realization has led to low-frequency stimulation of the PPN for treating patients with Parkinson disease (PD) who are refractory to other treatment modalities. However, the molecular mechanisms underlying PPN neuronal loss and the therapeutic substrate for the clinical benefits following PPN stimulation remain poorly characterized, hampering progress toward designing more efficient therapies aimed at restoring the PPN's normal functions during progressive parkinsonism. Here, we investigated postmortem pathological changes in the PPN of PD cases. Our study detected a loss of neurons producing gamma-aminobutyric acid (GABA) as their output and glycinergic neurons, along with the pronounced loss of cholinergic neurons. These losses were accompanied by altered somatic cell size that affected the remaining neurons of all neuronal subtypes studied here. Because studies showed that mitochondrial dysfunction exists in sporadic PD and in PD animal models, we investigated whether altered mitochondrial composition exists in the PPN. A significant up-regulation of several mitochondrial proteins was seen in GABAergic and glycinergic neurons; however, cholinergic neurons indicated down-regulation of the same proteins. Our findings suggest an imbalance in the activity of key neuronal subgroups of the PPN in PD, potentially because of abnormal inhibitory activity and altered cholinergic outflow. PMID:24099985

  6. miRNA profiling of high, low and non-producing CHO cells during biphasic fed-batch cultivation reveals process relevant targets for host cell engineering.

    PubMed

    Stiefel, Fabian; Fischer, Simon; Sczyrba, Alexander; Otte, Kerstin; Hesse, Friedemann

    2016-05-10

    Fed-batch cultivation of recombinant Chinese hamster ovary (CHO) cell lines is one of the most widely used production modes for commercial manufacturing of recombinant protein therapeutics. Furthermore, fed-batch cultivations are often conducted as biphasic processes where the culture temperature is decreased to maximize volumetric product yields. However, it remains to be elucidated which intracellular regulatory elements actually control the observed pro-productive phenotypes. Recently, several studies have revealed microRNAs (miRNAs) to be important molecular switches of cell phenotypes. In this study, we analyzed miRNA profiles of two different recombinant CHO cell lines (high and low producer), and compared them to a non-producing CHO DG44 host cell line during fed-batch cultivation at 37°C versus a temperature shift to 30°C. Taking advantage of next-generation sequencing combined with cluster, correlation and differential expression analyses, we could identify 89 different miRNAs, which were differentially expressed in the different cell lines and cultivation phases. Functional validation experiments using 19 validated target miRNAs confirmed that these miRNAs indeed induced changes in process relevant phenotypes. Furthermore, computational miRNA target prediction combined with functional clustering identified putative target genes and cellular pathways, which might be regulated by these miRNAs. This study systematically identified novel target miRNAs during different phases and conditions of a biphasic fed-batch production process and functionally evaluated their potential for host cell engineering.

  7. An artificial lncRNA targeting multiple miRNAs overcomes sorafenib resistance in hepatocellular carcinoma cells

    PubMed Central

    Tang, Shuyao; Tan, Gang; Jiang, Xian; Han, Peng; Zhai, Bo; Dong, Xuesong; Qiao, Haiquan; Jiang, Hongchi; Sun, Xueying

    2016-01-01

    Sorafenib resistance remains a major obstacle for the effective treatment of hepatocellular carcinoma (HCC), and a number of miRNAs contribute to this resistance. However, the regulatory networks of miRNAs are very complex, thus inhibiting a single miRNA may sequentially activate other compensatory pathways. In the present study, we generated an artificial long non-coding RNA (AlncRNA), which simultaneously targets multiple miRNAs including miR-21, miR-153, miR-216a, miR-217, miR-494 and miR-10a-5p. These miRNAs have been shown to be upregulated in sorafenib-resistant cells and participate in the mechanisms underlying sorafenib resistance. The AlncRNA contains tandem sequences of 6 copies of the complementary binding sequences to the target miRNAs and is expressed by an adenoviral vector (Ad5-AlncRNA). Infection of Ad5-AlncRNA into sorafenib-resistant HCC cells blocked the function of miRNAs, and sequentially inhibited the downregulation of PTEN and activation of AKT. Ad5-AlncRNA significantly inhibited proliferation and induced apoptosis of sorafenib-resistant cells and enhanced the effects of sorafenib in vitro and in animal models. Inhibition of autophagy decreased the sensitivity of sorafenib-resistant cells to Ad5-AlncRNA, while its induction had the