Science.gov

Sample records for aberrant chromatin remodeling

  1. Teaching resources. Chromatin remodeling.

    PubMed

    Lue, Neal F

    2005-07-26

    This Teaching Resource provides lecture notes and slides for a class covering chromatin remodeling mechanisms and is part of the course "Cell Signaling Systems: a Course for Graduate Students." The lecture begins with a discussion of chromatin organization and then proceeds to describe the process of chromatin remodeling through a review of chromatin remodeling complexes and methods used to study their function.

  2. Chromatin Remodeling and Plant Immunity.

    PubMed

    Chen, W; Zhu, Q; Liu, Y; Zhang, Q

    2017-01-01

    Chromatin remodeling, an important facet of the regulation of gene expression in eukaryotes, is performed by two major types of multisubunit complexes, covalent histone- or DNA-modifying complexes, and ATP-dependent chromosome remodeling complexes. Snf2 family DNA-dependent ATPases constitute the catalytic subunits of ATP-dependent chromosome remodeling complexes, which accounts for energy supply during chromatin remodeling. Increasing evidence indicates a critical role of chromatin remodeling in the establishment of long-lasting, even transgenerational immune memory in plants, which is supported by the findings that DNA methylation, histone deacetylation, and histone methylation can prime the promoters of immune-related genes required for disease defense. So what are the links between Snf2-mediated ATP-dependent chromosome remodeling and plant immunity, and what mechanisms might support its involvement in disease resistance?

  3. Impaired Contextual Fear Extinction Learning is Associated with Aberrant Regulation of CHD-Type Chromatin Remodeling Factors.

    PubMed

    Wille, Alexandra; Maurer, Verena; Piatti, Paolo; Whittle, Nigel; Rieder, Dietmar; Singewald, Nicolas; Lusser, Alexandra

    2015-01-01

    Successful attenuation of fearful memories is a cognitive process requiring initiation of highly coordinated transcription programs. Chromatin-modulating mechanisms such as DNA methylation and histone modifications, including acetylation, are key regulators of these processes. However, knowledge concerning the role of ATP-dependent chromatin remodeling factors (ChRFs) being required for successful fear extinction is lacking. Underscoring the potential importance of these factors that alter histone-DNA contacts within nucleosomes are recent genome-wide association studies linking several ChRFs to various human cognitive and psychiatric disorders. To better understand the role of ChRFs in the brain, and since to date little is known about ChRF expression in the brain, we performed a comprehensive survey of expression levels of 24 ATP-dependent remodelers across different brain areas, and we identified several distinct high molecular weight complexes by chromatographic methods. We next aimed to gain novel insight into the potential regulation of ChRFs in different brain regions in association with normal and impaired fear extinction learning. To this end, we established the 129S1/SvImJ (S1) laboratory mouse strain as a model for compromised contextual fear extinction learning that can be rescued by dietary zinc restriction (ZnR). Using this model along with genetically related but fear extinction-competent 129S6/SvEv (S6) mice as controls, we found that impaired fear extinction in S1 was associated with enhanced ventral hippocampal expression of CHD1 and reduced expression of CHD5 that was normalized following successful rescue of impaired fear extinction. Moreover, a select reduction in CHD3 expression was observed in the ventral hippocampus (vHC) following successful rescue of fear extinction in S1 mice. Taken together, these data provide novel insight into the regulation of specific ChRFs following an impaired cognitive process and its rescue, and they suggest that

  4. Chromatin remodeling: nucleosomes bulging at the seams.

    PubMed

    Peterson, Craig L

    2002-04-02

    ATP-dependent chromatin remodeling enzymes, such as SWI/SNF, hydrolyze thousands of ATPs to regulate gene expression on chromatin fibers. Recent mechanistic studies suggest that these enzymes generate localized changes in DNA topology that drive formation of multiple, remodeled nucleosomal states.

  5. The Chd Family of Chromatin Remodelers

    PubMed Central

    Marfella, Concetta G.A.; Imbalzano, Anthony N.

    2007-01-01

    Chromatin remodeling enzymes contribute to the dynamic changes that occur in chromatin structure during cellular processes such as transcription, recombination, repair, and replication. Members of the chromodomain helicase DNA-binding (Chd) family of enzymes belong to the SNF2 superfamily of ATP-dependent chromatin remodelers. The Chd proteins are distinguished by the presence of two N-terminal chromodomains that function as interaction surfaces for a variety of chromatin components. Genetic, biochemical, and structural studies demonstrate that Chd proteins are important regulators of transcription and play critical roles during developmental processes. Numerous Chd proteins are also implicated in human disease. PMID:17350655

  6. Nucleosome dynamics during chromatin remodeling in vivo.

    PubMed

    Ramachandran, Srinivas; Henikoff, Steven

    2016-01-01

    Precise positioning of nucleosomes around regulatory sites is achieved by the action of chromatin remodelers, which use the energy of ATP to slide, evict or change the composition of nucleosomes. Chromatin remodelers act to bind nucleosomes, disrupt histone-DNA interactions and translocate the DNA around the histone core to reposition nucleosomes. Hence, remodeling is expected to involve nucleosomal intermediates with a structural organization that is distinct from intact nucleosomes. We describe the identification of a partially unwrapped nucleosome structure using methods that map histone-DNA contacts genome-wide. This alternative nucleosome structure is likely formed as an intermediate or by-product during nucleosome remodeling by the RSC complex. Identification of the loss of histone-DNA contacts during chromatin remodeling by RSC in vivo has implications for the regulation of transcriptional initiation.

  7. Mechanisms of ATP Dependent Chromatin Remodeling

    PubMed Central

    Gangaraju, Vamsi K.; Bartholomew, Blaine

    2007-01-01

    The inter-relationship between DNA repair and ATP dependent chromatin remodeling has begun to become very apparent with recent discoveries. ATP dependent remodeling complexes mobilize nucleosomes along DNA, promote the exchange of histones, or completely displace nucleosomes from DNA. These remodeling complexes are often categorized based on the domain organization of their catalytic subunit. The biochemical properties and structural information of several of these remodeling complexes are reviewed. The different models for how these complexes are able to mobilize nucleosomes and alter nucleosome structure are presented incorporating several recent findings. Finally the role of histone tails and their respective modifications in ATP-dependent remodeling are discussed. PMID:17306844

  8. Chromatin Remodeling, DNA Damage Repair and Aging

    PubMed Central

    Liu, Baohua; Yip, Raymond KH; Zhou, Zhongjun

    2012-01-01

    Cells are constantly exposed to a variety of environmental and endogenous conditions causing DNA damage, which is detected and repaired by conserved DNA repair pathways to maintain genomic integrity. Chromatin remodeling is critical in this process, as the organization of eukaryotic DNA into compact chromatin presents a natural barrier to all DNA-related events. Studies on human premature aging syndromes together with normal aging have suggested that accumulated damages might lead to exhaustion of resources that are required for physiological functions and thus accelerate aging. In this manuscript, combining the present understandings and latest findings, we focus mainly on discussing the role of chromatin remodeling in the repair of DNA double-strand breaks (DSBs) and regulation of aging. PMID:23633913

  9. CHD chromatin remodelers and the transcription cycle.

    PubMed

    Murawska, Magdalena; Brehm, Alexander

    2011-01-01

    It is well established that ATP-dependent chromatin remodelers modulate DNA access of transcription factors and RNA polymerases by "opening" or "closing" chromatin structure. However, this view is far too simplistic. Recent findings have demonstrated that these enzymes not only set the stage for the transcription machinery to act but are actively involved at every step of the transcription process. As a consequence, they affect initiation, elongation, termination and RNA processing. In this review we will use the CHD family as a paradigm to illustrate the progress that has been made in revealing these new concepts.

  10. Overlapping chromatin-remodeling systems collaborate genome wide at dynamic chromatin transitions.

    PubMed

    Morris, Stephanie A; Baek, Songjoon; Sung, Myong-Hee; John, Sam; Wiench, Malgorzata; Johnson, Thomas A; Schiltz, R Louis; Hager, Gordon L

    2014-01-01

    ATP-dependent chromatin remodeling is an essential process required for the dynamic organization of chromatin structure. Here we describe the genome-wide location and activity of three remodeler proteins with diverse physiological functions in the mouse genome: Brg1, Chd4 and Snf2h. The localization patterns of all three proteins substantially overlap with one another and with regions of accessible chromatin. Furthermore, using inducible mutant variants, we demonstrate that the catalytic activity of these proteins contributes to the remodeling of chromatin genome wide and that each of these remodelers can independently regulate chromatin reorganization at distinct sites. Many regions require the activity of more than one remodeler to regulate accessibility. These findings provide a dynamic view of chromatin organization and highlight the differential contributions of remodelers to chromatin maintenance in higher eukaryotes.

  11. Maternal chromatin remodeling during maturation and after fertilization in mouse oocytes.

    PubMed

    Spinaci, Marcella; Seren, Eraldo; Mattioli, Mauro

    2004-10-01

    Immunofluorescence staining with antibodies against acetylated histone H4 and 5-methylcytosine was carried out to investigate female chromatin remodeling throughout oocyte maturation and chromatin rearrangement involving both male and female genomes after fertilization. Oocyte cytoplasm remodels female chromatin in preparation of the fertilizing event and the subsequent chromatin rearrangement. Histone H4 are in fact progressively deacetylated whereas demethylating enzymes do not seem to be active over this period. The acetylase/deacetylase balance seems to be cell cycle dependent as female chromatin is deacetylated during maturation and reacetylated at telophase II stage both after fertilization and activation. On the contrary, DNA demethylation seems to be strictly selective. It is in fact confined to the remodeling of paternal genome after fertilization of mature oocytes as the ooplasm is not effective in demethylating either paternal chromatin in germinal vesicle breakdown (GVBD) fertilized oocytes or maternal genome of partenogenetically activated oocytes. Surprisingly, we induced maternal chromatin demethylation after fertilization by treating oocytes with a combination of a methyltransferase inhibitor, 5-azacytidine (5-AzaC), and a reversible and specific inhibitor of histone deacetylase, trichostatin A (TSA). This treatment likely induces a hyperacetylation of histones (thus favoring the access to demethylating enzymes by opening female chromatin structure) associated with a block of reparative methylation by inhibiting methytransferases. This manipulation of chromatin remodeling may have applications regarding the biological significance of aberrant DNA methylation.

  12. Roles of chromatin remodellers in DNA double strand break repair.

    PubMed

    Jeggo, Penny A; Downs, Jessica A

    2014-11-15

    Now that we have a good understanding of the DNA double strand break (DSB) repair mechanisms and DSB-induced damage signalling, attention is focusing on the changes to the chromatin environment needed for efficient DSB repair. Mutations in chromatin remodelling complexes have been identified in cancers, making it important to evaluate how they impact upon genomic stability. Our current understanding of the DSB repair pathways suggests that each one has distinct requirements for chromatin remodelling. Moreover, restricting the extent of chromatin modifications could be a significant factor regulating the decision of pathway usage. In this review, we evaluate the distinct DSB repair pathways for their potential need for chromatin remodelling and review the roles of ATP-driven chromatin remodellers in the pathways.

  13. Mechanisms of ATP-Dependent Chromatin Remodeling Motors.

    PubMed

    Zhou, Coral Y; Johnson, Stephanie L; Gamarra, Nathan I; Narlikar, Geeta J

    2016-07-05

    Chromatin remodeling motors play essential roles in all DNA-based processes. These motors catalyze diverse outcomes ranging from sliding the smallest units of chromatin, known as nucleosomes, to completely disassembling chromatin. The broad range of actions carried out by these motors on the complex template presented by chromatin raises many stimulating mechanistic questions. Other well-studied nucleic acid motors provide examples of the depth of mechanistic understanding that is achievable from detailed biophysical studies. We use these studies as a guiding framework to discuss the current state of knowledge of chromatin remodeling mechanisms and highlight exciting open questions that would continue to benefit from biophysical analyses.

  14. Chromatin remodeling: from transcription to cancer.

    PubMed

    Yaniv, Moshe

    2014-09-01

    In this short review article, I have tried to trace the path that led my laboratory from the early studies of the structure of papova minichromosomes and transcription control to the investigation of chromatin remodeling complexes of the SWI/SNF family. I discuss briefly the genetic and biochemical studies that lead to the discovery of the SWI/SNF complex in yeast and drosophila and summarize some of the studies on the developmental role of the murine complex. The discovery of the tumor suppressor function of the SNF5/INI1/SMARCB1 gene in humans and the identification of frequent mutations in other subunits of this complex in different human tumors opened a fascinating field of research on this epigenetic regulator. The hope is to better understand tumor development and to develop novel treatments.

  15. Chromatin remodeling in DNA double-strand break repair.

    PubMed

    Bao, Yunhe; Shen, Xuetong

    2007-04-01

    ATP-dependent chromatin remodeling complexes use ATP hydrolysis to remodel nucleosomes and have well-established functions in transcription. However, emerging lines of evidence suggest that chromatin remodeling complexes are important players in DNA double-strand break (DSB) repair as well. The INO80 and SWI2 subfamilies of chromatin remodeling complexes have been found to be recruited to the double-strand lesions and to function directly in both homologous recombination and non-homologous end-joining, the two major conserved DSB repair pathways. Improperly repaired DSBs are implicated in cancer development in higher organisms. Understanding how chromatin remodeling complexes contribute to DSB repair should provide new insights into the mechanisms of carcinogenesis and might suggest new targets for cancer treatment.

  16. Chromatin remodelling: the industrial revolution of DNA around histones.

    PubMed

    Saha, Anjanabha; Wittmeyer, Jacqueline; Cairns, Bradley R

    2006-06-01

    Chromatin remodellers are specialized multi-protein machines that enable access to nucleosomal DNA by altering the structure, composition and positioning of nucleosomes. All remodellers have a catalytic ATPase subunit that is similar to known DNA-translocating motor proteins, suggesting DNA translocation as a unifying aspect of their mechanism. Here, we explore the diversity and specialization of chromatin remodellers, discuss how nucleosome modifications regulate remodeller activity and consider a model for the exposure of nucleosomal DNA that involves the use of directional DNA translocation to pump 'DNA waves' around the nucleosome.

  17. Chromatin dynamics: Interplay between remodeling enzymes and histone modifications

    PubMed Central

    Swygert, Sarah G.; Peterson, Craig L.

    2014-01-01

    Chromatin dynamics play an essential role in regulating the accessibility of genomic DNA for a variety of nuclear processes, including gene transcription and DNA repair. The posttranslational modification of the core histones and the action of ATP-dependent chromatin remodeling enzymes represent two primary mechanisms by which chromatin dynamics are controlled and linked to nuclear events. Although there are examples in which a histone modification or a remodeling enzyme may be sufficient to drive a chromatin transition, these mechanisms typically work in concert to integrate regulatory inputs, leading to a coordinated alteration in chromatin structure and function. Indeed, site-specific histone modifications can facilitate the recruitment of chromatin remodeling enzymes to particular genomic regions, or they can regulate the efficiency or the outcome of a chromatin remodeling reaction. Conversely, chromatin remodeling enzymes can also influence, and sometimes directly modulate, the modification state of histones. These functional interactions are generally complex, frequently transient, and often require the association of myriad additional factors. PMID:24583555

  18. Roles and activities of chromatin remodeling ATPases in plants.

    PubMed

    Han, Soon-Ki; Wu, Miin-Feng; Cui, Sujuan; Wagner, Doris

    2015-07-01

    Chromatin remodeling ATPases and their associated complexes can alter the accessibility of the genome in the context of chromatin by using energy derived from the hydrolysis of ATP to change the positioning, occupancy and composition of nucleosomes. In animals and plants, these remodelers have been implicated in diverse processes ranging from stem cell maintenance and differentiation to developmental phase transitions and stress responses. Detailed investigation of their roles in individual processes has suggested a higher level of selectivity of chromatin remodeling ATPase activity than previously anticipated, and diverse mechanisms have been uncovered that can contribute to the selectivity. This review summarizes recent advances in understanding the roles and activities of chromatin remodeling ATPases in plants.

  19. Balancing chromatin remodeling and histone modifications in transcription

    PubMed Central

    Petty, Emily; Pillus, Lorraine

    2013-01-01

    Chromatin remodelers use the energy of ATP hydrolysis to reposition or evict nucleosomes or to replace canonical histones with histone variants. By regulating nucleosome dynamics, remodelers gate access to the underlying DNA for replication, repair, and transcription. Nucleosomes are subject to extensive post-translational modifications that can recruit regulatory proteins or alter the local chromatin structure. Just as extensive cross-talk has been observed between different histone post-translational modifications, there is growing evidence for both coordinated and antagonistic functional relationships between nucleosome remodeling and modifying machineries. Defining the combined functions of the complexes that alter nucleosome interactions, position, and stability is key to understanding processes that require access to DNA, particularly with growing appreciation of their contributions to human health and disease. Here, we highlight recent advances in the interactions between histone modifications and the ISWI and CHD1 chromatin remodelers from studies in budding yeast, fission yeast, flies, and mammalian cells, with a focus on yeast. PMID:23870137

  20. Epigenetic remodeling of chromatin architecture: exploring tumor differentiation therapies in mesenchymal stem cells and sarcomas.

    PubMed

    Siddiqi, Sara; Mills, Joslyn; Matushansky, Igor

    2010-03-01

    Sarcomas are the mesenchymal-derived malignant tumors of connective tissues (e.g., fat, bone, and cartilage) presumed to arise from aberrant development or differentiation of mesenchymal stem cells (MSCs). Appropriate control of stem cell maintenance versus differentiation allows for normal connective tissue development. Current theories suggest that loss of this control--through accumulation of genetic lesions in MSCs at various points in the differentiation process--leads to development of sarcomas, including undifferentiated, high grade sarcoma tumors. The initiation of stem cell differentiation is highly associated with alteration of gene expression, which depends on chromatin remodeling. Epigenetic chromatin modifying agents have been shown to induce cancer cell differentiation and are currently being used clinically to treat cancer. This review will focus on the importance of epigenetic chromatin remodeling in the context of mesenchymal stem cells, sarcoma tumorigenesis and differentiation therapy.

  1. ATP dependent chromatin remodeling enzymes in embryonic stem cells.

    PubMed

    Saladi, Srinivas Vinod; de la Serna, Ivana L

    2010-03-01

    Embryonic stem (ES) cells are pluripotent cells that can self renew or be induced to differentiate into multiple cell lineages, and thus have the potential to be utilized in regenerative medicine. Key pluripotency specific factors (Oct 4/Sox2/Nanog/Klf4) maintain the pluripotent state by activating expression of pluripotency specific genes and by inhibiting the expression of developmental regulators. Pluripotent ES cells are distinguished from differentiated cells by a specialized chromatin state that is required to epigenetically regulate the ES cell phenotype. Recent studies show that in addition to pluripotency specific factors, chromatin remodeling enzymes play an important role in regulating ES cell chromatin and the capacity to self-renew and to differentiate. Here we review recent studies that delineate the role of ATP dependent chromatin remodeling enzymes in regulating ES cell chromatin structure.

  2. Chd5 orchestrates chromatin remodeling during sperm development

    PubMed Central

    Li, Wangzhi; Wu, Jie; Kim, Sang-Yong; Zhao, Ming; Hearn, Stephen A.; Zhang, Michael Q.; Meistrich, Marvin L.

    2014-01-01

    One of the most remarkable chromatin remodeling processes occurs during spermiogenesis, the post-meiotic phase of sperm development during which histones are replaced with sperm-specific protamines to repackage the genome into the highly compact chromatin structure of mature sperm. Here we identify Chromodomain helicase DNA binding protein 5 (Chd5) as a master regulator of the histone-to-protamine chromatin remodeling process. Chd5 deficiency leads to defective sperm chromatin compaction and male infertility in mice, mirroring the observation of low CHD5 expression in testes of infertile men. Chd5 orchestrates a cascade of molecular events required for histone removal and replacement, including histone 4 (H4) hyperacetylation, histone variant expression, nucleosome eviction, and DNA damage repair. Chd5 deficiency also perturbs expression of transition proteins (Tnp1/Tnp2) and protamines (Prm1/2). These findings define Chd5 as a multi-faceted mediator of histone-to-protamine replacement and depict the cascade of molecular events underlying chromatin remodeling during this process of extensive chromatin remodeling. PMID:24818823

  3. Dynamical DNA accessibility induced by chromatin remodeling and protein binding

    NASA Astrophysics Data System (ADS)

    Montel, F.; Faivre-Moskalenko, C.; Castelnovo, M.

    2014-11-01

    Chromatin remodeling factors are enzymes being able to alter locally chromatin structure at the nucleosomal level and they actively participate in the regulation of gene expression. Using simple rules for individual nucleosome motion induced by a remodeling factor, we designed simulations of the remodeling of oligomeric chromatin, in order to address quantitatively collective effects in DNA accessibility upon nucleosome mobilization. Our results suggest that accessibility profiles are inhomogeneous thanks to borders effects like protein binding. Remarkably, we show that the accessibility lifetime of DNA sequence is roughly doubled in the vicinity of borders as compared to its value in bulk regions far from the borders. These results are quantitatively interpreted as resulting from the confined diffusion of a large nucleosome depleted region.

  4. Multiple modes of chromatin remodeling by Forkhead box proteins.

    PubMed

    Lalmansingh, Avin S; Karmakar, Sudipan; Jin, Yetao; Nagaich, Akhilesh K

    2012-07-01

    Forkhead box (FOX) proteins represent a large family of transcriptional regulators unified by their DNA binding domain (DBD) known as a 'forkhead' or 'winged helix' domain. Over 40 FOX genes have been identified in the mammalian genome. FOX proteins share significant sequence similarities in the DBD which allow them to bind to a consensus DNA response element. However, their modes of action are quite diverse as they regulate gene expression by acting as pioneer factors, transcription factors, or both. This review focuses on the mechanisms of chromatin remodeling with an emphasis on three sub-classes-FOXA, FOXO, and FOXP members. FOXA proteins serve as pioneer factors to open up local chromatin structure and thereby increase accessibility of chromatin to factors regulating transcription. FOXP proteins, in contrast, function as classic transcription factors to recruit a variety of chromatin modifying enzymes to regulate gene expression. FOXO proteins represent a hybrid subclass having dual roles as pioneering factors and transcription factors. A subset of FOX proteins interacts with condensed mitotic chromatin and may function as 'bookmarking' agents to maintain transcriptional competence at specific genomic sites. The overall diversity in chromatin remodeling function by FOX proteins is related to unique structural motifs present within the DBD flanking regions that govern selective interactions with core histones and/or chromatin coregulatory proteins. This article is part of a Special Issue entitled: Chromatin in time and space.

  5. Chromatin remodeling and bivalent histone modifications in embryonic stem cells.

    PubMed

    Harikumar, Arigela; Meshorer, Eran

    2015-12-01

    Pluripotent embryonic stem cells (ESCs) are characterized by distinct epigenetic features including a relative enrichment of histone modifications related to active chromatin. Among these is tri-methylation of lysine 4 on histone H3 (H3K4me3). Several thousands of the H3K4me3-enriched promoters in pluripotent cells also contain a repressive histone mark, namely H3K27me3, a situation referred to as "bivalency". While bivalent promoters are not unique to pluripotent cells, they are relatively enriched in these cell types, largely marking developmental and lineage-specific genes which are silent but poised for immediate action. The H3K4me3 and H3K27me3 modifications are catalyzed by lysine methyltransferases which are usually found within, although not entirely limited to, the Trithorax group (TrxG) and Polycomb group (PcG) protein complexes, respectively, but these do not provide selective bivalent specificity. Recent studies highlight the family of ATP-dependent chromatin remodeling proteins as regulators of bivalent domains. Here, we discuss bivalency in general, describe the machineries that catalyze bivalent chromatin domains, and portray the emerging connection between bivalency and the action of different families of chromatin remodelers, namely INO80, esBAF, and NuRD, in pluripotent cells. We posit that chromatin remodeling proteins may enable "bivalent specificity", often selectively acting on, or selectively depleted from, bivalent domains.

  6. Frequent mutations of chromatin remodeling genes in transitional cell carcinoma of the bladder

    PubMed Central

    Gui, Yaoting; Guo, Guangwu; Huang, Yi; Hu, Xueda; Tang, Aifa; Gao, Shengjie; Wu, Renhua; Chen, Chao; Li, Xianxin; Zhou, Liang; He, Minghui; Li, Zesong; Sun, Xiaojuan; Jia, Wenlong; Chen, Jinnong; Yang, Shangming; Zhou, Fangjian; Zhao, Xiaokun; Wan, Shengqing; Ye, Rui; Liang, Chaozhao; Liu, Zhisheng; Huang, Peide; Liu, Chunxiao; Jiang, Hui; Wang, Yong; Zheng, Hancheng; Sun, Liang; Liu, Xingwang; Jiang, Zhimao; Feng, Dafei; Chen, Jing; Wu, Song; Zou, Jing; Zhang, Zhongfu; Yang, Ruilin; Zhao, Jun; Xu, Congjie; Yin, Weihua; Guan, Zhichen; Ye, Jiongxian; Zhang, Hong; Li, Jingxiang; Kristiansen, Karsten; Nickerson, Michael L; Theodorescu, Dan; Li, Yingrui; Zhang, Xiuqing; Li, Songgang; Wang, Jian; Yang, Huanming; Wang, Jun; Cai, Zhiming

    2017-01-01

    Transitional cell carcinoma (TCC) is the most common type of bladder cancer. Here we sequenced the exomes of nine individuals with TCC and screened all the somatically mutated genes in a prevalence set of 88 additional individuals with TCC with different tumor stages and grades. In our study, we discovered a variety of genes previously unknown to be mutated in TCC. Notably, we identified genetic aberrations of the chromatin remodeling genes (UTX, MLL-MLL3, CREBBP-EP300, NCOR1, ARID1A and CHD6) in 59% of our 97 subjects with TCC. Of these genes, we showed UTX to be altered substantially more frequently in tumors of low stages and grades, highlighting its potential role in the classification and diagnosis of bladder cancer. Our results provide an overview of the genetic basis of TCC and suggest that aberration of chromatin regulation might be a hallmark of bladder cancer. PMID:21822268

  7. Chromatin remodelling during male gametophyte development.

    PubMed

    Borg, Michael; Berger, Frédéric

    2015-07-01

    The plant life cycle alternates between a diploid sporophytic phase and haploid gametophytic phase, with the latter giving rise to the gametes. Male gametophyte development encompasses two mitotic divisions that results in a simple three-celled structure knows as the pollen grain, in which two sperm cells are encased within a larger vegetative cell. Both cell types exhibit a very different type of chromatin organization - highly condensed in sperm cell nuclei and highly diffuse in the vegetative cell. Distinct classes of histone variants have dynamic and differential expression in the two cell lineages of the male gametophyte. Here we review how the dynamics of histone variants are linked to reprogramming of chromatin activities in the male gametophyte, compaction of the sperm cell genome and zygotic transitions post-fertilization.

  8. Remodeling of chromatin under low intensity diffuse ultrasound.

    PubMed

    Noriega, Sandra; Budhiraja, Gaurav; Subramanian, Anuradha

    2012-08-01

    A variety of mechanotransduction pathways mediate the response of fibroblasts or chondrocytes to ultrasound stimulation. In addition, regulatory pathways that co-ordinate stimulus-specific cellular responses are likely to exist. In this study, analysis was confined to the hypothesis that ultrasound stimulation (US) influences the chromatin structure, and that these changes may reflect a regulatory pathway that connects nuclear architecture, chromatin structure and gene expression. Murine fibroblasts seeded on tissue culture plates were stimulated with US (5.0 MHz (14 kPa), 51-s per application) and the thermal denaturation profiles of nuclei isolated from fibroblasts were assessed by dynamic scanning calorimetry (DSC). When compared to the thermal profiles obtained from the nuclei of non-stimulated cells, the nuclei obtained from stimulated cells showed a change in peak profiles and peak areas, which is indicative of chromatin remodeling. Independently, US was also observed to impact the histone (H1):chromatin association as measured indirectly by DAPI staining. Based on our work, it appears plausible that US can produce a remodeling of chromatin, thus triggering signal cascade and other intracellular mechanisms.

  9. On the mechanochemical machinery underlying chromatin remodeling

    NASA Astrophysics Data System (ADS)

    Yusufaly, Tahir I.

    This dissertation discuss two recent efforts, via a unique combination of structural bioinformatics and density functional theory, to unravel some of the details concerning how molecular machinery within the eukaryotic cell nucleus controls chromatin architecture. The first, a study of the 5-methylation of cytosine in 5'-CG-3' : 5'-CG-3' base-pair steps, reveals that the methyl groups roughen the local elastic energy landscape of the DNA. This enhances the probability of the canonical B-DNA structure transitioning into the undertwisted A-like and overtwisted C-like forms seen in nucleosomes, or looped segments of DNA bound to histones. The second part focuses on the formation of salt bridges between arginine residues in histones and phosphate groups on the DNA backbone. The arginine residues are ob- served to apply a tunable mechanical load to the backbone, enabling precision-controlled activation of DNA deformations.

  10. Senataxin controls meiotic silencing through ATR activation and chromatin remodeling.

    PubMed

    Yeo, Abrey J; Becherel, Olivier J; Luff, John E; Graham, Mark E; Richard, Derek; Lavin, Martin F

    2015-01-01

    Senataxin, defective in ataxia oculomotor apraxia type 2, protects the genome by facilitating the resolution of RNA-DNA hybrids (R-loops) and other aspects of RNA processing. Disruption of this gene in mice causes failure of meiotic recombination and defective meiotic sex chromosome inactivation, leading to male infertility. Here we provide evidence that the disruption of Setx leads to reduced SUMOylation and disruption of protein localization across the XY body during meiosis. We demonstrate that senataxin and other DNA damage repair proteins, including ataxia telangiectasia and Rad3-related protein-interacting partner, are SUMOylated, and a marked downregulation of both ataxia telangiectasia and Rad3-related protein-interacting partner and TopBP1 leading to defective activation and signaling through ataxia telangiectasia and Rad3-related protein occurs in the absence of senataxin. Furthermore, chromodomain helicase DNA-binding protein 4, a component of the nucleosome remodeling and deacetylase chromatin remodeler that interacts with both ataxia telangiectasia and Rad3-related protein and senataxin was not recruited efficiently to the XY body, triggering altered histone acetylation and chromatin conformation in Setx (-/-) pachytene-staged spermatocytes. These results demonstrate that senataxin has a critical role in ataxia telangiectasia and Rad3-related protein- and chromodomain helicase DNA-binding protein 4-mediated transcriptional silencing and chromatin remodeling during meiosis providing greater insight into its critical role in gene regulation to protect against neurodegeneration.

  11. Senataxin controls meiotic silencing through ATR activation and chromatin remodeling

    PubMed Central

    Yeo, Abrey J; Becherel, Olivier J; Luff, John E; Graham, Mark E; Richard, Derek; Lavin, Martin F

    2015-01-01

    Senataxin, defective in ataxia oculomotor apraxia type 2, protects the genome by facilitating the resolution of RNA–DNA hybrids (R-loops) and other aspects of RNA processing. Disruption of this gene in mice causes failure of meiotic recombination and defective meiotic sex chromosome inactivation, leading to male infertility. Here we provide evidence that the disruption of Setx leads to reduced SUMOylation and disruption of protein localization across the XY body during meiosis. We demonstrate that senataxin and other DNA damage repair proteins, including ataxia telangiectasia and Rad3-related protein-interacting partner, are SUMOylated, and a marked downregulation of both ataxia telangiectasia and Rad3-related protein-interacting partner and TopBP1 leading to defective activation and signaling through ataxia telangiectasia and Rad3-related protein occurs in the absence of senataxin. Furthermore, chromodomain helicase DNA-binding protein 4, a component of the nucleosome remodeling and deacetylase chromatin remodeler that interacts with both ataxia telangiectasia and Rad3-related protein and senataxin was not recruited efficiently to the XY body, triggering altered histone acetylation and chromatin conformation in Setx−/− pachytene-staged spermatocytes. These results demonstrate that senataxin has a critical role in ataxia telangiectasia and Rad3-related protein- and chromodomain helicase DNA-binding protein 4-mediated transcriptional silencing and chromatin remodeling during meiosis providing greater insight into its critical role in gene regulation to protect against neurodegeneration. PMID:27462424

  12. Epigenetic regulation and chromatin remodeling in learning and memory

    PubMed Central

    Kim, Somi; Kaang, Bong-Kiun

    2017-01-01

    Understanding the underlying mechanisms of memory formation and maintenance has been a major goal in the field of neuroscience. Memory formation and maintenance are tightly controlled complex processes. Among the various processes occurring at different levels, gene expression regulation is especially crucial for proper memory processing, as some genes need to be activated while some genes must be suppressed. Epigenetic regulation of the genome involves processes such as DNA methylation and histone post-translational modifications. These processes edit genomic properties or the interactions between the genome and histone cores. They then induce structural changes in the chromatin and lead to transcriptional changes of different genes. Recent studies have focused on the concept of chromatin remodeling, which consists of 3D structural changes in chromatin in relation to gene regulation, and is an important process in learning and memory. In this review, we will introduce three major epigenetic processes involved in memory regulation: DNA methylation, histone methylation and histone acetylation. We will also discuss general mechanisms of long-term memory storage and relate the epigenetic control of learning and memory to chromatin remodeling. Finally, we will discuss how epigenetic mechanisms can contribute to the pathologies of neurological disorders and cause memory-related symptoms. PMID:28082740

  13. The Circadian NAD+ Metabolism: Impact on Chromatin Remodeling and Aging

    PubMed Central

    Bessho, Yasumasa

    2016-01-01

    Gene expression is known to be a stochastic phenomenon. The stochastic gene expression rate is thought to be altered by topological change of chromosome and/or by chromatin modifications such as acetylation and methylation. Changes in mechanical properties of chromosome/chromatin by soluble factors, mechanical stresses from the environment, or metabolites determine cell fate, regulate cellular functions, or maintain cellular homeostasis. Circadian clock, which drives the expression of thousands of genes with 24-hour rhythmicity, has been known to be indispensable for maintaining cellular functions/homeostasis. During the last decade, it has been demonstrated that chromatin also undergoes modifications with 24-hour rhythmicity and facilitates the fine-tuning of circadian gene expression patterns. In this review, we cover data which suggests that chromatin structure changes in a circadian manner and that NAD+ is the key metabolite for circadian chromatin remodeling. Furthermore, we discuss the relationship among circadian clock, NAD+ metabolism, and aging/age-related diseases. In addition, the interventions of NAD+ metabolism for the prevention and treatment of aging and age-related diseases are also discussed. PMID:28050554

  14. The Ino80 chromatin-remodeling complex restores chromatin structure during UV DNA damage repair

    PubMed Central

    Sarkar, Sovan; Kiely, Rhian

    2010-01-01

    Chromatin structure is modulated during deoxyribonucleic acid excision repair, but how this is achieved is unclear. Loss of the yeast Ino80 chromatin-remodeling complex (Ino80-C) moderately sensitizes cells to ultraviolet (UV) light. In this paper, we show that INO80 acts in the same genetic pathway as nucleotide excision repair (NER) and that the Ino80-C contributes to efficient UV photoproduct removal in a region of high nucleosome occupancy. Moreover, Ino80 interacts with the early NER damage recognition complex Rad4–Rad23 and is recruited to chromatin by Rad4 in a UV damage–dependent manner. Using a modified chromatin immunoprecipitation assay, we find that chromatin disruption during UV lesion repair is normal, whereas the restoration of nucleosome structure is defective in ino80 mutant cells. Collectively, our work suggests that Ino80 is recruited to sites of UV lesion repair through interactions with the NER apparatus and is required for the restoration of chromatin structure after repair. PMID:21135142

  15. Chromatin remodelers Isw1 and Chd1 maintain chromatin structure during transcription by preventing histone exchange

    PubMed Central

    Smolle, Michaela; Venkatesh, Swaminathan; Gogol, Madelaine M.; Li, Hua; Zhang, Ying; Florens, Laurence; Washburn, Michael P.; Workman, Jerry L.

    2012-01-01

    Set2-mediated methylation of histone H3 Lys36 (H3K36) is a mark associated with the coding sequences of actively transcribed genes, yet plays a negative role during transcription elongation. It prevents trans-histone exchange over coding regions and signals for histone deacetylation in the wake of RNA polymerase II (RNAPII) passage. We have found that in Saccharomyces cerevisiae the Isw1b chromatin-remodeling complex is specifically recruited to open reading frames (ORFs) by H3K36 methylation through the PWWP domain of its Ioc4 subunit in vivo and in vitro. Isw1b acts in conjunction with Chd1 to regulate chromatin structure by preventing trans-histone exchange from taking place over coding regions and thus maintains chromatin integrity during transcription elongation by RNA polymerase II. PMID:22922743

  16. JOINING THE DOTS: FROM CHROMATIN REMODELING TO NEURONAL PLASTICITY

    PubMed Central

    Zocchi, Loredana; Sassone-Corsi, Paolo

    2010-01-01

    SUMMARY In recent years spectacular advances in the field of epigenetics have taken place. Multiple lines of evidence that connect epigenetic regulation to brain functions have been accumulating. Neurons daily convert a variety of external stimuli into rapid or long-lasting changes in gene expression. Control is achieved through several post-translational modifications that occur both on DNA and chromatin. Specific modifications mediate many developmental processes and adult brain functions, such as synaptic plasticity and memory. In this review, we focus on critical chromatin remodeling events that mediate long-lasting neuronal responses. The challenging goal is to reach sufficient understanding of these epigenetic pathways in the brain so that they may be useful for future development of specific pharmacological strategies. PMID:20471240

  17. Sequence-targeted nucleosome sliding in vivo by a hybrid Chd1 chromatin remodeler

    PubMed Central

    Bowman, Gregory D.

    2016-01-01

    ATP-dependent chromatin remodelers regulate chromatin dynamics by modifying nucleosome positions and occupancy. DNA-dependent processes such as replication and transcription rely on chromatin to faithfully regulate DNA accessibility, yet how chromatin remodelers achieve well-defined nucleosome positioning in vivo is poorly understood. Here, we report a simple method for site-specifically altering nucleosome positions in live cells. By fusing the Chd1 remodeler to the DNA binding domain of the Saccharomyces cerevisiae Ume6 repressor, we have engineered a fusion remodeler that selectively positions nucleosomes on top of adjacent Ume6 binding motifs in a highly predictable and reproducible manner. Positioning of nucleosomes by the fusion remodeler recapitulates closed chromatin structure at Ume6-sensitive genes analogous to the endogenous Isw2 remodeler. Strikingly, highly precise positioning of single founder nucleosomes by either chimeric Chd1-Ume6 or endogenous Isw2 shifts phased chromatin arrays in cooperation with endogenous chromatin remodelers. Our results demonstrate feasibility of engineering precise nucleosome rearrangements through sequence-targeted chromatin remodeling and provide insight into targeted action and cooperation of endogenous chromatin remodelers in vivo. PMID:26993344

  18. Soluble Endoglin Modulates Aberrant Cerebral Vascular Remodeling

    PubMed Central

    Chen, Yongmei; Hao, Qi; Kim, Helen; Su, Hua; Letarte, Michelle; Karumanchi, S. Ananth; Lawton, Michael T.; Barbaro, Nicholas M.; Yang, Guo-Yuan; Young, William L.

    2009-01-01

    Objective Brain arteriovenous malformations (AVMs) are an important cause of neurological morbidity in young adults. The pathophysiology of these lesions is poorly understood. A soluble form of endoglin (sEng) has been shown to cause endothelial dysfunction and induce preeclampsia. We tested if sEng would be elevated in brain AVM tissues relative to epilepsy brain tissues, and also investigated whether sEng overexpression via gene transfer in the mouse brain would induce vascular dysplasia and associated changes in downstream signaling pathways. Methods Expression levels of sEng in surgical specimens were determined by Western blot assay and ELISA. Vascular dysplasia, levels of MMP and oxidative stress were determined by immunohistochemistry and gelatin zymography. Results Brain AVMs (n=33) had higher mean sEng levels (245 ± 175 vs 100 ± 60, % of control, P=0.04) compared with controls (n=8), as determined by Western blot. In contrast, membrane-bound Eng was not significantly different (108 ± 79 vs 100 ± 63, % of control, P=0.95). sEng gene transduction in the mouse brain induced abnormal vascular structures. It also increased matrix metalloproteinase (MMP) activity by 490 ± 30% (MMP-9), 220 ± 30% (MMP-2), and oxidants by 260 ± 20% (4-hydroxy-2-nonenal) at 2 weeks after injection, suggesting that MMPs and oxidative radicals may mediate sEng-induced pathological vascular remodeling. Interpretation The results suggest that elevated sEng may play a role in the generation of sporadic brain AVMs. Our findings may provide new targets for therapeutic intervention for patients with brain AVMs. PMID:19670444

  19. A SWI/SNF Chromatin Remodelling Protein Controls Cytokinin Production through the Regulation of Chromatin Architecture

    PubMed Central

    Jégu, Teddy; Domenichini, Séverine; Blein, Thomas; Ariel, Federico; Christ, Aurélie; Kim, Soon-Kap; Crespi, Martin; Boutet-Mercey, Stéphanie; Mouille, Grégory; Bourge, Mickaël; Hirt, Heribert; Bergounioux, Catherine; Raynaud, Cécile; Benhamed, Moussa

    2015-01-01

    Chromatin architecture determines transcriptional accessibility to DNA and consequently gene expression levels in response to developmental and environmental stimuli. Recently, chromatin remodelers such as SWI/SNF complexes have been recognized as key regulators of chromatin architecture. To gain insight into the function of these complexes during root development, we have analyzed Arabidopsis knock-down lines for one sub-unit of SWI/SNF complexes: BAF60. Here, we show that BAF60 is a positive regulator of root development and cell cycle progression in the root meristem via its ability to down-regulate cytokinin production. By opposing both the deposition of active histone marks and the formation of a chromatin regulatory loop, BAF60 negatively regulates two crucial target genes for cytokinin biosynthesis (IPT3 and IPT7) and one cell cycle inhibitor (KRP7). Our results demonstrate that SWI/SNF complexes containing BAF60 are key factors governing the equilibrium between formation and dissociation of a chromatin loop controlling phytohormone production and cell cycle progression. PMID:26457678

  20. Coupling Circadian Rhythms of Metabolism and Chromatin Remodeling

    PubMed Central

    Masri, Selma; Orozco-Solis, Ricardo; Aguilar-Arnal, Lorena; Cervantes, Marlene; Sassone-Corsi, Paolo

    2015-01-01

    The circadian clock controls a large variety of neuronal, endocrine, behavioral and physiological responses in mammals. This control is exerted in large part at the transcriptional level on genes expressed in a cyclic manner. A highly specialized transcriptional machinery based on clock regulatory factors organized in feedback autoregulatory loops governs a significant portion of the genome. These oscillations in gene expression are paralleled by critical events of chromatin remodeling that appear to provide plasticity to circadian regulation. Specifically, the NAD+-dependent deacetylases SIRT1 and SIRT6 have been linked to circadian control of gene expression. This, and additional accumulating evidence, shows that the circadian epigenome appears to share intimate links with cellular metabolic processes and has remarkable plasticity showing reprogramming in response to nutritional challenges. In addition to SIRT1 and SIRT6, a number of chromatin remodelers have been implicated in clock control, including the histone H3K4 tri-methyltransferase MLL1. Deciphering the molecular mechanisms that link metabolism, epigenetic control and circadian responses will provide valauble insights towards innovative strategies of therapeutic intervention. PMID:26332964

  1. Genomic Aberrations Frequently Alter Chromatin Regulatory Genes in Chordoma

    PubMed Central

    Wang, Lu; Zehir, Ahmet; Nafa, Khedoudja; Zhou, Nengyi; Berger, Michael F.; Casanova, Jacklyn; Sadowska, Justyna; Lu, Chao; Allis, C. David; Gounder, Mrinal; Chandhanayingyong, Chandhanarat; Ladanyi, Marc; Boland, Patrick J; Hameed, Meera

    2016-01-01

    Chordoma is a rare primary bone neoplasm that is resistant to standard chemotherapies. Despite aggressive surgical management, local recurrence and metastasis is not uncommon. To identify the specific genetic aberrations that play key roles in chordoma pathogenesis, we utilized a genome-wide high-resolution SNP-array and next generation sequencing (NGS)-based molecular profiling platform to study 24 patient samples with typical histopathologic features of chordoma. Matching normal tissues were available for 16 samples. SNP-array analysis revealed nonrandom copy number losses across the genome, frequently involving 3, 9p, 1p, 14, 10, and 13. In contrast, copy number gain is uncommon in chordomas. Two minimum deleted regions were observed on 3p within a ~8 Mb segment at 3p21.1–p21.31, which overlaps SETD2, BAP1 and PBRM1. The minimum deleted region on 9p was mapped to CDKN2A locus at 9p21.3, and homozygous deletion of CDKN2A was detected in 5/22 chordomas (~23%). NGS-based molecular profiling demonstrated an extremely low level of mutation rate in chordomas, with an average of 0.5 mutations per sample for the 16 cases with matched normal. When the mutated genes were grouped based on molecular functions, many of the mutation events (~40%) were found in chromatin regulatory genes. The combined copy number and mutation profiling revealed that SETD2 is the single gene affected most frequently in chordomas, either by deletion or by mutations. Our study demonstrated that chordoma belongs to the C-class (copy number changes) tumors whose oncogenic signature is non-random multiple copy number losses across the genome and genomic aberrations frequently alter chromatin regulatory genes. PMID:27072194

  2. The Chd1 chromatin remodeler shifts hexasomes unidirectionally

    PubMed Central

    Levendosky, Robert F; Sabantsev, Anton; Deindl, Sebastian; Bowman, Gregory D

    2016-01-01

    Despite their canonical two-fold symmetry, nucleosomes in biological contexts are often asymmetric: functionalized with post-translational modifications (PTMs), substituted with histone variants, and even lacking H2A/H2B dimers. Here we show that the Widom 601 nucleosome positioning sequence can produce hexasomes in a specific orientation on DNA, providing a useful tool for interrogating chromatin enzymes and allowing for the generation of nucleosomes with precisely defined asymmetry. Using this methodology, we demonstrate that the Chd1 chromatin remodeler from Saccharomyces cerevisiae requires H2A/H2B on the entry side for sliding, and thus, unlike the back-and-forth sliding observed for nucleosomes, Chd1 shifts hexasomes unidirectionally. Chd1 takes part in chromatin reorganization surrounding transcribing RNA polymerase II (Pol II), and using asymmetric nucleosomes we show that ubiquitin-conjugated H2B on the entry side stimulates nucleosome sliding by Chd1. We speculate that biased nucleosome and hexasome sliding due to asymmetry contributes to the packing of arrays observed in vivo. DOI: http://dx.doi.org/10.7554/eLife.21356.001 PMID:28032848

  3. Footprint traversal by adenosine-triphosphate-dependent chromatin remodeler motor

    NASA Astrophysics Data System (ADS)

    Garai, Ashok; Mani, Jesrael; Chowdhury, Debashish

    2012-04-01

    Adenosine-triphosphate (ATP)-dependent chromatin remodeling enzymes (CREs) are biomolecular motors in eukaryotic cells. These are driven by a chemical fuel, namely, ATP. CREs actively participate in many cellular processes that require accessibility of specific segments of DNA which are packaged as chromatin. The basic unit of chromatin is a nucleosome where 146 bp ˜ 50 nm of a double-stranded DNA (dsDNA) is wrapped around a spool formed by histone proteins. The helical path of histone-DNA contact on a nucleosome is also called “footprint.” We investigate the mechanism of footprint traversal by a CRE that translocates along the dsDNA. Our two-state model of a CRE captures effectively two distinct chemical (or conformational) states in the mechanochemical cycle of each ATP-dependent CRE. We calculate the mean time of traversal. Our predictions on the ATP dependence of the mean traversal time can be tested by carrying out in vitro experiments on mononucleosomes.

  4. Chromatin-Remodeling-Factor ARID1B Represses Wnt/β-Catenin Signaling

    PubMed Central

    Vasileiou, Georgia; Ekici, Arif B.; Uebe, Steffen; Zweier, Christiane; Hoyer, Juliane; Engels, Hartmut; Behrens, Jürgen; Reis, André; Hadjihannas, Michel V.

    2015-01-01

    The link of chromatin remodeling to both neurodevelopment and cancer has recently been highlighted by the identification of mutations affecting BAF chromatin-remodeling components, such as ARID1B, in individuals with intellectual disability and cancer. However, the underlying molecular mechanism(s) remains unknown. Here, we show that ARID1B is a repressor of Wnt/β-catenin signaling. Through whole-transcriptome analysis, we find that in individuals with intellectual disability and ARID1B loss-of-function mutations, Wnt/β-catenin target genes are upregulated. Using cellular models of low and high Wnt/β-catenin activity, we demonstrate that knockdown of ARID1B activates Wnt/β-catenin target genes and Wnt/β-catenin-dependent transcriptional reporters in a β-catenin-dependent manner. Reciprocally, forced expression of ARID1B inhibits Wnt/β-catenin signaling downstream of the β-catenin destruction complex. Both endogenous and exogenous ARID1B associate with β-catenin and repress Wnt/β-catenin-mediated transcription through the BAF core subunit BRG1. Accordingly, mutations in ARID1B leading to partial or complete deletion of its BRG1-binding domain, as is often observed in intellectual disability and cancers, compromise association with β-catenin, and the resultant ARID1B mutant proteins fail to suppress Wnt/β-catenin signaling. Finally, knockdown of ARID1B in mouse neuroblastoma cells leads to neurite outgrowth through β-catenin. The data suggest that aberrations in chromatin-remodeling factors, such as ARID1B, might contribute to neurodevelopmental abnormalities and cancer through deregulation of developmental and oncogenic pathways, such as the Wnt/β-catenin signaling pathway. PMID:26340334

  5. SWI/SNF-dependent chromatin remodeling of RNR3 requires TAFIIs and the general transcription machinery

    PubMed Central

    Sharma, Vishva Mitra; Li, Bing; Reese, Joseph C.

    2003-01-01

    Gene expression requires the recruitment of chromatin remodeling activities and general transcription factors (GTFs) to promoters. Whereas the role of activators in recruiting chromatin remodeling activities has been clearly demonstrated, the contributions of the transcription machinery have not been firmly established. Here we demonstrate that the remodeling of the RNR3 promoter requires a number of GTFs, mediator and RNA polymerase II. We also show that remodeling is dependent upon the SWI/SNF complex, and that TFIID and RNA polymerase II are required for its recruitment to the promoter. In contrast, Gcn5p-dependent histone acetylation occurs independently of TFIID and RNA polymerase II function, and we provide evidence that acetylation increases the extent of nucleosome remodeling, but is not required for SWI/SNF recruitment. Thus, the general transcription machinery can contribute to nucleosome remodeling by mediating the association of SWI/SNF with promoters, thereby revealing a novel pathway for the recruitment of chromatin remodeling activities. PMID:12600943

  6. Vitamin D receptor (VDR) promoter targeting through a novel chromatin remodeling complex.

    PubMed

    Kato, Shigeaki; Fujiki, Ryoji; Kitagawa, Hirochika

    2004-05-01

    We have purified nuclear complexes for Vitamin D receptor (VDR), and identified one of them as a novel ATP-dependent chromatine remodeling containing Williams syndrome transcription factor (WSTF), that is supposed to be responsible for Williams syndrome. This complex (WSTF including nucleosome assembly complex (WINAC)) exhibited an ATP-dependent chromatin remodeling activity in vitro. Transient expression assays revealed that WINAC potentiates ligand-induced function of VDR in gene activation and repression. Thus, this study describes a molecular basis of the VDR function on chromosomal DNA through chromatine remodeling.

  7. The Fun30 chromatin remodeler Fft3 controls nuclear organization and chromatin structure of insulators and subtelomeres in fission yeast.

    PubMed

    Steglich, Babett; Strålfors, Annelie; Khorosjutina, Olga; Persson, Jenna; Smialowska, Agata; Javerzat, Jean-Paul; Ekwall, Karl

    2015-03-01

    In eukaryotic cells, local chromatin structure and chromatin organization in the nucleus both influence transcriptional regulation. At the local level, the Fun30 chromatin remodeler Fft3 is essential for maintaining proper chromatin structure at centromeres and subtelomeres in fission yeast. Using genome-wide mapping and live cell imaging, we show that this role is linked to controlling nuclear organization of its targets. In fft3∆ cells, subtelomeres lose their association with the LEM domain protein Man1 at the nuclear periphery and move to the interior of the nucleus. Furthermore, genes in these domains are upregulated and active chromatin marks increase. Fft3 is also enriched at retrotransposon-derived long terminal repeat (LTR) elements and at tRNA genes. In cells lacking Fft3, these sites lose their peripheral positioning and show reduced nucleosome occupancy. We propose that Fft3 has a global role in mediating association between specific chromatin domains and the nuclear envelope.

  8. The poly(ADP-ribose)-dependent chromatin remodeler Alc1 induces local chromatin relaxation upon DNA damage

    PubMed Central

    Sellou, Hafida; Lebeaupin, Théo; Chapuis, Catherine; Smith, Rebecca; Hegele, Anna; Singh, Hari R.; Kozlowski, Marek; Bultmann, Sebastian; Ladurner, Andreas G.; Timinszky, Gyula; Huet, Sébastien

    2016-01-01

    Chromatin relaxation is one of the earliest cellular responses to DNA damage. However, what determines these structural changes, including their ATP requirement, is not well understood. Using live-cell imaging and laser microirradiation to induce DNA lesions, we show that the local chromatin relaxation at DNA damage sites is regulated by PARP1 enzymatic activity. We also report that H1 is mobilized at DNA damage sites, but, since this mobilization is largely independent of poly(ADP-ribosyl)ation, it cannot solely explain the chromatin relaxation. Finally, we demonstrate the involvement of Alc1, a poly(ADP-ribose)- and ATP-dependent remodeler, in the chromatin-relaxation process. Deletion of Alc1 impairs chromatin relaxation after DNA damage, while its overexpression strongly enhances relaxation. Altogether our results identify Alc1 as an important player in the fast kinetics of the NAD+- and ATP-dependent chromatin relaxation upon DNA damage in vivo. PMID:27733626

  9. Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells.

    PubMed

    de Dieuleveult, Maud; Yen, Kuangyu; Hmitou, Isabelle; Depaux, Arnaud; Boussouar, Fayçal; Bou Dargham, Daria; Jounier, Sylvie; Humbertclaude, Hélène; Ribierre, Florence; Baulard, Céline; Farrell, Nina P; Park, Bongsoo; Keime, Céline; Carrière, Lucie; Berlivet, Soizick; Gut, Marta; Gut, Ivo; Werner, Michel; Deleuze, Jean-François; Olaso, Robert; Aude, Jean-Christophe; Chantalat, Sophie; Pugh, B Franklin; Gérard, Matthieu

    2016-02-04

    ATP-dependent chromatin remodellers allow access to DNA for transcription factors and the general transcription machinery, but whether mammalian chromatin remodellers target specific nucleosomes to regulate transcription is unclear. Here we present genome-wide remodeller-nucleosome interaction profiles for the chromatin remodellers Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind one or both full nucleosomes that flank micrococcal nuclease (MNase)-defined nucleosome-free promoter regions (NFRs), where they separate divergent transcription. Surprisingly, large CpG-rich NFRs that extend downstream of annotated transcriptional start sites are nevertheless bound by non-nucleosomal or subnucleosomal histone variants (H3.3 and H2A.Z) and marked by H3K4me3 and H3K27ac modifications. RNA polymerase II therefore navigates hundreds of base pairs of altered chromatin in the sense direction before encountering an MNase-resistant nucleosome at the 3' end of the NFR. Transcriptome analysis after remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. Whereas at active genes individual remodellers have either positive or negative roles via altering nucleosome stability, at polycomb-enriched bivalent genes the same remodellers act in an opposite manner. These findings indicate that remodellers target specific nucleosomes at the edge of NFRs, where they regulate ES cell transcriptional programs.

  10. Akirin: a context-dependent link between transcription and chromatin remodeling.

    PubMed

    Nowak, Scott J; Baylies, Mary K

    2012-01-01

    Embryonic patterning relies upon an exquisitely timed program of gene regulation. While the regulation of this process via the action of transcription factor networks is well understood, new lines of study have highlighted the importance of a concurrently regulated program of chromatin remodeling during development. Chromatin remodeling refers to the manipulation of the chromatin architecture through rearrangement, repositioning, or restructuring of nucleosomes to either favor or hinder the expression of associated genes. While the role of chromatin remodeling pathways during tumor development and cancer progression are beginning to be clarified, the roles of these pathways in the course of tissue specification, morphogenesis and patterning remains relatively unknown. Further, relatively little is understood as to the mechanism whereby developmentally critical transcription factors coordinate with chromatin remodeling factors to optimize target gene loci for gene expression. Such a mechanism might involve direct transcription factor/chromatin remodeling factor interactions, or could likely be mediated via an unknown intermediary. Our group has identified the relatively unknown protein Akirin as a putative member of this latter group: a secondary cofactor that serves as an interface between a developmentally critical transcription factor and the chromatin remodeling machinery. This role for the Akirin protein suggests a novel regulatory mode for regulating gene expression during development.

  11. Kinetic Control of Nucleosome Displacement by ISWI/ACF Chromatin Remodelers

    NASA Astrophysics Data System (ADS)

    Florescu, Ana-Maria; Schiessel, Helmut; Blossey, Ralf

    2012-09-01

    Chromatin structure is dynamically organized by chromatin remodelers, motor protein complexes which move and remove nucleosomes. The regulation of remodeler action has recently been proposed to underlie a kinetic proofreading scheme which combines the recognition of histone-tail states and the ATP-dependent loosening of DNA around nucleosomes. Members of the ISWI-family of remodelers additionally recognize linker length between nucleosomes. Here, we show that the additional proofreading step involving linker length alone is sufficient to promote the formation of regular arrays of nucleosomes. ATP-dependent remodeling by bidirectional motors is shown to reinforce positioning as compared to statistical positioning.

  12. Relocalization of human chromatin remodeling cofactor TIP48 in mitosis

    SciTech Connect

    Sigala, Barbara; Edwards, Mina; Puri, Teena; Tsaneva, Irina R. . E-mail: tsaneva@biochem.ucl.ac.uk

    2005-11-01

    TIP48 is a highly conserved eukaryotic AAA{sup +} protein which is an essential cofactor for several complexes involved in chromatin acetylation and remodeling, transcriptional and developmental regulation and nucleolar organization and trafficking. We show that TIP48 abundance in HeLa cells did not change during the cell cycle, nor did its distribution in various biochemical fractions. However, we observed distinct changes in the subcellular localization of TIP48 during M phase using immunofluorescence microscopy. Our studies demonstrate that in interphase cells TIP48 was found mainly in the nucleus and exhibited a distinct localization in the nuclear periphery. As the cells entered mitosis, TIP48 was excluded from the condensing chromosomes but showed association with the mitotic apparatus. During anaphase, some TIP48 was detected in the centrosome colocalizing with tubulin but the strongest staining appeared in the mitotic equator associated with the midzone central spindle. Accumulation of TIP48 in the midzone and the midbody was observed in late telophase and cytokinesis. This redeployment of TIP48 during anaphase and cytokinesis was independent of microtubule assembly. The relocation of endogenous TIP48 to the midzone/midbody under physiological conditions suggests a novel and distinct function for TIP48 in mitosis and possible involvement in the exit of mitosis.

  13. Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells

    PubMed Central

    de Dieuleveult, Maud; Yen, Kuangyu; Hmitou, Isabelle; Depaux, Arnaud; Boussouar, Fayçal; Dargham, Daria Bou; Jounier, Sylvie; Humbertclaude, Hélène; Ribierre, Florence; Baulard, Céline; Farrell, Nina P.; Park, Bongsoo; Keime, Céline; Carrière, Lucie; Berlivet, Soizick; Gut, Marta; Gut, Ivo; Werner, Michel; Deleuze, Jean-François; Olaso, Robert; Aude, Jean-Christophe; Chantalat, Sophie; Pugh, B. Franklin; Gérard, Matthieu

    2015-01-01

    Summary ATP-dependent chromatin remodellers allow access to DNA for transcription factors and the general transcription machinery, but whether mammalian chromatin remodellers1–3 target specific nucleosomes to regulate transcription is unclear. Here, we present genome-wide remodeller-nucleosome interaction profiles for Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind one or both full nucleosomes that flank MNase-defined nucleosome-free promoter regions (NFRs), where they separate divergent transcription. Surprisingly, large CpG-rich NFRs that extend downstream of annotated transcriptional start sites (TSSs) are nevertheless chromatinized with non-nucleosomal or subnucleosomal histone variants (H3.3 and H2A.Z) and modifications (H3K4me3 and H3K27ac). RNA polymerase (pol) II therefore navigates hundreds of bp of altered chromatin in the sense direction before encountering an MNase-resistant nucleosome at the 3′ end of the NFR. Transcriptome analysis upon remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. Whereas at active genes individual remodellers play either positive or negative roles via altering nucleosome stability, at polycomb-enriched bivalent genes the same remodellers act in an opposite manner. These findings indicate that remodellers target specific nucleosomes at the edge of NFRs, where they regulate ES cell transcriptional programs. PMID:26814966

  14. Chromatin remodeller Fun30Fft3 induces nucleosome disassembly to facilitate RNA polymerase II elongation

    PubMed Central

    Lee, Junwoo; Shik Choi, Eun; David Seo, Hogyu; Kang, Keunsoo; Gilmore, Joshua M.; Florens, Laurence; Washburn, Michael P.; Choe, Joonho; Workman, Jerry L.; Lee, Daeyoup

    2017-01-01

    Previous studies have revealed that nucleosomes impede elongation of RNA polymerase II (RNAPII). Recent observations suggest a role for ATP-dependent chromatin remodellers in modulating this process, but direct in vivo evidence for this is unknown. Here using fission yeast, we identify Fun30Fft3 as a chromatin remodeller, which localizes at transcribing regions to promote RNAPII transcription. Fun30Fft3 associates with RNAPII and collaborates with the histone chaperone, FACT, which facilitates RNAPII elongation through chromatin, to induce nucleosome disassembly at transcribing regions during RNAPII transcription. Mutants, resulting in reduced nucleosome-barrier, such as deletion mutants of histones H3/H4 themselves and the genes encoding components of histone deacetylase Clr6 complex II suppress the defects in growth and RNAPII occupancy of cells lacking Fun30Fft3. These data suggest that RNAPII utilizes the chromatin remodeller, Fun30Fft3, to overcome the nucleosome barrier to transcription elongation. PMID:28218250

  15. Chd1 remodelers maintain open chromatin and regulate the epigenetics of differentiation

    SciTech Connect

    Persson, Jenna; Ekwall, Karl

    2010-05-01

    Eukaryotic DNA is packaged around octamers of histone proteins into nucleosomes, the basic unit of chromatin. In addition to enabling meters of DNA to fit within the confines of a nucleus, the structure of chromatin has functional implications for cell identity. Covalent chemical modifications to the DNA and to histones, histone variants, ATP-dependent chromatin remodelers, small noncoding RNAs and the level of chromatin compaction all contribute to chromosomal structure and to the activity or silencing of genes. These chromatin-level alterations are defined as epigenetic when they are heritable from mother to daughter cell. The great diversity of epigenomes that can arise from a single genome permits a single, totipotent cell to generate the hundreds of distinct cell types found in humans. Two recent studies in mouse and in fly have highlighted the importance of Chd1 chromatin remodelers for maintaining an open, active chromatin state. Based on evidence from fission yeast as a model system, we speculate that Chd1 remodelers are involved in the disassembly of nucleosomes at promoter regions, thus promoting active transcription and open chromatin. It is likely that these nucleosomes are specifically marked for disassembly by the histone variant H2A.Z.

  16. Defective ATM-Kap-1-mediated chromatin remodeling impairs DNA repair and accelerates senescence in progeria mouse model.

    PubMed

    Liu, Baohua; Wang, Zimei; Ghosh, Shrestha; Zhou, Zhongjun

    2013-04-01

    ATM-mediated phosphorylation of KAP-1 triggers chromatin remodeling and facilitates the loading and retention of repair proteins at DNA lesions. Mouse embryonic fibroblasts (MEFs) derived from Zmpste24(-/-) mice undergo early senescence, attributable to delayed recruitment of DNA repair proteins. Here, we show that ATM-Kap-1 signaling is compromised in Zmpste24(-/-) MEFs, leading to defective DNA damage-induced chromatin remodeling. Knocking down Kap-1 rescues impaired chromatin remodeling, defective DNA repair and early senescence in Zmpste24(-/-) MEFs. Thus, ATM-Kap-1-mediated chromatin remodeling plays a critical role in premature aging, carrying significant implications for progeria therapy.

  17. Mast cell desensitization inhibits calcium flux and aberrantly remodels actin

    PubMed Central

    Ang, W.X. Gladys; Church, Alison M.; Kulis, Mike; Choi, Hae Woong; Burks, A. Wesley

    2016-01-01

    Rush desensitization (DS) is a widely used and effective clinical strategy for the rapid inhibition of IgE-mediated anaphylactic responses. However, the cellular targets and underlying mechanisms behind this process remain unclear. Recent studies have implicated mast cells (MCs) as the primary target cells for DS. Here, we developed a murine model of passive anaphylaxis with demonstrated MC involvement and an in vitro assay to evaluate the effect of DS on MCs. In contrast with previous reports, we determined that functional IgE remains on the cell surface of desensitized MCs following DS. Despite notable reductions in MC degranulation following DS, the high-affinity IgE receptor FcεRI was still capable of transducing signals in desensitized MCs. Additionally, we found that displacement of the actin cytoskeleton and its continued association with FcεRI impede the capacity of desensitized MCs to evoke the calcium response that is essential for MC degranulation. Together, these findings suggest that reduced degranulation responses in desensitized MCs arise from aberrant actin remodeling, providing insights that may lead to improvement of DS treatments for anaphylactic responses. PMID:27669462

  18. PICKLE is a CHD subfamily II ATP-dependent chromatin remodeling factor.

    PubMed

    Ho, Kwok Ki; Zhang, Heng; Golden, Barbara L; Ogas, Joe

    2013-02-01

    PICKLE plays a critical role in repression of genes that regulate development identity in Arabidopsis thaliana. PICKLE codes for a putative ATP-dependent chromatin remodeler that exhibits sequence similarity to members of subfamily II of animal CHD remodelers, which includes remodelers such as CHD3/Mi-2 that also restrict expression of developmental regulators. Whereas animal CHD3 remodelers are a component of the Mi-2/NuRD complex that promotes histone deacetylation, PICKLE promotes trimethylation of histone H3 lysine 27 suggesting that it acts via a distinct epigenetic pathway. Here, we examine whether PICKLE is also a member of a multisubunit complex and characterize the biochemical properties of recombinant PICKLE protein. Phylogenetic analysis indicates that PICKLE-related proteins in plants share a common ancestor with members of subfamily II of animal CHD remodelers. Biochemical characterization of PICKLE in planta, however, reveals that PICKLE primarily exists as a monomer. Recombinant PICKLE protein is an ATPase that is stimulated by ssDNA and mononucleosomes and binds to both naked DNA and mononucleosomes. Furthermore, recombinant PICKLE exhibits ATP-dependent chromatin remodeling activity. These studies demonstrate that subfamily II CHD proteins in plants, such as PICKLE, retain ATP-dependent chromatin remodeling activity but act through a mechanism that does not involve the ubiquitous Mi-2/NuRD complex.

  19. Nucleosome recognition and spacing by chromatin remodelling factor ISW1a.

    PubMed

    Richmond, Timothy J

    2012-04-01

    Nucleosomes are actively positioned along DNA by ATP-dependent, chromatin remodelling factors. A structural model for the ISW1a chromatin remodelling factor from Saccharomyces cerevisiae in complex with a dinucleosome substrate was constructed from the X-ray structures of ISW1a (ΔATPase) with and without DNA bound, two different cryo-EM (cryo-electron microscopy) structures of ISW1a (ΔATPase) bound to a nucleosome, and site-directed photo-cross-linking analyses in solution. The X-ray structure of ISW1a (ΔATPase) with DNA bound suggests that DNA sequence may be involved in nucleosome recognition and thereby specificity of promoter interaction. The model suggests how the highly ordered nucleosome arrays observed by mapping nucleosomes in genes and their promoter regions could be generated by a chromatin remodelling factor.

  20. Chromatin structure and ionizing-radiation-induced chromosome aberrations

    SciTech Connect

    Muehlmann-Diaz, M.C.

    1993-01-01

    The possible influence of chromatic structure or activity on chromosomal radiosensitivity was studied. A cell line was isolated which contained some 10[sup 5] copies of an amplified plasmid in a single large mosquito artificial chromosome (MAC). This chromosome was hypersensitive to DNase I. Its radiosensitivity was some three fold greater than normal mosquito chromosomes in the same cell. In cultured human cells irradiated during G[sub 0], the initial breakage frequency in chromosome 4, 19 and the euchromatic and heterochromatic portions of the Y chromosome were measured over a wide range of doses by inducing Premature Chromosome Condensation (PCC) immediately after irradiation with Cs-137 gamma rays. No evidence was seen that Y heterochromatin or large fragments of it remained unbroken. The only significant deviation from the expected initial breakage frequency per Gy per unit length of chromosome was that observed for the euchromatic portion of the Y chromosome, with breakage nearly twice that expected. The development of aberrations involving X and Y chromosomes at the first mitosis after irradation was also studied. Normal female cells sustained about twice the frequency of aberrations involving X chromosomes for a dose of 7.3 Gy than the corresponding male cells. Fibroblasts from individuals with supernumerary X chromosomes did not show any further increase in X aberrations for this dos. The frequency of aberrations involving the heterochromatic portion of the long arm of the Y chromosome was about what would be expected for a similar length of autosome, but the euchromatic portion of the Y was about 3 times more radiosensitive per unit length. 5-Azacytidine treatment of cultured human female fibroblasts or fibroblasts from a 49,XXXXY individual, reduced the methylation of cytosine residues in DNA, and resulted in an increased chromosomal radiosensitivity in general, but it did not increase the frequency of aberrations involving the X chromosomes.

  1. In vitro chromatin remodelling by chromatin accessibility complex (CHRAC) at the SV40 origin of DNA replication.

    PubMed Central

    Alexiadis, V; Varga-Weisz, P D; Bonte, E; Becker, P B; Gruss, C

    1998-01-01

    DNA replication is initiated by binding of initiation factors to the origin of replication. Nucleosomes are known to inhibit the access of the replication machinery to origin sequences. Recently, nucleosome remodelling factors have been identified that increase the accessibility of nucleosomal DNA to transcription regulators. To test whether the initiation of DNA replication from an origin covered by nucleosomes would also benefit from the action of nucleosome remodelling factors, we reconstituted SV40 DNA into chromatin in Drosophila embryo extracts. In the presence of T-antigen and ATP, a chromatin-associated cofactor allowed efficient replication from a nucleosomal origin in vitro. In search of the energy-dependent cofactor responsible we found that purified 'chromatin accessibility complex' (CHRAC) was able to alter the nucleosomal structure at the origin allowing the binding of T-antigen and efficient initiation of replication. These experiments provide evidence for the involvement of a nucleosome remodelling machine in structural changes at the SV40 origin of DNA replication in vitro. PMID:9628878

  2. Regulatory motifs on ISWI chromatin remodelers: molecular mechanisms and kinetic proofreading

    NASA Astrophysics Data System (ADS)

    Brysbaert, Guillaume; Lensink, Marc F.; Blossey, Ralf

    2015-02-01

    Recently, kinetic proofreading scenarios have been proposed for the regulation of chromatin remodeling, first on purely theoretical grounds (Blossey and Schiessel 2008 HFSP J. 2 167-70) and deduced from experiments on the ISWI/ACF system (Narlikar 2010 Curr. Opin. Chem. Biol. 14 660). In the kinetic proofreading scenario of chromatin remodeling, the combination of the recognition of a histone tail state and ATP-hydrolysis in the remodeler motor act together to select (i.e. proofread) a nucleosomal substrate. ISWI remodelers have recently been shown to have an additional level of regulation as they contain auto-inhibitory motifs which need to be inactivated through an interaction with the nucleosome. In this paper we show that the auto-regulatory effect enhances substrate recognition in kinetic proofreading. We further report some suggestive additional insights into the molecular mechanism underlying ISWI-autoregulation.

  3. Conformational change in the chromatin remodelling protein MENT.

    PubMed

    Ong, Poh Chee; Golding, Sarah J; Pearce, Mary C; Irving, James A; Grigoryev, Sergei A; Pike, Debbie; Langendorf, Christopher G; Bashtannyk-Puhalovich, Tanya A; Bottomley, Stephen P; Whisstock, James C; Pike, Robert N; McGowan, Sheena

    2009-01-01

    Chromatin condensation to heterochromatin is a mechanism essential for widespread suppression of gene transcription, and the means by which a chromatin-associated protein, MENT, induces a terminally differentiated state in cells. MENT, a protease inhibitor of the serpin superfamily, is able to undergo conformational change in order to effect enzyme inhibition. Here, we sought to investigate whether conformational change in MENT is 'fine-tuned' in the presence of a bound ligand in an analogous manner to other serpins, such as antithrombin where such movements are reflected by a change in intrinsic tryptophan fluorescence. Using this technique, MENT was found to undergo structural shifts in the presence of DNA packaged into nucleosomes, but not naked DNA. The contribution of the four Trp residues of MENT to the fluorescence change was mapped using deconvolution analysis of variants containing single Trp to Phe mutations. The analysis indicated that the overall emission spectra is dominated by a helix-H tryptophan, but this residue did not dominate the conformational change in the presence of chromatin, suggesting that other Trp residues contained in the A-sheet and RCL regions contribute to the conformational change. Mutagenesis revealed that the conformational change requires the presence of the DNA-binding 'M-loop' and D-helix of MENT, but is independent of the protease specificity determining 'reactive centre loop'. The D-helix mutant of MENT, which is unable to condense chromatin, does not undergo a conformational change, despite being able to bind chromatin, indicating that the conformational change may contribute to chromatin condensation by the serpin.

  4. Chromatin Remodeling Mediated by Drosophila GAGA Factor and ISWI Activates fushi tarazu Gene Transcription In Vitro

    PubMed Central

    Okada, Masahiro; Hirose, Susumu

    1998-01-01

    GAGA factor is known to remodel the chromatin structure in concert with nucleosome-remodeling factor NURF in a Drosophila embryonic S150 extract. The promoter region of the Drosophila fushi tarazu (ftz) gene carries several binding sites for GAGA factor. Both the GAGA factor-binding sites and GAGA factor per se are necessary for the proper expression of ftz in vivo. We observed transcriptional activation of the ftz gene when a preassembled chromatin template was incubated with GAGA factor and the S150 extract. The chromatin structure within the ftz promoter was specifically disrupted by incubation of the preassembled chromatin with GAGA factor and the S150 extract. Both transcriptional activation and chromatin disruption were blocked by an antiserum raised against ISWI or by base substitutions in the GAGA factor-binding sites in the ftz promoter region. These results demonstrate that GAGA factor- and ISWI-mediated disruption of the chromatin structure within the promoter region of ftz activates transcription on the chromatin template. PMID:9566866

  5. Genome-wide chromatin remodeling modulates the Alu heat shock response.

    PubMed

    Kim, C; Rubin, C M; Schmid, C W

    2001-10-03

    During heat shock recovery in Hela cells, the level of Alu RNA transiently increases with kinetics that approximately parallel the transient expression of heat shock protein mRNAs. Coincidentally, there is a transient increase in the accessibility of Alu chromatin to restriction enzyme cleavage suggesting that an opening and re-closing of chromatin regulates the Alu stress response. Similar changes occur in alpha satellite and LINE1 chromatin showing that heat shock induces a genome-wide remodeling of chromatin structure which is independent of transcription. The increased accessibility of restriction sites within these repetitive sequences is inconsistent with a simple lengthening of the nucleosome linker region but instead suggests a scrambling of nucleosome positions. Chromatin structure and its dynamics account for many of the principal features of SINE transcriptional regulation potentially providing a functional rationale for the dispersion and high copy number of SINEs.

  6. Genetic variants in chromatin-remodeling pathway associated with lung cancer risk in a Chinese population.

    PubMed

    Geng, Liguo; Zhu, Meng; Wang, Yuzhuo; Cheng, Yang; Liu, Jia; Shen, Wei; Li, Zhihua; Zhang, Jiahui; Wang, Cheng; Jin, Guangfu; Ma, Hongxia; Shen, Hongbing; Hu, Zhibin; Dai, Juncheng

    2016-08-10

    Chromatin remodeling complexes utilize the energy of ATP hydrolysis to remodel nucleosomes and have essential roles in transcriptional modulation. Increasing evidences indicate that these complexes directly interact with numerous proteins and regulate the formation of cancer. However, few studies reported the association of polymorphisms in chromatin remodeling genes and lung cancer. We hypothesized that variants in critical genes of chromatin remodeling pathway might contribute to the susceptibility of lung cancer. To validate this hypothesis, we systematically screened 40 polymorphisms in six key chromatin remodeling genes (SMARCA5, SMARCC2, SMARCD2, ARID1A, NR3C1 and SATB1) and evaluated them with a case-control study including 1341 cases and 1982 controls. Logistic regression revealed that four variants in NR3C1 and SATB1 were significantly associated with lung cancer risk after false discovery rate (FDR) correction [For NR3C1, rs9324921: odds ratio (OR)=1.23, P for FDR=0.029; rs12521436: OR=0.85, P for FDR=0.040; rs4912913: OR=1.17, P for FDR=0.040; For SATB1, rs6808523: OR=1.33, P for FDR=0.040]. Combing analysis presented a significant allele-dosage tendency for the number of risk alleles and lung cancer risk (Ptrend<0.001). Moreover, expression quantitative trait loci (eQTL) analysis revealed that these two genes were differently expressed between lung tumor and adjacent normal tissues in the database of The Cancer Genome Atlas (TCGA) (P=0.009 for rs6808523). These findings suggested that genetic variants in key chromatin remodeling genes may contribute to lung cancer risk in Chinese population. Further large and well-designed studies are warranted to validate our results.

  7. Chromatin remodeling of human subtelomeres and TERRA promoters upon cellular senescence

    PubMed Central

    Thijssen, Peter E.; Tobi, Elmar W.; Balog, Judit; Schouten, Suzanne G.; Kremer, Dennis; El Bouazzaoui, Fatiha; Henneman, Peter; Putter, Hein; Eline Slagboom, P.; Heijmans, Bastiaan T.; Van der Maarel, Silvère M.

    2013-01-01

    Subtelomeres are patchworks of evolutionary conserved sequence blocks and harbor the transcriptional start sites for telomere repeat containing RNAs (TERRA). Recent studies suggest that the interplay between telomeres and subtelomeric chromatin is required for maintaining telomere function. To further characterize chromatin remodeling of subtelomeres in relation to telomere shortening and cellular senescence, we systematically quantified histone modifications and DNA methylation at the subtelomeres of chromosomes 7q and 11q in primary human WI-38 fibroblasts. Upon senescence, both subtelomeres were characterized by a decrease in markers of constitutive heterochromatin, suggesting relative chromatin relaxation. However, we did not find increased levels of markers of euchromatin or derepression of the 7q VIPR2 gene. The repressed state of the subtelomeres was maintained upon senescence, which could be attributed to a rise in levels of facultative heterochromatin markers at both subtelomeres. While senescence-induced subtelomeric chromatin remodeling was similar for both chromosomes, chromatin remodeling at TERRA promoters displayed chromosome-specific patterns. At the 7q TERRA promoter, chromatin structure was co-regulated with the more proximal subtelomere. In contrast, the 11q TERRA promoter, which was previously shown to be bound by CCCTC-binding factor CTCF, displayed lower levels of markers of constitutive heterochromatin that did not change upon senescence, whereas levels of markers of facultative heterochromatin decreased upon senescence. In line with the chromatin state data, transcription of 11q TERRA but not 7q TERRA was detected. Our study provides a detailed description of human subtelomeric chromatin dynamics and shows distinct regulation of the TERRA promoters of 7q and 11q upon cellular senescence. PMID:23644601

  8. The Mouse INO80 Chromatin-Remodeling Complex Is an Essential Meiotic Factor for Spermatogenesis1

    PubMed Central

    Serber, Daniel W.; Runge, John S.; Menon, Debashish U.; Magnuson, Terry

    2015-01-01

    The ability to faithfully transmit genetic information across generations via the germ cells is a critical aspect of mammalian reproduction. The process of germ cell development requires a number of large-scale modulations of chromatin within the nucleus. One such occasion arises during meiotic recombination, when hundreds of DNA double-strand breaks are induced and subsequently repaired, enabling the transfer of genetic information between homologous chromosomes. The inability to properly repair DNA damage is known to lead to an arrest in the developing germ cells and sterility within the animal. Chromatin-remodeling activity, and in particular the BRG1 subunit of the SWI/SNF complex, has been shown to be required for successful completion of meiosis. In contrast, remodeling complexes of the ISWI and CHD families are required for postmeiotic processes. Little is known regarding the contribution of the INO80 family of chromatin-remodeling complexes, which is a particularly interesting candidate due to its well described functions during DNA double-strand break repair. Here we show that INO80 is expressed in developing spermatocytes during the early stages of meiotic prophase I. Based on this information, we used a conditional allele to delete the INO80 core ATPase subunit, thereby eliminating INO80 chromatin-remodeling activity in this lineage. The loss of INO80 resulted in an arrest during meiosis associated with a failure to repair DNA damage during meiotic recombination. PMID:26607718

  9. Genome-Wide Chromatin Remodeling Identified at GC-Rich Long Nucleosome-Free Regions

    PubMed Central

    Hochreiter, Sepp

    2012-01-01

    To gain deeper insights into principles of cell biology, it is essential to understand how cells reorganize their genomes by chromatin remodeling. We analyzed chromatin remodeling on next generation sequencing data from resting and activated T cells to determine a whole-genome chromatin remodeling landscape. We consider chromatin remodeling in terms of nucleosome repositioning which can be observed most robustly in long nucleosome-free regions (LNFRs) that are occupied by nucleosomes in another cell state. We found that LNFR sequences are either AT-rich or GC-rich, where nucleosome repositioning was observed much more prominently in GC-rich LNFRs — a considerable proportion of them outside promoter regions. Using support vector machines with string kernels, we identified a GC-rich DNA sequence pattern indicating loci of nucleosome repositioning in resting T cells. This pattern appears to be also typical for CpG islands. We found out that nucleosome repositioning in GC-rich LNFRs is indeed associated with CpG islands and with binding sites of the CpG-island-binding ZF-CXXC proteins KDM2A and CFP1. That this association occurs prominently inside and also prominently outside of promoter regions hints at a mechanism governing nucleosome repositioning that acts on a whole-genome scale. PMID:23144837

  10. Akirin Links Twist-Regulated Transcription with the Brahma Chromatin Remodeling Complex during Embryogenesis

    PubMed Central

    Nowak, Scott J.; Aihara, Hitoshi; Gonzalez, Katie; Nibu, Yutaka; Baylies, Mary K.

    2012-01-01

    The activities of developmentally critical transcription factors are regulated via interactions with cofactors. Such interactions influence transcription factor activity either directly through protein–protein interactions or indirectly by altering the local chromatin environment. Using a yeast double-interaction screen, we identified a highly conserved nuclear protein, Akirin, as a novel cofactor of the key Drosophila melanogaster mesoderm and muscle transcription factor Twist. We find that Akirin interacts genetically and physically with Twist to facilitate expression of some, but not all, Twist-regulated genes during embryonic myogenesis. akirin mutant embryos have muscle defects consistent with altered regulation of a subset of Twist-regulated genes. To regulate transcription, Akirin colocalizes and genetically interacts with subunits of the Brahma SWI/SNF-class chromatin remodeling complex. Our results suggest that, mechanistically, Akirin mediates a novel connection between Twist and a chromatin remodeling complex to facilitate changes in the chromatin environment, leading to the optimal expression of some Twist-regulated genes during Drosophila myogenesis. We propose that this Akirin-mediated link between transcription factors and the Brahma complex represents a novel paradigm for providing tissue and target specificity for transcription factor interactions with the chromatin remodeling machinery. PMID:22396663

  11. Chromatin Folding, Fragile Sites, and Chromosome Aberrations Induced by Low- and High- LET Radiation

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Cox, Bradley; Asaithamby, Aroumougame; Chen, David J.; Wu, Honglu

    2013-01-01

    We previously demonstrated non-random distributions of breaks involved in chromosome aberrations induced by low- and high-LET radiation. To investigate the factors contributing to the break point distribution in radiation-induced chromosome aberrations, human epithelial cells were fixed in G1 phase. Interphase chromosomes were hybridized with a multicolor banding in situ hybridization (mBAND) probe for chromosome 3 which distinguishes six regions of the chromosome in separate colors. After the images were captured with a laser scanning confocal microscope, the 3-dimensional structure of interphase chromosome 3 was reconstructed at multimega base pair scale. Specific locations of the chromosome, in interphase, were also analyzed with bacterial artificial chromosome (BAC) probes. Both mBAND and BAC studies revealed non-random folding of chromatin in interphase, and suggested association of interphase chromatin folding to the radiation-induced chromosome aberration hotspots. We further investigated the distribution of genes, as well as the distribution of breaks found in tumor cells. Comparisons of these distributions to the radiation hotspots showed that some of the radiation hotspots coincide with the frequent breaks found in solid tumors and with the fragile sites for other environmental toxins. Our results suggest that multiple factors, including the chromatin structure and the gene distribution, can contribute to radiation-induced chromosome aberrations.

  12. Prothymosin alpha is a chromatin-remodelling protein in mammalian cells.

    PubMed Central

    Gomez-Marquez, J; Rodríguez, P

    1998-01-01

    Prothymosin alpha (ProTalpha) is an abundant mammalian acidic nuclear protein whose expression is related to cell proliferation. Here we report that in HL-60 cells overexpressing ProTalpha, the accessibility of micrococcal nuclease to chromatin is strongly increased. In the DNA ladder generated by the nuclease activity, the sizes of the mononucleosome (146 bp, the DNA fragment that is bound to the histone octamer) and its multimers correspond to nucleosomes lacking histone H1. The percentage of histone-H1-depleted chromatin (active chromatin) is also higher in the cells overexpressing ProTalpha. On the basis of these and previous findings, we propose a biological role for ProTalpha in the remodelling of chromatin fibres through its interaction with histone H1. PMID:9639554

  13. Arabidopsis chromatin remodeling factor PICKLE interacts with transcription factor HY5 to regulate hypocotyl cell elongation.

    PubMed

    Jing, Yanjun; Zhang, Dong; Wang, Xin; Tang, Weijiang; Wang, Wanqing; Huai, Junling; Xu, Gang; Chen, Dongqin; Li, Yunliang; Lin, Rongcheng

    2013-01-01

    Photomorphogenesis is a critical plant developmental process that involves light-mediated transcriptome changes, histone modifications, and inhibition of hypocotyl growth. However, the chromatin-based regulatory mechanism underlying this process remains largely unknown. Here, we identify ENHANCED PHOTOMORPHOGENIC1 (EPP1), previously known as PICKLE (PKL), an ATP-dependent chromatin remodeling factor of the chromodomain/helicase/DNA binding family, as a repressor of photomorphogenesis in Arabidopsis thaliana. We show that PKL/EPP1 expression is repressed by light in the hypocotyls in a photoreceptor-dependent manner. Furthermore, we reveal that the transcription factor ELONGATED HYPOCOTYL5 (HY5) binds to the promoters of cell elongation-related genes and recruits PKL/EPP1 through their physical interaction. PKL/EPP1 in turn negatively regulates HY5 by repressing trimethylation of histone H3 Lys 27 at the target loci, thereby regulating the expression of these genes and, thus, hypocotyl elongation. We also show that HY5 possesses transcriptional repression activity. Our study reveals a crucial role for a chromatin remodeling factor in repressing photomorphogenesis and demonstrates that transcription factor-mediated recruitment of chromatin-remodeling machinery is important for plant development in response to changing light environments.

  14. Mammalian SWI/SNF chromatin remodeling complexes and cancer: Mechanistic insights gained from human genomics

    PubMed Central

    Kadoch, Cigall; Crabtree, Gerald R.

    2015-01-01

    Over the past 4 years, nearly 100 exome sequencing studies have revealed the high frequency of mutations in the genes encoding the subunits of ATP-dependent chromatin remodelers in human cancer. Most of these mutations are within the genes encoding subunits of the BAF (Brg/Brahma-associated factors) or mSWI/SNF complex, which is one of two dozen predicted ATP-dependent chromatin remodeling complexes in mammals. Considering BAF complexes as a single entity, the 15 subunits encoded by 29 genes are mutated in >20% of human cancer, across a broad range of tumor types. These observations demonstrate that there is little redundancy in the oncogenic function of BAF complexes with the other remodeling complexes, underscoring their unique roles. Several important conclusions emerge from these genomic data: specific subunits appear to be mutated in specific cancers, highlighting tissue-specific protective roles; mutations can function as tumor suppressors or oncogenes; mutations can be homozygous or, more commonly, heterozygous, implying their dosage-sensitive roles in an unknown yet fundamental process used to suppress the genesis of cancer. These new human genetic findings paired with biochemical studies are challenging old ideas on how chromatin remodeling complexes function, generating new hypotheses with respect to their normal and oncogenic mechanisms and highlighting potential avenues for therapeutic intervention in human cancer. PMID:26601204

  15. Adr1 and Cat8 Mediate Coactivator Recruitment and Chromatin Remodeling at Glucose-Regulated Genes

    PubMed Central

    Biddick, Rhiannon K.; Law, G. Lynn; Young, Elton T.

    2008-01-01

    Background Adr1 and Cat8 co-regulate numerous glucose-repressed genes in S. cerevisiae, presenting a unique opportunity to explore their individual roles in coactivator recruitment, chromatin remodeling, and transcription. Methodology/Principal Findings We determined the individual contributions of Cat8 and Adr1 on the expression of a cohort of glucose-repressed genes and found three broad categories: genes that need both activators for full derepression, genes that rely mostly on Cat8 and genes that require only Adr1. Through combined expression and recruitment data, along with analysis of chromatin remodeling at two of these genes, ADH2 and FBP1, we clarified how these activators achieve this wide range of co-regulation. We find that Adr1 and Cat8 are not intrinsically different in their abilities to recruit coactivators but rather, promoter context appears to dictate which activator is responsible for recruitment to specific genes. These promoter-specific contributions are also apparent in the chromatin remodeling that accompanies derepression: ADH2 requires both Adr1 and Cat8, whereas, at FBP1, significant remodeling occurs with Cat8 alone. Although over-expression of Adr1 can compensate for loss of Cat8 at many genes in terms of both activation and chromatin remodeling, this over-expression cannot complement all of the cat8Δ phenotypes. Conclusions/Significance Thus, at many of the glucose-repressed genes, Cat8 and Adr1 appear to have interchangeable roles and promoter architecture may dictate the roles of these activators. PMID:18197247

  16. Localized recruitment of a chromatin-remodeling activity by an activator in vivo drives transcriptional elongation

    PubMed Central

    Corey, Laura L.; Weirich, Christine S.; Benjamin, Ivor J.; Kingston, Robert E.

    2003-01-01

    To understand the role of chromatin-remodeling activities in transcription, it is necessary to understand how they interact with transcriptional activators in vivo to regulate the different steps of transcription. Human heat shock factor 1 (HSF1) stimulates both transcriptional initiation and elongation. We replaced mouse HSF1 in fibroblasts with wild-type and mutant human HSF1 constructs and characterized regulation of an endogenous mouse hsp70 gene. A mutation that diminished transcriptional initiation led to twofold reductions in hsp70 mRNA induction and recruitment of a SWI/SNF remodeling complex. In contrast, a mutation that diminished transcriptional elongation abolished induction of full-length mRNA, SWI/SNF recruitment, and chromatin remodeling, but minimally impaired initiation from the hsp70 promoter. Another remodeling factor, SNF2h, is constitutively present at the promoter irrespective of the genotype of HSF1. These data suggest that localized recruitment of SWI/SNF drives a specialized remodeling reaction necessary for the production of full-length hsp70 mRNA. PMID:12782657

  17. Low 17beta-estradiol levels in CNR1 knock-out mice affect spermatid chromatin remodeling by interfering with chromatin reorganization.

    PubMed

    Cacciola, Giovanna; Chioccarelli, Teresa; Altucci, Lucia; Ledent, Catherine; Mason, J Ian; Fasano, Silvia; Pierantoni, Riccardo; Cobellis, Gilda

    2013-06-01

    The type 1-cannabinoid receptor, CNR1, regulates differentiation of spermatids. Indeed, we have recently reported that the genetic inactivation of Cnr1 in mice influenced chromatin remodeling of spermatids, by reducing histone displacement and then sperm chromatin quality indices (chromatin condensation and DNA integrity). Herein, we have studied, at both central and testicular levels, the molecular signals potentially involved in histone displacement. In particular, investigation of the neuroendocrine axis involved in estrogen production demonstrated down-regulation of the axis supporting FSH/estrogen secretion in Cnr1-knockout male mice. Conversely, Cnr1-knockout male mice treated with 17beta-estradiol showed a weak increase of pituitary Fsh-beta subunit mRNA levels and a rescue of sperm chromatin quality indices demonstrating that estrogens, possibly in combination with FSH secretion, play an important role in regulating chromatin remodeling of spermatids.

  18. ATP-dependent chromatin remodeling and DNA double-strand break repair.

    PubMed

    van Attikum, Haico; Gasser, Susan M

    2005-08-01

    The repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic stability. Two pathways for the repair of DBSs, nonhomologous end-joining (NHEJ) and homologous recombination (HR), have evolved in eukaryotes. These pathways, like processes such as transcription and replication, act on DNA that is embedded in nucleosomes. Recent studies have shown that DNA repair, like transcription, is facilitated both by histone tail modification and by ATP-dependent chromatin remodeling. This review emphasizes recent reports that demonstrate a function for the ATP-dependent chromatin remodeling complexes INO80 and RSC in NHEJ and HR. We also discuss the possible role of SWR1- and TIP60-mediated nucleosomal histone exchange in DNA repair.

  19. Chromatin remodeling and stem cell theory of relativity.

    PubMed

    Cerny, Jan; Quesenberry, Peter J

    2004-10-01

    The field of stem cell biology is currently being redefined. Stem cell (hematopoietic and non-hematopoietic) differentiation has been considered hierarchical in nature, but recent data suggest that there is no progenitor/stem cell hierarchy, but rather a reversible continuum. The stem cell (hematopoietic and non-hematopoietic) phenotype, the total differentiation capacity (hematopoietic and non-hematopoietic), gene expression as well as other stem cell functional characteristics (homing, receptor and adhesion molecule expression) vary throughout a cell-cycle transit widely. This seems to be dependent on shifting chromatin and gene expression with cell-cycle transit. The published data on DNA methylation, histone acetylation, and also RNAi, the major regulators of gene expression, conjoins very well and provides an explanation for the major issues of stem cell biology. Those features of stem cells mentioned above can be rather difficult to apprehend when a classical hierarchy biology view is applied, but they become clear and easier to understand once they are correlated with the underlining epigenetic changes. We are entering a new era of stem cell biology the era of "chromatinomics." We are one step closer to the practical use of cellular therapy for degenerative diseases.

  20. Comparative analysis of ATRX, a chromatin remodeling protein.

    PubMed

    Park, Daniel J; Pask, Andrew J; Huynh, Kim; Renfree, Marilyn B; Harley, Vincent R; Graves, Jennifer A Marshall

    2004-09-15

    The ATRX protein, associated with X-linked alpha-thalassaemia, mental retardation and developmental abnormalities including genital dysgenesis, has been proposed to function as a global transcriptional regulator within a multi-protein complex. However, an understanding of the composition and mechanics of this machinery has remained elusive. We applied inter-specific comparative analysis to identify conserved elements which may be involved in regulating the conformation of chromatin. As part of this study, we cloned and sequenced the entire translatable coding region (7.4 kb) of the ATRX gene from a model marsupial (tammar wallaby, Macropus eugenii). We identify an ATRX ancestral core, conserved between plants, fish and mammals, comprising the cysteine-rich and SWI2/SNF2 helicase-like regions and protein interaction domains. Our data are consistent with the model of the cysteine-rich region as a DNA-binding zinc finger adjacent to a protein-binding (plant homeodomain-like) domain. Alignment of vertebrate ATRX sequences highlights other conserved elements, including a negatively charged mammalian sequence which we propose to be involved in binding of positively charged histone tails.

  1. High-throughput small molecule screen identifies inhibitors of aberrant chromatin accessibility

    PubMed Central

    Pattenden, Samantha G.; Simon, Jeremy M.; Wali, Aminah; Jayakody, Chatura N.; Troutman, Jacob; McFadden, Andrew W.; Wooten, Joshua; Wood, Cameron C.; Frye, Stephen V.; Janzen, William P.; Davis, Ian J.

    2016-01-01

    Mutations in chromatin-modifying proteins and transcription factors are commonly associated with a wide variety of cancers. Through gain- or loss-of-function, these mutations may result in characteristic alterations of accessible chromatin, indicative of shifts in the landscape of regulatory elements genome-wide. The identification of compounds that reverse a specific chromatin signature could lead to chemical probes or potential therapies. To explore whether chromatin accessibility could serve as a platform for small molecule screening, we adapted formaldehyde-assisted isolation of regulatory elements (FAIRE), a chemical method to enrich for nucleosome-depleted genomic regions, as a high-throughput, automated assay. After demonstrating the validity and robustness of this approach, we applied this method to screen an epigenetically targeted small molecule library by evaluating regions of aberrant nucleosome depletion mediated by EWSR1-FLI1, the chimeric transcription factor critical for the bone and soft tissue tumor Ewing sarcoma. As a class, histone deacetylase inhibitors were greatly overrepresented among active compounds. These compounds resulted in diminished accessibility at targeted sites by disrupting transcription of EWSR1-FLI1. Capitalizing on precise differences in chromatin accessibility for drug discovery efforts offers significant advantages because it does not depend on the a priori selection of a single molecular target and may detect novel biologically relevant pathways. PMID:26929321

  2. Mutations in two non-canonical Arabidopsis SWI2/SNF2 chromatin remodeling ATPases cause embryogenesis and stem cell maintenance defects

    PubMed Central

    Sang, Yi; Silva-Ortega, Claudia O.; Wu, Shuang; Yamaguchi, Nobutoshi; Wu, Miin-Feng; Pfluger, Jennifer; Gillmor, C. Stewart; Gallagher, Kimberly L.; Wagner, Doris

    2012-01-01

    Summary SWI2/SNF2 chromatin remodeling ATPases play important roles in plant and metazoan development. While metazoans generally encode one or two SWI2/SNF2 ATPase genes, Arabidopsis encodes four such chromatin regulators: the well-studied BRAHMA and SPLAYED ATPases as well as two closely related non-canonical SWI2/SNF2 ATPases, CHR12 and CHR23. No developmental role has as yet been described for CHR12 and CHR23. Here we show that while strong single chr12 or chr23 mutants are morphologically indistinguishable from the wild type, chr12 chr23 double mutants cause embryonic lethality. The double mutant embryos fail to initiate root and shoot meristems and display few and aberrant cell division. Weak double mutant embryos give rise to viable seedlings with dramatic defects in the maintenance of both the shoot and the root stem cell populations. Paradoxically, the stem cell defects are correlated with increased expression of the stem cell markers WUSCHEL and WOX5. During subsequent development, the meristem defects are partially overcome to allow for the formation of very small, bushy adult plants. Based on the observed morphological defects we named the two chromatin remodelers MINUSCULE 1 and 2. Possible links between minu1 minu2 defects and defects in hormone signaling and replication-coupled chromatin assembly are discussed. PMID:23062007

  3. Chromatin remodeling complexes in the assembly of long noncoding RNA-dependent nuclear bodies.

    PubMed

    Kawaguchi, Tetsuya; Hirose, Tetsuro

    2015-01-01

    Paraspeckles are subnuclear structures that assemble on nuclear paraspeckle assembly transcript 1 (NEAT1) long noncoding (lnc)RNA. Paraspeckle formation requires appropriate NEAT1 biogenesis and subsequent assembly with multiple prion-like domain (PLD) containing RNA-binding proteins. We found that SWI/SNF chromatin remodeling complexes function as paraspeckle components that interact with paraspeckle proteins (PSPs) and NEAT1. SWI/SNF complexes play an essential role in paraspeckle formation that does not require their ATP-dependent chromatin remodeling activity. Instead, SWI/SNF complexes facilitate organization of the PSP interaction network required for intact paraspeckle assembly. SWI/SNF complexes may collectively bind multiple PSPs to recruit them onto NEAT1. SWI/SNF complexes are also required for Sat III (Satellite III) lncRNA-dependent formation of nuclear stress bodies under heat shock conditions. Organization of the lncRNA-dependent omega speckle in Drosophila also depends on the chromatin remodeling complex. These findings raise the possibility that a common mechanism controls the formation of lncRNA-dependent nuclear body architecture.

  4. Chromatin remodeling during the in vivo glial differentiation in early Drosophila embryos

    PubMed Central

    Ye, Youqiong; Gu, Liang; Chen, Xiaolong; Shi, Jiejun; Zhang, Xiaobai; Jiang, Cizhong

    2016-01-01

    Chromatin remodeling plays a critical role in gene regulation and impacts many biological processes. However, little is known about the relationship between chromatin remodeling dynamics and in vivo cell lineage commitment. Here, we reveal the patterns of histone modification change and nucleosome positioning dynamics and their epigenetic regulatory roles during the in vivo glial differentiation in early Drosophila embryos. The genome-wide average H3K9ac signals in promoter regions are decreased in the glial cells compared to the neural progenitor cells. However, H3K9ac signals are increased in a group of genes that are up-regulated in glial cells and involved in gliogenesis. There occurs extensive nucleosome remodeling including shift, loss, and gain. Nucleosome depletion regions (NDRs) form in both promoters and enhancers. As a result, the associated genes are up-regulated. Intriguingly, NDRs form in two fashions: nucleosome shift and eviction. Moreover, the mode of NDR formation is independent of the original chromatin state of enhancers in the neural progenitor cells. PMID:27634414

  5. Remodelers Organize Cellular Chromatin by Counteracting Intrinsic Histone-DNA Sequence Preferences in a Class-Specific Manner

    PubMed Central

    Chalkley, Gillian E.; Kan, Tsung Wai; Reddy, B. Ashok; Ozgur, Zeliha; van Ijcken, Wilfred F. J.; Dekkers, Dick H. W.; Demmers, Jeroen A.; Travers, Andrew A.

    2012-01-01

    The nucleosome is the fundamental repeating unit of eukaryotic chromatin. Here, we assessed the interplay between DNA sequence and ATP-dependent chromatin-remodeling factors (remodelers) in the nucleosomal organization of a eukaryotic genome. We compared the genome-wide distribution of Drosophila NURD, (P)BAP, INO80, and ISWI, representing the four major remodeler families. Each remodeler has a unique set of genomic targets and generates distinct chromatin signatures. Remodeler loci have characteristic DNA sequence features, predicted to influence nucleosome formation. Strikingly, remodelers counteract DNA sequence-driven nucleosome distribution in two distinct ways. NURD, (P)BAP, and INO80 increase histone density at their target sequences, which intrinsically disfavor positioned nucleosome formation. In contrast, ISWI promotes open chromatin at sites that are propitious for precise nucleosome placement. Remodelers influence nucleosome organization genome-wide, reflecting their high genomic density and the propagation of nucleosome redistribution beyond remodeler binding sites. In transcriptionally silent early embryos, nucleosome organization correlates with intrinsic histone-DNA sequence preferences. Following differential expression of the genome, however, this relationship diminishes and eventually disappears. We conclude that the cellular nucleosome landscape is the result of the balance between DNA sequence-driven nucleosome placement and active nucleosome repositioning by remodelers and the transcription machinery. PMID:22124157

  6. The chromatin remodeling factor CHD7 controls cerebellar development by regulating reelin expression

    PubMed Central

    Whittaker, Danielle E.; Riegman, Kimberley L.H.; Kasah, Sahrunizam; Mohan, Conor; Yu, Tian; Sala, Blanca Pijuan; Hebaishi, Husam; Caruso, Angela; Marques, Ana Claudia; Michetti, Caterina; Smachetti, María Eugenia Sanz; Shah, Apar; Sabbioni, Mara; Kulhanci, Omer; Tee, Wee-Wei; Reinberg, Danny; Scattoni, Maria Luisa; McGonnell, Imelda; Wardle, Fiona C.; Fernandes, Cathy

    2017-01-01

    The mechanisms underlying the neurodevelopmental deficits associated with CHARGE syndrome, which include cerebellar hypoplasia, developmental delay, coordination problems, and autistic features, have not been identified. CHARGE syndrome has been associated with mutations in the gene encoding the ATP-dependent chromatin remodeler CHD7. CHD7 is expressed in neural stem and progenitor cells, but its role in neurogenesis during brain development remains unknown. Here we have shown that deletion of Chd7 from cerebellar granule cell progenitors (GCps) results in reduced GCp proliferation, cerebellar hypoplasia, developmental delay, and motor deficits in mice. Genome-wide expression profiling revealed downregulated expression of the gene encoding the glycoprotein reelin (Reln) in Chd7-deficient GCps. Recessive RELN mutations have been associated with severe cerebellar hypoplasia in humans. We found molecular and genetic evidence that reductions in Reln expression contribute to GCp proliferative defects and cerebellar hypoplasia in GCp-specific Chd7 mouse mutants. Finally, we showed that CHD7 is necessary for maintaining an open, accessible chromatin state at the Reln locus. Taken together, this study shows that Reln gene expression is regulated by chromatin remodeling, identifies CHD7 as a previously unrecognized upstream regulator of Reln, and provides direct in vivo evidence that a mammalian CHD protein can control brain development by modulating chromatin accessibility in neuronal progenitors. PMID:28165338

  7. miR-93 regulates Msk2-mediated chromatin remodelling in diabetic nephropathy

    PubMed Central

    Badal, Shawn S.; Wang, Yin; Long, Jianyin; Corcoran, David L.; Chang, Benny H.; Truong, Luan D.; Kanwar, Yashpal S.; Overbeek, Paul A.; Danesh, Farhad R.

    2016-01-01

    How the kidney responds to the metabolic cues from the environment remains a central question in kidney research. This question is particularly relevant to the pathogenesis of diabetic nephropathy (DN) in which evidence suggests that metabolic events in podocytes regulate chromatin structure. Here, we show that miR-93 is a critical metabolic/epigenetic switch in the diabetic milieu linking the metabolic state to chromatin remodelling. Mice with inducible overexpression of a miR-93 transgene exclusively in podocytes exhibit significant improvements in key features of DN. We identify miR-93 as a regulator of nucleosomal dynamics in podocytes. miR-93 has a critical role in chromatin reorganization and progression of DN by modulating its target Msk2, a histone kinase, and its substrate H3S10. These findings implicate a central role for miR-93 in high glucose-induced chromatin remodelling in the kidney, and provide evidence for a previously unrecognized role for Msk2 as a target for DN therapy. PMID:27350436

  8. Chromatin associated mechanisms in base excision repair - nucleosome remodeling and DNA transcription, two key players.

    PubMed

    Menoni, Hervé; Di Mascio, Paolo; Cadet, Jean; Dimitrov, Stefan; Angelov, Dimitar

    2016-12-20

    Genomic DNA is prone to a large number of insults by a myriad of endogenous and exogenous agents. The base excision repair (BER) is the major mechanism used by cells for the removal of various DNA lesions spontaneously or environmentally induced and the maintenance of genome integrity. The presence of persistent DNA damage is not compatible with life, since abrogation of BER leads to early embryonic lethality in mice. There are several lines of evidences showing existence of a link between deficient BER, cancer proneness and ageing, thus illustrating the importance of this DNA repair pathway in human health. Although the enzymology of BER mechanisms has been largely elucidated using chemically defined DNA damage substrates and purified proteins, the complex interplay of BER with another vital process like transcription or when DNA is in its natural state (i.e. wrapped in nucleosome and assembled in chromatin fiber is largely unexplored. Cells use chromatin remodeling factors to overcome the general repression associated with the nucleosomal organization. It is broadly accepted that energy-dependent nucleosome remodeling factors disrupt histones-DNA interactions at the expense of ATP hydrolysis to favor transcription as well as DNA repair. Importantly, unlike transcription, BER is not part of a regulated developmental process but represents a maintenance system that should be efficient anytime and anywhere in the genome. In this review we will discuss how BER can deal with chromatin organization to maintain genetic information. Emphasis will be placed on the following challenging question: how BER is initiated within chromatin?

  9. The Chromodomains of the Chd1 Chromatin Remodeler Regulate DNA Access to the ATPase Motor

    SciTech Connect

    Hauk, G.; McKnight, J; Nodelman, I; Bowman, G

    2010-01-01

    Chromatin remodelers are ATP-driven machines that assemble, slide, and remove nucleosomes from DNA, but how the ATPase motors of remodelers are regulated is poorly understood. Here we show that the double chromodomain unit of the Chd1 remodeler blocks DNA binding and activation of the ATPase motor in the absence of nucleosome substrates. The Chd1 crystal structure reveals that an acidic helix joining the chromodomains can pack against a DNA-binding surface of the ATPase motor. Disruption of the chromodomain-ATPase interface prevents discrimination between nucleosomes and naked DNA and reduces the reliance on the histone H4 tail for nucleosome sliding. We propose that the chromodomains allow Chd1 to distinguish between nucleosomes and naked DNA by physically gating access to the ATPase motor, and we hypothesize that related ATPase motors may employ a similar strategy to discriminate among DNA-containing substrates.

  10. p38 pathway targets SWI-SNF chromatin-remodeling complex to muscle-specific loci.

    PubMed

    Simone, Cristiano; Forcales, Sonia Vanina; Hill, David A; Imbalzano, Anthony N; Latella, Lucia; Puri, Pier Lorenzo

    2004-07-01

    During skeletal myogenesis, genomic reprogramming toward terminal differentiation is achieved by recruiting chromatin-modifying enzymes to muscle-specific loci. The relative contribution of extracellular signaling cascades in targeting these enzymes to individual genes is unknown. Here we show that the differentiation-activated p38 pathway targets the SWI-SNF chromatin-remodeling complex to myogenic loci. Upon differentiation, p38 kinases were recruited to the chromatin of muscle-regulatory elements. Blockade of p38 alpha/beta repressed the transcription of muscle genes by preventing recruitment of the SWI-SNF complex at these elements without affecting chromatin binding of muscle-regulatory factors and acetyltransferases. The SWI-SNF subunit BAF60 could be phosphorylated by p38 alpha-beta in vitro, and forced activation of p38 alpha/beta in myoblasts by expression of a constitutively active MKK6 (refs. 5,6,7) promoted unscheduled SWI-SNF recruitment to the myogenin promoter. Conversely, inactivation of SWI-SNF enzymatic subunits abrogated MKK6-dependent induction of muscle gene expression. These results identify an unexpected function of differentiation-activated p38 in converting external cues into chromatin modifications at discrete loci, by selectively targeting SWI-SNF to muscle-regulatory elements.

  11. LXCXE-independent chromatin remodeling by Rb/E2f mediates neuronal quiescence.

    PubMed

    Andrusiak, Matthew G; Vandenbosch, Renaud; Dick, Fred A; Park, David S; Slack, Ruth S

    2013-05-01

    Neuronal survival is dependent upon the retinoblastoma family members, Rb1 (Rb) and Rb2 (p130). Rb is thought to regulate gene repression, in part, through direct recruitment of chromatin modifying enzymes to its conserved LXCXE binding domain. We sought to examine the mechanisms that Rb employs to mediate cell cycle gene repression in terminally differentiated cortical neurons. Here, we report that Rb loss converts chromatin at the promoters of E2f-target genes to an activated state. We established a mouse model system in which Rb-LXCXE interactions could be induciblely disabled. Surprisingly, this had no effect on survival or gene silencing in neuronal quiescence. Absence of the Rb LXCXE-binding domain in neurons is compatible with gene repression and long-term survival, unlike Rb deficiency. Finally, we are able to show that chromatin activation following Rb deletion occurs at the level of E2fs. Blocking E2f-mediated transcription downstream of Rb loss is sufficient to maintain chromatin in an inactive state. Taken together our results suggest a model whereby Rb-E2f interactions are sufficient to maintain gene repression irrespective of LXCXE-dependent chromatin remodeling.

  12. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling.

    PubMed

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook; Baek, Songjoon; Sung, Myong-Hee; Zhao, Li; Park, Jeong Won; Nielsen, Ronni; Walker, Robert L; Zhu, Yuelin J; Meltzer, Paul S; Hager, Gordon L; Cheng, Sheue-yann

    2015-04-28

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated gene transcription. Genome-wide footprinting analysis using DNase-seq provides little evidence for TR footprints both in the absence and presence of hormone, suggesting that unliganded TR engagement with repressive complexes on chromatin is, similar to activating receptor complexes, a highly dynamic process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand.

  13. LXCXE-independent chromatin remodeling by Rb/E2f mediates neuronal quiescence

    PubMed Central

    Andrusiak, Matthew G.; Vandenbosch, Renaud; Dick, Fred A.; Park, David S.; Slack, Ruth S.

    2013-01-01

    Neuronal survival is dependent upon the retinoblastoma family members, Rb1 (Rb) and Rb2 (p130). Rb is thought to regulate gene repression, in part, through direct recruitment of chromatin modifying enzymes to its conserved LXCXE binding domain. We sought to examine the mechanisms that Rb employs to mediate cell cycle gene repression in terminally differentiated cortical neurons. Here, we report that Rb loss converts chromatin at the promoters of E2f-target genes to an activated state. We established a mouse model system in which Rb-LXCXE interactions could be induciblely disabled. Surprisingly, this had no effect on survival or gene silencing in neuronal quiescence. Absence of the Rb LXCXE-binding domain in neurons is compatible with gene repression and long-term survival, unlike Rb deficiency. Finally, we are able to show that chromatin activation following Rb deletion occurs at the level of E2fs. Blocking E2f-mediated transcription downstream of Rb loss is sufficient to maintain chromatin in an inactive state. Taken together our results suggest a model whereby Rb-E2f interactions are sufficient to maintain gene repression irrespective of LXCXE-dependent chromatin remodeling. PMID:23574720

  14. The Drosophila Mi-2 Chromatin-Remodeling Factor Regulates Higher-Order Chromatin Structure and Cohesin Dynamics In Vivo

    PubMed Central

    Fasulo, Barbara; Deuring, Renate; Murawska, Magdalena; Gause, Maria; Dorighi, Kristel M.; Schaaf, Cheri A.; Dorsett, Dale; Brehm, Alexander; Tamkun, John W.

    2012-01-01

    dMi-2 is a highly conserved ATP-dependent chromatin-remodeling factor that regulates transcription and cell fates by altering the structure or positioning of nucleosomes. Here we report an unanticipated role for dMi-2 in the regulation of higher-order chromatin structure in Drosophila. Loss of dMi-2 function causes salivary gland polytene chromosomes to lose their characteristic banding pattern and appear more condensed than normal. Conversely, increased expression of dMi-2 triggers decondensation of polytene chromosomes accompanied by a significant increase in nuclear volume; this effect is relatively rapid and is dependent on the ATPase activity of dMi-2. Live analysis revealed that dMi-2 disrupts interactions between the aligned chromatids of salivary gland polytene chromosomes. dMi-2 and the cohesin complex are enriched at sites of active transcription; fluorescence-recovery after photobleaching (FRAP) assays showed that dMi-2 decreases stable association of cohesin with polytene chromosomes. These findings demonstrate that dMi-2 is an important regulator of both chromosome condensation and cohesin binding in interphase cells. PMID:22912596

  15. Fokker-Planck description of single nucleosome repositioning by dimeric chromatin remodelers

    NASA Astrophysics Data System (ADS)

    Vandecan, Yves; Blossey, Ralf

    2013-07-01

    Recent experiments have demonstrated that the ATP-utilizing chromatin assembly and remodeling factor (ACF) is a dimeric, processive motor complex which can move a nucleosome more efficiently towards longer flanking DNA than towards shorter flanking DNA strands, thereby centering an initially ill-positioned nucleosome on DNA substrates. We give a Fokker-Planck description for the repositioning process driven by transitions between internal chemical states of the remodelers. In the chemical states of ATP hydrolysis during which the repositioning takes place a power stroke is considered. The slope of the effective driving potential is directly related to ATP hydrolysis and leads to the unidirectional motion of the nucleosome-remodeler complex along the DNA strand. The Einstein force relation allows us to deduce the ATP-concentration dependence of the diffusion constant of the nucleosome-remodeler complex. We have employed our model to study the efficiency of positioning of nucleosomes as a function of the ATP sampling rate between the two motors which shows that the synchronization between the motors is crucial for the remodeling mechanism to work.

  16. Structural analyses of the chromatin remodeling enzymes INO80-C and SWR-C

    PubMed Central

    Watanabe, Shinya; Tan, Dongyan; Lakshminarasimhan, Mahadevan; Washburn, Michael P.; Hong, Eun-Jin Erica; Walz, Thomas; Peterson, Craig L.

    2015-01-01

    INO80-C and SWR-C are conserved members of a subfamily of ATP-dependent chromatin remodeling enzymes that function in transcription and genome-maintenance pathways. A crucial role for these enzymes is to control chromosomal distribution of the H2A.Z histone variant. Here we use electron microscopy (EM) and two-dimensional (2D) class averaging to demonstrate that these remodeling enzymes have similar overall architectures. Each enzyme is characterized by a dynamic ‘tail’ domain and a compact ‘head’ that contains Rvb1/Rvb2 subunits organized as hexameric rings. EM class averages and mass spectrometry support the existence of single heterohexameric rings in both SWR-C and INO80-C. EM studies define the position of the Arp8/Arp4/Act1 module within INO80-C, and we find that this module enhances nucleosome binding affinity but is largely dispensable for remodeling activities. In contrast, the Ies6/Arp5 module is essential for INO80-C remodeling, and furthermore this module controls conformational changes that may couple nucleosome binding to remodeling. PMID:25964121

  17. Impact of the Chromatin Remodeling Factor CHD1 on Gut Microbiome Composition of Drosophila melanogaster

    PubMed Central

    Krogsdam, Anne; Orth-Höller, Dorothea; Trajanoski, Zlatko; Lusser, Alexandra

    2016-01-01

    The composition of the intestinal microbiota of Drosophila has been studied in some detail in recent years. Environmental, developmental and host-specific genetic factors influence microbiome composition in the fly. Our previous work has indicated that intestinal bacterial load can be affected by chromatin-targeted regulatory mechanisms. Here we studied a potential role of the conserved chromatin assembly and remodeling factor CHD1 in the shaping of the gut microbiome in Drosophila melanogaster. Using high-throughput sequencing of 16S rRNA gene amplicons, we found that Chd1 deletion mutant flies exhibit significantly reduced microbial diversity compared to rescued control strains. Specifically, although Acetobacteraceae dominated the microbiota of both Chd1 wild-type and mutant guts, Chd1 mutants were virtually monoassociated with this bacterial family, whereas in control flies other bacterial taxa constituted ~20% of the microbiome. We further show age-linked differences in microbial load and microbiota composition between Chd1 mutant and control flies. Finally, diet supplementation experiments with Lactobacillus plantarum revealed that, in contrast to wild-type flies, Chd1 mutant flies were unable to maintain higher L. plantarum titres over time. Collectively, these data provide evidence that loss of the chromatin remodeler CHD1 has a major impact on the gut microbiome of Drosophila melanogaster. PMID:27093431

  18. Impact of the Chromatin Remodeling Factor CHD1 on Gut Microbiome Composition of Drosophila melanogaster.

    PubMed

    Sebald, Johanna; Willi, Michaela; Schoberleitner, Ines; Krogsdam, Anne; Orth-Höller, Dorothea; Trajanoski, Zlatko; Lusser, Alexandra

    2016-01-01

    The composition of the intestinal microbiota of Drosophila has been studied in some detail in recent years. Environmental, developmental and host-specific genetic factors influence microbiome composition in the fly. Our previous work has indicated that intestinal bacterial load can be affected by chromatin-targeted regulatory mechanisms. Here we studied a potential role of the conserved chromatin assembly and remodeling factor CHD1 in the shaping of the gut microbiome in Drosophila melanogaster. Using high-throughput sequencing of 16S rRNA gene amplicons, we found that Chd1 deletion mutant flies exhibit significantly reduced microbial diversity compared to rescued control strains. Specifically, although Acetobacteraceae dominated the microbiota of both Chd1 wild-type and mutant guts, Chd1 mutants were virtually monoassociated with this bacterial family, whereas in control flies other bacterial taxa constituted ~20% of the microbiome. We further show age-linked differences in microbial load and microbiota composition between Chd1 mutant and control flies. Finally, diet supplementation experiments with Lactobacillus plantarum revealed that, in contrast to wild-type flies, Chd1 mutant flies were unable to maintain higher L. plantarum titres over time. Collectively, these data provide evidence that loss of the chromatin remodeler CHD1 has a major impact on the gut microbiome of Drosophila melanogaster.

  19. Chromatin remodelers clear nucleosomes from intrinsically unfavorable sites to establish nucleosome-depleted regions at promoters.

    PubMed

    Tolkunov, Denis; Zawadzki, Karl A; Singer, Cara; Elfving, Nils; Morozov, Alexandre V; Broach, James R

    2011-06-15

    Most promoters in yeast contain a nucleosome-depleted region (NDR), but the mechanisms by which NDRs are established and maintained in vivo are currently unclear. We have examined how genome-wide nucleosome placement is altered in the absence of two distinct types of nucleosome remodeling activity. In mutants of both SNF2, which encodes the ATPase component of the Swi/Snf remodeling complex, and ASF1, which encodes a histone chaperone, distinct sets of gene promoters carry excess nucleosomes in their NDRs relative to wild-type. In snf2 mutants, excess promoter nucleosomes correlate with reduced gene expression. In both mutants, the excess nucleosomes occupy DNA sequences that are energetically less favorable for nucleosome formation, indicating that intrinsic histone-DNA interactions are not sufficient for nucleosome positioning in vivo, and that Snf2 and Asf1 promote thermodynamic equilibration of nucleosomal arrays. Cells lacking SNF2 or ASF1 still accomplish the changes in promoter nucleosome structure associated with large-scale transcriptional reprogramming. However, chromatin reorganization in the mutants is reduced in extent compared to wild-type cells, even though transcriptional changes proceed normally. In summary, active remodeling is required for distributing nucleosomes to energetically favorable positions in vivo and for reorganizing chromatin in response to changes in transcriptional activity.

  20. Aberrant Pulmonary Vascular Growth and Remodeling in Bronchopulmonary Dysplasia

    PubMed Central

    Alvira, Cristina M.

    2016-01-01

    In contrast to many other organs, a significant portion of lung development occurs after birth during alveolarization, thus rendering the lung highly susceptible to injuries that may disrupt this developmental process. Premature birth heightens this susceptibility, with many premature infants developing the chronic lung disease, bronchopulmonary dysplasia (BPD), a disease characterized by arrested alveolarization. Over the past decade, tremendous progress has been made in the elucidation of mechanisms that promote postnatal lung development, including extensive data suggesting that impaired pulmonary angiogenesis contributes to the pathogenesis of BPD. Moreover, in addition to impaired vascular growth, patients with BPD also frequently demonstrate alterations in pulmonary vascular remodeling and tone, increasing the risk for persistent hypoxemia and the development of pulmonary hypertension. In this review, an overview of normal lung development will be presented, and the pathologic features of arrested development observed in BPD will be described, with a specific emphasis on the pulmonary vascular abnormalities. Key pathways that promote normal pulmonary vascular development will be reviewed, and the experimental and clinical evidence demonstrating alterations of these essential pathways in BPD summarized. PMID:27243014

  1. MRN1 Implicates Chromatin Remodeling Complexes and Architectural Factors in mRNA Maturation

    PubMed Central

    Düring, Louis; Thorsen, Michael; Petersen, Darima Sophia Njama; Køster, Brian; Jensen, Torben Heick; Holmberg, Steen

    2012-01-01

    A functional relationship between chromatin structure and mRNA processing events has been suggested, however, so far only a few involved factors have been characterized. Here we show that rsc nhp6ΔΔ mutants, deficient for the function of the chromatin remodeling factor RSC and the chromatin architectural proteins Nhp6A/Nhp6B, accumulate intron-containing pre-mRNA at the restrictive temperature. In addition, we demonstrate that rsc8-ts16 nhp6ΔΔ cells contain low levels of U6 snRNA and U4/U6 di-snRNA that is further exacerbated after two hours growth at the restrictive temperature. This change in U6 snRNA and U4/U6 di-snRNA levels in rsc8-ts16 nhp6ΔΔ cells is indicative of splicing deficient conditions. We identify MRN1 (multi-copy suppressor of rsc nhp6ΔΔ) as a growth suppressor of rsc nhp6ΔΔ synthetic sickness. Mrn1 is an RNA binding protein that localizes both to the nucleus and cytoplasm. Genetic interactions are observed between 2 µm-MRN1 and the splicing deficient mutants snt309Δ, prp3, prp4, and prp22, and additional genetic analyses link MRN1, SNT309, NHP6A/B, SWI/SNF, and RSC supporting the notion of a role of chromatin structure in mRNA processing. PMID:23028530

  2. Downregulation of SWI/SNF chromatin remodeling factor subunits modulates cisplatin cytotoxicity

    SciTech Connect

    Kothandapani, Anbarasi; Gopalakrishnan, Kathirvel; Kahali, Bhaskar; Reisman, David; Patrick, Steve M.

    2012-10-01

    Chromatin remodeling complex SWI/SNF plays important roles in many cellular processes including transcription, proliferation, differentiation and DNA repair. In this report, we investigated the role of SWI/SNF catalytic subunits Brg1 and Brm in the cellular response to cisplatin in lung cancer and head/neck cancer cells. Stable knockdown of Brg1 and Brm enhanced cellular sensitivity to cisplatin. Repair kinetics of cisplatin DNA adducts revealed that downregulation of Brg1 and Brm impeded the repair of both intrastrand adducts and interstrand crosslinks (ICLs). Cisplatin ICL-induced DNA double strand break repair was also decreased in Brg1 and Brm depleted cells. Altered checkpoint activation with enhanced apoptosis as well as impaired chromatin relaxation was observed in Brg1 and Brm deficient cells. Downregulation of Brg1 and Brm did not affect the recruitment of DNA damage recognition factor XPC to cisplatin DNA lesions, but affected ERCC1 recruitment, which is involved in the later stages of DNA repair. Based on these results, we propose that SWI/SNF chromatin remodeling complex modulates cisplatin cytotoxicity by facilitating efficient repair of the cisplatin DNA lesions. -- Highlights: Black-Right-Pointing-Pointer Stable knockdown of Brg1 and Brm enhances cellular sensitivity to cisplatin. Black-Right-Pointing-Pointer Downregulation of Brg1 and Brm impedes the repair of cisplatin intrastrand adducts and interstrand crosslinks. Black-Right-Pointing-Pointer Brg1 and Brm deficiency results in impaired chromatin relaxation, altered checkpoint activation as well as enhanced apoptosis. Black-Right-Pointing-Pointer Downregulation of Brg1 and Brm affects recruitment of ERCC1, but not XPC to cisplatin DNA lesions.

  3. Reversible phosphorylation and regulation of mammalian oocyte meiotic chromatin remodeling and segregation.

    PubMed

    Swain, J E; Smith, G D

    2007-01-01

    The mammalian oocyte is notorious for high rates of chromosomal abnormalities. This results in subsequent embryonic aneuploidy, resulting in infertility and congenital defects. Therefore, understanding regulatory mechanisms involved in chromatin remodeling and chromosome segregation during oocyte meiotic maturation is imperative to fully understand the complex process and establish potential therapies. This review will focus on major events occurring during oocyte meiosis, critical to ensure proper cellular ploidy. Mechanistic and cellular events such as chromosome condensation, meiotic spindle formation, as well as cohesion of homologues and sister chromatids will be discussed, focusing on the role of reversible phosphorylation in control of these processes.

  4. Structure of a RSC-nucleosome complex and insights into chromatin remodeling.

    PubMed

    Chaban, Yuriy; Ezeokonkwo, Chukwudi; Chung, Wen-Hsiang; Zhang, Fan; Kornberg, Roger D; Maier-Davis, Barbara; Lorch, Yahli; Asturias, Francisco J

    2008-12-01

    ATP-dependent chromatin-remodeling complexes, such as RSC, can reposition, evict or restructure nucleosomes. A structure of a RSC-nucleosome complex with a nucleosome determined by cryo-EM shows the nucleosome bound in a central RSC cavity. Extensive interaction of RSC with histones and DNA seems to destabilize the nucleosome and lead to an overall ATP-independent rearrangement of its structure. Nucleosomal DNA appears disordered and largely free to bulge out into solution as required for remodeling, but the structure of the RSC-nucleosome complex indicates that RSC is unlikely to displace the octamer from the nucleosome to which it is bound. Consideration of the RSC-nucleosome structure and published biochemical information suggests that ATP-dependent DNA translocation by RSC may result in the eviction of histone octamers from adjacent nucleosomes.

  5. Chromatin-remodeling factor SMARCD2 regulates transcriptional networks controlling differentiation of neutrophil granulocytes.

    PubMed

    Witzel, Maximilian; Petersheim, Daniel; Fan, Yanxin; Bahrami, Ehsan; Racek, Tomas; Rohlfs, Meino; Puchałka, Jacek; Mertes, Christian; Gagneur, Julien; Ziegenhain, Christoph; Enard, Wolfgang; Stray-Pedersen, Asbjørg; Arkwright, Peter D; Abboud, Miguel R; Pazhakh, Vahid; Lieschke, Graham J; Krawitz, Peter M; Dahlhoff, Maik; Schneider, Marlon R; Wolf, Eckhard; Horny, Hans-Peter; Schmidt, Heinrich; Schäffer, Alejandro A; Klein, Christoph

    2017-04-03

    We identify SMARCD2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 2), also known as BAF60b (BRG1/Brahma-associated factor 60b), as a critical regulator of myeloid differentiation in humans, mice, and zebrafish. Studying patients from three unrelated pedigrees characterized by neutropenia, specific granule deficiency, myelodysplasia with excess of blast cells, and various developmental aberrations, we identified three homozygous loss-of-function mutations in SMARCD2. Using mice and zebrafish as model systems, we showed that SMARCD2 controls early steps in the differentiation of myeloid-erythroid progenitor cells. In vitro, SMARCD2 interacts with the transcription factor CEBPɛ and controls expression of neutrophil proteins stored in specific granules. Defective expression of SMARCD2 leads to transcriptional and chromatin changes in acute myeloid leukemia (AML) human promyelocytic cells. In summary, SMARCD2 is a key factor controlling myelopoiesis and is a potential tumor suppressor in leukemia.

  6. INO80 and gamma-H2AX interaction links ATP-dependent chromatin remodeling to DNA damage repair.

    PubMed

    Morrison, Ashby J; Highland, Jessica; Krogan, Nevan J; Arbel-Eden, Ayelet; Greenblatt, Jack F; Haber, James E; Shen, Xuetong

    2004-12-17

    While the role of ATP-dependent chromatin remodeling in transcription is well established, a link between chromatin remodeling and DNA repair has remained elusive. We have found that the evolutionarily conserved INO80 chromatin remodeling complex directly participates in the repair of a double-strand break (DSB) in yeast. The INO80 complex is recruited to a HO endonuclease-induced DSB through a specific interaction with the DNA damage-induced phosphorylated histone H2A (gamma-H2AX). This interaction requires Nhp10, an HMG-like subunit of the INO80 complex. The loss of Nhp10 or gamma-H2AX results in reduced INO80 recruitment to the DSB. Finally, components of the INO80 complex show synthetic genetic interactions with the RAD52 DNA repair pathway, the main pathway for DSB repair in yeast. Our findings reveal a new role of ATP-dependent chromatin remodeling in nuclear processes and suggest that an ATP-dependent chromatin remodeling complex can read a DNA repair histone code.

  7. Chromatin remodeling during in vivo neural stem cells differentiating to neurons in early Drosophila embryos

    PubMed Central

    Ye, Youqiong; Li, Min; Gu, Liang; Chen, Xiaolong; Shi, Jiejun; Zhang, Xiaobai; Jiang, Cizhong

    2017-01-01

    Neurons are a key component of the nervous system and differentiate from multipotent neural stem cells (NSCs). Chromatin remodeling has a critical role in the differentiation process. However, its in vivo epigenetic regulatory role remains unknown. We show here that nucleosome depletion regions (NDRs) form in both proximal promoters and distal enhancers during NSCs differentiating into neurons in the early Drosophila embryonic development. NDR formation in the regulatory regions involves nucleosome shift and eviction. Nucleosome occupancy in promoter NDRs is inversely proportional to the gene activity. Genes with promoter NDR formation during differentiation are enriched for functions related to neuron development and maturation. Active histone-modification signals (H3K4me3 and H3K9ac) in promoters are gained in neurons in two modes: de novo establishment to high levels or increase from the existing levels in NSCs. The gene sets corresponding to the two modes have different neuron-related functions. Dynamic changes of H3K27ac and H3K9ac signals in enhancers and promoters synergistically repress genes associated with neural stem or progenitor cell-related pluripotency and upregulate genes associated with neuron projection morphogenesis, neuron differentiation, and so on. Our results offer new insights into chromatin remodeling during in vivo neuron development and lay a foundation for its epigenetic regulatory mechanism study of other lineage specification. PMID:27858939

  8. Chromatin remodeling regulates catalase expression during cancer cells adaptation to chronic oxidative stress.

    PubMed

    Glorieux, Christophe; Sandoval, Juan Marcelo; Fattaccioli, Antoine; Dejeans, Nicolas; Garbe, James C; Dieu, Marc; Verrax, Julien; Renard, Patricia; Huang, Peng; Calderon, Pedro Buc

    2016-10-01

    Regulation of ROS metabolism plays a major role in cellular adaptation to oxidative stress in cancer cells, but the molecular mechanism that regulates catalase, a key antioxidant enzyme responsible for conversion of hydrogen peroxide to water and oxygen, remains to be elucidated. Therefore, we investigated the transcriptional regulatory mechanism controlling catalase expression in three human mammary cell lines: the normal mammary epithelial 250MK primary cells, the breast adenocarcinoma MCF-7 cells and an experimental model of MCF-7 cells resistant against oxidative stress resulting from chronic exposure to H2O2 (Resox), in which catalase was overexpressed. Here we identify a novel promoter region responsible for the regulation of catalase expression at -1518/-1226 locus and the key molecules that interact with this promoter and affect catalase transcription. We show that the AP-1 family member JunB and retinoic acid receptor alpha (RARα) mediate catalase transcriptional activation and repression, respectively, by controlling chromatin remodeling through a histone deacetylases-dependent mechanism. This regulatory mechanism plays an important role in redox adaptation to chronic exposure to H2O2 in breast cancer cells. Our study suggests that cancer adaptation to oxidative stress may be regulated by transcriptional factors through chromatin remodeling, and reveals a potential new mechanism to target cancer cells.

  9. The Chromatin Remodeler CHD8 Is Required for Activation of Progesterone Receptor-Dependent Enhancers

    PubMed Central

    Giannopoulou, Eugenia G.; Soronellas, Daniel; Vázquez-Chávez, Elena; Vicent, Guillermo P.; Elemento, Olivier; Beato, Miguel; Reyes, José C.

    2015-01-01

    While the importance of gene enhancers in transcriptional regulation is well established, the mechanisms and the protein factors that determine enhancers activity have only recently begun to be unravelled. Recent studies have shown that progesterone receptor (PR) binds regions that display typical features of gene enhancers. Here, we show by ChIP-seq experiments that the chromatin remodeler CHD8 mostly binds promoters under proliferation conditions. However, upon progestin stimulation, CHD8 re-localizes to PR enhancers also enriched in p300 and H3K4me1. Consistently, CHD8 depletion severely impairs progestin-dependent gene regulation. CHD8 binding is PR-dependent but independent of the pioneering factor FOXA1. The SWI/SNF chromatin-remodelling complex is required for PR-dependent gene activation. Interestingly, we show that CHD8 interacts with the SWI/SNF complex and that depletion of BRG1 and BRM, the ATPases of SWI/SNF complex, impairs CHD8 recruitment. We also show that CHD8 is not required for H3K27 acetylation, but contributes to increase accessibility of the enhancer to DNaseI. Furthermore, CHD8 was required for RNAPII recruiting to the enhancers and for transcription of enhancer-derived RNAs (eRNAs). Taken together our data demonstrate that CHD8 is involved in late stages of PR enhancers activation. PMID:25894978

  10. Newly identified prion linked to the chromatin-remodeling factor Swi1 in Saccharomyces cerevisiae.

    PubMed

    Du, Zhiqiang; Park, Kyung-Won; Yu, Haijing; Fan, Qing; Li, Liming

    2008-04-01

    SWI/SNF, an evolutionarily conserved ATP-dependent chromatin-remodeling complex, has an important role in transcriptional regulation. In Saccharomyces cerevisiae, SWI/SNF regulates the expression of approximately 6% of total genes through activation or repression. Swi1, a subunit of SWI/SNF, contains an N-terminal region rich in glutamine and asparagine, a notable feature shared by all characterized yeast prions--a group of unique proteins capable of self-perpetuating changes in conformation and function. Here we provide evidence that Swi1 can become a prion, [SWI+]. Swi1 aggregates in [SWI+] cells but not in nonprion cells. Cells bearing [SWI+] show a partial loss-of-function phenotype of SWI/SNF. [SW+] can be eliminated by guanidine hydrochloride treatment, HSP104 deletion or loss of Swi1. Moreover, we show [SWI+] is dominantly and cytoplasmically transmitted. Our findings reveal a novel mechanism of 'protein-only' inheritance that results in modification of chromatin-remodeling and, ultimately, global gene regulation.

  11. Compact tomato seedlings and plants upon overexpression of a tomato chromatin remodelling ATPase gene.

    PubMed

    Folta, Adam; Bargsten, Joachim W; Bisseling, Ton; Nap, Jan-Peter; Mlynarova, Ludmila

    2016-02-01

    Control of plant growth is an important aspect of crop productivity and yield in agriculture. Overexpression of the AtCHR12/23 genes in Arabidopsis thaliana reduced growth habit without other morphological changes. These two genes encode Snf2 chromatin remodelling ATPases. Here, we translate this approach to the horticultural crop tomato (Solanum lycopersicum). We identified and cloned the single tomato ortholog of the two Arabidopsis Snf2 genes, designated SlCHR1. Transgenic tomato plants (cv. Micro-Tom) that constitutively overexpress the coding sequence of SlCHR1 show reduced growth in all developmental stages of tomato. This confirms that SlCHR1 combines the functions of both Arabidopsis genes in tomato. Compared to the wild type, the transgenic seedlings of tomato have significantly shorter roots, hypocotyls and reduced cotyledon size. Transgenic plants have a much more compact growth habit with markedly reduced plant height, severely compacted reproductive structures with smaller flowers and smaller fruits. The results indicate that either GMO-based or non-GMO-based approaches to modulate the expression of chromatin remodelling ATPase genes could develop into methods to control plant growth, for example to replace the use of chemical growth retardants. This approach is likely to be applicable and attractive for any crop for which growth habit reduction has added value.

  12. The Chromatin-Remodeling Factor PICKLE Integrates Brassinosteroid and Gibberellin Signaling during Skotomorphogenic Growth in Arabidopsis.

    PubMed

    Zhang, Dong; Jing, Yanjun; Jiang, Zhimin; Lin, Rongcheng

    2014-06-01

    Plant cell elongation is controlled by endogenous hormones, including brassinosteroid (BR) and gibberellin (GA), and by environmental factors, such as light/darkness. The molecular mechanisms underlying the convergence of these signals that govern cell growth remain largely unknown. We previously showed that the chromatin-remodeling factor PICKLE/ENHANCED PHOTOMORPHOGENIC1 (PKL/EPP1) represses photomorphogenesis in Arabidopsis thaliana. Here, we demonstrated that PKL physically interacted with PHYTOCHROME-INTERACTING FACTOR3 (PIF3) and BRASSINAZOLE-RESISTANT1 (BZR1), key components of the light and BR signaling pathways, respectively. Also, this interaction promoted the association of PKL with cell elongation-related genes. We found that PKL, PIF3, and BZR1 coregulate skotomorphogenesis by repressing the trimethylation of histone H3 Lys-27 (H3K27me3) on target promoters. Moreover, DELLA proteins interacted with PKL and attenuated its binding ability. Strikingly, brassinolide and GA3 inhibited H3K27me3 modification of histones associated with cell elongation-related loci in a BZR1- and DELLA-mediated manner, respectively. Our findings reveal that the PKL chromatin-remodeling factor acts as a critical node that integrates light/darkness, BR, and GA signals to epigenetically regulate plant growth and development. This work also provides a molecular framework by which hormone signals regulate histone modification in concert with light/dark environmental cues.

  13. The CKH2/PKL chromatin remodeling factor negatively regulates cytokinin responses in Arabidopsis calli.

    PubMed

    Furuta, Kaori; Kubo, Minoru; Sano, Kiyomi; Demura, Taku; Fukuda, Hiroo; Liu, Yao-Guang; Shibata, Daisuke; Kakimoto, Tatsuo

    2011-04-01

    Cytokinins promote cell division and chloroplast development in tissue culture. We previously isolated two mutants of Arabidopsis thaliana, ckh1 (cytokinin-hypersensitive 1) and ckh2, which produce rapidly growing green calli in response to lower levels of cytokinins than those found in the wild type. Here we report that the product of the CKH2 gene is PICKLE, a protein resembling the CHD3 class of SWI/SNF chromatin remodeling factors. We also show that inhibition of histone deacetylase by trichostatin A (TSA) partially substituted for cytokinins, but not for auxin, in the promotion of callus growth, indicating that chromatin remodeling and histone deacetylation are intimately related to cytokinin-induced callus growth. A microarray experiment revealed that either the ckh1 mutation or the ckh2 mutation caused hypersensitivity to cytokinins in terms of gene expression, especially of photosynthesis-related genes. The ckh1 and ckh2 mutations up-regulated nuclear-encoded genes, but not plastid-encoded genes, whereas TSA deregulated both nuclear- and plastid-encoded genes. The ckh1 ckh2 double mutant showed synergistic phenotypes: the callus grew with a green color independently of exogenous cytokinins. A yeast two-hybrid experiment showed protein interaction between CKH1/EER4/AtTAF12b and CKH2/PKL. These results suggest that CKH1/EER4/AtTAF12b and CKH2/PKL may act together on cytokinin-regulated genes.

  14. Arabidopsis FORGETTER1 mediates stress-induced chromatin memory through nucleosome remodeling

    PubMed Central

    Brzezinka, Krzysztof; Altmann, Simone; Czesnick, Hjördis; Nicolas, Philippe; Gorka, Michal; Benke, Eileen; Kabelitz, Tina; Jähne, Felix; Graf, Alexander; Kappel, Christian; Bäurle, Isabel

    2016-01-01

    Plants as sessile organisms can adapt to environmental stress to mitigate its adverse effects. As part of such adaptation they maintain an active memory of heat stress for several days that promotes a more efficient response to recurring stress. We show that this heat stress memory requires the activity of the FORGETTER1 (FGT1) locus, with fgt1 mutants displaying reduced maintenance of heat-induced gene expression. FGT1 encodes the Arabidopsis thaliana orthologue of Strawberry notch (Sno), and the protein globally associates with the promoter regions of actively expressed genes in a heat-dependent fashion. FGT1 interacts with chromatin remodelers of the SWI/SNF and ISWI families, which also display reduced heat stress memory. Genomic targets of the BRM remodeler overlap significantly with FGT1 targets. Accordingly, nucleosome dynamics at loci with altered maintenance of heat-induced expression are affected in fgt1. Together, our results suggest that by modulating nucleosome occupancy, FGT1 mediates stress-induced chromatin memory. DOI: http://dx.doi.org/10.7554/eLife.17061.001 PMID:27680998

  15. The Chromatin Remodelling Enzymes SNF2H and SNF2L Position Nucleosomes adjacent to CTCF and Other Transcription Factors

    PubMed Central

    Wiechens, Nicola; Gkikopoulos, Triantaffyllos; Schofield, Pieta; Rocha, Sonia; Owen-Hughes, Tom

    2016-01-01

    Within the genomes of metazoans, nucleosomes are highly organised adjacent to the binding sites for a subset of transcription factors. Here we have sought to investigate which chromatin remodelling enzymes are responsible for this. We find that the ATP-dependent chromatin remodelling enzyme SNF2H plays a major role organising arrays of nucleosomes adjacent to the binding sites for the architectural transcription factor CTCF sites and acts to promote CTCF binding. At many other factor binding sites SNF2H and the related enzyme SNF2L contribute to nucleosome organisation. The action of SNF2H at CTCF sites is functionally important as depletion of CTCF or SNF2H affects transcription of a common group of genes. This suggests that chromatin remodelling ATPase’s most closely related to the Drosophila ISWI protein contribute to the function of many human gene regulatory elements. PMID:27019336

  16. Sfh1p, a component of a novel chromatin-remodeling complex, is required for cell cycle progression.

    PubMed Central

    Cao, Y; Cairns, B R; Kornberg, R D; Laurent, B C

    1997-01-01

    Several eukaryotic multiprotein complexes, including the Saccharomyces cerevisiae Snf/Swi complex, remodel chromatin for transcription. In contrast to the Snf/Swi proteins, Sfh1p, a new Snf5p paralog, is essential for viability. The evolutionarily conserved domain of Sfh1p is sufficient for normal function, and Sfh1p interacts functionally and physically with an essential Snf2p paralog in a novel nucleosome-restructuring complex called RSC (for remodels the structure of chromatin). A temperature-sensitive sfh1 allele arrests cells in the G2/M phase of the cell cycle, and the Sfh1 protein is specifically phosphorylated in the G1 phase. Together, these results demonstrate a link between chromatin remodeling and progression through the cell division cycle, providing genetic clues to possible targets for RSC function. PMID:9154831

  17. A Novel Mechanism of Antagonism between ATP-Dependent Chromatin Remodeling Complexes Regulates RNR3 Expression▿

    PubMed Central

    Tomar, Raghuvir S.; Psathas, James N.; Zhang, Hesheng; Zhang, Zhengjian; Reese, Joseph C.

    2009-01-01

    Gene expression depends upon the antagonistic actions of chromatin remodeling complexes. While this has been studied extensively for the enzymes that covalently modify the tails of histones, the mechanism of how ATP-dependent remodeling complexes antagonize each other to maintain the proper level of gene activity is not known. The gene encoding a large subunit of ribonucleotide reductase, RNR3, is regulated by ISW2 and SWI/SNF, complexes that repress and activate transcription, respectively. Here, we studied the functional interactions of these two complexes at RNR3. Deletion of ISW2 causes constitutive recruitment of SWI/SNF, and conditional reexpression of ISW2 causes the repositioning of nucleosomes and reduced SWI/SNF occupancy at RNR3. Thus, ISW2 is required for restriction of access of SWI/SNF to the RNR3 promoter under the uninduced condition. Interestingly, the binding of sequence-specific DNA binding factors and the general transcription machinery are unaffected by the status of ISW2, suggesting that disruption of nucleosome positioning does not cause a nonspecific increase in cross-linking of all factors to RNR3. We provide evidence that ISW2 does not act on SWI/SNF directly but excludes its occupancy by positioning nucleosomes over the promoter. Genetic disruption of nucleosome positioning by other means led to a similar phenotype, linking repressed chromatin structure to SWI/SNF exclusion. Thus, incorporation of promoters into a repressive chromatin structure is essential for prevention of the opportunistic actions of nucleosome-disrupting activities in vivo, providing a novel mechanism for maintaining tight control of gene expression. PMID:19349301

  18. Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells.

    PubMed

    Laurette, Patrick; Strub, Thomas; Koludrovic, Dana; Keime, Céline; Le Gras, Stéphanie; Seberg, Hannah; Van Otterloo, Eric; Imrichova, Hana; Siddaway, Robert; Aerts, Stein; Cornell, Robert A; Mengus, Gabrielle; Davidson, Irwin

    2015-03-24

    Microphthalmia-associated transcription factor (MITF) is the master regulator of the melanocyte lineage. To understand how MITF regulates transcription, we used tandem affinity purification and mass spectrometry to define a comprehensive MITF interactome identifying novel cofactors involved in transcription, DNA replication and repair, and chromatin organisation. We show that MITF interacts with a PBAF chromatin remodelling complex comprising BRG1 and CHD7. BRG1 is essential for melanoma cell proliferation in vitro and for normal melanocyte development in vivo. MITF and SOX10 actively recruit BRG1 to a set of MITF-associated regulatory elements (MAREs) at active enhancers. Combinations of MITF, SOX10, TFAP2A, and YY1 bind between two BRG1-occupied nucleosomes thus defining both a signature of transcription factors essential for the melanocyte lineage and a specific chromatin organisation of the regulatory elements they occupy. BRG1 also regulates the dynamics of MITF genomic occupancy. MITF-BRG1 interplay thus plays an essential role in transcription regulation in melanoma.

  19. Widespread Collaboration of Isw2 and Sin3-Rpd3 Chromatin Remodeling Complexes in Transcriptional Repression

    PubMed Central

    Fazzio, Thomas G.; Kooperberg, Charles; Goldmark, Jesse P.; Neal, Cassandra; Basom, Ryan; Delrow, Jeffrey; Tsukiyama, Toshio

    2001-01-01

    The yeast Isw2 chromatin remodeling complex functions in parallel with the Sin3-Rpd3 histone deacetylase complex to repress early meiotic genes upon recruitment by Ume6p. For many of these genes, the effect of an isw2 mutation is partially masked by a functional Sin3-Rpd3 complex. To identify the full range of genes repressed or activated by these factors and uncover hidden targets of Isw2-dependent regulation, we performed full genome expression analyses using cDNA microarrays. We find that the Isw2 complex functions mainly in repression of transcription in a parallel pathway with the Sin3-Rpd3 complex. In addition to Ume6 target genes, we find that many Ume6-independent genes are derepressed in mutants lacking functional Isw2 and Sin3-Rpd3 complexes. Conversely, we find that ume6 mutants, but not isw2 sin3 or isw2 rpd3 double mutants, have reduced fidelity of mitotic chromosome segregation, suggesting that one or more functions of Ume6p are independent of Sin3-Rpd3 and Isw2 complexes. Chromatin structure analyses of two nonmeiotic genes reveals increased DNase I sensitivity within their regulatory regions in an isw2 mutant, as seen previously for one meiotic locus. These data suggest that the Isw2 complex functions at Ume6-dependent and -independent loci to create DNase I-inaccessible chromatin structure by regulating the positioning or placement of nucleosomes. PMID:11533234

  20. Essential roles of the chromatin remodeling factor BRG1 in spermatogenesis in mice.

    PubMed

    Wang, Jianguan; Gu, Honggang; Lin, Haifan; Chi, Tian

    2012-06-01

    Mammalian spermatogenesis is a complex process that involves spatiotemporal regulation of gene expression and meiotic recombination, both of which require the modulation of chromatin structure. Proteins important for chromatin regulation during spermatogenesis remain poorly understood. Here we addressed the role of BRG1, the catalytic subunit of the mammalian Swi/Snf-like BAF chromatin-remodeling complex, during spermatogenesis in mice. BRG1 expression is dynamically regulated in the male germline, being weakly detectable in spermatogonia, highly expressed in pachytene spermatocytes, and turned off in maturing round spermatids. This expression pattern overlaps that of Brm, the Brg1 homolog. While Brm knockout males are known to be fertile, germline-specific Brg1 deletion completely arrests spermatogenesis at the midpachytene stage, which is associated with spermatocyte apoptosis and apparently also with impaired homologous recombination and meiotic sex chromosome inactivation. However, Brg1 is dispensable for gammaH2AX formation during meiotic recombination, contrary to its reported role in DNA repair in somatic cells. Our study reveals the essential role of Brg1 in meiosis and underscores the differences in the mechanisms of DNA repair between germ cells and somatic cells.

  1. Chromatin Remodeling, Cell Proliferation and Cell Death in Valproic Acid-Treated HeLa Cells

    PubMed Central

    Felisbino, Marina Barreto; Tamashiro, Wirla M. S. C.; Mello, Maria Luiza S.

    2011-01-01

    Background Valproic acid (VPA) is a potent anticonvulsant that inhibits histone deacetylases. Because of this inhibitory action, we investigated whether VPA would affect chromatin supraorganization, mitotic indices and the frequency of chromosome abnormalities and cell death in HeLa cells. Methodology/Principal Findings Image analysis was performed by scanning microspectrophotometry for cells cultivated for 24 h, treated with 0.05, 0.5 or 1.0 mM VPA for 1–24 h, and subjected to the Feulgen reaction. TSA-treated cells were used as a predictable positive control. DNA fragmentation was investigated with the TUNEL assay. Chromatin decondensation was demonstrated under TSA and all VPA treatments, but no changes in chromosome abnormalities, mitotic indices or morphologically identified cell death were found with the VPA treatment conditions mentioned above, although decreased mitotic indices were detected under higher VPA concentration and longer exposure time. The frequency of DNA fragmentation identified with the TUNEL assay in HeLa cells increased after a 24-h VPA treatment, although this fragmentation occurred much earlier after treatment with TSA. Conclusions/Significance The inhibition of histone deacetylases by VPA induces chromatin remodeling in HeLa cells, which suggests an association to altered gene expression. Under VPA doses close to the therapeutic antiepileptic plasma range no changes in cell proliferation or chromosome abnormalities are elicited. The DNA fragmentation results indicate that a longer exposure to VPA or a higher VPA concentration is required for the induction of cell death. PMID:22206001

  2. Understanding the role of chromatin remodeling in the regulation of circadian transcription in Drosophila

    PubMed Central

    Kwok, Rosanna S.; Lam, Vu H.; Chiu, Joanna C.

    2015-01-01

    ABSTRACT Circadian clocks enable organisms to anticipate daily changes in the environment and coordinate temporal rhythms in physiology and behavior with the 24-h day-night cycle. The robust cycling of circadian gene expression is critical for proper timekeeping, and is regulated by transcription factor binding, RNA polymerase II (RNAPII) recruitment and elongation, and post-transcriptional mechanisms. Recently, it has become clear that dynamic alterations in chromatin landscape at the level of histone posttranslational modification and nucleosome density facilitate rhythms in transcription factor recruitment and RNAPII activity, and are essential for progression through activating and repressive phases of circadian transcription. Here, we discuss the characterization of the BRAHMA (BRM) chromatin-remodeling protein in Drosophila in the context of circadian clock regulation. By dissecting its catalytic vs. non-catalytic activities, we propose a model in which the non-catalytic activity of BRM functions to recruit repressive factors to limit the transcriptional output of CLOCK (CLK) during the active phase of circadian transcription, while the primary function of the ATP-dependent catalytic activity is to tune and prevent over-recruitment of negative regulators by increasing nucleosome density. Finally, we divulge ongoing efforts and investigative directions toward a deeper mechanistic understanding of transcriptional regulation of circadian gene expression at the chromatin level. PMID:26926115

  3. Glucocorticoid-induced p11 over-expression and chromatin remodeling: a novel molecular mechanism of traumatic stress?

    PubMed

    Zhang, Lei; Li, He; Hu, Xianzhang; Li, Xiao Xia; Smerin, Stanley; Ursano, Robert

    2011-06-01

    While the actions of glucocorticoids on brain function have been comprehensively studied, understanding of the underlying genomic mechanisms is advancing slowly. Recently, it was found that p11 is associated with traumatic stress and depression, and glucocorticoids regulate expression of the p11 gene. The ligand-activated glucocorticoid receptor (GR) interacts with two glucocorticoid response elements (GREs) in the p11 promoter region to up-regulate the p11 gene. RU486, a glucocorticoid receptor antagonist, and mutation of GREs both block glucocorticoid-induced p11 over-expression, suggesting that glucocorticoid-induced p11 over-expression is mediated by GR and GREs. Thus, the p11 gene can be transcriptionally activated. There is evidence that this transcriptional activation is mediated by the remodeling of chromatin complexes in response to glucocorticoid receptor-regulated promotors. The regulation of eukaryotic gene expression by chromatin remodeling is complex and is essential for numerous cellular processes. The association of linker-histone, non-histone and heterochromatin-specific proteins plays a key role in the generation of higher-order chromatin structures. Understanding the chromatin remodeling involved in the glucocorticoid-mediated increase of p11 expression by stress may clarify stress-induced over-expression of p11 and also identify a new therapeutic target for post-traumatic disorder and depressive disorders, i.e., chromatin remodeling.

  4. Long-Term Effects of Chromatin Remodeling and DNA Damage in Stem Cells Induced by Environmental and Dietary Agents

    PubMed Central

    Bariar, Bhawana; Vestal, C. Greer; Richardson, Christine

    2014-01-01

    The presence of histones acts as a barrier to protein access; thus chromatin remodeling must occur for essential processes such as transcription and replication. In conjunction with histone modifications, DNA methylation plays critical roles in gene silencing through chromatin remodeling. Chromatin remodeling is also interconnected with the DNA damage response, maintenance of stem cell properties, and cell differentiation programs. Chromatin modifications have increasingly been shown to produce long-lasting alterations in chromatin structure and transcription. Recent studies have shown environmental exposures in utero have the potential to alter normal developmental signaling networks, physiologic responses, and disease susceptibility later in life during a process known as developmental reprogramming. In this review we discuss the long-term impact of exposure to environmental compounds, the chromatin modifications that they induce, and the differentiation and developmental programs of multiple stem and progenitor cell types altered by exposure. The main focus is to highlight agents present in the human lifestyle that have the potential to promote epigenetic changes that impact developmental programs of specific cell types, may promote tumorigenesis through altering epigenetic marks, and may be transgenerational, for example, those able to be transmitted through multiple cell divisions. PMID:24579784

  5. Genome-Wide Mapping Targets of the Metazoan Chromatin Remodeling Factor NURF Reveals Nucleosome Remodeling at Enhancers, Core Promoters and Gene Insulators

    PubMed Central

    Kwon, So Yeon; Grisan, Valentina; Jang, Boyun; Herbert, John; Badenhorst, Paul

    2016-01-01

    NURF is a conserved higher eukaryotic ISWI-containing chromatin remodeling complex that catalyzes ATP-dependent nucleosome sliding. By sliding nucleosomes, NURF is able to alter chromatin dynamics to control transcription and genome organization. Previous biochemical and genetic analysis of the specificity-subunit of Drosophila NURF (Nurf301/Enhancer of Bithorax (E(bx)) has defined NURF as a critical regulator of homeotic, heat-shock and steroid-responsive gene transcription. It has been speculated that NURF controls pathway specific transcription by co-operating with sequence-specific transcription factors to remodel chromatin at dedicated enhancers. However, conclusive in vivo demonstration of this is lacking and precise regulatory elements targeted by NURF are poorly defined. To address this, we have generated a comprehensive map of in vivo NURF activity, using MNase-sequencing to determine at base pair resolution NURF target nucleosomes, and ChIP-sequencing to define sites of NURF recruitment. Our data show that, besides anticipated roles at enhancers, NURF interacts physically and functionally with the TRF2/DREF basal transcription factor to organize nucleosomes downstream of active promoters. Moreover, we detect NURF remodeling and recruitment at distal insulator sites, where NURF functionally interacts with and co-localizes with DREF and insulator proteins including CP190 to establish nucleosome-depleted domains. This insulator function of NURF is most apparent at subclasses of insulators that mark the boundaries of chromatin domains, where multiple insulator proteins co-associate. By visualizing the complete repertoire of in vivo NURF chromatin targets, our data provide new insights into how chromatin remodeling can control genome organization and regulatory interactions. PMID:27046080

  6. The PBAP remodeling complex is required for histone H3.3 replacement at chromatin boundaries and for boundary functions.

    PubMed

    Nakayama, Takahiro; Shimojima, Tsukasa; Hirose, Susumu

    2012-12-01

    Establishment and maintenance of epigenetic memories are essential for development. Replacement of canonical histone H3 by its variant H3.3 has been implicated in cellular memory. Drosophila sequence-specific DNA-binding protein GAGA factor and a chromatin factor FACT direct H3.3 replacement in conjunction with H3.3-specific chaperone HIRA at chromatin boundaries to counteract the spreading of silent chromatin. However, little is known about which ATP-driven chromatin remodeling factor is responsible for the H3.3 replacement at chromatin boundaries. Here, we report that GAGA factor associates with the Polybromo-associated Brm (PBAP) remodeling complex, which consists of many Trithorax group proteins, and recruits this complex to chromatin boundaries d1 (which is downstream of w), the Fab-7 DNase-hypersensitive site (HS) 1 of Abd-B and the bxd region of Ubx. Trl-encoding GAGA factor, brm and polybromo/bap180 mutations compromise the H3.3 replacement and boundary functions in a synergistic manner. Furthermore, Polybromo is necessary for generation of the DNase HS at d1, and HIRA functions to restore the alteration. Taken together, we propose that FACT and PBAP complexes are recruited to chromatin boundaries in a GAGA factor-dependent manner, and are needed for H3.3 replacement to execute boundary functions. Our results provide new insight into the function of the trithorax group during development.

  7. Antagonistic roles of Drosophila Tctp and Brahma in chromatin remodelling and stabilizing repeated sequences

    PubMed Central

    Hong, Sung-Tae; Choi, Kwang-Wook

    2016-01-01

    Genome stability is essential for all organisms. Translationally controlled tumour protein (TCTP) is a conserved protein associated with cancers. TCTP is involved in multiple intracellular functions, but its role in transcription and genome stability is poorly understood. Here, we demonstrate new functions of Drosophila TCTP (Tctp) in transcription and the stability of repeated sequences (rDNA and pericentromeric heterochromatin). Tctp binds Brahma (Brm) chromatin remodeler to negatively modulate its activity. Tctp mutants show abnormally high levels of transcription in a large set of genes and transposons. These defects are ameliorated by brm mutations. Furthermore, Tctp promotes the stability of repeated sequences by opposing the Brm function. Additional regulation of pericentromeric heterochromatin by Tctp is mediated by su(var)3-9 transcriptional regulation. Altogether, Tctp regulates transcription and the stability of repeated sequences by antagonizing excess Brm activity. This study provides insights into broader nuclear TCTP functions for the maintenance of genome stability. PMID:27687497

  8. Coordination of cell signaling, chromatin remodeling, histone modifications, and regulator recruitment in human matrix metalloproteinase 9 gene transcription.

    PubMed

    Ma, Zhendong; Shah, Reesha C; Chang, Mi Jung; Benveniste, Etty N

    2004-06-01

    Transcriptional activation of eukaryotic genes depends on the precise and ordered recruitment of activators, chromatin modifiers/remodelers, coactivators, and general transcription factors to the promoters of target genes. Using the human matrix metalloproteinase 9 (MMP-9) gene as a model system, we investigated the sequential assembly and dynamic formation of transcription complexes on a human promoter under the influence of mitogen signaling. We find that, coincident with activation of the MMP-9 gene, activators, chromatin remodeling complexes, and coactivators are recruited to the preassembled MMP-9 promoter in a stepwise and coordinated order, which is dependent on activation of MEK-1/extracellular signal-regulated kinase and NF-kappa B signaling pathways. Conversely, corepressor complexes are released from the MMP-9 promoter after transcriptional activation. Histone modifications shift from repressive to permissive modifications concurrent with activation of the MMP-9 gene. Chromatin remodeling induced by Brg-1 is required for MMP-9 gene transcription, which is concomitant with initiation of transcription. Therefore, coordination of cell signaling, chromatin remodeling, histone modifications, and stepwise recruitment of transcription regulators is critical to precisely regulate MMP-9 gene transcription in a temporally and spatially dependent manner. Given the important role of MMP-9 in both normal development and pathological conditions, understanding MMP-9 gene regulation is of great relevance.

  9. Gibberellin Signaling Requires Chromatin Remodeler PICKLE to Promote Vegetative Growth and Phase Transitions1[OPEN

    PubMed Central

    Nguyen, Khoa Thi; Ogas, Joe; Choi, Giltsu

    2017-01-01

    PICKLE (PKL) is an ATP-dependent chromodomain-helicase-DNA-binding domain (CHD3) chromatin remodeling enzyme in Arabidopsis (Arabidopsis thaliana). Previous studies showed that PKL promotes embryonic-to-vegetative transition by inhibiting expression of seed-specific genes during seed germination. The pkl mutants display a low penetrance of the “pickle root” phenotype, with a thick and green primary root that retains embryonic characteristics. The penetrance of this pickle root phenotype in pkl is dramatically increased in gibberellin (GA)-deficient conditions. At adult stages, the pkl mutants are semidwarfs with delayed flowering time, which resemble reduced GA-signaling mutants. These findings suggest that PKL may play a positive role in regulating GA signaling. A recent biochemical analysis further showed that PKL and GA signaling repressors DELLAs antagonistically regulate hypocotyl cell elongation genes by direct protein-protein interaction. To elucidate further the role of PKL in GA signaling and plant development, we studied the genetic interaction between PKL and DELLAs using the hextuple mutant containing pkl and della pentuple (dP) mutations. Here, we show that PKL is required for most of GA-promoted developmental processes, including vegetative growth such as hypocotyl, leaf, and inflorescence stem elongation, and phase transitions such as juvenile-to-adult leaf and vegetative-to-reproductive phase. The removal of all DELLA functions (in the dP background) cannot rescue these phenotypes in pkl. RNA-sequencing analysis using the ga1 (a GA-deficient mutant), pkl, and the ga1 pkl double mutant further shows that expression of 80% of GA-responsive genes in seedlings is PKL dependent, including genes that function in cell elongation, cell division, and phase transitions. These results indicate that the CHD3 chromatin remodeler PKL is required for regulating gene expression during most of GA-regulated developmental processes. PMID:28057895

  10. Gibberellin Signaling Requires Chromatin Remodeler PICKLE to Promote Vegetative Growth and Phase Transitions.

    PubMed

    Park, Jeongmoo; Oh, Dong-Ha; Dassanayake, Maheshi; Nguyen, Khoa Thi; Ogas, Joe; Choi, Giltsu; Sun, Tai-Ping

    2017-02-01

    PICKLE (PKL) is an ATP-dependent chromodomain-helicase-DNA-binding domain (CHD3) chromatin remodeling enzyme in Arabidopsis (Arabidopsis thaliana). Previous studies showed that PKL promotes embryonic-to-vegetative transition by inhibiting expression of seed-specific genes during seed germination. The pkl mutants display a low penetrance of the "pickle root" phenotype, with a thick and green primary root that retains embryonic characteristics. The penetrance of this pickle root phenotype in pkl is dramatically increased in gibberellin (GA)-deficient conditions. At adult stages, the pkl mutants are semidwarfs with delayed flowering time, which resemble reduced GA-signaling mutants. These findings suggest that PKL may play a positive role in regulating GA signaling. A recent biochemical analysis further showed that PKL and GA signaling repressors DELLAs antagonistically regulate hypocotyl cell elongation genes by direct protein-protein interaction. To elucidate further the role of PKL in GA signaling and plant development, we studied the genetic interaction between PKL and DELLAs using the hextuple mutant containing pkl and della pentuple (dP) mutations. Here, we show that PKL is required for most of GA-promoted developmental processes, including vegetative growth such as hypocotyl, leaf, and inflorescence stem elongation, and phase transitions such as juvenile-to-adult leaf and vegetative-to-reproductive phase. The removal of all DELLA functions (in the dP background) cannot rescue these phenotypes in pkl RNA-sequencing analysis using the ga1 (a GA-deficient mutant), pkl, and the ga1 pkl double mutant further shows that expression of 80% of GA-responsive genes in seedlings is PKL dependent, including genes that function in cell elongation, cell division, and phase transitions. These results indicate that the CHD3 chromatin remodeler PKL is required for regulating gene expression during most of GA-regulated developmental processes.

  11. SWR-C and INO80 chromatin remodelers recognize nucleosome-free regions near +1 nucleosomes.

    PubMed

    Yen, Kuangyu; Vinayachandran, Vinesh; Pugh, B Franklin

    2013-09-12

    SWR-C/SWR1 and INO80 are multisubunit complexes that catalyze the deposition and removal, respectively, of histone variant H2A.Z from the first nucleosome at the start of genes. How they target and engage these +1 nucleosomes is unclear. Using ChIP-exo, we identified the subnucleosomal placement of 20 of their subunits across the yeast genome. The Swc2 subunit of SWR-C bound a narrowly defined region in the adjacent nucleosome-free region (NFR), where it positioned the Swr1 subunit over one of two sites of H2A.Z deposition at +1. The genomic binding maps suggest that many subunits have a rather plastic organization that allows subunits to exchange between the two complexes. One outcome of promoting H2A/H2A.Z exchange was an enhanced turnover of entire nucleosomes, thereby creating dynamic chromatin at the start of genes. Our findings provide unifying concepts on how these two opposing chromatin remodeling complexes function selectively at the +1 nucleosome of nearly all genes.

  12. A Mutation in the Mouse Chd2 Chromatin Remodeling Enzyme Results in a Complex Renal Phenotype

    PubMed Central

    Marfella, Concetta G.A.; Henninger, Nils; LeBlanc, Scott E.; Krishnan, Namrata; Garlick, David S.; Holzman, Lawrence B.; Imbalzano, Anthony N.

    2009-01-01

    Background and Aims Glomerular diseases are the third leading cause of kidney failure worldwide, behind only diabetes and hypertension. The molecular mechanisms underlying the cause of glomerular diseases are still largely unknown. The identification and characterization of new molecules associated with glomerular function should provide new insights into understanding the diverse group of glomerular diseases. The Chd2 protein belongs to a family of enzymes involved in ATP-dependent chromatin remodeling, suggesting that it likely functions as an epigenetic regulator of gene expression via the modification of chromatin structure. Methods In this study, we present a detailed histomorphologic characterization of mice containing a mutation in the chromodomain helicase DNA-binding protein 2 (Chd2). Results We show that Chd2-mutant mice present with glomerulopathy, proteinuria, and significantly impaired kidney function. Additionally, serum analysis revealed decreased hemoglobin and hematocrit levels in Chd2-mutant mice, suggesting that the glomerulopathy observed in these mice is associated with anemia. Conclusion Collectively, the data suggest a role for the Chd2 protein in the maintenance of kidney function. PMID:19142019

  13. Chromatin remodeling as a mechanism for circadian prolactin transcription: rhythmic NONO and SFPQ recruitment to HLTF.

    PubMed

    Guillaumond, Fabienne; Boyer, Benedicte; Becquet, Denis; Guillen, Severine; Kuhn, Lauriane; Garin, Jerome; Belghazi, Maya; Bosler, Olivier; Franc, Jean-Louis; François-Bellan, Anne-Marie

    2011-08-01

    Most clock-controlled genes (CCGs) lack the specific E-box response element necessary for direct circadian regulation. This is the case for the prolactin (Prl) gene, the expression of which oscillates in individual lactotrope pituitary cells. To characterize the processes underlying this oscillation, we used a lactotrope cell line (GH4C1 cells). In these cells, Prl gene expression fluctuated significantly during 24 h (P=0.0418). Circadian Prl transcription depended on an interaction between the pituitary-specific transcription factor, PIT-1, and the helicase-like transcription factor (HLTF), a SWI/SNF chromatin remodeler, shown here to bind the Prl promoter on an E-box that differs from the specific E-box preferentially bound by clock proteins. Circadian Prl transcription was further accompanied by marked daily chromatin transitions. While neither HLTF nor PIT-1 was rhythmically expressed, NONO and SFPQ, identified as HLTF-associated proteins by mass spectrometry, displayed a circadian pattern and bound rhythmically to the Prl promoter. Furthermore, NONO and SFPQ were functionally involved in circadian Prl transcription since overexpression of both proteins greatly reduced Prl promoter activity (P<0.001) and disrupted its circadian pattern. A mechanism involving a rhythm in paraspeckle protein recruitment is proposed to explain how the core oscillator can generate a circadian pattern of CCGs lacking the specific E-box response element.

  14. The SWI/SNF chromatin remodelling complex is required for maintenance of lineage specific enhancers

    PubMed Central

    Alver, Burak H.; Kim, Kimberly H.; Lu, Ping; Wang, Xiaofeng; Manchester, Haley E.; Wang, Weishan; Haswell, Jeffrey R.; Park, Peter J.; Roberts, Charles W. M.

    2017-01-01

    Genes encoding subunits of SWI/SNF (BAF) chromatin remodelling complexes are collectively altered in over 20% of human malignancies, but the mechanisms by which these complexes alter chromatin to modulate transcription and cell fate are poorly understood. Utilizing mouse embryonic fibroblast and cancer cell line models, here we show via ChIP-seq and biochemical assays that SWI/SNF complexes are preferentially targeted to distal lineage specific enhancers and interact with p300 to modulate histone H3 lysine 27 acetylation. We identify a greater requirement for SWI/SNF at typical enhancers than at most super-enhancers and at enhancers in untranscribed regions than in transcribed regions. Our data further demonstrate that SWI/SNF-dependent distal enhancers are essential for controlling expression of genes linked to developmental processes. Our findings thus establish SWI/SNF complexes as regulators of the enhancer landscape and provide insight into the roles of SWI/SNF in cellular fate control. PMID:28262751

  15. Protooncogene Ski cooperates with the chromatin-remodeling factor Satb2 in specifying callosal neurons.

    PubMed

    Baranek, Constanze; Dittrich, Manuela; Parthasarathy, Srinivas; Bonnon, Carine Gaiser; Britanova, Olga; Lanshakov, Dmitriy; Boukhtouche, Fatiha; Sommer, Julia E; Colmenares, Clemencia; Tarabykin, Victor; Atanasoski, Suzana

    2012-02-28

    First insights into the molecular programs orchestrating the progression from neural stem cells to cortical projection neurons are emerging. Loss of the transcriptional regulator Ski has been linked to the human 1p36 deletion syndrome, which includes central nervous system defects. Here, we report critical roles for Ski in the maintenance of the neural stem cell pool and the specification of callosal neurons. Ski-deficient callosal neurons lose their identity and ectopically express the transcription factor Ctip2. The misspecified callosal neurons largely fail to form the corpus callosum and instead redirect their axons toward subcortical targets. We identify the chromatin-remodeling factor Satb2 as a partner of Ski, and show that both proteins are required for transcriptional repression of Ctip2 in callosal neurons. We propose a model in which Satb2 recruits Ski to the Ctip2 locus, and Ski attracts histone deacetylases, thereby enabling the formation of a functional nucleosome remodeling and deacetylase repressor complex. Our findings establish a central role for Ski-Satb2 interactions in regulating transcriptional mechanisms of callosal neuron specification.

  16. Protooncogene Ski cooperates with the chromatin-remodeling factor Satb2 in specifying callosal neurons

    PubMed Central

    Baranek, Constanze; Dittrich, Manuela; Parthasarathy, Srinivas; Bonnon, Carine Gaiser; Britanova, Olga; Lanshakov, Dmitriy; Boukhtouche, Fatiha; Sommer, Julia E.; Colmenares, Clemencia; Tarabykin, Victor; Atanasoski, Suzana

    2012-01-01

    First insights into the molecular programs orchestrating the progression from neural stem cells to cortical projection neurons are emerging. Loss of the transcriptional regulator Ski has been linked to the human 1p36 deletion syndrome, which includes central nervous system defects. Here, we report critical roles for Ski in the maintenance of the neural stem cell pool and the specification of callosal neurons. Ski-deficient callosal neurons lose their identity and ectopically express the transcription factor Ctip2. The misspecified callosal neurons largely fail to form the corpus callosum and instead redirect their axons toward subcortical targets. We identify the chromatin-remodeling factor Satb2 as a partner of Ski, and show that both proteins are required for transcriptional repression of Ctip2 in callosal neurons. We propose a model in which Satb2 recruits Ski to the Ctip2 locus, and Ski attracts histone deacetylases, thereby enabling the formation of a functional nucleosome remodeling and deacetylase repressor complex. Our findings establish a central role for Ski–Satb2 interactions in regulating transcriptional mechanisms of callosal neuron specification. PMID:22334647

  17. Essential Role of Chromatin Remodeling Protein Bptf in Early Mouse Embryos and Embryonic Stem Cells

    PubMed Central

    Landry, Joseph; Sharov, Alexei A.; Piao, Yulan; Sharova, Lioudmila V.; Xiao, Hua; Southon, Eileen; Matta, Jennifer; Tessarollo, Lino; Zhang, Ying E.; Ko, Minoru S. H.; Kuehn, Michael R.; Yamaguchi, Terry P.; Wu, Carl

    2008-01-01

    We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor), the largest subunit of NURF (Nucleosome Remodeling Factor) in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf−/− embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf−/− embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo. PMID:18974875

  18. The ISW1 and CHD1 ATP-dependent chromatin remodelers compete to set nucleosome spacing in vivo

    PubMed Central

    Ocampo, Josefina; Chereji, Răzvan V.; Eriksson, Peter R.; Clark, David J.

    2016-01-01

    Adenosine triphosphate-dependent chromatin remodeling machines play a central role in gene regulation by manipulating chromatin structure. Most genes have a nucleosome-depleted region at the promoter and an array of regularly spaced nucleosomes phased relative to the transcription start site. In vitro, the three known yeast nucleosome spacing enzymes (CHD1, ISW1 and ISW2) form arrays with different spacing. We used genome-wide nucleosome sequencing to determine whether these enzymes space nucleosomes differently in vivo. We find that CHD1 and ISW1 compete to set the spacing on most genes, such that CHD1 dominates genes with shorter spacing and ISW1 dominates genes with longer spacing. In contrast, ISW2 plays a minor role, limited to transcriptionally inactive genes. Heavily transcribed genes show weak phasing and extreme spacing, either very short or very long, and are depleted of linker histone (H1). Genes with longer spacing are enriched in H1, which directs chromatin folding. We propose that CHD1 directs short spacing, resulting in eviction of H1 and chromatin unfolding, whereas ISW1 directs longer spacing, allowing H1 to bind and condense the chromatin. Thus, competition between the two remodelers to set the spacing on each gene may result in a highly dynamic chromatin structure. PMID:26861626

  19. The tumour suppressor CHD5 forms a NuRD-type chromatin remodelling complex.

    PubMed

    Kolla, Venkatadri; Naraparaju, Koumudi; Zhuang, Tiangang; Higashi, Mayumi; Kolla, Sriharsha; Blobel, Gerd A; Brodeur, Garrett M

    2015-06-01

    Eukaryotic gene expression is developmentally regulated, in part by chromatin remodelling, and its dysregulation has been linked to cancer. CHD5 (chromodomain helicase DNA-binding protein 5) is a tumour suppressor gene (TSG) that maps to a region of consistent deletion on 1p36.31 in neuroblastomas (NBs) and other tumour types. CHD5 encodes a protein with chromatin remodelling, helicase and DNA-binding motifs that is preferentially expressed in neural and testicular tissues. CHD5 is highly homologous to CHD3 and CHD4, which are the core subunits of nucleosome remodelling and deacetylation (NuRD) complexes. To determine if CHD5 forms a similar complex, we performed studies on nuclear extracts from NBLS, SY5Y (both with endogenous CHD5 expression), NLF (CHD5 null) and NLF cells stably transfected with CHD5 cDNA (wild-type and V5-histidine-tagged). Immunoprecipitation (IP) was performed with either CHD5 antibody or antibody to V5/histidine-tagged protein. We identified NuRD components both by GST-FOG1 (Friend Of GATA1) pull-down and by IP. We also performed MS/MS analysis to confirm the presence of CHD5 or other protein components of the NuRD complex, as well as to identify other novel proteins. CHD5 was clearly associated with all canonical NuRD components, including metastasis-associated protein (MTA)1/2, GATA zinc finger domain containing 2A (GATAD2A), histone deacetylase (HDAC)1/2, retinoblastoma-binding protein (RBBP)4/7 and methyl DNA-binding domain protein (MBD)2/3, as determined by Western blotting and MS/MS. Our data suggest CHD5 forms a NuRD complex similar to CHD4. However, CHD5-NuRD may also have unique protein associations that confer functional specificity and may contribute to normal development and to tumour suppression in NB and other cancers.

  20. Histone H3 lysine 14 (H3K14) acetylation facilitates DNA repair in a positioned nucleosome by stabilizing the binding of the chromatin Remodeler RSC (Remodels Structure of Chromatin).

    PubMed

    Duan, Ming-Rui; Smerdon, Michael J

    2014-03-21

    Histone H3 acetylation is induced by UV damage in yeast and may play an important role in regulating the repair of UV photolesions in nucleosome-loaded genomic loci. However, it remains elusive how H3 acetylation facilitates repair. We generated a strongly positioned nucleosome containing homogeneously acetylated H3 at Lys-14 (H3K14ac) and investigated possible mechanisms by which H3K14 acetylation modulates repair. We show that H3K14ac does not alter nucleosome unfolding dynamics or enhance the repair of UV-induced cyclobutane pyrimidine dimers by UV photolyase. Importantly, however, nucleosomes with H3K14ac have a higher affinity for purified chromatin remodeling complex RSC (Remodels the Structure of Chromatin) and show greater cyclobutane pyrimidine dimer repair compared with unacetylated nucleosomes. Our study indicates that, by anchoring RSC, H3K14 acetylation plays an important role in the unfolding of strongly positioned nucleosomes during repair of UV damage.

  1. SHORT HYPOCOTYL1 Encodes a SMARCA3-Like Chromatin Remodeling Factor Regulating Elongation1[OPEN

    PubMed Central

    Bo, Kailiang; Behera, Tusar K.; Pandey, Sudhakar; Wen, Changlong; Wang, Yuhui; Simon, Philipp W.; Li, Yuhong

    2016-01-01

    In Arabidopsis (Arabidopsis thaliana), the UVR8-mediated signaling pathway is employed to attain UVB protection and acclimation to deal with low-dosage UVB (LDUVB)-induced stresses. Here, we identified SHORT HYPOCOTYL1 (SH1) in cucumber (Cucumis sativus), which regulates LDUVB-dependent hypocotyl elongation by modulating the UVR8 signaling pathway. We showed that hypocotyl elongation in cucumbers carrying the recessive sh1 allele was LDUVB insensitive and that Sh1 encoded a human SMARCA3-like chromatin remodeling factor. The allele frequency and distribution pattern at this locus among natural populations supported the wild cucumber origin of sh1 for local adaptation, which was under selection during domestication. The cultivated cucumber carries predominantly the Sh1 allele; the sh1 allele is nearly fixed in the semiwild Xishuangbanna cucumber, and the wild cucumber population is largely at Hardy-Weinberg equilibrium for the two alleles. The SH1 protein sequence was highly conserved among eukaryotic organisms, but its regulation of hypocotyl elongation in cucumber seems to be a novel function. While Sh1 expression was inhibited by LDUVB, its transcript abundance was highly correlated with hypocotyl elongation rate and the expression level of cell-elongation-related genes. Expression profiling of key regulators in the UVR8 signaling pathway revealed significant differential expression of CsHY5 between two near isogenic lines of Sh1. Sh1 and CsHY5 acted antagonistically at transcriptional level. A working model was proposed in which Sh1 regulates LDUVB-dependent hypocotyl elongation in cucumber through changing the chromatin states and thus the accessibility of CsHY5 in the UVR8 signaling pathway to promoters of LDUVB-responsive genes for hypocotyl elongation. PMID:27559036

  2. Mutations and Modeling of the Chromatin Remodeler CHD8 Define an Emerging Autism Etiology

    PubMed Central

    Barnard, Rebecca A.; Pomaville, Matthew B.; O'Roak, Brian J.

    2015-01-01

    Autism Spectrum Disorder (ASD) is a common neurodevelopmental disorder with a strong but complex genetic component. Recent family based exome-sequencing strategies have identified recurrent de novo mutations at specific genes, providing strong evidence for ASD risk, but also highlighting the extreme genetic heterogeneity of the disorder. However, disruptions in these genes converge on key molecular pathways early in development. In particular, functional enrichment analyses have found that there is a bias toward genes involved in transcriptional regulation, such as chromatin modifiers. Here we review recent genetic, animal model, co-expression network, and functional genomics studies relating to the high confidence ASD risk gene, CHD8. CHD8, a chromatin remodeling factor, may serve as a “master regulator” of a common ASD etiology. Individuals with a CHD8 mutation show an ASD subtype that includes similar physical characteristics, such as macrocephaly and prolonged GI problems including recurrent constipation. Similarly, animal models of CHD8 disruption exhibit enlarged head circumference and reduced gut motility phenotypes. Systems biology approaches suggest CHD8 and other candidate ASD risk genes are enriched during mid-fetal development, which may represent a critical time window in ASD etiology. Transcription and CHD8 binding site profiles from cell and primary tissue models of early development indicate that CHD8 may also positively regulate other candidate ASD risk genes through both direct and indirect means. However, continued study is needed to elucidate the mechanism of regulation as well as identify which CHD8 targets are most relevant to ASD risk. Overall, these initial studies suggest the potential for common ASD etiologies and the development of personalized treatments in the future. PMID:26733790

  3. SHORT HYPOCOTYL1 Encodes a SMARCA3-Like Chromatin Remodeling Factor Regulating Elongation.

    PubMed

    Bo, Kailiang; Wang, Hui; Pan, Yupeng; Behera, Tusar K; Pandey, Sudhakar; Wen, Changlong; Wang, Yuhui; Simon, Philipp W; Li, Yuhong; Chen, Jinfeng; Weng, Yiqun

    2016-10-01

    In Arabidopsis (Arabidopsis thaliana), the UVR8-mediated signaling pathway is employed to attain UVB protection and acclimation to deal with low-dosage UVB (LDUVB)-induced stresses. Here, we identified SHORT HYPOCOTYL1 (SH1) in cucumber (Cucumis sativus), which regulates LDUVB-dependent hypocotyl elongation by modulating the UVR8 signaling pathway. We showed that hypocotyl elongation in cucumbers carrying the recessive sh1 allele was LDUVB insensitive and that Sh1 encoded a human SMARCA3-like chromatin remodeling factor. The allele frequency and distribution pattern at this locus among natural populations supported the wild cucumber origin of sh1 for local adaptation, which was under selection during domestication. The cultivated cucumber carries predominantly the Sh1 allele; the sh1 allele is nearly fixed in the semiwild Xishuangbanna cucumber, and the wild cucumber population is largely at Hardy-Weinberg equilibrium for the two alleles. The SH1 protein sequence was highly conserved among eukaryotic organisms, but its regulation of hypocotyl elongation in cucumber seems to be a novel function. While Sh1 expression was inhibited by LDUVB, its transcript abundance was highly correlated with hypocotyl elongation rate and the expression level of cell-elongation-related genes. Expression profiling of key regulators in the UVR8 signaling pathway revealed significant differential expression of CsHY5 between two near isogenic lines of Sh1 Sh1 and CsHY5 acted antagonistically at transcriptional level. A working model was proposed in which Sh1 regulates LDUVB-dependent hypocotyl elongation in cucumber through changing the chromatin states and thus the accessibility of CsHY5 in the UVR8 signaling pathway to promoters of LDUVB-responsive genes for hypocotyl elongation.

  4. The mammalian INO80 chromatin remodeling complex is required for replication stress recovery

    PubMed Central

    Vassileva, Ivelina; Yanakieva, Iskra; Peycheva, Michaela; Gospodinov, Anastas; Anachkova, Boyka

    2014-01-01

    A number of studies have implicated the yeast INO80 chromatin remodeling complex in DNA replication, but the function of the human INO80 complex during S phase remains poorly understood. Here, we have systematically investigated the involvement of the catalytic subunit of the human INO80 complex during unchallenged replication and under replication stress by following the effects of its depletion on cell survival, S-phase checkpoint activation, the fate of individual replication forks, and the consequences of fork collapse. We report that INO80 was specifically needed for efficient replication elongation, while it was not required for initiation of replication. In the absence of the Ino80 protein, cells became hypersensitive to hydroxyurea and displayed hyperactive ATR-Chk1 signaling. Using bulk and fiber labeling of DNA, we found that cells deficient for Ino80 and Arp8 had impaired replication restart after treatment with replication inhibitors and accumulated double-strand breaks as evidenced by the formation of γ-H2AX and Rad51 foci. These data indicate that under conditions of replication stress mammalian INO80 protects stalled forks from collapsing and allows their subsequent restart. PMID:25016522

  5. Chromatin remodeling gene EZH2 involved in the genetic etiology of autism in Chinese Han population.

    PubMed

    Li, Jun; You, Yang; Yue, Weihua; Yu, Hao; Lu, Tianlan; Wu, Zhiliu; Jia, Meixiang; Ruan, Yanyan; Liu, Jing; Zhang, Dai; Wang, Lifang

    2016-01-01

    Autism spectrum disorder (ASD) is a group of severe neurodevelopmental disorders. Epigenetic factors play a critical role in the etiology of ASD. Enhancer of zest homolog 2 (EZH2), which encodes a histone methyltransferase, plays an important role in the process of chromatin remodeling during neurodevelopment. Further, EZH2 is located in chromosome 7q35-36, which is one of the linkage regions for autism. However, the genetic relationship between autism and EZH2 remains unclear. To investigate the association between EZH2 and autism in Chinese Han population, we performed a family-based association study between autism and three tagged single nucleotide polymorphisms (SNPs) that covered 95.4% of the whole region of EZH2. In the discovery cohort of 239 trios, two SNPs (rs740949 and rs6464926) showed a significant association with autism. To decrease false positive results, we expanded the sample size to 427 trios. A SNP (rs6464926) was significantly associated with autism even after Bonferroni correction (p=0.008). Haplotype G-T (rs740949 and rs6464926) was a risk factor for autism (Z=2.655, p=0.008, Global p=0.024). In silico function prediction for SNPs indicated that these two SNPs might be regulatory SNPs. Expression pattern of EZH2 showed that it is highly expressed in human embryonic brains. In conclusion, our findings demonstrate that EZH2 might contribute to the genetic etiology of autism in Chinese Han population.

  6. Molecular architecture of the ATP-dependent chromatin-remodeling complex SWR1.

    PubMed

    Nguyen, Vu Q; Ranjan, Anand; Stengel, Florian; Wei, Debbie; Aebersold, Ruedi; Wu, Carl; Leschziner, Andres E

    2013-09-12

    The ATP-dependent chromatin-remodeling complex SWR1 exchanges a variant histone H2A.Z/H2B dimer for a canonical H2A/H2B dimer at nucleosomes flanking histone-depleted regions, such as promoters. This localization of H2A.Z is conserved throughout eukaryotes. SWR1 is a 1 megadalton complex containing 14 different polypeptides, including the AAA+ ATPases Rvb1 and Rvb2. Using electron microscopy, we obtained the three-dimensional structure of SWR1 and mapped its major functional components. Our data show that SWR1 contains a single heterohexameric Rvb1/Rvb2 ring that, together with the catalytic subunit Swr1, brackets two independently assembled multisubunit modules. We also show that SWR1 undergoes a large conformational change upon engaging a limited region of the nucleosome core particle. Our work suggests an important structural role for the Rvbs and a distinct substrate-handling mode by SWR1, thereby providing a structural framework for understanding the complex dimer-exchange reaction.

  7. Camk2a-Cre-mediated conditional deletion of chromatin remodeler Brg1 causes perinatal hydrocephalus.

    PubMed

    Cao, Mou; Wu, Jiang I

    2015-06-15

    Mammalian SWI/SNF-like BAF chromatin remodeling complexes are essential for many aspects of neural development. Mutations in the genes encoding the core subunit Brg1/SmarcA4 or other complex components cause neurodevelopmental diseases and are associated with autism. Congenital hydrocephalus is a serious brain disorder often experienced by these patients. We report a role of Brg1 in the pathogenesis of hydrocephalus disorder. We discovered an unexpected early activity of mouse Camk2a-Cre transgene, which mediates Brg1 deletion in a subset of forebrain neurons beginning in the late embryonic stage. Brg1 deletion in these neurons led to severe congenital hydrocephalus with enlargement of the lateral ventricles and attenuation of the cerebral cortex. The Brg1-deficient mice had significantly smaller subcommissural organs and narrower Sylvian aqueducts than mice that express normal levels of Brg1. Effects were non-cell autonomous and may be responsible for the development of the congenital hydrocephalus phenotype. Our study provides evidence indicating that abnormalities in Brg1 function result in defects associated with neurodevelopmental disorders and autism.

  8. The chromatin remodeler DDM1 promotes hybrid vigor by regulating salicylic acid metabolism.

    PubMed

    Zhang, Qingzhu; Li, Yanqiang; Xu, Tao; Srivastava, Ashish Kumar; Wang, Dong; Zeng, Liang; Yang, Lan; He, Li; Zhang, Heng; Zheng, Zhimin; Yang, Dong-Lei; Zhao, Cheng; Dong, Juan; Gong, Zhizhong; Liu, Renyi; Zhu, Jian-Kang

    2016-01-01

    In plants, hybrid vigor is influenced by genetic and epigenetic mechanisms; however, the molecular pathways are poorly understood. We investigated the potential contributions of epigenetic regulators to heterosis in Arabidposis and found that the chromatin remodeler DECREASED DNA METHYLATION 1 (DDM1) affects early seedling growth heterosis in Col/C24 hybrids. ddm1 mutants showed impaired heterosis and increased expression of non-additively expressed genes related to salicylic acid metabolism. Interestingly, our data suggest that salicylic acid is a hormetic regulator of seedling growth heterosis, and that hybrid vigor arises from crosses that produce optimal salicylic acid levels. Although DNA methylation failed to correlate with differential non-additively expressed gene expression, we uncovered DDM1 as an epigenetic link between salicylic acid metabolism and heterosis, and propose that the endogenous salicylic acid levels of parental plants can be used to predict the heterotic outcome. Salicylic acid protects plants from pathogens and abiotic stress. Thus, our findings suggest that stress-induced hormesis, which has been associated with increased longevity in other organisms, may underlie specific hybrid vigor traits.

  9. Genomic and proteomic characterization of ARID1A chromatin remodeller in ampullary tumors

    PubMed Central

    Nastase, Anca; Teo, Jin Yao; Heng, Hong Lee; Ng, Cedric Chuan Young; Myint, Swe Swe; Rajasegaran, Vikneswari; Loh, Jia Liang; Lee, Ser Yee; Ooi, London Lucien; Chung, Alexander Yaw Fui; Chow, Pierce Kah Hoe; Cheow, Peng Chung; Wan, Wei Keat; Azhar, Rafy; Khoo, Avery; Xiu, Sam Xin; Alkaff, Syed Muhammad Fahmy; Cutcutache, Ioana; Lim, Jing Quan; Ong, Choon Kiat; Herlea, Vlad; Dima, Simona; Duda, Dan G; Teh, Bin Tean; Popescu, Irinel; Lim, Tony Kiat Hon

    2017-01-01

    AT rich interactive domain 1A (ARID1A) is one of the most commonly mutated genes in a broad variety of tumors. The mechanisms that involve ARID1A in ampullary cancer progression remains elusive. Here, we evaluated the frequency of ARID1A and KRAS mutations in ampullary adenomas and adenocarcinomas and in duodenal adenocarcinomas from two cohorts of patients from Singapore and Romania, correlated with clinical and pathological tumor features, and assessed the functional role of ARID1A. In the ampullary adenocarcinomas, the frequency of KRAS and ARID1A mutations was 34.7% and 8.2% respectively, with a loss or reduction of ARID1A protein in 17.2% of the cases. ARID1A mutational status was significantly correlated with ARID1A protein expression level (P=0.023). There was a significant difference in frequency of ARID1A mutation between Romania and Singapore (2.7% versus 25%, P=0.04), suggestive of different etiologies. One somatic mutation was detected in the ampullary adenoma group. In vitro studies indicated the tumor suppressive role of ARID1A. Our results warrant further investigation of this chromatin remodeller as a potential early biomarker of the disease, as well as identification of therapeutic targets in ARID1A mutated ampullary cancers.

  10. Regulation of Vegetative Phase Change by SWI2/SNF2 Chromatin Remodeling ATPase BRAHMA.

    PubMed

    Xu, Yunmin; Guo, Changkui; Zhou, Bingying; Li, Chenlong; Wang, Huasen; Zheng, Ben; Ding, Han; Zhu, Zhujun; Peragine, Angela; Cui, Yuhai; Poethig, Scott; Wu, Gang

    2016-12-01

    Plants progress from a juvenile vegetative phase of development to an adult vegetative phase of development before they enter the reproductive phase. miR156 has been shown to be the master regulator of the juvenile-to-adult transition in plants. However, the mechanism of how miR156 is transcriptionally regulated still remains elusive. In a forward genetic screen, we identified that a mutation in the SWI2/SNF2 chromatin remodeling ATPase BRAHMA (BRM) exhibited an accelerated vegetative phase change phenotype by reducing the expression of miR156, which in turn caused a corresponding increase in the levels of SQUAMOSA PROMOTER BINDING PROTEIN LIKE genes. BRM regulates miR156 expression by directly binding to the MIR156A promoter. Mutations in BRM not only increased occupancy of the -2 and +1 nucleosomes proximal to the transcription start site at the MIR156A locus but also the levels of trimethylated histone H3 at Lys 27. The precocious phenotype of brm mutant was partially suppressed by a second mutation in SWINGER (SWN), but not by a mutation in CURLEY LEAF, both of which are key components of the Polycomb Group Repressive Complex 2 in plants. Our results indicate that BRM and SWN act antagonistically at the nucleosome level to fine-tune the temporal expression of miR156 to regulate vegetative phase change in Arabidopsis.

  11. The chromatin remodeler DDM1 promotes hybrid vigor by regulating salicylic acid metabolism

    PubMed Central

    Zhang, Qingzhu; Li, Yanqiang; Xu, Tao; Srivastava, Ashish Kumar; Wang, Dong; Zeng, Liang; Yang, Lan; He, Li; Zhang, Heng; Zheng, Zhimin; Yang, Dong-Lei; Zhao, Cheng; Dong, Juan; Gong, Zhizhong; Liu, Renyi; Zhu, Jian-Kang

    2016-01-01

    In plants, hybrid vigor is influenced by genetic and epigenetic mechanisms; however, the molecular pathways are poorly understood. We investigated the potential contributions of epigenetic regulators to heterosis in Arabidposis and found that the chromatin remodeler DECREASED DNA METHYLATION 1 (DDM1) affects early seedling growth heterosis in Col/C24 hybrids. ddm1 mutants showed impaired heterosis and increased expression of non-additively expressed genes related to salicylic acid metabolism. Interestingly, our data suggest that salicylic acid is a hormetic regulator of seedling growth heterosis, and that hybrid vigor arises from crosses that produce optimal salicylic acid levels. Although DNA methylation failed to correlate with differential non-additively expressed gene expression, we uncovered DDM1 as an epigenetic link between salicylic acid metabolism and heterosis, and propose that the endogenous salicylic acid levels of parental plants can be used to predict the heterotic outcome. Salicylic acid protects plants from pathogens and abiotic stress. Thus, our findings suggest that stress-induced hormesis, which has been associated with increased longevity in other organisms, may underlie specific hybrid vigor traits. PMID:27551435

  12. The INO80 Complex Requires the Arp5-Ies6 Subcomplex for Chromatin Remodeling and Metabolic Regulation

    PubMed Central

    Yao, Wei; King, Devin A.; Beckwith, Sean L.; Gowans, Graeme J.; Yen, Kuangyu; Zhou, Coral

    2016-01-01

    ATP-dependent chromatin remodeling complexes are essential for transcription regulation, and yet it is unclear how these multisubunit complexes coordinate their activities to facilitate diverse transcriptional responses. In this study, we found that the conserved Arp5 and Ies6 subunits of the Saccharomyces cerevisiae INO80 chromatin-remodeler form an abundant and distinct subcomplex in vivo and stimulate INO80-mediated activity in vitro. Moreover, our genomic studies reveal that the relative occupancy of Arp5-Ies6 correlates with nucleosome positioning at transcriptional start sites and expression levels of >1,000 INO80-regulated genes. Notably, these genes are significantly enriched in energy metabolism pathways. Specifically, arp5Δ, ies6Δ, and ino80Δ mutants demonstrate decreased expression of genes involved in glycolysis and increased expression of genes in the oxidative phosphorylation pathway. Deregulation of these metabolic pathways results in constitutively elevated mitochondrial potential and oxygen consumption. Our results illustrate the dynamic nature of the INO80 complex assembly and demonstrate for the first time that a chromatin remodeler regulates glycolytic and respiratory capacity, thereby maintaining metabolic stability. PMID:26755556

  13. Histone density is maintained during transcription mediated by the chromatin remodeler RSC and histone chaperone NAP1 in vitro

    PubMed Central

    Kuryan, Benjamin G.; Kim, Jessica; Tran, Nancy Nga H.; Lombardo, Sarah R.; Venkatesh, Swaminathan; Workman, Jerry L.; Carey, Michael

    2012-01-01

    ATPases and histone chaperones facilitate RNA polymerase II (pol II) elongation on chromatin. In vivo, the coordinated action of these enzymes is necessary to permit pol II passage through a nucleosome while restoring histone density afterward. We have developed a biochemical system recapitulating this basic process. Transcription through a nucleosome in vitro requires the ATPase remodels structure of chromatin (RSC) and the histone chaperone nucleosome assembly protein 1 (NAP1). In the presence of NAP1, RSC generates a hexasome. Despite the propensity of RSC to evict histones, NAP1 reprograms the reaction such that the hexasome is retained on the template during multiple rounds of transcription. This work has implications toward understanding the mechanism of pol II elongation on chromatin. PMID:22308335

  14. Chromatin remodelling and DNA repair genes are frequently mutated in endometrioid endometrial carcinoma.

    PubMed

    García-Sanz, Pablo; Triviño, Juan Carlos; Mota, Alba; Pérez López, María; Colás, Eva; Rojo-Sebastián, Alejandro; García, Ángel; Gatius, Sonia; Ruiz, María; Prat, Jaime; López-López, Rafael; Abal, Miguel; Gil-Moreno, Antonio; Reventós, Jaume; Matias-Guiu, Xavier; Moreno-Bueno, Gema

    2017-04-01

    In developed countries, endometrial carcinoma is the most common cancer that affects the female genital tract. Endometrial carcinoma is divided into two main histological types, type I or endometrioid and type II or non-endometrioid, each of which have characteristic, although not exclusive, molecular alterations and mutational profiles. Nevertheless, information about the implication and relevance of some of these genes in this disease is lacking. We sought here to identify new recurrently mutated genes in endometrioid cancers that play a role in tumourigenesis and that influence the clinical outcome. We focused on low-grade, non-ultramutated tumours as these tumours have a worse prognosis than the ultramutated POLE-positive endometrioid endometrial carcinomas (EECs). We performed exome-sequencing of 11 EECs with matched normal tissue and subsequently validated 15 candidate genes in 76 samples. For the first time, we show that mutations in chromatin remodelling-related genes (KMT2D, KMT2C, SETD1B and BCOR) and in DNA-repair-related genes (BRCA1, BRCA2, RAD50 and CHD4) are frequent in this subtype of endometrial cancer. The alterations to these genes occurred with frequencies ranging from 35.5% for KMT2D to 10.5% for BRCA1 and BCOR, with some showing a tendency toward co-occurrence (RAD50-KMT2D and RAD50-SETD1B). All these genes harboured specific mutational hotspots. In addition, the mutational status of KMT2C, KMT2D and SETD1B helps to predict the degree of myometrial invasion, a critical prognostic feature. These results highlight the possible implication of these genes in this disease, creating opportunities for new therapeutic approaches.

  15. Histone H1 Phosphorylation by Cdk2 Selectively Modulates Mouse Mammary Tumor Virus Transcription through Chromatin Remodeling

    PubMed Central

    Bhattacharjee, Rabindra N.; Banks, Geoffrey C.; Trotter, Kevin W.; Lee, Huay-Leng; Archer, Trevor K.

    2001-01-01

    Transcriptional activation of the mouse mammary tumor virus (MMTV) promoter by ligand-bound glucocorticoid receptor (GR) is transient. Previously, we demonstrated that prolonged hormone exposure results in displacement of the transcription factor nuclear factor 1 (NF1) and the basal transcription complex from the promoter, the dephosphorylation of histone H1, and the establishment of a repressive chromatin structure. We have explored the mechanistic link between histone H1 dephosphorylation and silencing of the MMTV promoter by describing the putative kinase responsible for H1 phosphorylation. Both in vitro kinase assays and in vivo protein expression studies suggest that in hormone-treated cells the ability of cdk2 to phosphorylate histone H1 is decreased and the cdk2 inhibitory p21 protein level is increased. To address the role of cdk2 and histone H1 dephosphorylation in the silencing of the MMTV promoter, we used potent cdk2 inhibitors, Roscovitine and CVT-313, to generate an MMTV promoter which is associated predominantly with the dephosphorylated form of histone H1. Both Roscovitine and CVT-313 block phosphorylation of histone H1 and, under these conditions, the GR is unable to remodel chromatin, recruit transcription factors to the promoter, or stimulate MMTV mRNA accumulation. These results suggest a model where cdk2-directed histone H1 phosphorylation is a necessary condition to permit GR-mediated chromatin remodeling and activation of the MMTV promoter in vivo. PMID:11463824

  16. SWI/SNF chromatin remodeling enzymes are associated with cardiac hypertrophy in a genetic rat model of hypertension.

    PubMed

    Mehrotra, Aanchal; Joe, Bina; de la Serna, Ivana L

    2013-12-01

    Pathological cardiac hypertrophy is characterized by a sustained increase in cardiomyocyte size and re-activation of the fetal cardiac gene program. Previous studies implicated SWI/SNF chromatin remodeling enzymes as regulators of the fetal cardiac gene program in surgical models of cardiac hypertrophy. Although hypertension is a common risk factor for developing cardiac hypertrophy, there has not yet been any investigation into the role of SWI/SNF enzymes in cardiac hypertrophy using genetic models of hypertension. In this study, we tested the hypothesis that components of the SWI/SNF complex are activated and recruited to promoters that regulate the fetal cardiac gene program in hearts that become hypertrophic as a result of salt induced hypertension. Utilizing the Dahl salt-sensitive (S) rat model, we found that the protein levels of several SWI/SNF subunits required for heart development, Brg1, Baf180, and Baf60c, are elevated in hypertrophic hearts from S rats fed a high salt diet compared with normotensive hearts from Dahl salt-resistant (R) rats fed the same diet. Furthermore, we detected significantly higher levels of SWI/SNF subunit enrichment as well as evidence of more accessible chromatin structure on two fetal cardiac gene promoters in hearts from S rats compared with R rats. Our data implicate SWI/SNF chromatin remodeling enzymes as regulators of gene expression in cardiac hypertrophy resulting from salt induced hypertension. Thus we provide novel insights into the epigenetic mechanisms by which salt induced hypertension leads to cardiac hypertrophy.

  17. Distinct roles for SWR1 and INO80 chromatin remodeling complexes at chromosomal double-strand breaks

    PubMed Central

    van Attikum, Haico; Fritsch, Olivier; Gasser, Susan M

    2007-01-01

    INO80 and SWR1 are two closely related ATP-dependent chromatin remodeling complexes that share several subunits. Ino80 was reported to be recruited to the HO endonuclease-induced double-strand break (DSB) at the budding yeast mating-type locus, MAT. We find Swr1 similarly recruited in a manner dependent on the phosphorylation of H2A (γH2AX). This is not unique to cleavage at MAT; both Swr1 and Ino80 bind near an induced DSB on chromosome XV. Whereas Swr1 incorporates the histone variant H2A.Z into chromatin at promoters, H2A.Z levels do not increase at DSBs. Instead, H2A.Z, γH2AX and core histones are coordinately removed near the break in an INO80-dependent, but SWR1-independent, manner. Mutations in INO80-specific subunits Arp8 or Nhp10 impair the binding of Mre11 nuclease, yKu80 and ATR-related Mec1 kinase at the DSB, resulting in defective end-processing and checkpoint activation. In contrast, Mre11 binding, end-resection and checkpoint activation were normal in the swr1 strain, but yKu80 loading and error-free end-joining were impaired. Thus, these two related chromatin remodelers have distinct roles in DSB repair and checkpoint activation. PMID:17762868

  18. The ATP-dependent chromatin remodeling enzyme Fun30 represses transcription by sliding promoter-proximal nucleosomes.

    PubMed

    Byeon, Boseon; Wang, Wei; Barski, Artem; Ranallo, Ryan T; Bao, Kan; Schones, Dustin E; Zhao, Keji; Wu, Carl; Wu, Wei-Hua

    2013-08-09

    The evolutionarily conserved ATP-dependent chromatin remodeling enzyme Fun30 has recently been shown to play important roles in heterochromatin silencing and DNA repair. However, how Fun30 remodels nucleosomes is not clear. Here we report a nucleosome sliding activity of Fun30 and its role in transcriptional repression. We observed that Fun30 repressed the expression of genes involved in amino acid and carbohydrate metabolism, the stress response, and meiosis. In addition, Fun30 was localized at the 5' and 3' ends of genes and within the open reading frames of its targets. Consistent with its role in gene repression, we observed that Fun30 target genes lacked histone modifications often associated with gene activation and showed an increased level of ubiquitinated histone H2B. Furthermore, a genome-wide nucleosome mapping analysis revealed that the length of the nucleosome-free region at the 5' end of a subset of genes was changed in Fun30-depleted cells. In addition, the positions of the -1, +2, and +3 nucleosomes at the 5' end of target genes were shifted significantly, whereas the position of the +1 nucleosome remained largely unchanged in the fun30Δ mutant. Finally, we demonstrated that affinity-purified, single-component Fun30 exhibited a nucleosome sliding activity in an ATP-dependent manner. These results define a role for Fun30 in the regulation of transcription and indicate that Fun30 remodels chromatin at the 5' end of genes by sliding promoter-proximal nucleosomes.

  19. Neuron-specific chromatin remodeling: a missing link in epigenetic mechanisms underlying synaptic plasticity, memory, and intellectual disability disorders.

    PubMed

    Vogel-Ciernia, Annie; Wood, Marcelo A

    2014-05-01

    Long-term memory formation requires the coordinated regulation of gene expression. Until recently nucleosome remodeling, one of the major epigenetic mechanisms for controlling gene expression, had been largely unexplored in the field of neuroscience. Nucleosome remodeling is carried out by chromatin remodeling complexes (CRCs) that interact with DNA and histones to physically alter chromatin structure and ultimately regulate gene expression. Human exome sequencing and gene wide association studies have linked mutations in CRC subunits to intellectual disability disorders, autism spectrum disorder and schizophrenia. However, how mutations in CRC subunits were related to human cognitive disorders was unknown. There appears to be both developmental and adult specific roles for the neuron specific CRC nBAF (neuronal Brg1/hBrm Associated Factor). nBAF regulates gene expression required for dendritic arborization during development, and in the adult, contributes to long-term potentiation, a form of synaptic plasticity, and long-term memory. We propose that the nBAF complex is a novel epigenetic mechanism for regulating transcription required for long-lasting forms of synaptic plasticity and memory processes and that impaired nBAF function may result in human cognitive disorders.

  20. Seed dormancy cycling in Arabidopsis: chromatin remodelling and regulation of DOG1 in response to seasonal environmental signals

    PubMed Central

    Footitt, Steven; Müller, Kerstin; Kermode, Allison R; Finch-Savage, William E

    2015-01-01

    The involvement of chromatin remodelling in dormancy cycling in the soil seed bank (SSB) is poorly understood. Natural variation between the winter and summer annual Arabidopsis ecotypes Cvi and Bur was exploited to investigate the expression of genes involved in chromatin remodelling via histone 2B (H2B) ubiquitination/de-ubiquitination and histone acetylation/deacetylation, the repressive histone methyl transferases CURLY LEAF (CLF) and SWINGER (SWN), and the gene silencing repressor ROS1 (REPRESSOR OF SILENCING1) and promoter of silencing KYP/SUVH4 (KRYPTONITE), during dormancy cycling in the SSB. ROS1 expression was positively correlated with dormancy while the reverse was observed for CLF and KYP/SUVH4. We propose ROS1 dependent repression of silencing and a sequential requirement of CLF and KYP/SUVH4 dependent gene repression and silencing for the maintenance and suppression of dormancy during dormancy cycling. Seasonal expression of H2B modifying genes was correlated negatively with temperature and positively with DOG1 expression, as were histone acetyltransferase genes, with histone deacetylases positively correlated with temperature. Changes in the histone marks H3K4me3 and H3K27me3 were seen on DOG1 (DELAY OF GERMINATION1) in Cvi during dormancy cycling. H3K4me3 activating marks remained stable along DOG1. During relief of dormancy, H3K27me3 repressive marks slowly accumulated and accelerated on exposure to light completing dormancy loss. We propose that these marks on DOG1 serve as a thermal sensing mechanism during dormancy cycling in preparation for light repression of dormancy. Overall, chromatin remodelling plays a vital role in temporal sensing through regulation of gene expression. PMID:25439058

  1. Seed dormancy cycling in Arabidopsis: chromatin remodelling and regulation of DOG1 in response to seasonal environmental signals.

    PubMed

    Footitt, Steven; Müller, Kerstin; Kermode, Allison R; Finch-Savage, William E

    2015-02-01

    The involvement of chromatin remodelling in dormancy cycling in the soil seed bank (SSB) is poorly understood. Natural variation between the winter and summer annual Arabidopsis ecotypes Cvi and Bur was exploited to investigate the expression of genes involved in chromatin remodelling via histone 2B (H2B) ubiquitination/de-ubiquitination and histone acetylation/deacetylation, the repressive histone methyl transferases CURLY LEAF (CLF) and SWINGER (SWN), and the gene silencing repressor ROS1 (REPRESSOR OF SILENCING1) and promoter of silencing KYP/SUVH4 (KRYPTONITE), during dormancy cycling in the SSB. ROS1 expression was positively correlated with dormancy while the reverse was observed for CLF and KYP/SUVH4. We propose ROS1 dependent repression of silencing and a sequential requirement of CLF and KYP/SUVH4 dependent gene repression and silencing for the maintenance and suppression of dormancy during dormancy cycling. Seasonal expression of H2B modifying genes was correlated negatively with temperature and positively with DOG1 expression, as were histone acetyltransferase genes, with histone deacetylases positively correlated with temperature. Changes in the histone marks H3K4me3 and H3K27me3 were seen on DOG1 (DELAY OF GERMINATION1) in Cvi during dormancy cycling. H3K4me3 activating marks remained stable along DOG1. During relief of dormancy, H3K27me3 repressive marks slowly accumulated and accelerated on exposure to light completing dormancy loss. We propose that these marks on DOG1 serve as a thermal sensing mechanism during dormancy cycling in preparation for light repression of dormancy. Overall, chromatin remodelling plays a vital role in temporal sensing through regulation of gene expression.

  2. ATM-mediated phosphorylation of the chromatin remodeling enzyme BRG1 modulates DNA double-strand break repair.

    PubMed

    Kwon, S-J; Park, J-H; Park, E-J; Lee, S-A; Lee, H-S; Kang, S W; Kwon, J

    2015-01-15

    ATP-dependent chromatin remodeling complexes such as SWI/SNF (SWItch/Sucrose NonFermentable) have been implicated in DNA double-strand break (DSB) repair and damage responses. However, the regulatory mechanisms that control the function of chromatin remodelers in DNA damage response are largely unknown. Here, we show that ataxia telangiectasia mutated (ATM) mediates the phosphorylation of BRG1, the catalytic ATPase of the SWI/SNF complex that contributes to DSB repair by binding γ-H2AX-containing nucleosomes via interaction with acetylated histone H3 and stimulating γ-H2AX formation, at Ser-721 in response to DNA damage. ATM-mediated phosphorylation of BRG1 occurs rapidly and transiently after DNA damage. Phosphorylated BRG1 binds γ-H2AX-containing nucleosomes to form the repair foci. The Ser-721 phosphorylation of BRG1 is critical for binding γ-H2AX-containing nucleosomes and stimulating γ-H2AX formation and DSB repair. BRG1 binds to acetylated H3 peptides much better after phosphorylation at Ser-721 by DNA damage. However, the phosphorylation of Ser-721 does not significantly affect the ATPase and transcriptional activities of BRG1. These results, establishing BRG1 as a novel and functional ATM substrate, suggest that the ATM-mediated phosphorylation of BRG1 facilitates DSB repair by stimulating the association of this remodeler with γ-H2AX nucleosomes via enhancing the affinity to acetylated H3. Our work also suggests that the mechanism of BRG1 stimulation of DNA repair is independent of the remodeler's enzymatic or transcriptional activities.

  3. MiRNA-Mediated Regulation of the SWI/SNF Chromatin Remodeling Complex Controls Pluripotency and Endodermal Differentiation in Human ESCs.

    PubMed

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

    2015-10-01

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

  4. Stress and the Emerging Roles of Chromatin Remodeling in Signal Integration and Stable Transmission of Reversible Phenotypes

    PubMed Central

    Weaver, Ian C. G.; Korgan, Austin C.; Lee, Kristen; Wheeler, Ryan V.; Hundert, Amos S.; Goguen, Donna

    2017-01-01

    The influence of early life experience and degree of parental-infant attachment on emotional development in children and adolescents has been comprehensively studied. Structural and mechanistic insight into the biological foundation and maintenance of mammalian defensive systems (metabolic, immune, nervous and behavioral) is slowly advancing through the emerging field of developmental molecular (epi)genetics. Initial evidence revealed that differential nurture early in life generates stable differences in offspring hypothalamic-pituitary-adrenal (HPA) regulation, in part, through chromatin remodeling and changes in DNA methylation of specific genes expressed in the brain, revealing physical, biochemical and molecular paths for the epidemiological concept of gene-environment interactions. Herein, a primary molecular mechanism underpinning the early developmental programming and lifelong maintenance of defensive (emotional) responses in the offspring is the alteration of chromatin domains of specific genomic regions from a condensed state (heterochromatin) to a transcriptionally accessible state (euchromatin). Conversely, DNA methylation promotes the formation of heterochromatin, which is essential for gene silencing, genomic integrity and chromosome segregation. Therefore, inter-individual differences in chromatin modifications and DNA methylation marks hold great potential for assessing the impact of both early life experience and effectiveness of intervention programs—from guided psychosocial strategies focused on changing behavior to pharmacological treatments that target chromatin remodeling and DNA methylation enzymes to dietary approaches that alter cellular pools of metabolic intermediates and methyl donors to affect nutrient bioavailability and metabolism. In this review article, we discuss the potential molecular mechanism(s) of gene regulation associated with chromatin modeling and programming of endocrine (e.g., HPA and metabolic or cardiovascular) and

  5. CHD3 chromatin-remodeling factor PICKLE regulates floral transition partially via modulating LEAFY expression at the chromatin level in Arabidopsis.

    PubMed

    Fu, Xing; Li, Chaonan; Liang, Qing; Zhou, Yangyang; He, Hang; Fan, Liu-Min

    2016-05-01

    PICKLE (PKL), a putative CHD3 chromatin remodeling factor, has been suggested to be involved in multiple processes in Arabidopsis. Here, we confirmed the late-flowering phenotype caused by pkl mutation with pkl mutants in two different ecotypes, and investigated the possible mechanisms that account for PKL regulation of flowering time. Quantitative RT-PCR and RNA-seq assays showed that expression of the LEAFY gene (LFY) and a number of LFY-regulated floral homeotic genes were down-regulated in seedlings of the pkl mutants. As predicted, overexpression of LFY restored normal flowering time of pkl mutants. Our results suggest that PKL may be involved in regulating flowering time via LFY expression. To uncover the underlying mechanism, ChIP-PCR using anti-PKL was performed on materials from three developmental stages of seedlings. Our results showed that PKL associated with the genomic sequences of LFY, particularly at 10-day and 25-day after germination. We also showed that loss of PKL affected H3K27me3 level at the promoter of LFY. Taken together, our data suggest that transcriptional regulation of LFY at the chromatin level by PKL may at least partially account for the late-flowering phenotype of pkl mutants.

  6. The forkhead transcription factor FoxI1 remains bound to condensed mitotic chromosomes and stably remodels chromatin structure.

    PubMed

    Yan, Jizhou; Xu, Lisha; Crawford, Gregory; Wang, Zenfeng; Burgess, Shawn M

    2006-01-01

    All forkhead (Fox) proteins contain a highly conserved DNA binding domain whose structure is remarkably similar to the winged-helix structures of histones H1 and H5. Little is known about Fox protein binding in the context of higher-order chromatin structure in living cells. We created a stable cell line expressing FoxI1-green fluorescent protein (GFP) or FoxI1-V5 fusion proteins under control of the reverse tetracycline-controlled transactivator doxycycline inducible system and found that unlike most transcription factors, FoxI1 remains bound to the condensed chromosomes during mitosis. To isolate DNA fragments directly bound by the FoxI1 protein within living cells, we performed chromatin immunoprecipitation assays (ChIPs) with antibodies to either enhanced GFP or the V5 epitope and subcloned the FoxI1-enriched DNA fragments. Sequence analyses indicated that 88% (106/121) of ChIP sequences contain the consensus binding sites for all Fox proteins. Testing ChIP sequences with a quantitative DNase I hypersensitivity assay showed that FoxI1 created stable DNase I sensitivity changes in condensed chromosomes. The majority of ChIP targets and random targets increased in resistance to DNase I in FoxI1-expressing cells, but a small number of targets became more accessible to DNase I. Consistently, the accessibility of micrococcal nuclease to chromatin was generally inhibited. Micrococcal nuclease partial digestion generated a ladder in which all oligonucleosomes were slightly longer than those observed with the controls. On the basis of these findings, we propose that FoxI1 is capable of remodeling chromatin higher-order structure and can stably create site-specific changes in chromatin to either stably create or remove DNase I hypersensitive sites.

  7. Global analysis of SUMO-binding proteins identifies SUMOylation as a key regulator of the INO80 chromatin remodeling complex.

    PubMed

    Cox, Eric; Hwang, Woochang; Uzoma, Ijeoma; Hu, Jianfei; Guzzo, Catherine; Jeong, Junseop; Matunis, Michael; Qian, Jiang; Zhu, Heng; Blackshaw, Seth

    2017-03-02

    SUMOylation is a critical regulator of a broad range of cellular processes, and is thought to do so in part by modulation of protein interaction. To comprehensively identify human proteins whose interaction is modulated by SUMOylation, we developed an in vitro binding assay using human proteome microarrays to identify targets of SUMO1 and SUMO2. We then integrated these results with protein SUMOylation and protein-protein interaction data to perform network motif analysis. We focused on a single network motif we termed a SUMOmodPPI (SUMO-modulated Protein-Protein Interaction) that included the INO80 chromatin remodeling complex subunits TFPT and INO80E. We validated the SUMO-binding activity of INO80E, and showed that TFPT is a SUMO substrate both in vitro and in vivo. We then demonstrated a key role for SUMOylation in mediating the interaction between these two proteins, both in vitro and in vivo. By demonstrating a key role for SUMOylation in regulating the INO80 chromatin-remodeling complex, this work illustrates the power of integrated analysis of large datasets in predicting novel biological phenomena.

  8. Selection on a Subunit of the NURF Chromatin Remodeler Modifies Life History Traits in a Domesticated Strain of Caenorhabditis elegans

    PubMed Central

    Large, Edward E.; Zhao, Yuehui; Long, Lijiang; Butcher, Rebecca A.; Andersen, Erik C.; McGrath, Patrick T.

    2016-01-01

    Evolutionary life history theory seeks to explain how reproductive and survival traits are shaped by selection through allocations of an individual’s resources to competing life functions. Although life-history traits evolve rapidly, little is known about the genetic and cellular mechanisms that control and couple these tradeoffs. Here, we find that two laboratory-adapted strains of C. elegans descended from a single common ancestor that lived in the 1950s have differences in a number of life-history traits, including reproductive timing, lifespan, dauer formation, growth rate, and offspring number. We identified a quantitative trait locus (QTL) of large effect that controls 24%–75% of the total trait variance in reproductive timing at various timepoints. Using CRISPR/Cas9-induced genome editing, we show this QTL is due in part to a 60 bp deletion in the 3’ end of the nurf-1 gene, which is orthologous to the human gene encoding the BPTF component of the NURF chromatin remodeling complex. Besides reproduction, nurf-1 also regulates growth rate, lifespan, and dauer formation. The fitness consequences of this deletion are environment specific—it increases fitness in the growth conditions where it was fixed but decreases fitness in alternative laboratory growth conditions. We propose that chromatin remodeling, acting through nurf-1, is a pleiotropic regulator of life history trade-offs underlying the evolution of multiple traits across different species. PMID:27467070

  9. The RSC chromatin remodeling complex bears an essential fungal-specific protein module with broad functional roles.

    PubMed

    Wilson, Boris; Erdjument-Bromage, Hediye; Tempst, Paul; Cairns, Bradley R

    2006-02-01

    RSC is an essential and abundant ATP-dependent chromatin remodeling complex from Saccharomyces cerevisiae. Here we show that the RSC components Rsc7/Npl6 and Rsc14/Ldb7 interact physically and/or functionally with Rsc3, Rsc30, and Htl1 to form a module important for a broad range of RSC functions. A strain lacking Rsc7 fails to properly assemble RSC, which confers sensitivity to temperature and to agents that cause DNA damage, microtubule depolymerization, or cell wall stress (likely via transcriptional misregulation). Cells lacking Rsc14 display sensitivity to cell wall stress and are deficient in the assembly of Rsc3 and Rsc30. Interestingly, certain rsc7delta and rsc14delta phenotypes are suppressed by an increased dosage of Rsc3, an essential RSC member with roles in cell wall integrity and spindle checkpoint pathways. Thus, Rsc7 and Rsc14 have different roles in the module as well as sharing physical and functional connections to Rsc3. Using a genetic array of nonessential null mutations (SGA) we identified mutations that are sick/lethal in combination with the rsc7delta mutation, which revealed connections to a surprisingly large number of chromatin remodeling complexes and cellular processes. Taken together, we define a protein module on the RSC complex with links to a broad spectrum of cellular functions.

  10. Ectopic histone H3S10 phosphorylation causes chromatin structure remodeling in Drosophila.

    PubMed

    Deng, Huai; Bao, Xiaomin; Cai, Weili; Blacketer, Melissa J; Belmont, Andrew S; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M

    2008-02-01

    Histones are subject to numerous post-translational modifications that correlate with the state of higher-order chromatin structure and gene expression. However, it is not clear whether changes in these epigenetic marks are causative regulatory factors in chromatin structure changes or whether they play a mainly reinforcing or maintenance role. In Drosophila phosphorylation of histone H3S10 in euchromatic chromatin regions by the JIL-1 tandem kinase has been implicated in counteracting heterochromatization and gene silencing. Here we show, using a LacI-tethering system, that JIL-1 mediated ectopic histone H3S10 phosphorylation is sufficient to induce a change in higher-order chromatin structure from a condensed heterochromatin-like state to a more open euchromatic state. This effect was absent when a ;kinase dead' LacI-JIL-1 construct without histone H3S10 phosphorylation activity was expressed. Instead, the 'kinase dead' construct had a dominant-negative effect, leading to a disruption of chromatin structure that was associated with a global repression of histone H3S10 phosphorylation levels. These findings provide direct evidence that the epigenetic histone tail modification of H3S10 phosphorylation at interphase can function as a causative regulator of higher-order chromatin structure in Drosophila in vivo.

  11. IL-10 transcription is negatively regulated by BAF180, a component of the SWI/SNF chromatin remodeling enzyme

    PubMed Central

    2012-01-01

    Background SWI/SNF chromatin remodeling enzymes play a critical role in the development of T helper lymphocytes, including Th2 cells, and directly program chromatin structure at Th2 cytokine genes. Different versions of SWI/SNF complexes, including BAF and PBAF, have been described based on unique subunit composition. However, the relative role of BAF and PBAF in Th cell function and cytokine expression has not been reported. Results Here we examine the role of the PBAF SWI/SNF complex in Th cell development and gene expression using mice deficient for a PBAF-specific component, BAF180. We find that T cell development in the thymus and lymphoid periphery is largely normal when the BAF180 gene is deleted late in thymic development. However, BAF180-deficient Th2 cells express high levels of the immunoregulatory cytokine IL-10. BAF180 binds directly to regulatory elements in the Il-10 locus but is replaced by BAF250 BAF complexes in the absence of BAF180, resulting in increased histone acetylation and CBP recruitment to the IL-10 locus. Conclusions These results demonstrate that BAF180 is a repressor of IL-10 transcription in Th2 cells and suggest that the differential recruitment of different SWI/SNF subtypes can have direct consequences on chromatin structure and gene transcription. PMID:22336179

  12. Mutant p53 cooperates with the SWI/SNF chromatin remodeling complex to regulate VEGFR2 in breast cancer cells.

    PubMed

    Pfister, Neil T; Fomin, Vitalay; Regunath, Kausik; Zhou, Jeffrey Y; Zhou, Wen; Silwal-Pandit, Laxmi; Freed-Pastor, William A; Laptenko, Oleg; Neo, Suat Peng; Bargonetti, Jill; Hoque, Mainul; Tian, Bin; Gunaratne, Jayantha; Engebraaten, Olav; Manley, James L; Børresen-Dale, Anne-Lise; Neilsen, Paul M; Prives, Carol

    2015-06-15

    Mutant p53 impacts the expression of numerous genes at the level of transcription to mediate oncogenesis. We identified vascular endothelial growth factor receptor 2 (VEGFR2), the primary functional VEGF receptor that mediates endothelial cell vascularization, as a mutant p53 transcriptional target in multiple breast cancer cell lines. Up-regulation of VEGFR2 mediates the role of mutant p53 in increasing cellular growth in two-dimensional (2D) and three-dimensional (3D) culture conditions. Mutant p53 binds near the VEGFR2 promoter transcriptional start site and plays a role in maintaining an open conformation at that location. Relatedly, mutant p53 interacts with the SWI/SNF complex, which is required for remodeling the VEGFR2 promoter. By both querying individual genes regulated by mutant p53 and performing RNA sequencing, the results indicate that >40% of all mutant p53-regulated gene expression is mediated by SWI/SNF. We surmise that mutant p53 impacts transcription of VEGFR2 as well as myriad other genes by promoter remodeling through interaction with and likely regulation of the SWI/SNF chromatin remodeling complex. Therefore, not only might mutant p53-expressing tumors be susceptible to anti VEGF therapies, impacting SWI/SNF tumor suppressor function in mutant p53 tumors may also have therapeutic potential.

  13. Microbiota modulate transcription in the intestinal epithelium without remodeling the accessible chromatin landscape

    PubMed Central

    Camp, J. Gray; Frank, Christopher L.; Lickwar, Colin R.; Guturu, Harendra; Rube, Tomas; Wenger, Aaron M.; Chen, Jenny; Bejerano, Gill; Crawford, Gregory E.

    2014-01-01

    Microbiota regulate intestinal physiology by modifying host gene expression along the length of the intestine, but the underlying regulatory mechanisms remain unresolved. Transcriptional specificity occurs through interactions between transcription factors (TFs) and cis-regulatory regions (CRRs) characterized by nucleosome-depleted accessible chromatin. We profiled transcriptome and accessible chromatin landscapes in intestinal epithelial cells (IECs) from mice reared in the presence or absence of microbiota. We show that regional differences in gene transcription along the intestinal tract were accompanied by major alterations in chromatin accessibility. Surprisingly, we discovered that microbiota modify host gene transcription in IECs without significantly impacting the accessible chromatin landscape. Instead, microbiota regulation of host gene transcription might be achieved by differential expression of specific TFs and enrichment of their binding sites in nucleosome-depleted CRRs near target genes. Our results suggest that the chromatin landscape in IECs is preprogrammed by the host in a region-specific manner to permit responses to microbiota through binding of open CRRs by specific TFs. PMID:24963153

  14. Domain Requirements of the JIL-1 Tandem Kinase for Histone H3 Serine 10 Phosphorylation and Chromatin Remodeling in Vivo*

    PubMed Central

    Li, Yeran; Cai, Weili; Wang, Chao; Yao, Changfu; Bao, Xiaomin; Deng, Huai; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M.

    2013-01-01

    The JIL-1 kinase localizes to Drosophila polytene chromosome interbands and phosphorylates histone H3 at interphase, counteracting histone H3 lysine 9 dimethylation and gene silencing. JIL-1 can be divided into four main domains, including an NH2-terminal domain, two separate kinase domains, and a COOH-terminal domain. In this study, we characterize the domain requirements of the JIL-1 kinase for histone H3 serine 10 (H3S10) phosphorylation and chromatin remodeling in vivo. We show that a JIL-1 construct without the NH2-terminal domain is without H3S10 phosphorylation activity despite the fact that it localizes properly to polytene interband regions and that it contains both kinase domains. JIL-1 is a double kinase, and we demonstrate that both kinase domains of JIL-1 are required to be catalytically active for H3S10 phosphorylation to occur. Furthermore, we provide evidence that JIL-1 is phosphorylated at serine 424 and that this phosphorylation is necessary for JIL-1 H3S10 phosphorylation activity. Thus, these data are compatible with a model where the NH2-terminal domain of JIL-1 is required for chromatin complex interactions that position the kinase domain(s) for catalytic activity in the context of the state of higher order nucleosome packaging and chromatin structure and where catalytic H3S10 phosphorylation activity mediated by the first kinase domain is dependent on autophosphorylation of serine 424 by the second kinase domain. Furthermore, using a lacO repeat tethering system to target mutated JIL-1 constructs with or without catalytic activity, we show that the epigenetic H3S10 phosphorylation mark itself functions as a causative regulator of chromatin structure independently of any structural contributions from the JIL-1 protein. PMID:23723094

  15. Reciprocal nuclear shuttling of two antagonizing Zn finger proteins modulates Tup family corepressor function to repress chromatin remodeling.

    PubMed

    Hirota, Kouji; Hoffman, Charles S; Ohta, Kunihiro

    2006-12-01

    The Schizosaccharomyces pombe global corepressors Tup11 and Tup12, which are orthologs of Saccharomyces cerevisiae Tup1, are involved in glucose-dependent transcriptional repression and chromatin alteration of the fbp1+ gene. The fbp1+ promoter contains two regulatory elements, UAS1 and UAS2, one of which (UAS2) serves as a binding site for two antagonizing C2H2 Zn finger transcription factors, the Rst2 activator and the Scr1 repressor. In this study, we analyzed the role of Tup proteins and Scr1 in chromatin remodeling at fbp1+ during glucose repression. We found that Scr1, cooperating with Tup11 and Tup12, functions to maintain the chromatin of the fbp1+ promoter in a transcriptionally inactive state under glucose-rich conditions. Consistent with this notion, Scr1 is quickly exported from the nucleus to the cytoplasm at the initial stage of derepression, immediately after glucose starvation, at which time Rst2 is known to be imported into the nucleus. In addition, chromatin immunoprecipitation assays revealed a switching of Scr1 to Rst2 bound at UAS2 during glucose derepression. On the other hand, Tup11 and Tup12 persist in the nucleus and bind to the fbp1+ promoter under both derepressed and repressed conditions. These observations suggest that Tup1-like proteins recruited to the fbp1+ promoter are controlled by either of two antagonizing C2H2 Zn finger proteins. We propose that the actions of Tup11 and Tup12 are regulated by reciprocal nuclear shuttling of the two antagonizing Zn finger proteins in response to the extracellular glucose concentration. This notion provides new insights into the molecular mechanisms of the Tup family corepressors in gene regulation.

  16. Structural Modeling of GR Interactions with the SWI/SNF Chromatin Remodeling Complex and C/EBP.

    PubMed

    Muratcioglu, Serena; Presman, Diego M; Pooley, John R; Grøntved, Lars; Hager, Gordon L; Nussinov, Ruth; Keskin, Ozlem; Gursoy, Attila

    2015-09-15

    The glucocorticoid receptor (GR) is a steroid-hormone-activated transcription factor that modulates gene expression. Transcriptional regulation by the GR requires dynamic receptor binding to specific target sites located across the genome. This binding remodels the chromatin structure to allow interaction with other transcription factors. Thus, chromatin remodeling is an essential component of GR-mediated transcriptional regulation, and understanding the interactions between these molecules at the structural level provides insights into the mechanisms of how GR and chromatin remodeling cooperate to regulate gene expression. This study suggests models for the assembly of the SWI/SNF-A (SWItch/Sucrose-NonFermentable) complex and its interaction with the GR. We used the PRISM algorithm (PRotein Interactions by Structural Matching) to predict the three-dimensional complex structures of the target proteins. The structural models indicate that BAF57 and/or BAF250 mediate the interaction between the GR and the SWI/SNF-A complex, corroborating experimental data. They further suggest that a BAF60a/BAF155 and/or BAF60a/BAF170 interaction is critical for association between the core and variant subunits. Further, we model the interaction between GR and CCAAT-enhancer-binding proteins (C/EBPs), since the GR can regulate gene expression indirectly by interacting with other transcription factors like C/EBPs. We observe that GR can bind to bZip domains of the C/EBPα homodimer as both a monomer and dimer of the DNA-binding domain. In silico mutagenesis of the predicted interface residues confirm the importance of these residues in binding. In vivo analysis of the computationally suggested mutations reveals that double mutations of the leucine residues (L317D+L335D) may disrupt the interaction between GR and C/EBPα. Determination of the complex structures of the GR is of fundamental relevance to understanding its interactions and functions, since the function of a protein or a

  17. Actin Family Proteins in the Human INO80 Chromatin Remodeling Complex Exhibit Functional Roles in the Induction of Heme Oxygenase-1 with Hemin.

    PubMed

    Takahashi, Yuichiro; Murakami, Hirokazu; Akiyama, Yusuke; Katoh, Yasutake; Oma, Yukako; Nishijima, Hitoshi; Shibahara, Kei-Ichi; Igarashi, Kazuhiko; Harata, Masahiko

    2017-01-01

    Nuclear actin family proteins, comprising of actin and actin-related proteins (Arps), are essential functional components of the multiple chromatin remodeling complexes. The INO80 chromatin remodeling complex, which is evolutionarily conserved and has roles in transcription, DNA replication and repair, consists of actin and actin-related proteins Arp4, Arp5, and Arp8. We generated Arp5 knockout (KO) and Arp8 KO cells from the human Nalm-6 pre-B cell line and used these KO cells to examine the roles of Arp5 and Arp8 in the transcriptional regulation mediated by the INO80 complex. In both of Arp5 KO and Arp8 KO cells, the oxidative stress-induced expression of HMOX1 gene, encoding for heme oxygenase-1 (HO-1), was significantly impaired. Consistent with these observations, chromatin immunoprecipitation (ChIP) assay revealed that oxidative stress caused an increase in the binding of the INO80 complex to the regulatory sites of HMOX1 in wild-type cells. The binding of INO80 complex to chromatin was reduced in Arp8 KO cells compared to that in the wild-type cells. On the other hand, the binding of INO80 complex to chromatin in Arp5 KO cells was similar to that in the wild-type cells even under the oxidative stress condition. However, both remodeling of chromatin at the HMOX1 regulatory sites and binding of a transcriptional activator to these sites were impaired in Arp5 KO cells, indicating that Arp5 is required for the activation of the INO80 complex. Collectively, these results suggested that these nuclear Arps play indispensable roles in the function of the INO80 chromatin remodeling complex.

  18. Actin Family Proteins in the Human INO80 Chromatin Remodeling Complex Exhibit Functional Roles in the Induction of Heme Oxygenase-1 with Hemin

    PubMed Central

    Takahashi, Yuichiro; Murakami, Hirokazu; Akiyama, Yusuke; Katoh, Yasutake; Oma, Yukako; Nishijima, Hitoshi; Shibahara, Kei-ichi; Igarashi, Kazuhiko; Harata, Masahiko

    2017-01-01

    Nuclear actin family proteins, comprising of actin and actin-related proteins (Arps), are essential functional components of the multiple chromatin remodeling complexes. The INO80 chromatin remodeling complex, which is evolutionarily conserved and has roles in transcription, DNA replication and repair, consists of actin and actin-related proteins Arp4, Arp5, and Arp8. We generated Arp5 knockout (KO) and Arp8 KO cells from the human Nalm-6 pre-B cell line and used these KO cells to examine the roles of Arp5 and Arp8 in the transcriptional regulation mediated by the INO80 complex. In both of Arp5 KO and Arp8 KO cells, the oxidative stress-induced expression of HMOX1 gene, encoding for heme oxygenase-1 (HO-1), was significantly impaired. Consistent with these observations, chromatin immunoprecipitation (ChIP) assay revealed that oxidative stress caused an increase in the binding of the INO80 complex to the regulatory sites of HMOX1 in wild-type cells. The binding of INO80 complex to chromatin was reduced in Arp8 KO cells compared to that in the wild-type cells. On the other hand, the binding of INO80 complex to chromatin in Arp5 KO cells was similar to that in the wild-type cells even under the oxidative stress condition. However, both remodeling of chromatin at the HMOX1 regulatory sites and binding of a transcriptional activator to these sites were impaired in Arp5 KO cells, indicating that Arp5 is required for the activation of the INO80 complex. Collectively, these results suggested that these nuclear Arps play indispensable roles in the function of the INO80 chromatin remodeling complex. PMID:28270832

  19. Quantitative analysis of the chromatin proteome in disease reveals remodeling principles and identifies high mobility group protein B2 as a regulator of hypertrophic growth.

    PubMed

    Franklin, Sarah; Chen, Haodong; Mitchell-Jordan, Scherise; Ren, Shuxun; Wang, Yibin; Vondriska, Thomas M

    2012-06-01

    A fundamental question in biology is how genome-wide changes in gene expression are enacted in response to a finite stimulus. Recent studies have mapped changes in nucleosome localization, determined the binding preferences for individual transcription factors, and shown that the genome adopts a nonrandom structure in vivo. What remains unclear is how global changes in the proteins bound to DNA alter chromatin structure and gene expression. We have addressed this question in the mouse heart, a system in which global gene expression and massive phenotypic changes occur without cardiac cell division, making the mechanisms of chromatin remodeling centrally important. To determine factors controlling genomic plasticity, we used mass spectrometry to measure chromatin-associated proteins. We have characterized the abundance of 305 chromatin-associated proteins in normal cells and measured changes in 108 proteins that accompany the progression of heart disease. These studies were conducted on a high mass accuracy instrument and confirmed in multiple biological replicates, facilitating statistical analysis and allowing us to interrogate the data bioinformatically for modules of proteins involved in similar processes. Our studies reveal general principles for global shifts in chromatin accessibility: altered linker to core histone ratio; differing abundance of chromatin structural proteins; and reprogrammed histone post-translational modifications. Using small interfering RNA-mediated loss-of-function in isolated cells, we demonstrate that the non-histone chromatin structural protein HMGB2 (but not HMGB1) suppresses pathologic cell growth in vivo and controls a gene expression program responsible for hypertrophic cell growth. Our findings reveal the basis for alterations in chromatin structure necessary for genome-wide changes in gene expression. These studies have fundamental implications for understanding how global chromatin remodeling occurs with specificity and

  20. Knockdown Brm and Baf170, components of chromatin remodeling complex, facilitates reprogramming of somatic cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The SWI/SNF (SWItch/Sucrose NonFermentable or BAF, Brg/Brahma-associated factors) complexes are epigenetic modifiers of chromatin structure and undergo progressive changes in subunit composition during cellular differentiation. For example, in embryonic stem cells (ESCs) esBAF contains Brg1 and Baf...

  1. SWR1 and INO80 chromatin remodelers contribute to DNA double-strand break perinuclear anchorage site choice.

    PubMed

    Horigome, Chihiro; Oma, Yukako; Konishi, Tatsunori; Schmid, Roger; Marcomini, Isabella; Hauer, Michael H; Dion, Vincent; Harata, Masahiko; Gasser, Susan M

    2014-08-21

    Persistent DNA double-strand breaks (DSBs) are recruited to the nuclear periphery in budding yeast. Both the Nup84 pore subcomplex and Mps3, an inner nuclear membrane (INM) SUN domain protein, have been implicated in DSB binding. It was unclear what, if anything, distinguishes the two potential sites of repair. Here, we characterize and distinguish the two binding sites. First, DSB-pore interaction occurs independently of cell-cycle phase and requires neither the chromatin remodeler INO80 nor recombinase Rad51 activity. In contrast, Mps3 binding is S and G2 phase specific and requires both factors. SWR1-dependent incorporation of Htz1 (H2A.Z) is necessary for break relocation to either site in both G1- and S-phase cells. Importantly, functional assays indicate that mutations in the two sites have additive repair defects, arguing that the two perinuclear anchorage sites define distinct survival pathways.

  2. Multi-omic data integration links Deleted in Breast Cancer 1 (DBC1) Degradation to Chromatin Remodeling in Inflammatory Response

    SciTech Connect

    Nakayasu, Ernesto S.; Brown, Roslyn N.; Ansong, Charles; Sydor, Michael A.; Imtiaz, Sayed; Mihai, Cosmin; Sontag, Ryan L.; Hixson, Kim K.; Monroe, Matthew E.; Sobreira, Tiago; Orr, Galya; Petyuk, Vladislav A.; Yang, Feng; Smith, Richard D.; Adkins, Joshua N.

    2013-08-12

    Ubiquitination is a common protein post-translational modification that regulates many key cellular functions. Here we investigated the dynamics of ubiquitinated proteins after an inflammatory stimulation of RAW264.7 macrophage-like cells with bacterial lipopolysaccharide. We demonstrate that levels of global ubiquitination, and K48 and K63 polyubiquitination change after lipopolysaccharide stimulation. A quantitative proteomic analysis identified 1199 ubiquitinated proteins, 78 of which had significantly changed ubiquitination levels after lipopolysaccharide stimulation. We next identified a subset of proteins that were targeted for degradation after lipopolysaccharide stimulation, by integrating the ubiquitinome data with global proteomics and transcriptomics results. Using cellular assays and western blot analyses we biochemically validated DBC1, a histone deacetylase inhibitor not previously linked to inflammation, as a degradation substrate, which is targeted via an orchestrated mechanism utilizing caspases and the proteasome. The degradation of DBC1 releases histone deacetylase activity, linking lipopolysaccharide activation to chromatin remodeling in caspase- and proteasome-mediated signaling.

  3. Acetylome Analysis Identifies SIRT1 Targets in mRNA-Processing and Chromatin-Remodeling in Mouse Liver

    PubMed Central

    Tang, Hui; Han, Weiping; Zhang, Kangling; Xu, Feng

    2015-01-01

    Lysine acetylation is a post-translational modification found on numerous proteins, a strategy used in cell signaling to change protein activity in response to internal or external cues. Sirtuin 1 (SIRT1) is a central lysine deacetylase involved in a variety of cellular processes including metabolism, apoptosis, and DNA repair. Here we characterize the lysine acetylome in mouse liver, and by using a model of Sirt1-/-knockout mouse, show that SIRT1 regulates the deacetylation of 70 proteins in the liver in-vivo. Amongst these SIRT1-regulated proteins, we find that four RNA-processing proteins and a chromatin-remodeling protein can be deacetylated by SIRT1 directly in-vitro. The discovery that SIRT1 has a potential role in RNA-processing suggests a new layer of regulation in the variety of functions performed by SIRT1. PMID:26468954

  4. PICKLE is a CHD3 chromatin-remodeling factor that regulates the transition from embryonic to vegetative development in Arabidopsis

    PubMed Central

    Ogas, Joe; Kaufmann, Scott; Henderson, Jim; Somerville, Chris

    1999-01-01

    The life cycle of angiosperms is punctuated by a dormant phase that separates embryonic and postembryonic development of the sporophyte. In the pickle (pkl) mutant of Arabidopsis, embryonic traits are expressed after germination. The penetrance of the pkl phenotype is strongly enhanced by inhibitors of gibberellin biosynthesis. Map-based cloning of the PKL locus revealed that it encodes a CHD3 protein. CHD3 proteins have been implicated as chromatin-remodeling factors involved in repression of transcription. PKL is necessary for repression of LEC1, a gene implicated as a critical activator of embryo development. We propose that PKL is a component of a gibberellin-modulated developmental switch that functions during germination to prevent reexpression of the embryonic developmental state. PMID:10570159

  5. PICKLE is a CHD3 chromatin-remodeling factor that regulates the transition from embryonic to vegetative development in Arabidopsis.

    PubMed

    Ogas, J; Kaufmann, S; Henderson, J; Somerville, C

    1999-11-23

    The life cycle of angiosperms is punctuated by a dormant phase that separates embryonic and postembryonic development of the sporophyte. In the pickle (pkl) mutant of Arabidopsis, embryonic traits are expressed after germination. The penetrance of the pkl phenotype is strongly enhanced by inhibitors of gibberellin biosynthesis. Map-based cloning of the PKL locus revealed that it encodes a CHD3 protein. CHD3 proteins have been implicated as chromatin-remodeling factors involved in repression of transcription. PKL is necessary for repression of LEC1, a gene implicated as a critical activator of embryo development. We propose that PKL is a component of a gibberellin-modulated developmental switch that functions during germination to prevent reexpression of the embryonic developmental state.

  6. The SWI/SNF chromatin remodeling complex regulates myocardin-induced smooth muscle-specific gene expression

    PubMed Central

    Zhou, Jiliang; Zhang, Min; Fang, Hong; El-Mounayri, Omar; Rodenberg, Jennifer M.; Imbalzano, Anthony N.; Herring, B. Paul

    2009-01-01

    Objective Transcription regulatory complexes comprising myocardin and serum response factor (SRF) are critical for the transcriptional regulation of many smooth muscle-specific genes. However, little is known about the epigenetic mechanisms that regulate the activity of these complexes. In the current study, we investigated the role of SWI/SNF ATP-dependent chromatin remodeling enzymes in regulating the myogenic activity of myocardin. Methods and Results We found that both Brg1 and Brm are required for maintaining expression of several smooth muscle-specific genes in primary cultures of aortic smooth muscle cells. Furthermore, the ability of myocardin to induce expression of smooth muscle-specific genes is abrogated in cells expressing dominant negative Brg1. In SW13 cells, that lack endogenous Brg1 and Brm1, myocardin is unable to induce expression of smooth muscle-specific genes. Whereas, reconstitution of wild type, or bromodomain mutant forms Brg1 or Brm1, into SW13 cells restored their responsiveness to myocardin. SWI/SNF complexes were found to be required for myocardin to increase SRF binding to the promoters of smooth muscle-specific genes. Brg1 and Brm directly bind to the N-terminus of myocardin, in vitro, through their ATPase domains and Brg1 forms a complex with SRF and myocardin in vivo in smooth muscle cells. Conclusion These data demonstrate that the ability of myocardin to induce smooth muscle-specific gene expression is dependent on its interaction with SWI/SNF ATP-dependent chromatin remodeling complexes. PMID:19342595

  7. SWI/SNF chromatin remodeling regulates alcohol response behaviors in Caenorhabditis elegans and is associated with alcohol dependence in humans.

    PubMed

    Mathies, Laura D; Blackwell, GinaMari G; Austin, Makeda K; Edwards, Alexis C; Riley, Brien P; Davies, Andrew G; Bettinger, Jill C

    2015-03-10

    Alcohol abuse is a widespread and serious problem. Understanding the factors that influence the likelihood of abuse is important for the development of effective therapies. There are both genetic and environmental influences on the development of abuse, but it has been difficult to identify specific liability factors, in part because of both the complex genetic architecture of liability and the influences of environmental stimuli on the expression of that genetic liability. Epigenetic modification of gene expression can underlie both genetic and environmentally sensitive variation in expression, and epigenetic regulation has been implicated in the progression to addiction. Here, we identify a role for the switching defective/sucrose nonfermenting (SWI/SNF) chromatin-remodeling complex in regulating the behavioral response to alcohol in the nematode Caenorhabditis elegans. We found that SWI/SNF components are required in adults for the normal behavioral response to ethanol and that different SWI/SNF complexes regulate different aspects of the acute response to ethanol. We showed that the SWI/SNF subunits SWSN-9 and SWSN-7 are required in neurons and muscle for the development of acute functional tolerance to ethanol. Examination of the members of the SWI/SNF complex for association with a diagnosis of alcohol dependence in a human population identified allelic variation in a member of the SWI/SNF complex, suggesting that variation in the regulation of SWI/SNF targets may influence the propensity to develop abuse disorders. Together, these data strongly implicate the chromatin remodeling associated with SWI/SNF complex members in the behavioral responses to alcohol across phyla.

  8. Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope.

    PubMed

    Tsai, Shang-Yi A; Chuang, Jian-Ying; Tsai, Meng-Shan; Wang, Xiao-Fei; Xi, Zheng-Xiong; Hung, Jan-Jong; Chang, Wen-Chang; Bonci, Antonello; Su, Tsung-Ping

    2015-11-24

    The sigma-1 receptor (Sig-1R) chaperone at the endoplasmic reticulum (ER) plays important roles in cellular regulation. Here we found a new function of Sig-1R, in that it translocates from the ER to the nuclear envelope (NE) to recruit chromatin-remodeling molecules and regulate the gene transcription thereof. Sig-1Rs mainly reside at the ER-mitochondrion interface. However, on stimulation by agonists such as cocaine, Sig-1Rs translocate from ER to the NE, where Sig-1Rs bind NE protein emerin and recruit chromatin-remodeling molecules, including lamin A/C, barrier-to-autointegration factor (BAF), and histone deacetylase (HDAC), to form a complex with the gene repressor specific protein 3 (Sp3). Knockdown of Sig-1Rs attenuates the complex formation. Cocaine was found to suppress the gene expression of monoamine oxidase B (MAOB) in the brain of wild-type but not Sig-1R knockout mouse. A single dose of cocaine (20 mg/kg) in rats suppresses the level of MAOB at nuclear accumbens without affecting the level of dopamine transporter. Daily injections of cocaine in rats caused behavioral sensitization. Withdrawal from cocaine in cocaine-sensitized rats induced an apparent time-dependent rebound of the MAOB protein level to about 200% over control on day 14 after withdrawal. Treatment of cocaine-withdrawn rats with the MAOB inhibitor deprenyl completely alleviated the behavioral sensitization to cocaine. Our results demonstrate a role of Sig-1R in transcriptional regulation and suggest cocaine may work through this newly discovered genomic action to achieve its addictive action. Results also suggest the MAOB inhibitor deprenyl as a therapeutic agent to block certain actions of cocaine during withdrawal.

  9. Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope

    PubMed Central

    Tsai, Shang-Yi A.; Chuang, Jian-Ying; Tsai, Meng-Shan; Wang, Xiao-fei; Hung, Jan-Jong; Chang, Wen-Chang; Bonci, Antonello; Su, Tsung-Ping

    2015-01-01

    The sigma-1 receptor (Sig-1R) chaperone at the endoplasmic reticulum (ER) plays important roles in cellular regulation. Here we found a new function of Sig-1R, in that it translocates from the ER to the nuclear envelope (NE) to recruit chromatin-remodeling molecules and regulate the gene transcription thereof. Sig-1Rs mainly reside at the ER–mitochondrion interface. However, on stimulation by agonists such as cocaine, Sig-1Rs translocate from ER to the NE, where Sig-1Rs bind NE protein emerin and recruit chromatin-remodeling molecules, including lamin A/C, barrier-to-autointegration factor (BAF), and histone deacetylase (HDAC), to form a complex with the gene repressor specific protein 3 (Sp3). Knockdown of Sig-1Rs attenuates the complex formation. Cocaine was found to suppress the gene expression of monoamine oxidase B (MAOB) in the brain of wild-type but not Sig-1R knockout mouse. A single dose of cocaine (20 mg/kg) in rats suppresses the level of MAOB at nuclear accumbens without affecting the level of dopamine transporter. Daily injections of cocaine in rats caused behavioral sensitization. Withdrawal from cocaine in cocaine-sensitized rats induced an apparent time-dependent rebound of the MAOB protein level to about 200% over control on day 14 after withdrawal. Treatment of cocaine-withdrawn rats with the MAOB inhibitor deprenyl completely alleviated the behavioral sensitization to cocaine. Our results demonstrate a role of Sig-1R in transcriptional regulation and suggest cocaine may work through this newly discovered genomic action to achieve its addictive action. Results also suggest the MAOB inhibitor deprenyl as a therapeutic agent to block certain actions of cocaine during withdrawal. PMID:26554014

  10. The chromatin remodeling and mRNA splicing functions of the Brahma (SWI/SNF) complex are mediated by the SNR1/SNF5 regulatory subunit.

    PubMed

    Zraly, Claudia B; Dingwall, Andrew K

    2012-07-01

    Nucleosome remodeling catalyzed by the ATP-dependent SWI/SNF complex is essential for regulated gene expression. Transcriptome profiling studies in flies and mammals identified cell cycle and hormone responsive genes as important targets of remodeling complex activities. Loss of chromatin remodeling function has been linked to developmental abnormalities and aggressive cancers. The Drosophila Brahma (Brm) SWI/SNF complex assists in reprogramming and coordinating gene expression in response to ecdysone hormone signaling at critical points during development. We used RNAi knockdown in cultured cells and transgenic flies, and conditional mutant alleles to identify unique and important functions of two conserved Brm complex core subunits, SNR1/SNF5 and BRM/SNF2-SWI2, on target gene regulation. Unexpectedly, we found that incorporation of a loss of function SNR1 subunit led to alterations in RNA polymerase elongation, pre-mRNA splicing regulation and chromatin accessibility of ecdysone hormone regulated genes, revealing that SNR1 functions to restrict BRM-dependent nucleosome remodeling activities downstream of the promoter region. Our results reveal critically important roles of the SNR1/SNF5 subunit and the Brm chromatin remodeling complex in transcription regulation during elongation by RNA Polymerase II and completion of pre-mRNA transcripts that are dependent on hormone signaling in late development.

  11. SWI/SNF chromatin remodeling complex is critical for the expression of microphthalmia-associated transcription factor in melanoma cells

    SciTech Connect

    Vachtenheim, Jiri; Ondrusova, Lubica; Borovansky, Jan

    2010-02-12

    The microphthalmia-associated transcription factor (MITF) is required for melanocyte development, maintenance of the melanocyte-specific transcription, and survival of melanoma cells. MITF positively regulates expression of more than 25 genes in pigment cells. Recently, it has been demonstrated that expression of several MITF downstream targets requires the SWI/SNF chromatin remodeling complex, which contains one of the two catalytic subunits, Brm or Brg1. Here we show that the expression of MITF itself critically requires active SWI/SNF. In several Brm/Brg1-expressing melanoma cell lines, knockdown of Brg1 severely compromised MITF expression with a concomitant dowregulation of MITF targets and decreased cell proliferation. Although Brm was able to substitute for Brg1 in maintaining MITF expression and melanoma cell proliferation, sequential knockdown of both Brm and Brg1 in 501mel cells abolished proliferation. In Brg1-null SK-MEL-5 melanoma cells, depletion of Brm alone was sufficient to abrogate MITF expression and cell proliferation. Chromatin immunoprecipitation confirmed the binding of Brg1 or Brm to the promoter of MITF. Together these results demonstrate the essential role of SWI/SNF for expression of MITF and suggest that SWI/SNF may be a promissing target in melanoma therapy.

  12. Chromatin Remodeling Protein SMAR1 Is a Critical Regulator of T Helper Cell Differentiation and Inflammatory Diseases

    PubMed Central

    Mirlekar, Bhalchandra; Gautam, Dipendra; Chattopadhyay, Samit

    2017-01-01

    T cell differentiation from naïve T cells to specialized effector subsets of mature cells is determined by the iterative action of transcription factors. At each stage of specific T cell lineage differentiation, transcription factor interacts not only with nuclear proteins such as histone and histone modifiers but also with other factors that are bound to the chromatin and play a critical role in gene expression. In this review, we focus on one of such nuclear protein known as tumor suppressor and scaffold matrix attachment region-binding protein 1 (SMAR1) in CD4+ T cell differentiation. SMAR1 facilitates Th1 differentiation by negatively regulating T-bet expression via recruiting HDAC1–SMRT complex to its gene promoter. In contrast, regulatory T (Treg) cell functions are dependent on inhibition of Th17-specific genes mainly IL-17 and STAT3 by SMAR1. Here, we discussed a critical role of chromatin remodeling protein SMAR1 in maintaining a fine-tuned balance between effector CD4+ T cells and Treg cells by influencing the transcription factors during allergic and autoimmune inflammatory diseases. PMID:28232831

  13. Protein markers of synaptic behavior and chromatin remodeling of the neo-XY body in phyllostomid bats.

    PubMed

    Rahn, Mónica I; Noronha, Renata C; Nagamachi, Cleusa Y; Pieczarka, Julio C; Solari, Alberto J; Sciurano, Roberta B

    2016-09-01

    The XX/XY system is the rule among mammals. However, many exceptions from this general pattern have been discovered since the last decades. One of these non-conventional sex chromosome mechanisms is the multiple sex chromosome system, which is evolutionary fixed among many bat species of the family Phyllostomidae, and has arisen by a translocation between one original gonosome (X or Y chromosome), and an autosome, giving rise to a "neo-XY body." The aim of this work is to study the synaptic behavior and the chromatin remodeling of multiple sex chromosomes in different species of phyllostomid bats using electron microscopy and molecular markers. Testicular tissues from adult males of the species Artibeus lituratus, Artibeus planirostris, Uroderma bilobatum, and Vampyrodes caraccioli from the eastern Amazonia were analyzed by optical/electron microscopy and immunofluorescence of meiotic proteins involved in synapsis (SYCP3 and SYCE3), sister-chromatid cohesion (SMC3), and chromatin silencing (BRCA1, γ-H2AX, and RNApol 2). The presence of asynaptic axes-labeled by BRCA1 and γ-H2AX-at meiotic prophase in testes that have a normal development of spermatogenesis, suggests that the basic mechanism that arrests spreading of transcriptional silencing (meiotic sex chromosome inactivation (MSCI)) to the autosomal segments may be per se the formation of a functional synaptonemal complex between homologous or non-homologous regions, and thus, this SC barrier might be probably related to the preservation of fertility in these systems.

  14. A Direct Link between Abscisic Acid Sensing and the Chromatin-Remodeling ATPase BRAHMA via Core ABA Signaling Pathway Components.

    PubMed

    Peirats-Llobet, Marta; Han, Soon-Ki; Gonzalez-Guzman, Miguel; Jeong, Cheol Woong; Rodriguez, Lesia; Belda-Palazon, Borja; Wagner, Doris; Rodriguez, Pedro L

    2016-01-04

    Optimal response to drought is critical for plant survival and will affect biodiversity and crop performance during climate change. Mitotically heritable epigenetic or dynamic chromatin state changes have been implicated in the plant response to the drought stress hormone abscisic acid (ABA). The Arabidopsis SWI/SNF chromatin-remodeling ATPase BRAHMA (BRM) modulates response to ABA by preventing premature activation of stress response pathways during germination. We show that core ABA signaling pathway components physically interact with BRM and post-translationally modify BRM by phosphorylation/dephosphorylation. Genetic evidence suggests that BRM acts downstream of SnRK2.2/2.3 kinases, and biochemical studies identified phosphorylation sites in the C-terminal region of BRM at SnRK2 target sites that are evolutionarily conserved. Finally, the phosphomimetic BRM(S1760D S1762D) mutant displays ABA hypersensitivity. Prior studies showed that BRM resides at target loci in the ABA pathway in the presence and absence of the stimulus, but is only active in the absence of ABA. Our data suggest that SnRK2-dependent phosphorylation of BRM leads to its inhibition, and PP2CA-mediated dephosphorylation of BRM restores the ability of BRM to repress ABA response. These findings point to the presence of a rapid phosphorylation-based switch to control BRM activity; this property could be potentially harnessed to improve drought tolerance in plants.

  15. Mi-2/NuRD chromatin remodeling complexes regulate B and T-lymphocyte development and function

    PubMed Central

    Dege, Carissa; Hagman, James

    2015-01-01

    Summary Mi-2/nucleosomal remodeling and deacetylase (NuRD) complexes are important epigenetic regulators of chromatin structure and gene expression. Mi-2/NuRD complexes are an assemblage of proteins that combine key epigenetic regulators necessary for (i) histone deacetylation and demethylation, (ii) binding to methylated DNA, (iii) mobilization of nucleosomes, and (iv) recruitment of additional regulatory proteins. Depending on their context in chromatin, Mi-2/NuRD complexes either activate or repress gene transcription. In this regard, they are important regulators of hematopoiesis and lymphopoiesis. Mi-2/NuRD complexes maintain pools of hematopoietic stem cells. Specifically, components of these complexes control multiple stages of B-cell development by regulating B-cell-specific transcription. With one set of components, they inhibit terminal differentiation of germinal center B cells into plasma B cells. They also mediate gene repression together with Blimp-1 during plasma cell differentiation. In cooperation with Ikaros, Mi-2/NuRD complexes also play important roles in T-cell development, including CD4 versus CD8 fate decisions and peripheral T-cell responses. Dysregulation of NuRD during lymphopoiesis promotes leukemogenesis. Here, we review general properties of Mi-2/NuRD complexes and focus on their functions in gene regulation and development of lymphocytes. PMID:25123281

  16. The CHD3 chromatin remodeler PICKLE and polycomb group proteins antagonistically regulate meristem activity in the Arabidopsis root.

    PubMed

    Aichinger, Ernst; Villar, Corina B R; Di Mambro, Riccardo; Sabatini, Sabrina; Köhler, Claudia

    2011-03-01

    The chromatin modifying Polycomb group (PcG) and trithorax group (trxG) proteins are central regulators of cell identity that maintain a tightly controlled balance between cell proliferation and cell differentiation. The opposing activities of PcG and trxG proteins ensure the correct expression of specific transcriptional programs at defined developmental stages. Here, we report that the chromatin remodeling factor PICKLE (PKL) and the PcG protein CURLY LEAF (CLF) antagonistically determine root meristem activity. Whereas loss of PKL function caused a decrease in meristematic activity, loss of CLF function increased meristematic activity. Alterations of meristematic activity in pkl and clf mutants were not connected with changes in auxin concentration but correlated with decreased or increased expression of root stem cell and meristem marker genes, respectively. Root stem cell and meristem marker genes are modified by the PcG-mediated trimethylation of histone H3 on lysine 27 (H3K27me3). Decreased expression levels of root stem cell and meristem marker genes in pkl correlated with increased levels of H3K27me3, indicating that root meristem activity is largely controlled by the antagonistic activity of PcG proteins and PKL.

  17. Chromatin organization and remodeling of interstitial telomeric sites during meiosis in the Mongolian gerbil (Meriones unguiculatus).

    PubMed

    de la Fuente, Roberto; Manterola, Marcia; Viera, Alberto; Parra, María Teresa; Alsheimer, Manfred; Rufas, Julio S; Page, Jesús

    2014-08-01

    Telomeric DNA repeats are key features of chromosomes that allow the maintenance of integrity and stability in the telomeres. However, interstitial telomere sites (ITSs) can also be found along the chromosomes, especially near the centromere, where they may appear following chromosomal rearrangements like Robertsonian translocations. There is no defined role for ITSs, but they are linked to DNA damage-prone sites. We were interested in studying the structural organization of ITSs during meiosis, a kind of cell division in which programmed DNA damage events and noticeable chromatin reorganizations occur. Here we describe the presence of highly amplified ITSs in the pericentromeric region of Mongolian gerbil (Meriones unguiculatus) chromosomes. During meiosis, ITSs show a different chromatin conformation than DNA repeats at telomeres, appearing more extended and accumulating heterochromatin markers. Interestingly, ITSs also recruit the telomeric proteins RAP1 and TRF1, but in a stage-dependent manner, appearing mainly at late prophase I stages. We did not find a specific accumulation of DNA repair factors to the ITSs, such as γH2AX or RAD51 at these stages, but we could detect the presence of MLH1, a marker for reciprocal recombination. However, contrary to previous reports, we did not find a specific accumulation of crossovers at ITSs. Intriguingly, some centromeric regions of metacentric chromosomes may bind the nuclear envelope through the association to SUN1 protein, a feature usually performed by telomeres. Therefore, ITSs present a particular and dynamic chromatin configuration in meiosis, which could be involved in maintaining their genetic stability, but they additionally retain some features of distal telomeres, provided by their capability to associate to telomere-binding proteins.

  18. The role of HDAC2 in chromatin remodelling and response to chemotherapy in ovarian cancer

    PubMed Central

    Huang, Rui; Langdon, Simon P; Tse, Matthew; Mullen, Peter; Um, In Hwa; Faratian, Dana; Harrison, David J

    2016-01-01

    Chromatin undergoes structural changes in response to extracellular and environmental signals. We observed changes in nuclear morphology in cancer tissue biopsied after chemotherapy and hypothesised that these DNA damage-induced changes are mediated by histone deacetylases (HDACs). Nuclear morphological changes in cell lines (PE01 and PE04 models) and a xenograft model (OV1002) were measured in response to platinum chemotherapy by image analysis of nuclear texture. HDAC2 expression increased in PEO1 cells treated with cisplatin at 24h, which was accompanied by increased expression of heterochromatin protein 1 (HP1). HDAC2 and HP1 expression were also increased after carboplatin treatment in the OV1002 carboplatin-sensitive xenograft model but not in the insensitive HOX424 model. Expression of DNA damage response pathways (pBRCA1, γH2AX, pATM, pATR) showed time-dependent changes after cisplatin treatment. HDAC2 knockdown by siRNA reduced HP1 expression, induced DNA double strand breaks (DSB) measured by γH2AX, and interfered with the activation of DNA damage response induced by cisplatin. Furthermore, HDAC2 depletion affected γH2AX foci formation, cell cycle distribution, and apoptosis triggered by cisplatin, and was additive to the inhibitory effect of cisplatin in cell lines. By inhibiting expression of HDAC2, reversible alterations in chromatin patterns during cisplatin treatment were observed. These results demonstrate quantifiable alterations in nuclear morphology after chemotherapy, and implicate HDAC2 in higher order chromatin changes and cellular DNA damage responses in ovarian cancer cells in vitro and in vivo. PMID:26683361

  19. The Core Subunit of A Chromatin-Remodeling Complex, ZmCHB101, Plays Essential Roles in Maize Growth and Development

    PubMed Central

    Yu, Xiaoming; Jiang, Lili; Wu, Rui; Meng, Xinchao; Zhang, Ai; Li, Ning; Xia, Qiong; Qi, Xin; Pang, Jinsong; Xu, Zheng-Yi; Liu, Bao

    2016-01-01

    ATP-dependent chromatin remodeling complexes play essential roles in the regulation of diverse biological processes by formulating a DNA template that is accessible to the general transcription apparatus. Although the function of chromatin remodelers in plant development has been studied in A. thaliana, how it affects growth and development of major crops (e.g., maize) remains uninvestigated. Combining genetic, genomic and bioinformatic analyses, we show here that the maize core subunit of chromatin remodeling complex, ZmCHB101, plays essential roles in growth and development of maize at both vegetative and reproductive stages. Independent ZmCHB101 RNA interference plant lines displayed abaxially curling leaf phenotype due to increase of bulliform cell numbers, and showed impaired development of tassel and cob. RNA-seq-based transcriptome profiling revealed that ZmCHB101 dictated transcriptional reprogramming of a significant set of genes involved in plant development, photosynthesis, metabolic regulation, stress response and gene expressional regulation. Intriguingly, we found that ZmCHB101 was required for maintaining normal nucleosome density and 45 S rDNA compaction. Our findings suggest that the SWI3 protein, ZmCHB101, plays pivotal roles in maize normal growth and development via regulation of chromatin structure. PMID:27917953

  20. The Core Subunit of A Chromatin-Remodeling Complex, ZmCHB101, Plays Essential Roles in Maize Growth and Development.

    PubMed

    Yu, Xiaoming; Jiang, Lili; Wu, Rui; Meng, Xinchao; Zhang, Ai; Li, Ning; Xia, Qiong; Qi, Xin; Pang, Jinsong; Xu, Zheng-Yi; Liu, Bao

    2016-12-05

    ATP-dependent chromatin remodeling complexes play essential roles in the regulation of diverse biological processes by formulating a DNA template that is accessible to the general transcription apparatus. Although the function of chromatin remodelers in plant development has been studied in A. thaliana, how it affects growth and development of major crops (e.g., maize) remains uninvestigated. Combining genetic, genomic and bioinformatic analyses, we show here that the maize core subunit of chromatin remodeling complex, ZmCHB101, plays essential roles in growth and development of maize at both vegetative and reproductive stages. Independent ZmCHB101 RNA interference plant lines displayed abaxially curling leaf phenotype due to increase of bulliform cell numbers, and showed impaired development of tassel and cob. RNA-seq-based transcriptome profiling revealed that ZmCHB101 dictated transcriptional reprogramming of a significant set of genes involved in plant development, photosynthesis, metabolic regulation, stress response and gene expressional regulation. Intriguingly, we found that ZmCHB101 was required for maintaining normal nucleosome density and 45 S rDNA compaction. Our findings suggest that the SWI3 protein, ZmCHB101, plays pivotal roles in maize normal growth and development via regulation of chromatin structure.

  1. BRG1 and BRM chromatin-remodeling complexes regulate the hypoxia response by acting as coactivators for a subset of hypoxia-inducible transcription factor target genes.

    PubMed

    Sena, Johnny A; Wang, Liyi; Hu, Cheng-Jun

    2013-10-01

    Chromatin remodeling is an active process, which represses or enables the access of transcription machinery to genes in response to external stimuli, including hypoxia. However, in hypoxia, the specific requirement, as well as the molecular mechanism by which the chromatin-remodeling complexes regulate gene expression, remains unclear. In this study, we report that the Brahma (BRM) and Brahma-related gene 1 (BRG1) ATPase-containing SWI/SNF chromatin-remodeling complexes promote the expression of the hypoxia-inducible transcription factor 1α (HIF1α) and HIF2α genes and also promote hypoxic induction of a subset of HIF1 and HIF2 target genes. We show that BRG1 or BRM knockdown in Hep3B and RCC4T cells reduces hypoxic induction of HIF target genes, while reexpression of BRG1 or BRM in BRG1/BRM-deficient SW13 cells increases HIF target gene activation. Mechanistically, HIF1 and HIF2 increase the hypoxic induction of HIF target genes by recruiting BRG1 complexes to HIF target gene promoters, which promotes nucleosome remodeling of HIF target gene promoters in a BRG1 ATPase-dependent manner. Importantly, we found that the function of BRG1 complexes in hypoxic SW13 and RCC4T cells is dictated by the HIF-mediated hypoxia response and could be opposite from their function in normoxic SW13 and RCC4T cells.

  2. Nuclear Matrix protein SMAR1 represses HIV-1 LTR mediated transcription through chromatin remodeling

    SciTech Connect

    Sreenath, Kadreppa; Pavithra, Lakshminarasimhan; Singh, Sandeep; Sinha, Surajit; Dash, Prasanta K.; Siddappa, Nagadenahalli B.; Ranga, Udaykumar; Mitra, Debashis; Chattopadhyay, Samit

    2010-04-25

    Nuclear Matrix and MARs have been implicated in the transcriptional regulation of host as well as viral genes but their precise role in HIV-1 transcription remains unclear. Here, we show that > 98% of HIV sequences contain consensus MAR element in their promoter. We show that SMAR1 binds to the LTR MAR and reinforces transcriptional silencing by tethering the LTR MAR to nuclear matrix. SMAR1 associated HDAC1-mSin3 corepressor complex is dislodged from the LTR upon cellular activation by PMA/TNFalpha leading to an increase in the acetylation and a reduction in the trimethylation of histones, associated with the recruitment of RNA Polymerase II on the LTR. Overexpression of SMAR1 lead to reduction in LTR mediated transcription, both in a Tat dependent and independent manner, resulting in a decreased virion production. These results demonstrate the role of SMAR1 in regulating viral transcription by alternative compartmentalization of LTR between the nuclear matrix and chromatin.

  3. Early programming of the oocyte epigenome temporally controls late prophase I transcription and chromatin remodelling

    PubMed Central

    Navarro-Costa, Paulo; McCarthy, Alicia; Prudêncio, Pedro; Greer, Christina; Guilgur, Leonardo G.; Becker, Jörg D.; Secombe, Julie; Rangan, Prashanth; Martinho, Rui G.

    2016-01-01

    Oocytes are arrested for long periods of time in the prophase of the first meiotic division (prophase I). As chromosome condensation poses significant constraints to gene expression, the mechanisms regulating transcriptional activity in the prophase I-arrested oocyte are still not entirely understood. We hypothesized that gene expression during the prophase I arrest is primarily epigenetically regulated. Here we comprehensively define the Drosophila female germ line epigenome throughout oogenesis and show that the oocyte has a unique, dynamic and remarkably diversified epigenome characterized by the presence of both euchromatic and heterochromatic marks. We observed that the perturbation of the oocyte's epigenome in early oogenesis, through depletion of the dKDM5 histone demethylase, results in the temporal deregulation of meiotic transcription and affects female fertility. Taken together, our results indicate that the early programming of the oocyte epigenome primes meiotic chromatin for subsequent functions in late prophase I. PMID:27507044

  4. A Specificity and Targeting Subunit of a Human SWI/SNF Family-Related Chromatin-Remodeling Complex

    PubMed Central

    Nie, Zuqin; Xue, Yutong; Yang, Dafeng; Zhou, Sharleen; Deroo, Bonnie J.; Archer, Trevor K.; Wang, Weidong

    2000-01-01

    The SWI/SNF family of chromatin-remodeling complexes facilitates gene activation by assisting transcription machinery to gain access to targets in chromatin. This family includes BAF (also called hSWI/SNF-A) and PBAF (hSWI/SNF-B) from humans and SWI/SNF and Rsc from Saccharomyces cerevisiae. However, the relationship between the human and yeast complexes is unclear because all human subunits published to date are similar to those of both yeast SWI/SNF and Rsc. Also, the two human complexes have many identical subunits, making it difficult to distinguish their structures or functions. Here we describe the cloning and characterization of BAF250, a subunit present in human BAF but not PBAF. BAF250 contains structural motifs conserved in yeast SWI1 but not in any Rsc components, suggesting that BAF is related to SWI/SNF. BAF250 is also a homolog of the Drosophila melanogaster Osa protein, which has been shown to interact with a SWI/SNF-like complex in flies. BAF250 possesses at least two conserved domains that could be important for its function. First, it has an AT-rich DNA interaction-type DNA-binding domain, which can specifically bind a DNA sequence known to be recognized by a SWI/SNF family-related complex at the β-globin locus. Second, BAF250 stimulates glucocorticoid receptor-dependent transcriptional activation, and the stimulation is sharply reduced when the C-terminal region of BAF250 is deleted. This region of BAF250 is capable of interacting directly with the glucocorticoid receptor in vitro. Our data suggest that BAF250 confers specificity to the human BAF complex and may recruit the complex to its targets through either protein-DNA or protein-protein interactions. PMID:11073988

  5. Context-dependent role for chromatin remodeling component PBRM1/BAF180 in clear cell renal cell carcinoma

    PubMed Central

    Murakami, A; Wang, L; Kalhorn, S; Schraml, P; Rathmell, W K; Tan, A C; Nemenoff, R; Stenmark, K; Jiang, B-H; Reyland, M E; Heasley, L; Hu, C-J

    2017-01-01

    A subset of clear cell renal cell carcinoma (ccRCC) tumors exhibit a HIF1A gene mutation, yielding two ccRCC tumor types, H1H2 type expressing both HIF1α and HIF2α, and H2 type expressing HIF2α, but not functional HIF1α protein. However, it is unclear how the H1H2 type ccRCC tumors escape HIF1's tumor-suppressive activity. The polybromo-1 (PBRM1) gene coding for the BAF180 protein, a component of the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex, is inactivated in 40% ccRCCs, the function and mechanism of BAF180 mutation is unknown. Our previous study indicates that BAF180-containing SWI/SNF chromatin remodeling complex is a co-activator for transcription factor HIF to induce HIF target genes. Thus, our questions are if BAF180 is involved in HIF-mediated hypoxia response and if PBRM1/BAF180 mutation has any association with the HIF1A retention in H1H2 type ccRCC. We report here that BAF180 is mutated in H1H2 ccRCC cell lines and tumors, and BAF180 re-expression in H1H2 ccRCC cell lines reduced cell proliferation/survival, indicating that BAF180 has tumor-suppressive role in these cells. However, BAF180 is expressed in HIF1-deficient H2 ccRCC cell lines and tumors, and BAF180 knockdown in H2 type ccRCC cell lines reduced cell proliferation/survival, indicating that BAF180 has tumor-promoting activity in these cells. In addition, our data show that BAF180 functions as co-activator for HIF1- and HIF2-mediated transcriptional response, and BAF180's tumor-suppressive and -promoting activity in ccRCC cell lines depends on co-expression of HIF1 and HIF2, respectively. Thus, our studies reveal that BAF180 function in ccRCC is context dependent, and that mutation of PBRM1/BAF180 serves as an alternative strategy for ccRCC tumors to reduce HIF1 tumor-suppressive activity in H1H2 ccRCC tumors. Our studies define distinct functional subgroups of ccRCCs based on expression of BAF180, and suggest that BAF180 inhibition may be a novel therapeutic

  6. Context-dependent role for chromatin remodeling component PBRM1/BAF180 in clear cell renal cell carcinoma.

    PubMed

    Murakami, A; Wang, L; Kalhorn, S; Schraml, P; Rathmell, W K; Tan, A C; Nemenoff, R; Stenmark, K; Jiang, B-H; Reyland, M E; Heasley, L; Hu, C-J

    2017-01-16

    A subset of clear cell renal cell carcinoma (ccRCC) tumors exhibit a HIF1A gene mutation, yielding two ccRCC tumor types, H1H2 type expressing both HIF1α and HIF2α, and H2 type expressing HIF2α, but not functional HIF1α protein. However, it is unclear how the H1H2 type ccRCC tumors escape HIF1's tumor-suppressive activity. The polybromo-1 (PBRM1) gene coding for the BAF180 protein, a component of the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex, is inactivated in 40% ccRCCs, the function and mechanism of BAF180 mutation is unknown. Our previous study indicates that BAF180-containing SWI/SNF chromatin remodeling complex is a co-activator for transcription factor HIF to induce HIF target genes. Thus, our questions are if BAF180 is involved in HIF-mediated hypoxia response and if PBRM1/BAF180 mutation has any association with the HIF1A retention in H1H2 type ccRCC. We report here that BAF180 is mutated in H1H2 ccRCC cell lines and tumors, and BAF180 re-expression in H1H2 ccRCC cell lines reduced cell proliferation/survival, indicating that BAF180 has tumor-suppressive role in these cells. However, BAF180 is expressed in HIF1-deficient H2 ccRCC cell lines and tumors, and BAF180 knockdown in H2 type ccRCC cell lines reduced cell proliferation/survival, indicating that BAF180 has tumor-promoting activity in these cells. In addition, our data show that BAF180 functions as co-activator for HIF1- and HIF2-mediated transcriptional response, and BAF180's tumor-suppressive and -promoting activity in ccRCC cell lines depends on co-expression of HIF1 and HIF2, respectively. Thus, our studies reveal that BAF180 function in ccRCC is context dependent, and that mutation of PBRM1/BAF180 serves as an alternative strategy for ccRCC tumors to reduce HIF1 tumor-suppressive activity in H1H2 ccRCC tumors. Our studies define distinct functional subgroups of ccRCCs based on expression of BAF180, and suggest that BAF180 inhibition may be a novel therapeutic

  7. Synthetic essentiality of chromatin remodelling factor CHD1 in PTEN-deficient cancer.

    PubMed

    Zhao, Di; Lu, Xin; Wang, Guocan; Lan, Zhengdao; Liao, Wenting; Li, Jun; Liang, Xin; Chen, Jasper Robin; Shah, Sagar; Shang, Xiaoying; Tang, Ming; Deng, Pingna; Dey, Prasenjit; Chakravarti, Deepavali; Chen, Peiwen; Spring, Denise J; Navone, Nora M; Troncoso, Patricia; Zhang, Jianhua; Wang, Y Alan; DePinho, Ronald A

    2017-02-23

    Synthetic lethality and collateral lethality are two well-validated conceptual strategies for identifying therapeutic targets in cancers with tumour-suppressor gene deletions. Here, we explore an approach to identify potential synthetic-lethal interactions by screening mutually exclusive deletion patterns in cancer genomes. We sought to identify 'synthetic-essential' genes: those that are occasionally deleted in some cancers but are almost always retained in the context of a specific tumour-suppressor deficiency. We also posited that such synthetic-essential genes would be therapeutic targets in cancers that harbour specific tumour-suppressor deficiencies. In addition to known synthetic-lethal interactions, this approach uncovered the chromatin helicase DNA-binding factor CHD1 as a putative synthetic-essential gene in PTEN-deficient cancers. In PTEN-deficient prostate and breast cancers, CHD1 depletion profoundly and specifically suppressed cell proliferation, cell survival and tumorigenic potential. Mechanistically, functional PTEN stimulates the GSK3β-mediated phosphorylation of CHD1 degron domains, which promotes CHD1 degradation via the β-TrCP-mediated ubiquitination-proteasome pathway. Conversely, PTEN deficiency results in stabilization of CHD1, which in turn engages the trimethyl lysine-4 histone H3 modification to activate transcription of the pro-tumorigenic TNF-NF-κB gene network. This study identifies a novel PTEN pathway in cancer and provides a framework for the discovery of 'trackable' targets in cancers that harbour specific tumour-suppressor deficiencies.

  8. Synapsis, recombination, and chromatin remodeling in the XY body of armadillos.

    PubMed

    Sciurano, Roberta B; Rahn, Mónica I; Rossi, Luis; Luaces, Juan Pablo; Merani, María Susana; Solari, Alberto J

    2012-02-01

    Three xenarthrans species Chaetophractus villosus, Chaetophractus vellerosus, and Zaedyus pichiy have been used for the analysis of the structure, behavior, and immunochemical features of the XY body during pachytene. In all these species, the sex chromosomes form an XY body easily identifiable in thin sections by the special and regular packing of the chromatin fibers of the internal region of the XY body ("differential" regions) and those of the peripheral region (synaptic region). Spermatocyte spreads show a complete synapsis between the X- and the Y-axis, which lasts up to the end of pachytene. From the early pachytene substages to the late ones, the X-axis develops prominent branches, which in late pachytene span the synaptic region. Synapsis is regular as shown by SYCP1 labeling. Axial development is followed by SYCP3 labeling and in the asynaptic region of the X-axis by BRCA1. Gamma-H2AX labels exclusively the differential (asynaptic) region of the X chromosome. A single focus is labeled by MLH1 in the synaptic region. The location of this MLH1 focus spans from 0.3 to 1.6 μm from the telomere in the analyzed xenarthrans, covering approximately half of the Y-axis length. It is concluded that xenarthrans, as basal placental mammals, harbor the largest pseudoautosomal regions of presently analyzed mammals, and shows the typical features of meiotic sex chromosome inactivation (MSCI).

  9. Interaction of the Chromatin Remodeling Protein hINO80 with DNA

    PubMed Central

    Jain, Shruti; Kaur, Taniya; Brahmachari, Vani

    2016-01-01

    The presence of a highly conserved DNA binding domain in INO80 subfamily predicted that INO80 directly interacts with DNA and we demonstrated its DNA binding activity in vitro. Here we report the consensus motif recognized by the DBINO domain identified by SELEX method and demonstrate the specific interaction of INO80 with the consensus motif. We show that INO80 significantly down regulates the reporter gene expression through its binding motif, and the repression is dependent on the presence of INO80 but not YY1 in the cell. The interaction is lost if specific residues within the consensus motif are altered. We identify a large number of potential target sites of INO80 in the human genome through in silico analysis that can grouped into three classes; sites that contain the recognition sequence for INO80 and YY1, only YY1 and only INO80. We demonstrate the binding of INO80 to a representative set of sites in HEK cells and the correlated repressive histone modifications around the binding motif. In the light of the role of INO80 in homeotic gene regulation in Drosophila as an Enhancer of trithorax and polycomb protein (ETP) that can modify the effect of both repressive complexes like polycomb as well as the activating complex like trithorax, it remains to be seen if INO80 can act as a recruiter of chromatin modifying complexes. PMID:27428271

  10. Phosphorylation of the chromatin remodeling factor DPF3a induces cardiac hypertrophy through releasing HEY repressors from DNA.

    PubMed

    Cui, Huanhuan; Schlesinger, Jenny; Schoenhals, Sophia; Tönjes, Martje; Dunkel, Ilona; Meierhofer, David; Cano, Elena; Schulz, Kerstin; Berger, Michael F; Haack, Timm; Abdelilah-Seyfried, Salim; Bulyk, Martha L; Sauer, Sascha; Sperling, Silke R

    2016-04-07

    DPF3 (BAF45c) is a member of the BAF chromatin remodeling complex. Two isoforms have been described, namely DPF3a and DPF3b. The latter binds to acetylated and methylated lysine residues of histones. Here, we elaborate on the role of DPF3a and describe a novel pathway of cardiac gene transcription leading to pathological cardiac hypertrophy. Upon hypertrophic stimuli, casein kinase 2 phosphorylates DPF3a at serine 348. This initiates the interaction of DPF3a with the transcriptional repressors HEY, followed by the release of HEY from the DNA. Moreover, BRG1 is bound by DPF3a, and is thus recruited to HEY genomic targets upon interaction of the two components. Consequently, the transcription of downstream targets such as NPPA and GATA4 is initiated and pathological cardiac hypertrophy is established. In human, DPF3a is significantly up-regulated in hypertrophic hearts of patients with hypertrophic cardiomyopathy or aortic stenosis. Taken together, we show that activation of DPF3a upon hypertrophic stimuli switches cardiac fetal gene expression from being silenced by HEY to being activated by BRG1. Thus, we present a novel pathway for pathological cardiac hypertrophy, whose inhibition is a long-term therapeutic goal for the treatment of the course of heart failure.

  11. Regulation of nucleosome positioning by a CHD Type III chromatin remodeler and its relationship to developmental gene expression in Dictyostelium

    PubMed Central

    Platt, James L.; Kent, Nicholas A.; Kimmel, Alan R.

    2017-01-01

    Nucleosome placement and repositioning can direct transcription of individual genes; however, the precise interactions of these events are complex and largely unresolved at the whole-genome level. The Chromodomain-Helicase-DNA binding (CHD) Type III proteins are a subfamily of SWI2/SNF2 proteins that control nucleosome positioning and are associated with several complex human disorders, including CHARGE syndrome and autism. Type III CHDs are required for multicellular development of animals and Dictyostelium but are absent in plants and yeast. These CHDs can mediate nucleosome translocation in vitro, but their in vivo mechanism is unknown. Here, we use genome-wide analysis of nucleosome positioning and transcription profiling to investigate the in vivo relationship between nucleosome positioning and gene expression during development of wild-type (WT) Dictyostelium and mutant cells lacking ChdC, a Type III CHD protein ortholog. We demonstrate major nucleosome positional changes associated with developmental gene regulation in WT. Loss of chdC caused an increase of intragenic nucleosome spacing and misregulation of gene expression, affecting ∼50% of the genes that are repositioned during WT development. These analyses demonstrate active nucleosome repositioning during Dictyostelium multicellular development, establish an in vivo function of CHD Type III chromatin remodeling proteins in this process, and reveal the detailed relationship between nucleosome positioning and gene regulation, as cells transition between developmental states. PMID:28330902

  12. Chromatin remodelling and antisense-mediated up-regulation of the developmental switch gene eud-1 control predatory feeding plasticity

    PubMed Central

    Serobyan, Vahan; Xiao, Hua; Namdeo, Suryesh; Rödelsperger, Christian; Sieriebriennikov, Bogdan; Witte, Hanh; Röseler, Waltraud; Sommer, Ralf J.

    2016-01-01

    Phenotypic plasticity has been suggested to act through developmental switches, but little is known about associated molecular mechanisms. In the nematode Pristionchus pacificus, the sulfatase eud-1 was identified as part of a developmental switch controlling mouth-form plasticity governing a predatory versus bacteriovorous mouth-form decision. Here we show that mutations in the conserved histone-acetyltransferase Ppa-lsy-12 and the methyl-binding-protein Ppa-mbd-2 mimic the eud-1 phenotype, resulting in the absence of one mouth-form. Mutations in both genes cause histone modification defects and reduced eud-1 expression. Surprisingly, Ppa-lsy-12 mutants also result in the down-regulation of an antisense-eud-1 RNA. eud-1 and antisense-eud-1 are co-expressed and further experiments suggest that antisense-eud-1 acts through eud-1 itself. Indeed, overexpression of the antisense-eud-1 RNA increases the eud-1-sensitive mouth-form and extends eud-1 expression. In contrast, this effect is absent in eud-1 mutants indicating that antisense-eud-1 positively regulates eud-1. Thus, chromatin remodelling and antisense-mediated up-regulation of eud-1 control feeding plasticity in Pristionchus. PMID:27487725

  13. Exome sequencing of serous endometrial tumors identifies recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes.

    PubMed

    Le Gallo, Matthieu; O'Hara, Andrea J; Rudd, Meghan L; Urick, Mary Ellen; Hansen, Nancy F; O'Neil, Nigel J; Price, Jessica C; Zhang, Suiyuan; England, Bryant M; Godwin, Andrew K; Sgroi, Dennis C; Hieter, Philip; Mullikin, James C; Merino, Maria J; Bell, Daphne W

    2012-12-01

    Endometrial cancer is the sixth most commonly diagnosed cancer in women worldwide, causing ~74,000 deaths annually. Serous endometrial cancers are a clinically aggressive subtype with a poorly defined genetic etiology. We used whole-exome sequencing to comprehensively search for somatic mutations within ~22,000 protein-encoding genes in 13 primary serous endometrial tumors. We subsequently resequenced 18 genes, which were mutated in more than 1 tumor and/or were components of an enriched functional grouping, from 40 additional serous tumors. We identified high frequencies of somatic mutations in CHD4 (17%), EP300 (8%), ARID1A (6%), TSPYL2 (6%), FBXW7 (29%), SPOP (8%), MAP3K4 (6%) and ABCC9 (6%). Overall, 36.5% of serous tumors had a mutated chromatin-remodeling gene, and 35% had a mutated ubiquitin ligase complex gene, implicating frequent mutational disruption of these processes in the molecular pathogenesis of one of the deadliest forms of endometrial cancer.

  14. SWP73 Subunits of Arabidopsis SWI/SNF Chromatin Remodeling Complexes Play Distinct Roles in Leaf and Flower Development

    PubMed Central

    Sacharowski, Sebastian P.; Gratkowska, Dominika M.; Sarnowska, Elzbieta A.; Kondrak, Paulina; Jancewicz, Iga; Porri, Aimone; Bucior, Ernest; Rolicka, Anna T.; Franzen, Rainer; Kowalczyk, Justyna; Pawlikowska, Katarzyna; Huettel, Bruno; Torti, Stefano; Schmelzer, Elmon; Coupland, George; Jerzmanowski, Andrzej; Koncz, Csaba; Sarnowski, Tomasz J.

    2015-01-01

    Arabidopsis thaliana SWP73A and SWP73B are homologs of mammalian BRAHMA-associated factors (BAF60s) that tether SWITCH/SUCROSE NONFERMENTING chromatin remodeling complexes to transcription factors of genes regulating various cell differentiation pathways. Here, we show that Arabidopsis thaliana SWP73s modulate several important developmental pathways. While undergoing normal vegetative development, swp73a mutants display reduced expression of FLOWERING LOCUS C and early flowering in short days. By contrast, swp73b mutants are characterized by retarded growth, severe defects in leaf and flower development, delayed flowering, and male sterility. MNase-Seq, transcript profiling, and ChIP-Seq studies demonstrate that SWP73B binds the promoters of ASYMMETRIC LEAVES1 and 2, KANADI1 and 3, and YABBY2, 3, and 5 genes, which regulate leaf development and show coordinately altered transcription in swp73b plants. Lack of SWP73B alters the expression patterns of APETALA1, APETALA3, and the MADS box gene AGL24, whereas other floral organ identity genes show reduced expression correlating with defects in flower development. Consistently, SWP73B binds to the promoter regions of APETALA1 and 3, SEPALLATA3, LEAFY, UNUSUAL FLORAL ORGANS, TERMINAL FLOWER1, AGAMOUS-LIKE24, and SUPPRESSOR OF CONSTANS OVEREXPRESSION1 genes, and the swp73b mutation alters nucleosome occupancy on most of these loci. In conclusion, SWP73B acts as important modulator of major developmental pathways, while SWP73A functions in flowering time control. PMID:26106148

  15. MicroRNA-939 restricts Hepatitis B virus by targeting Jmjd3-mediated and C/EBPα-coordinated chromatin remodeling

    PubMed Central

    Chen, Cuncun; Wu, Min; Zhang, Wen; Lu, Wei; Zhang, Min; Zhang, Zhanqing; Zhang, Xiaonan; Yuan, Zhenghong

    2016-01-01

    Multi-layered mechanisms of virus host interaction exist for chronic hepatitis B virus (HBV) infection, which have been typically manifested at the microRNA level. Our previous study suggested that miRNA-939 (miR-939) may play a potential role in regulating HBV replication. Here we further investigated the mechanism by which miR-939 regulates HBV life cycle. We found that miR-939 inhibited the abundance of viral RNAs without direct miRNA-mRNA base pairing, but via host factors. Expression profiling and functional validation identified Jmjd3 as a target responsible for miR-939 induced anti-HBV effect. Jmjd3 appeared to enhance the transcription efficiency of HBV enhancer II/core promoter (En II) in a C/EBPα-dependent manner. However, the demethylase activity of Jmjd3 was not required in this process. Rather, Jmjd3’s transactivation activity depended on its interaction with C/EBPα. This coordinated action further recruited the Brm containing SWI/SNF chromatin remodeling complex which promoted the transcription of HBV RNAs. Taken together, we propose that the miR-939-Jmjd3 axis perturbs the accessibility of En II promoter to essential nuclear factors (C/EBPα and SWI/SNF complex) therefore leading to compromised viral RNA synthesis and hence restricted viral multiplication. PMID:27779233

  16. Genetic analysis of brahma: the Drosophila homolog of the yeast chromatin remodeling factor SWI2/SNF2.

    PubMed

    Elfring, L K; Daniel, C; Papoulas, O; Deuring, R; Sarte, M; Moseley, S; Beek, S J; Waldrip, W R; Daubresse, G; DePace, A; Kennison, J A; Tamkun, J W

    1998-01-01

    The Drosophila brahma (brm) gene encodes an activator of homeotic genes related to the yeast chromatin remodeling factor SWI2/SNF2. Here, we report the phenotype of null and dominant-negative brm mutations. Using mosaic analysis, we found that the complete loss of brm function decreases cell viability and causes defects in the peripheral nervous system of the adult. A dominant-negative brm mutation was generated by replacing a conserved lysine in the ATP-binding site of the BRM protein with an arginine. This mutation eliminates brm function in vivo but does not affect assembly of the 2-MD BRM complex. Expression of the dominant-negative BRM protein caused peripheral nervous system defects, homeotic transformations, and decreased viability. Consistent with these findings, the BRM protein is expressed at relatively high levels in nuclei throughout the developing organism. Site-directed mutagenesis was used to investigate the functions of conserved regions of the BRM protein. Domain II is essential for brm function and is required for the assembly or stability of the BRM complex. In spite of its conservation in numerous eukaryotic regulatory proteins, the deletion of the bromodomain of the BRM protein has no discernible phenotype.

  17. Genetic analysis of brahma: the Drosophila homolog of the yeast chromatin remodeling factor SWI2/SNF2.

    PubMed Central

    Elfring, L K; Daniel, C; Papoulas, O; Deuring, R; Sarte, M; Moseley, S; Beek, S J; Waldrip, W R; Daubresse, G; DePace, A; Kennison, J A; Tamkun, J W

    1998-01-01

    The Drosophila brahma (brm) gene encodes an activator of homeotic genes related to the yeast chromatin remodeling factor SWI2/SNF2. Here, we report the phenotype of null and dominant-negative brm mutations. Using mosaic analysis, we found that the complete loss of brm function decreases cell viability and causes defects in the peripheral nervous system of the adult. A dominant-negative brm mutation was generated by replacing a conserved lysine in the ATP-binding site of the BRM protein with an arginine. This mutation eliminates brm function in vivo but does not affect assembly of the 2-MD BRM complex. Expression of the dominant-negative BRM protein caused peripheral nervous system defects, homeotic transformations, and decreased viability. Consistent with these findings, the BRM protein is expressed at relatively high levels in nuclei throughout the developing organism. Site-directed mutagenesis was used to investigate the functions of conserved regions of the BRM protein. Domain II is essential for brm function and is required for the assembly or stability of the BRM complex. In spite of its conservation in numerous eukaryotic regulatory proteins, the deletion of the bromodomain of the BRM protein has no discernible phenotype. PMID:9475737

  18. Preferential occupancy of histone variant H2AZ at inactive promoters influences local histone modifications and chromatin remodeling

    PubMed Central

    Li, Bing; Pattenden, Samantha G.; Lee, Daeyoup; Gutiérrez, José; Chen, Jie; Seidel, Chris; Gerton, Jennifer; Workman, Jerry L.

    2005-01-01

    The yeast histone variant H2AZ (Htz1) is implicated in transcription activation, prevention of the ectopic spread of heterochromatin, and genome integrity. Our genome-wide localization analysis revealed that Htz1 is widely, but nonrandomly, distributed throughout the genome in an SWR1-dependent manner. We found that Htz1 is enriched in intergenic regions compared with coding regions. Its occupancy is inversely proportional to transcription rates and the enrichment of the RNA polymerase II under different growth conditions. However, Htz1 does not seem to directly regulate transcription repression genome-wide; instead, the presence of Htz1 under the inactivated condition is essential for optimal activation of a subset of genes. In addition, Htz1 is not generally responsible for nucleosome positioning, even at those promoters where Htz1 is highly enriched. Finally, using a biochemical approach, we demonstrate that incorporation of Htz1 into nucleosomes inhibits activities of histone modifiers associated with transcription, Dot1, Set2, and NuA4 and reduces the nucleosome mobilization driven by chromatin remodeling complexes. These lines of evidence collectively suggest that Htz1 may serve to mark quiescent promoters for proper activation. PMID:16344463

  19. Evidence for chromatin-remodeling complex PBAP-controlled maintenance of the Drosophila ovarian germline stem cells.

    PubMed

    He, Jie; Xuan, Tao; Xin, Tianchi; An, Hongbo; Wang, Jinye; Zhao, Gengchun; Li, Mingfa

    2014-01-01

    In the Drosophila oogenesis, germline stem cells (GSCs) continuously self-renew and differentiate into daughter cells for consecutive germline lineage commitment. This developmental process has become an in vivo working platform for studying adult stem cell fate regulation. An increasing number of studies have shown that while concerted actions of extrinsic signals from the niche and intrinsic regulatory machineries control GSC self-renewal and germline differentiation, epigenetic regulation is implicated in the process. Here, we report that Brahma (Brm), the ATPase subunit of the Drosophila SWI/SNF chromatin-remodeling complexes, is required for maintaining GSC fate. Removal or knockdown of Brm function in either germline or niche cells causes a GSC loss, but does not disrupt normal germline differentiation within the germarium evidenced at the molecular and morphological levels. There are two Drosophila SWI/SNF complexes: the Brm-associated protein (BAP) complex and the polybromo-containing BAP (PBAP) complex. More genetic studies reveal that mutations in polybromo/bap180, rather than gene encoding Osa, the BAP complex-specific subunit, elicit a defect in GSC maintenance reminiscent of the brm mutant phenotype. Further genetic interaction test suggests a functional association between brm and polybromo in controlling GSC self-renewal. Taken together, studies in this paper provide the first demonstration that Brm in the form of the PBAP complex functions in the GSC fate regulation.

  20. Histone Acetylation and Chromatin Remodeling Are Required for UV-B–Dependent Transcriptional Activation of Regulated Genes in Maize[W

    PubMed Central

    Casati, Paula; Campi, Mabel; Chu, Feixia; Suzuki, Nagi; Maltby, David; Guan, Shenheng; Burlingame, Alma L.; Walbot, Virginia

    2008-01-01

    The nuclear proteomes of maize (Zea mays) lines that differ in UV-B tolerance were compared by two-dimensional gel electrophoresis after UV light treatment. Differential accumulation of chromatin proteins, particularly histones, constituted the largest class identified by mass spectrometry. UV-B–tolerant landraces and the B73 inbred line show twice as many protein changes as the UV-B–sensitive b, pl W23 inbred line and transgenic maize expressing RNA interference constructs directed against chromatin factors. Mass spectrometic analysis of posttranslational modifications on histone proteins demonstrates that UV-B–tolerant lines exhibit greater acetylation on N-terminal tails of histones H3 and H4 after irradiation. These acetylated histones are enriched in the promoter and transcribed regions of the two UV-B–upregulated genes examined; radiation-sensitive lines lack this enrichment. DNase I and micrococcal nuclease hypersensitivity assays indicate that chromatin adopts looser structures around the selected genes in the UV-B–tolerant samples. Chromatin immunoprecipitation experiments identified additional chromatin factor changes associated with the nfc102 test gene after UV-B treatment in radiation-tolerant lines. Chromatin remodeling is thus shown to be a key process in acclimation to UV-B, and lines deficient in this process are more sensitive to UV-B. PMID:18398050

  1. Methylation specific targeting of a chromatin remodeling complex from sponges to humans

    PubMed Central

    Cramer, Jason M.; Pohlmann, Deborah; Gomez, Fernando; Mark, Leslie; Kornegay, Benjamin; Hall, Chelsea; Siraliev-Perez, Edhriz; Walavalkar, Ninad M.; Sperlazza, M. Jeannette; Bilinovich, Stephanie; Prokop, Jeremy W.; Hill, April L.; Williams Jr., David C.

    2017-01-01

    DNA cytosine methylation and methyl-cytosine binding domain (MBD) containing proteins are found throughout all vertebrate species studied to date. However, both the presence of DNA methylation and pattern of methylation varies among invertebrate species. Invertebrates generally have only a single MBD protein, MBD2/3, that does not always contain appropriate residues for selectively binding methylated DNA. Therefore, we sought to determine whether sponges, one of the most ancient extant metazoan lineages, possess an MBD2/3 capable of recognizing methylated DNA and recruiting the associated nucleosome remodeling and deacetylase (NuRD) complex. We find that Ephydatia muelleri has genes for each of the NuRD core components including an EmMBD2/3 that selectively binds methylated DNA. NMR analyses reveal a remarkably conserved binding mode, showing almost identical chemical shift changes between binding to methylated and unmethylated CpG dinucleotides. In addition, we find that EmMBD2/3 and EmGATAD2A/B proteins form a coiled-coil interaction known to be critical for the formation of NuRD. Finally, we show that knockdown of EmMBD2/3 expression disrupts normal cellular architecture and development of E. muelleri. These data support a model in which the MBD2/3 methylation-dependent functional role emerged with the earliest multicellular organisms and has been maintained to varying degrees across animal evolution. PMID:28094816

  2. Using Atomic Force Microscopy To Study Chromatin Structure and Nucleosome Remodeling

    PubMed Central

    Lohr, D.; Bash, R.; Wang, H.; Yodh, J.; Lindsay, S.

    2007-01-01

    Atomic Force Microscopy (AFM) is a technique that can directly image single molecules in solution and it therefore provides a powerful tool for obtaining unique insights into the basic properties of biological materials and the functional processes in which they are involved. We have used AFM to analyze basic features of nucleosomes in arrays, such as DNA-histone binding strength, cooperativity in template occupation, nucleosome stabilities, nucleosome locations and the effects of acetylation, to compare these features in different types of arrays and to track the response of array nucleosomes to the action of the human Swi-Snf ATP-dependent nucleosome remodeling complex. These experiments required several specific adaptations of basic AFM methods, such as repetitive imaging of the same fields of molecules in liquid, the ability to change the environmental conditions of the sample being imaged and detection of specific types of molecules within compositionally complex samples. Here we describe the techniques that allowed such analyses to be carried out. PMID:17309844

  3. Belinostat, a potent HDACi, exerts antileukaemic effect in human acute promyelocytic leukaemia cells via chromatin remodelling

    PubMed Central

    Valiuliene, Giedre; Stirblyte, Ieva; Cicenaite, Dovile; Kaupinis, Algirdas; Valius, Mindaugas; Navakauskiene, Ruta

    2015-01-01

    Epigenetic changes play a significant role in leukaemia pathogenesis, therefore histone deacetylases (HDACis) are widely accepted as an attractive strategy for acute promyelocytic leukaemia (APL) treatment. Belinostat (Bel, PXD101), a hydroxamate-type HDACi, has proved to be a promising cure in clinical trials for solid tumours and haematological malignancies. However, insight into molecular effects of Bel on APL, is still lacking. In this study, we investigated the effect of Bel alone and in combination with differentiation inducer retinoic acid (RA) on human promyelocytic leukaemia NB4 and HL-60 cells. We found that treatment with Bel, depending on the dosage used, inhibits cell proliferation, whereas in combination with RA enhances and accelerates granulocytic leukaemia cell differentiation. We also evaluated the effect of used treatments with Bel and RA on certain epigenetic modifiers (HDAC1, HDAC2, PCAF) as well as cell cycle regulators (p27) gene expression and protein level modulation. We showed that Bel in combination with RA up-regulates basal histone H4 hyperacetylation level more strongly compared to Bel or RA alone. Furthermore, chromatin immunoprecipitation assay indicated that Bel induces the accumulation of hyperacetylated histone H4 at the p27 promoter region. Mass spectrometry analysis revealed that in control NB4 cells, hyperacetylated histone H4 is mainly found in association with proteins involved in DNA replication and transcription, whereas after Bel treatment it is found with proteins implicated in pro-apoptotic processes, in defence against oxidative stress and tumour suppression. Summarizing, our study provides some novel insights into the molecular mechanisms of HDACi Bel action on APL cells. PMID:25864732

  4. Belinostat, a potent HDACi, exerts antileukaemic effect in human acute promyelocytic leukaemia cells via chromatin remodelling.

    PubMed

    Valiuliene, Giedre; Stirblyte, Ieva; Cicenaite, Dovile; Kaupinis, Algirdas; Valius, Mindaugas; Navakauskiene, Ruta

    2015-07-01

    Epigenetic changes play a significant role in leukaemia pathogenesis, therefore histone deacetylases (HDACis) are widely accepted as an attractive strategy for acute promyelocytic leukaemia (APL) treatment. Belinostat (Bel, PXD101), a hydroxamate-type HDACi, has proved to be a promising cure in clinical trials for solid tumours and haematological malignancies. However, insight into molecular effects of Bel on APL, is still lacking. In this study, we investigated the effect of Bel alone and in combination with differentiation inducer retinoic acid (RA) on human promyelocytic leukaemia NB4 and HL-60 cells. We found that treatment with Bel, depending on the dosage used, inhibits cell proliferation, whereas in combination with RA enhances and accelerates granulocytic leukaemia cell differentiation. We also evaluated the effect of used treatments with Bel and RA on certain epigenetic modifiers (HDAC1, HDAC2, PCAF) as well as cell cycle regulators (p27) gene expression and protein level modulation. We showed that Bel in combination with RA up-regulates basal histone H4 hyperacetylation level more strongly compared to Bel or RA alone. Furthermore, chromatin immunoprecipitation assay indicated that Bel induces the accumulation of hyperacetylated histone H4 at the p27 promoter region. Mass spectrometry analysis revealed that in control NB4 cells, hyperacetylated histone H4 is mainly found in association with proteins involved in DNA replication and transcription, whereas after Bel treatment it is found with proteins implicated in pro-apoptotic processes, in defence against oxidative stress and tumour suppression. Summarizing, our study provides some novel insights into the molecular mechanisms of HDACi Bel action on APL cells.

  5. A Genetic Screen and Transcript Profiling Reveal a Shared Regulatory Program for Drosophila Linker Histone H1 and Chromatin Remodeler CHD1

    PubMed Central

    Kavi, Harsh; Lu, Xingwu; Xu, Na; Bartholdy, Boris A.; Vershilova, Elena; Skoultchi, Arthur I.; Fyodorov, Dmitry V.

    2015-01-01

    Chromatin structure and activity can be modified through ATP-dependent repositioning of nucleosomes and posttranslational modifications of core histone tails within nucleosome core particles and by deposition of linker histones into the oligonucleosome fiber. The linker histone H1 is essential in metazoans. It has a profound effect on organization of chromatin into higher-order structures and on recruitment of histone-modifying enzymes to chromatin. Here, we describe a genetic screen for modifiers of the lethal phenotype caused by depletion of H1 in Drosophila melanogaster. We identify 41 mis-expression alleles that enhance and 20 that suppress the effect of His1 depletion in vivo. Most of them are important for chromosome organization, transcriptional regulation, and cell signaling. Specifically, the reduced viability of H1-depleted animals is strongly suppressed by ubiquitous mis-expression of the ATP-dependent chromatin remodeling enzyme CHD1. Comparison of transcript profiles in H1-depleted and Chd1 null mutant larvae revealed that H1 and CHD1 have common transcriptional regulatory programs in vivo. H1 and CHD1 share roles in repression of numerous developmentally regulated and extracellular stimulus-responsive transcripts, including immunity-related and stress response-related genes. Thus, linker histone H1 participates in various regulatory programs in chromatin to alter gene expression. PMID:25628309

  6. miR-151-5p, targeting chromatin remodeler SMARCA5, as a marker for the BRCAness phenotype

    PubMed Central

    Tommasi, Stefania; Pinto, Rosamaria; Danza, Katia; Pilato, Brunella; Palumbo, Orazio; Micale, Lucia; Summa, Simona De

    2016-01-01

    In recent years, the assessment of biomarkers useful for “precision medicine” has been a hot topic in research. The involvement of microRNAs in the pathogenesis of breast cancer has been highly investigated with the aim of being able to molecularly stratify this highly heterogeneous disease. Our aim was to identify microRNAs targeting DNA repair machinery, through Affymetrix GeneChip miRNA Arrays, in a cohort of BRCA-related and sporadic breast cancers. Moreover, we analyzed microRNA expression taking into account our previous results on the expression of PARP1, because of its importance in targeted therapy. miR-361-5p and miR-151-5p were found to be overexpressed in PARP1-upregulating BRCA-germline mutated and sporadic breast tumors. Pathway enrichment analysis was performed to identify potential target genes to be analyzed in the validation step in an independent cohort. Our results confirmed the overexpression of miR-151-5p and, interestingly, its role in the targeting of SMARCA5, a chromatin remodeler. This result was also confirmed in vitro, both through luciferase assay and by analyzing endogenous levels of SMARCA5 in MCF-7 cell lines using miR-151-5p mimic and inhibitor. In conclusion, our data showed the possibility of considering the overexpression of PARP1 and miR-151-5p as biomarkers useful to correctly treat sporadic breast cancers, which eventually could be considered as BRCAness tumors, with PARP-inhibitors. PMID:27385001

  7. miR-151-5p, targeting chromatin remodeler SMARCA5, as a marker for the BRCAness phenotype.

    PubMed

    Tommasi, Stefania; Pinto, Rosamaria; Danza, Katia; Pilato, Brunella; Palumbo, Orazio; Micale, Lucia; De Summa, Simona

    2016-12-06

    In recent years, the assessment of biomarkers useful for "precision medicine" has been a hot topic in research. The involvement of microRNAs in the pathogenesis of breast cancer has been highly investigated with the aim of being able to molecularly stratify this highly heterogeneous disease. Our aim was to identify microRNAs targeting DNA repair machinery, through Affymetrix GeneChip miRNA Arrays, in a cohort of BRCA-related and sporadic breast cancers. Moreover, we analyzed microRNA expression taking into account our previous results on the expression of PARP1, because of its importance in targeted therapy. miR-361-5p and miR-151-5p were found to be overexpressed in PARP1-upregulating BRCA-germline mutated and sporadic breast tumors. Pathway enrichment analysis was performed to identify potential target genes to be analyzed in the validation step in an independent cohort. Our results confirmed the overexpression of miR-151-5p and, interestingly, its role in the targeting of SMARCA5, a chromatin remodeler. This result was also confirmed in vitro, both through luciferase assay and by analyzing endogenous levels of SMARCA5 in MCF-7 cell lines using miR-151-5p mimic and inhibitor. In conclusion, our data showed the possibility of considering the overexpression of PARP1 and miR-151-5p as biomarkers useful to correctly treat sporadic breast cancers, which eventually could be considered as BRCAness tumors, with PARP-inhibitors.

  8. Functional Interplay of Two Paralogs Encoding SWI/SNF Chromatin-Remodeling Accessory Subunits During Caenorhabditis elegans Development.

    PubMed

    Ertl, Iris; Porta-de-la-Riva, Montserrat; Gómez-Orte, Eva; Rubio-Peña, Karinna; Aristizábal-Corrales, David; Cornes, Eric; Fontrodona, Laura; Osteikoetxea, Xabier; Ayuso, Cristina; Askjaer, Peter; Cabello, Juan; Cerón, Julián

    2016-03-01

    SWI/SNF ATP-dependent chromatin-remodeling complexes have been related to several cellular processes such as transcription, regulation of chromosomal stability, and DNA repair. The Caenorhabditis elegans gene ham-3 (also known as swsn-2.1) and its paralog swsn-2.2 encode accessory subunits of SWI/SNF complexes. Using RNA interference (RNAi) assays and diverse alleles we investigated whether ham-3 and swsn-2.2 have different functions during C. elegans development since they encode proteins that are probably mutually exclusive in a given SWI/SNF complex. We found that ham-3 and swsn-2.2 display similar functions in vulva specification, germline development, and intestinal cell proliferation, but have distinct roles in embryonic development. Accordingly, we detected functional redundancy in some developmental processes and demonstrated by RNA sequencing of RNAi-treated L4 animals that ham-3 and swsn-2.2 regulate the expression of a common subset of genes but also have specific targets. Cell lineage analyses in the embryo revealed hyper-proliferation of intestinal cells in ham-3 null mutants whereas swsn-2.2 is required for proper cell divisions. Using a proteomic approach, we identified SWSN-2.2-interacting proteins needed for early cell divisions, such as SAO-1 and ATX-2, and also nuclear envelope proteins such as MEL-28. swsn-2.2 mutants phenocopy mel-28 loss-of-function, and we observed that SWSN-2.2 and MEL-28 colocalize in mitotic and meiotic chromosomes. Moreover, we demonstrated that SWSN-2.2 is required for correct chromosome segregation and nuclear reassembly after mitosis including recruitment of MEL-28 to the nuclear periphery.

  9. Functional Interplay of Two Paralogs Encoding SWI/SNF Chromatin-Remodeling Accessory Subunits During Caenorhabditis elegans Development

    PubMed Central

    Ertl, Iris; Porta-de-la-Riva, Montserrat; Gómez-Orte, Eva; Rubio-Peña, Karinna; Aristizábal-Corrales, David; Cornes, Eric; Fontrodona, Laura; Osteikoetxea, Xabier; Ayuso, Cristina; Askjaer, Peter; Cabello, Juan; Cerón, Julián

    2016-01-01

    SWI/SNF ATP-dependent chromatin-remodeling complexes have been related to several cellular processes such as transcription, regulation of chromosomal stability, and DNA repair. The Caenorhabditis elegans gene ham-3 (also known as swsn-2.1) and its paralog swsn-2.2 encode accessory subunits of SWI/SNF complexes. Using RNA interference (RNAi) assays and diverse alleles we investigated whether ham-3 and swsn-2.2 have different functions during C. elegans development since they encode proteins that are probably mutually exclusive in a given SWI/SNF complex. We found that ham-3 and swsn-2.2 display similar functions in vulva specification, germline development, and intestinal cell proliferation, but have distinct roles in embryonic development. Accordingly, we detected functional redundancy in some developmental processes and demonstrated by RNA sequencing of RNAi-treated L4 animals that ham-3 and swsn-2.2 regulate the expression of a common subset of genes but also have specific targets. Cell lineage analyses in the embryo revealed hyper-proliferation of intestinal cells in ham-3 null mutants whereas swsn-2.2 is required for proper cell divisions. Using a proteomic approach, we identified SWSN-2.2-interacting proteins needed for early cell divisions, such as SAO-1 and ATX-2, and also nuclear envelope proteins such as MEL-28. swsn-2.2 mutants phenocopy mel-28 loss-of-function, and we observed that SWSN-2.2 and MEL-28 colocalize in mitotic and meiotic chromosomes. Moreover, we demonstrated that SWSN-2.2 is required for correct chromosome segregation and nuclear reassembly after mitosis including recruitment of MEL-28 to the nuclear periphery. PMID:26739451

  10. The chromatin remodeling complex NuRD establishes the poised state of rRNA genes characterized by bivalent histone modifications and altered nucleosome positions.

    PubMed

    Xie, Wenbing; Ling, Te; Zhou, Yonggang; Feng, Weijun; Zhu, Qiaoyun; Stunnenberg, Henk G; Grummt, Ingrid; Tao, Wei

    2012-05-22

    rRNA genes (rDNA) exist in two distinct epigenetic states, active promoters being unmethylated and marked by euchromatic histone modifications, whereas silent ones are methylated and exhibit heterochromatic features. Here we show that the nucleosome remodeling and deacetylation (NuRD) complex establishes a specific chromatin structure at rRNA genes that are poised for transcription activation. The promoter of poised rRNA genes is unmethylated, associated with components of the preinitiation complex, marked by bivalent histone modifications and covered by a nucleosome in the "off" position, which is refractory to transcription initiation. Repression of rDNA transcription in growth-arrested and differentiated cells correlates with elevated association of NuRD and increased levels of poised rRNA genes. Reactivation of transcription requires resetting the promoter-bound nucleosome into the "on" position by the DNA-dependent ATPase CSB (Cockayne syndrome protein B). The results uncover a unique mechanism by which ATP-dependent chromatin remodeling complexes with opposing activities establish a specific chromatin state and regulate transcription.

  11. Aberrant lung remodeling in a mouse model of surfactant dysregulation induced by modulation of the Abca3 gene.

    PubMed

    Beers, Michael F; Knudsen, Lars; Tomer, Yaniv; Maronn, Julian; Zhao, Ming; Ochs, Matthias; Mulugeta, Surafel

    2017-03-01

    The lipid transporter, ATP binding cassette class A3 (ABCA3), plays a critical role in the biogenesis of alveolar type 2 (AT2) cell lamellar bodies (LBs). A relatively large number of mutations in the ABCA3 gene have been identified in association with diffuse parenchymal lung disease (DPLD), the most common of which is a missense mutation (valine substitution for lysine at residue 292 (ABCA3(E292V))) that leads to functional impairment of the transporter in vitro. The consequences of ABCA3(E292)(V) gene expression in vivo are unknown. To address this question, we developed mouse models expressing ABCA3(E292V) knocked-in to the endogenous mouse locus. The parental (F1) mouse line (mAbca3(E292)(V)) that retained an intronic pgk-Neo selection cassette (inserted in reverse orientation) (mAbca3(E292)(V)-rNeo) demonstrated an allele dependent extracellular surfactant phospholipid (PL) deficiency. We hypothesize that this PL deficiency leads to aberrant parenchymal remodeling contributing to the pathophysiology of the DPLD phenotype. Compared to wild type littermates, baseline studies of mice homozygous for the pgk-Neo insert (mAbca3(E292)(V)-rNeo(+/)(+)) revealed nearly 50% reduction in bronchoalveolar lavage (BAL) PL content that was accompanied by quantitative reduction in AT2 LB size with a compensatory increase in LB number. The phenotypic alteration in surfactant lipid homeostasis resulted in an early macrophage predominant alveolitis which peaked at 8 weeks of age. This was followed by age-dependent development of histological DPLD characterized initially by peribronchial inflammatory cell infiltration and culminating in both an emphysema-like phenotype (which included stereologically quantifiable reductions in both alveolar septal surface area and volume of septal wall tissue) plus foci of trichrome-positive collagen deposition together with substantial proliferation of hyperplastic AT2 cells. In addition to spontaneous lung remodeling, mABCA3(E292V)-rNeo mice

  12. Distinct roles for the RSC and Swi/Snf ATP-dependent chromatin remodelers in DNA double-strand break repair.

    PubMed

    Chai, Bob; Huang, Jian; Cairns, Bradley R; Laurent, Brehon C

    2005-07-15

    The failure of cells to repair damaged DNA can result in genomic instability and cancer. To efficiently repair chromosomal DNA lesions, the repair machinery must gain access to the damaged DNA in the context of chromatin. Here we report that both the RSC and Swi/Snf ATP-dependent chromatin-remodeling complexes play key roles in double-strand break (DSB) repair, specifically by homologous recombination (HR). RSC and Swi/Snf are each recruited to an in vivo DSB site but with distinct kinetics. We show that Swi/Snf is required earlier, at or preceding the strand invasion step of HR, while RSC is required following synapsis for completion of the recombinational repair event.

  13. IUGR increases chromatin-remodeling factor Brg1 expression and binding to GR exon 1.7 promoter in newborn male rat hippocampus.

    PubMed

    Ke, Xingrao; McKnight, Robert A; Gracey Maniar, Lia E; Sun, Ying; Callaway, Christopher W; Majnik, Amber; Lane, Robert H; Cohen, Susan S

    2015-07-15

    Intrauterine growth restriction (IUGR) increases the risk for neurodevelopment delay and neuroendocrine reprogramming in both humans and rats. Neuroendocrine reprogramming involves the glucocorticoid receptor (GR) gene that is epigenetically regulated in the hippocampus. Using a well-characterized rodent model, we have previously shown that IUGR increases GR exon 1.7 mRNA variant and total GR expressions in male rat pup hippocampus. Epigenetic regulation of GR transcription may involve chromatin remodeling of the GR gene. A key chromatin remodeler is Brahma-related gene-1(Brg1), a member of the ATP-dependent SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex. Brg1 regulates gene expression by affecting nucleosome repositioning and recruiting transcriptional components to target promoters. We hypothesized that IUGR would increase hippocampal Brg1 expression and binding to GR exon 1.7 promoter, as well as alter nucleosome positioning over GR promoters in newborn male pups. Further, we hypothesized that IUGR would lead to accumulation of specificity protein 1 (Sp1) and RNA pol II at GR exon 1.7 promoter. Indeed, we found that IUGR increased Brg1 expression and binding to GR exon 1.7 promoter. We also found that increased Brg1 binding to GR exon 1.7 promoter was associated with accumulation of Sp1 and RNA pol II carboxy terminal domain pSer-5 (a marker of active transcription). Furthermore, the transcription start site of GR exon 1.7 was located within a nucleosome-depleted region. We speculate that changes in hippocampal Brg1 expression mediate GR expression and subsequently trigger neuroendocrine reprogramming in male IUGR rats.

  14. Downregulation of ARID1A, a component of the SWI/SNF chromatin remodeling complex, in breast cancer

    PubMed Central

    Takao, Chika; Morikawa, Akemi; Ohkubo, Hiroshi; Kito, Yusuke; Saigo, Chiemi; Sakuratani, Takuji; Futamura, Manabu; Takeuchi, Tamotsu; Yoshida, Kazuhiro

    2017-01-01

    Recent studies unraveled that AT-rich interactive domain-containing protein 1A (ARID1A), a subunit of the mammary SWI/SNF chromatin remodeling complex, acts as a tumor suppressor in various cancers. In this study, we first evaluated ARID1A expression by immunohistochemistry in invasive breast cancer tissue specimens and assessed the correlation with the prognosis of patients with breast cancer. Non-tumorous mammary duct epithelial cells exhibited strong nuclear ARID1A staining, whereas different degrees of loss in ARID1A immunoreactivity were observed in many invasive breast cancer cells. We scored ARID1A immunoreactivity based on the sum of the percentage score in invasive cancer cells (on a scale of 0 to 5) and the intensity score (on a scale of 0 to 3), for a possible total score of 0 to 8. Interestingly, partial loss of ARID1A expression, score 2 to 3, was significantly correlated with poor disease free survival of the patients. Subsequently, we performed siRNA-mediated ARID1A knockdown in cultured breast cancer cells followed by comprehensive gene profiling and quantitative RT-PCR. Interestingly, many genes were downregulated by partial loss of ARID1A, whereas RAB11FIP1 gene expression was significantly upregulated by partial loss of ARID1A expression in breast cancer cells. In contrast, a more than 50% reduction in ARID1A mRNA decreased RAB11FIP1gene expression. Immunoblotting also demonstrated that partial downregulation of ARID1A mRNA at approximately 20% reduction significantly increased the expression of RAB11FIP1 protein in MCF-7 cells, whereas, over 50% reduction of ARID1A mRNA resulted in reduction of RAB11FIP1 protein in cultured breast cancer cells. Recent studies reveal that RAB11FIP1 overexpression leads to breast cancer progression. Altogether, the present findings indicated that partial loss of ARID1A expression is linked to unfavorable outcome for patients with breast cancer, possibly due to increased RAB11FIP1 expression. PMID:28123592

  15. Dendritic Cell-Associated miRNAs Are Modulated via Chromatin Remodeling in Response to Different Environments

    PubMed Central

    Mei, Shiyue; Liu, Yuanhang; Bao, Yue; Zhang, Yuan; Min, Siping; Liu, Yifei; Huang, Yun; Yuan, Xidi; Feng, Yue; Shi, Jiandang; Yang, Rongcun

    2014-01-01

    Introduction Epigenetic modification plays a critical role in regulating gene expression. To understand how epigenetic modification alters miRNA expression in monocyte-derived dendritic cells (moDCs) in different environments, we analyzed the connections between H3K4me3 and H3K27me3 modification and the expression of miRNAs in LPS- and TGF-β-conditioned moDCs. Results In moDCs, H3K4me3 modification was strongly associated with the expression of activating miRNAs, whereas H3K27me3 was related to repressive miRNAs. The regulation of miRNA expression by H3K4me3 and H3K27me3 was further confirmed by silencing or inhibiting methyltransferases or methylation-associated factors in LPS- and TGF-β-conditioned moDCs. siRNAs targeting H3K4me3-associated mixed lineage leukemia (MLL) and retinoblastoma binding protein 5 (RBBP5) reduced H3K4me3 enrichment and downregulated miRNA expression; conversely, silencing H3K27me3-associated enhancer of zeste homolog 2 (EZH2) and embryonic ectoderm development (EED) genes upregulated the DC-associated miRNAs. However, LPS-mediated miRNAs were often associated with H3K4me3 redistribution from the transcription start site (TSS) to the miRNA-coding region. Silencing LPS-associated NF-κB p65 and CBP/p300 not only inhibited H3K4m3 redistribution but also reduced miRNA expression. LPS-upregulated RBBP4 and RBBP7, which are involved in chromatin remodeling, also affected the redistribution of H3K4me3 and reduced the expression of miRNAs. Conclusion In LPS- and TGF-β-conditioned moDCs, miRNAs may be modulated not only by H3K4m3 and H3K27me3 modification but also by redistribution of H3K4me3 around the transcriptional start site of miRNAs. Thus, H3K4me3 and H3K27me3 epigenetic modification may play an important role in regulating DC differentiation and function in the presence of tumor or inflammatory environments. PMID:24699235

  16. Remodeling somatic nuclei in Xenopus laevis egg extracts: molecular mechanisms for the selective release of histones H1 and H1(0) from chromatin and the acquisition of transcriptional competence.

    PubMed Central

    Dimitrov, S; Wolffe, A P

    1996-01-01

    The molecular mechanisms responsible for the remodeling of entire somatic erythrocyte nuclei in Xenopus laevis egg cytoplasm have been examined. These transitions in chromosomal composition are associated with the capacity to activate new patterns of gene expression and the re-acquisition of replication competence. Somatic linker histone variants H1 and H1 (0) are released from chromatin in egg cytoplasm, whereas the oocyte-specific linker histone B4 and HMG1 are efficiently incorporated into remodeled chromatin. Histone H1 (0) is released from chromatin preferentially in comparison with histone H1. Core histones H2A and H4 in the somatic nucleus are phosphorylated during this remodeling process. These transitions recapitulate the chromosomal environment found within the nuclei of the early Xenopus embryo. Phosphorylation of somatic linker histone variants is demonstrated not to direct their release from chromatin, nor does direct competition with cytoplasmic stores of linker histone B4 determine their release. However, the molecular chaperone nucleoplasmin does have an important role in the selective removal of linker histones from somatic nuclei. For Xenopus erythrocyte nuclei, this disruption of chromatin structure leads to activation of the 5S rRNA genes. These results provide a molecular explanation for the remodeling of chromatin in Xenopus egg cytoplasm and indicate the capacity of molecular chaperones to disrupt a natural chromosomal environment, thereby facilitating transcription. Images PMID:8918467

  17. BAF250B-associated SWI/SNF chromatin-remodeling complex is required to maintain undifferentiated mouse embryonic stem cells

    PubMed Central

    Yan, Zhijiang; Wang, Zhong; Sharova, Lioudmila; Sharov, Alexei A.; Ling, Chen; Piao, Yulan; Aiba, Kazuhiro; Matoba, Ryo; Wang, Weidong; Ko, Minoru S.H.

    2008-01-01

    Whether SWI/SNF chromatin remodeling complexes play roles in embryonic stem (ES) cells remains unknown. Here we show that SWI/SNF complexes are present in mouse ES cells, and their composition is dynamically regulated upon induction of ES cell differentiation. For example, the SWI/SNF purified from undifferentiated ES cells contains a high level of BAF155 and a low level of BAF170 (both of which are homologs of yeast SWI3 protein), whereas that from differentiated cells contains near equal amounts of both. Moreover, the levels of BAF250A and BAF250B decrease, whereas that of BRM increases, during the differentiation of ES cells. The altered expression of SWI/SNF components hinted that these complexes could play roles in ES cell maintenance or differentiation. We therefore generated ES cells with biallelic inactivation of BAF250B, and found that these cells display a reduced proliferation rate and an abnormal cell cycle. Importantly, these cells are deficient in self-renewal capacity of undifferentiated ES cells, and exhibit certain phenotypes of differentiated cells, including reduced expression of several pluripotency-related genes, and increased expression of some differentiation-related genes. These data suggest that the BAF250B-associated SWI/SNF is essential for mouse ES cells to maintain its normal proliferation and pluripotency. The work presented here underscores the importance of SWI/SNF chromatin remodeling complexes in pluripotent stem cells. PMID:18323406

  18. Roles of chromatin remodelers in maintenance mechanisms of multipotency of mouse trunk neural crest cells in the formation of neural crest-derived stem cells.

    PubMed

    Fujita, Kyohei; Ogawa, Ryuhei; Kawawaki, Syunsaku; Ito, Kazuo

    2014-08-01

    We analyzed roles of two chromatin remodelers, Chromodomain Helicase DNA-binding protein 7 (CHD7) and SWItch/Sucrose NonFermentable-B (SWI/SNF-B), and Bone Morphogenetic Protein (BMP)/Wnt signaling in the maintenance of the multipotency of mouse trunk neural crest cells, leading to the formation of mouse neural crest-derived stem cells (mouse NCSCs). CHD7 was expressed in the undifferentiated neural crest cells and in the dorsal root ganglia (DRG) and sciatic nerve, typical tissues containing NCSCs. BMP/Wnt signaling stimulated the expression of CHD7 and participated in maintaining the multipotency of neural crest cells. Furthermore, the promotion of CHD7 expression maintained the multipotency of these cells. The inhibition of CHD7 and SWI/SNF-B expression significantly suppressed the maintenance of the multipotency of these cells. In addition, BMP/Wnt treatment promoted CHD7 expression and caused the increase of the percentage of multipotent cells in DRG. Thus, the present data suggest that the chromatin remodelers as well as BMP/Wnt signaling play essential roles in the maintenance of the multipotency of mouse trunk neural crest cells and in the formation of mouse NCSCs.

  19. Arabidopsis BREVIPEDICELLUS interacts with the SWI2/SNF2 chromatin remodeling ATPase BRAHMA to regulate KNAT2 and KNAT6 expression in control of inflorescence architecture.

    PubMed

    Zhao, Minglei; Yang, Songguang; Chen, Chia-Yang; Li, Chenlong; Shan, Wei; Lu, Wangjin; Cui, Yuhai; Liu, Xuncheng; Wu, Keqiang

    2015-03-01

    BREVIPEDICELLUS (BP or KNAT1), a class-I KNOTTED1-like homeobox (KNOX) transcription factor in Arabidopsis thaliana, contributes to shaping the normal inflorescence architecture through negatively regulating other two class-I KNOX genes, KNAT2 and KNAT6. However, the molecular mechanism of BP-mediated transcription regulation remains unclear. In this study, we showed that BP directly interacts with the SWI2/SNF2 chromatin remodeling ATPase BRAHMA (BRM) both in vitro and in vivo. Loss-of-function BRM mutants displayed inflorescence architecture defects, with clustered inflorescences, horizontally orientated pedicels, and short pedicels and internodes, a phenotype similar to the bp mutants. Furthermore, the transcript levels of KNAT2 and KNAT6 were elevated in brm-3, bp-9 and brm-3 bp-9 double mutants. Increased histone H3 lysine 4 tri-methylation (H3K4me3) levels were detected in brm-3, bp-9 and brm-3 bp-9 double mutants. Moreover, BRM and BP co-target to KNAT2 and KNAT6 genes, and BP is required for the binding of BRM to KNAT2 and KNAT6. Taken together, our results indicate that BP interacts with the chromatin remodeling factor BRM to regulate the expression of KNAT2 and KNAT6 in control of inflorescence architecture.

  20. Use of chromatin remodeling ATPases as RNAi targets for parental control of western corn rootworm (Diabrotica virgifera virgifera) and Neotropical brown stink bug (Euschistus heros).

    PubMed

    Fishilevich, Elane; Vélez, Ana M; Khajuria, Chitvan; Frey, Meghan L F; Hamm, Ronda L; Wang, Haichuan; Schulenberg, Greg A; Bowling, Andrew J; Pence, Heather E; Gandra, Premchand; Arora, Kanika; Storer, Nicholas P; Narva, Kenneth E; Siegfried, Blair D

    2016-04-01

    RNA interference (RNAi) is a gene silencing mechanism that is present in animals and plants and is triggered by double stranded RNA (dsRNA) or small interfering RNA (siRNA), depending on the organism. In the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), RNAi can be achieved by feeding rootworms dsRNA added to artificial diet or plant tissues transformed to express dsRNA. The effect of RNAi depends on the targeted gene function and can range from an absence of phenotypic response to readily apparent responses, including lethality. Furthermore, RNAi can directly affect individuals that consume dsRNA or the effect may be transferred to the next generation. Our previous work described the potential use of genes involved in embryonic development as a parental RNAi technology for the control of WCR. In this study, we describe the use of chromatin-remodeling ATPases as target genes to achieve parental gene silencing in two insect pests, a coleopteran, WCR, and a hemipteran, the Neotropical brown stink bug, Euschistus heros Fabricius (Hemiptera: Pentatomidae). Our results show that dsRNA targeting chromatin-remodeling ATPase transcripts, brahma, mi-2, and iswi strongly reduced the fecundity of the exposed females in both insect species. Additionally, knockdown of chd1 reduced the fecundity of E. heros.

  1. Haploinsufficiency of ARID1B, a Member of the SWI/SNF-A Chromatin-Remodeling Complex, Is a Frequent Cause of Intellectual Disability

    PubMed Central

    Hoyer, Juliane; Ekici, Arif B.; Endele, Sabine; Popp, Bernt; Zweier, Christiane; Wiesener, Antje; Wohlleber, Eva; Dufke, Andreas; Rossier, Eva; Petsch, Corinna; Zweier, Markus; Göhring, Ina; Zink, Alexander M.; Rappold, Gudrun; Schröck, Evelin; Wieczorek, Dagmar; Riess, Olaf; Engels, Hartmut; Rauch, Anita; Reis, André

    2012-01-01

    Intellectual disability (ID) is a clinically and genetically heterogeneous common condition that remains etiologically unresolved in the majority of cases. Although several hundred diseased genes have been identified in X-linked, autosomal-recessive, or syndromic types of ID, the establishment of an etiological basis remains a difficult task in unspecific, sporadic cases. Just recently, de novo mutations in SYNGAP1, STXBP1, MEF2C, and GRIN2B were reported as relatively common causes of ID in such individuals. On the basis of a patient with severe ID and a 2.5 Mb microdeletion including ARID1B in chromosomal region 6q25, we performed mutational analysis in 887 unselected patients with unexplained ID. In this cohort, we found eight (0.9%) additional de novo nonsense or frameshift mutations predicted to cause haploinsufficiency. Our findings indicate that haploinsufficiency of ARID1B, a member of the SWI/SNF-A chromatin-remodeling complex, is a common cause of ID, and they add to the growing evidence that chromatin-remodeling defects are an important contributor to neurodevelopmental disorders. PMID:22405089

  2. Human INO80 chromatin-remodelling complex contributes to DNA double-strand break repair via the expression of Rad54B and XRCC3 genes.

    PubMed

    Park, Eun-Jung; Hur, Shin-Kyoung; Kwon, Jongbum

    2010-10-15

    Recent studies have shown that the SWI/SNF family of ATP-dependent chromatin-remodelling complexes play important roles in DNA repair as well as in transcription. The INO80 complex, the most recently described member of this family, has been shown in yeast to play direct role in DNA DSB (double-strand break) repair without affecting the expression of the genes involved in this process. However, whether this function of the INO80 complex is conserved in higher eukaryotes has not been investigated. In the present study, we found that knockdown of hINO80 (human INO80) confers DNA-damage hypersensitivity and inefficient DSB repair. Microarray analysis and other experiments have identified the Rad54B and XRCC3 (X-ray repair complementing defective repair in Chinese-hamster cells 3) genes, implicated in DSB repair, to be repressed by hINO80 deficiency. Chromatin immunoprecipitation studies have shown that hINO80 binds to the promoters of the Rad54B and XRCC3 genes. Re-expression of the Rad54B and XRCC3 genes rescues the DSB repair defect in hINO80-deficient cells. These results suggest that hINO80 assists DSB repair by positively regulating the expression of the Rad54B and XRCC3 genes. Therefore, unlike yeast INO80, hINO80 can contribute to DSB repair indirectly via gene expression, suggesting that the mechanistic role of this chromatin remodeller in DSB repair is evolutionarily diversified.

  3. The Catalytic and Non-catalytic Functions of the Brahma Chromatin-Remodeling Protein Collaborate to Fine-Tune Circadian Transcription in Drosophila

    PubMed Central

    Kwok, Rosanna S.; Li, Ying H.; Lei, Anna J.; Edery, Isaac; Chiu, Joanna C.

    2015-01-01

    Daily rhythms in gene expression play a critical role in the progression of circadian clocks, and are under regulation by transcription factor binding, histone modifications, RNA polymerase II (RNAPII) recruitment and elongation, and post-transcriptional mechanisms. Although previous studies have shown that clock-controlled genes exhibit rhythmic chromatin modifications, less is known about the functions performed by chromatin remodelers in animal clockwork. Here we have identified the Brahma (Brm) complex as a regulator of the Drosophila clock. In Drosophila, CLOCK (CLK) is the master transcriptional activator driving cyclical gene expression by participating in an auto-inhibitory feedback loop that involves stimulating the expression of the main negative regulators, period (per) and timeless (tim). BRM functions catalytically to increase nucleosome density at the promoters of per and tim, creating an overall restrictive chromatin landscape to limit transcriptional output during the active phase of cycling gene expression. In addition, the non-catalytic function of BRM regulates the level and binding of CLK to target promoters and maintains transient RNAPII stalling at the per promoter, likely by recruiting repressive and pausing factors. By disentangling its catalytic versus non-catalytic functions at the promoters of CLK target genes, we uncovered a multi-leveled mechanism in which BRM fine-tunes circadian transcription. PMID:26132408

  4. Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators.

    PubMed

    Polstein, Lauren R; Perez-Pinera, Pablo; Kocak, D Dewran; Vockley, Christopher M; Bledsoe, Peggy; Song, Lingyun; Safi, Alexias; Crawford, Gregory E; Reddy, Timothy E; Gersbach, Charles A

    2015-08-01

    Genome engineering technologies based on the CRISPR/Cas9 and TALE systems are enabling new approaches in science and biotechnology. However, the specificity of these tools in complex genomes and the role of chromatin structure in determining DNA binding are not well understood. We analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators in human cells using ChIP-seq to assess DNA-binding specificity and RNA-seq to measure the specificity of perturbing the transcriptome. Additionally, DNase-seq was used to assess genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these transcription factors are highly specific in both DNA binding and gene regulation and are able to open targeted regions of closed chromatin independent of gene activation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function.

  5. Promoter chromatin remodeling of immediate-early genes is mediated through H3 phosphorylation at either serine 28 or 10 by the MSK1 multi-protein complex

    PubMed Central

    Drobic, Bojan; Pérez-Cadahía, Beatriz; Yu, Jenny; Kung, Sam Kam-Pun; Davie, James R.

    2010-01-01

    Upon activation of the ERK and p38 MAPK pathways, the MSK1/2-mediated nucleosomal response, including H3 phosphorylation at serine 28 or 10, is coupled with the induction of immediate-early (IE) gene transcription. The outcome of this response, varying with the stimuli and cellular contexts, ranges from neoplastic transformation to neuronal synaptic plasticity. Here, we used sequential co-immunoprecipitation assays and sequential chromatin immunoprecipitation (ChIP) assays on mouse fibroblast 10T1/2 and MSK1 knockdown 10T1/2 cells to show that H3 serine 28 and 10 phosphorylation leads to promoter remodeling. MSK1, in complexes with phospho-serine adaptor 14-3-3 proteins and BRG1 the ATPase subunit of the SWI/SNF remodeler, is recruited to the promoter of target genes by transcription factors such as Elk-1 or NF-κB. Following MSK1-mediated H3 phosphorylation, BRG1 associates with the promoter of target genes via 14-3-3 proteins, which act as scaffolds. The recruited SWI/SNF remodels nucleosomes at the promoter of IE genes enabling the binding of transcription factors like JUN and the onset of transcription. PMID:20129940

  6. Synergistic activation of Arg1 gene by retinoic acid and IL-4 involves chromatin remodeling for transcription initiation and elongation coupling

    PubMed Central

    Lee, Bomi; Wu, Cheng-Ying; Lin, Yi-Wei; Park, Sung Wook; Wei, Li-Na

    2016-01-01

    All-trans Retinoic acid (RA) and its derivatives are potent therapeutics for immunological functions including wound repair. However, the molecular mechanism of RA modulation in innate immunity is poorly understood, especially in macrophages. We found that topical application of RA significantly improves wound healing and that RA and IL-4 synergistically activate Arg1, a critical gene for tissue repair, in M2 polarized macrophages. This involves feed forward regulation of Raldh2, a rate-limiting enzyme for RA biosynthesis, and requires Med25 to coordinate RAR, STAT6 and chromatin remodeler, Brg1 to remodel the +1 nucleosome of Arg1 for transcription initiation. By recruiting elongation factor TFIIS, Med25 also facilitates transcriptional initiation-elongation coupling. This study uncovers synergistic activation of Arg1 by RA and IL-4 in M2 macrophages that involves feed forward regulation of RA synthesis and dual functions of Med25 in nucleosome remodeling and transcription initiation-elongation coupling that underlies robust modulatory activity of RA in innate immunity. PMID:27166374

  7. Remodeling sperm chromatin in Xenopus laevis egg extracts: the role of core histone phosphorylation and linker histone B4 in chromatin assembly

    PubMed Central

    1994-01-01

    We find that the remodeling of the condensed Xenopus laevis sperm nucleus into the paternal pronucleus in egg extracts is associated with phosphorylation of the core histones H2A, H2A.X and H4, and uptake of a linker histone B4 and a HMG 2 protein. Histone B4 is required for the assembly of chromatosome structures in the pronucleus. However neither B4 nor core histone phosphorylation are required for the assembly of spaced nucleosomal arrays. We suggest that the spacing of nucleosomal arrays is determined by interaction between adjacent histone octamers under physiological assembly conditions. PMID:8045925

  8. Changes in chromatin accessibility across the GM-CSF promoter upon T cell activation are dependent on nuclear factor kappaB proteins.

    PubMed

    Holloway, Adele F; Rao, Sudha; Chen, Xinxin; Shannon, M Frances

    2003-02-17

    Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a key cytokine in myelopoiesis and aberrant expression is associated with chronic inflammatory disease and myeloid leukemias. This aberrant expression is often associated with constitutive nuclear factor (NF)-kappaB activation. To investigate the relationship between NF-kappaB and GM-CSF transcription in a chromatin context, we analyzed the chromatin structure of the GM-CSF gene in T cells and the role of NF-kappaB proteins in chromatin remodeling. We show here that chromatin remodeling occurs across a region of the GM-CSF gene between -174 and +24 upon T cell activation, suggesting that remodeling is limited to a single nucleosome encompassing the proximal promoter. Nuclear NF-kappaB levels appear to play a critical role in this process. In addition, using an immobilized template assay we found that the ATPase component of the SWI/SNF chromatin remodeling complex, brg1, is recruited to the GM-CSF proximal promoter in an NF-kappaB-dependent manner in vitro. These results suggest that chromatin remodeling across the GM-CSF promoter in T cells is a result of recruitment of SWI/SNF type remodeling complexes by NF-kappaB proteins binding to the CD28 response region of the promoter.

  9. Chromatin-prebound Crm1 recruits Nup98-HoxA9 fusion to induce aberrant expression of Hox cluster genes.

    PubMed

    Oka, Masahiro; Mura, Sonoko; Yamada, Kohji; Sangel, Percival; Hirata, Saki; Maehara, Kazumitsu; Kawakami, Koichi; Tachibana, Taro; Ohkawa, Yasuyuki; Kimura, Hiroshi; Yoneda, Yoshihiro

    2016-01-07

    The nucleoporin Nup98 is frequently rearranged to form leukemogenic Nup98-fusion proteins with various partners. However, their function remains largely elusive. Here, we show that Nup98-HoxA9, a fusion between Nup98 and the homeobox transcription factor HoxA9, forms nuclear aggregates that frequently associate with facultative heterochromatin. We demonstrate that stable expression of Nup98-HoxA9 in mouse embryonic stem cells selectively induces the expression of Hox cluster genes. Genome-wide binding site analysis revealed that Nup98-HoxA9 is preferentially targeted and accumulated at Hox cluster regions where the export factor Crm1 is originally prebound. In addition, leptomycin B, an inhibitor of Crm1, disassembled nuclear Nup98-HoxA9 dots, resulting in the loss of chromatin binding of Nup98-HoxA9 and Nup98-HoxA9-mediated activation of Hox genes. Collectively, our results indicate that highly selective targeting of Nup98-fusion proteins to Hox cluster regions via prebound Crm1 induces the formation of higher order chromatin structures that causes aberrant Hox gene regulation.

  10. Chromatin-prebound Crm1 recruits Nup98-HoxA9 fusion to induce aberrant expression of Hox cluster genes

    PubMed Central

    Oka, Masahiro; Mura, Sonoko; Yamada, Kohji; Sangel, Percival; Hirata, Saki; Maehara, Kazumitsu; Kawakami, Koichi; Tachibana, Taro; Ohkawa, Yasuyuki; Kimura, Hiroshi; Yoneda, Yoshihiro

    2016-01-01

    The nucleoporin Nup98 is frequently rearranged to form leukemogenic Nup98-fusion proteins with various partners. However, their function remains largely elusive. Here, we show that Nup98-HoxA9, a fusion between Nup98 and the homeobox transcription factor HoxA9, forms nuclear aggregates that frequently associate with facultative heterochromatin. We demonstrate that stable expression of Nup98-HoxA9 in mouse embryonic stem cells selectively induces the expression of Hox cluster genes. Genome-wide binding site analysis revealed that Nup98-HoxA9 is preferentially targeted and accumulated at Hox cluster regions where the export factor Crm1 is originally prebound. In addition, leptomycin B, an inhibitor of Crm1, disassembled nuclear Nup98-HoxA9 dots, resulting in the loss of chromatin binding of Nup98-HoxA9 and Nup98-HoxA9-mediated activation of Hox genes. Collectively, our results indicate that highly selective targeting of Nup98-fusion proteins to Hox cluster regions via prebound Crm1 induces the formation of higher order chromatin structures that causes aberrant Hox gene regulation. DOI: http://dx.doi.org/10.7554/eLife.09540.001 PMID:26740045

  11. The SWI2/SNF2 Chromatin Remodeling ATPase BRAHMA Represses Abscisic Acid Responses in the Absence of the Stress Stimulus in Arabidopsis[W

    PubMed Central

    Han, Soon-Ki; Sang, Yi; Rodrigues, Americo; Wu, Miin-Feng; Rodriguez, Pedro L.; Wagner, Doris

    2012-01-01

    The survival of plants as sessile organisms depends on their ability to cope with environmental challenges. Of key importance in this regard is the phytohormone abscisic acid (ABA). ABA not only promotes seed dormancy but also triggers growth arrest in postgermination embryos that encounter water stress. This is accompanied by increased desiccation tolerance. Postgermination ABA responses in Arabidopsis thaliana are mediated in large part by the ABA-induced basic domain/leucine zipper transcription factor ABA INSENSITIVE5 (ABI5). Here, we show that loss of function of the SWI2/SNF2 chromatin remodeling ATPase BRAHMA (BRM) causes ABA hypersensitivity during postgermination growth arrest. ABI5 expression was derepressed in brm mutants in the absence of exogenous ABA and accumulated to high levels upon ABA sensing. This effect was likely direct; chromatin immunoprecipitation revealed BRM binding to the ABI5 locus. Moreover, loss of BRM activity led to destabilization of a nucleosome likely to repress ABI5 transcription. Finally, the abi5 null mutant was epistatic to BRM in postgermination growth arrest. In addition, vegetative growth defects typical of brm mutants in the absence of ABA treatment could be partially overcome by reduction of ABA responses, and brm mutants displayed increased drought tolerance. We propose a role for BRM in the balance between growth or stress responses. PMID:23209114

  12. Dietary phenolic acids attenuate multiple stages of protein glycation and high-glucose-stimulated proinflammatory IL-1beta activation by interfering with chromatin remodeling and transcription in monocytes.

    PubMed

    Wu, Chi-Hao; Yeh, Chi-Tai; Shih, Ping-Hsiao; Yen, Gow-Chin

    2010-07-01

    This study examined the effects of dietary phenolic acids on individual stages of protein glycation and utilized monocyte cultures to assess whether these phytochemicals modulate the activation of proinflammatory cytokine under high glucose (HG, 15 mmol/L) conditions mimicking diabetes. In vitro glycation assays showed that a number of phenolic acids exerted inhibitory effects on the glycation reaction and its subsequent crosslinking. Phenolic acids, especially methoxyphenolic acids, prevented increase in both levels of the interleukin-1beta (IL-1beta) and oxidative stress caused by HG. The effect appeared to be mediated by modulation of the protein kinase C/nuclear factor-kappaB axis. Chromatin immunoprecipitation demonstrated for the first time that HG increased the recruitment of nuclear factor-kappaB p65 and CREB-binding protein to the IL-1beta promoter. Interestingly, HG also increased histone acetylation and methylation within the IL-1beta promoter and decreased histone deacetylase activities in monocytes, thus facilitating chromatin remodeling and transcription. Such inappropriate inflammatory responses were found to be controlled effectively by treatment with methoxyphenolic compounds. In conclusion, this study suggests that phenolic acids could exert their anti-inflammatory activities as antiglycation agents and as modifiers of signaling pathways. It provides evidence for a novel mechanism by which phenolics supplementation might have additional protective effects against diabetic complications.

  13. A genome-wide IR-induced RAD51 foci RNAi screen identifies CDC73 involved in chromatin remodeling for DNA repair

    PubMed Central

    Herr, Patrick; Lundin, Cecilia; Evers, Bastiaan; Ebner, Daniel; Bauerschmidt, Christina; Kingham, Guy; Palmai-Pallag, Timea; Mortusewicz, Oliver; Frings, Oliver; Sonnhammer, Erik; Helleday, Thomas

    2015-01-01

    To identify new regulators of homologous recombination repair, we carried out a genome-wide short-interfering RNA screen combined with ionizing irradiation using RAD51 foci formation as readout. All candidates were confirmed by independent short-interfering RNAs and validated in secondary assays like recombination repair activity and RPA foci formation. Network analysis of the top modifiers identified gene clusters involved in recombination repair as well as components of the ribosome, the proteasome and the spliceosome, which are known to be required for effective DNA repair. We identified and characterized the RNA polymerase II-associated protein CDC73/Parafibromin as a new player in recombination repair and show that it is critical for genomic stability. CDC73 interacts with components of the SCF/Cullin and INO80/NuA4 chromatin-remodeling complexes to promote Histone ubiquitination. Our findings indicate that CDC73 is involved in local chromatin decondensation at sites of DNA damage to promote DNA repair. This function of CDC73 is related to but independent of its role in transcriptional elongation. PMID:27462432

  14. Lack of telomerase gene expression in alternative lengthening of telomere cells is associated with chromatin remodeling of the hTR and hTERT gene promoters.

    PubMed

    Atkinson, Stuart P; Hoare, Stacey F; Glasspool, Rosalind M; Keith, W Nicol

    2005-09-01

    The presence of active telomere maintenance mechanisms in immortal cells allows the bypass of senescence by maintaining telomere length. In most immortal cell lines and tumors, telomere maintenance is attributable to telomerase reactivation. However, a number of immortal cell lines and tumors can achieve telomere maintenance in the absence of detectable telomerase activity by the alternative lengthening of telomere (ALT) mechanism. Epigenetic mechanisms have been implicated in the regulation of telomerase expression. We show that specific modifications within the chromatin environment of the hTR and hTERT promoters correlate with expression of hTR and hTERT in ALT, normal and telomerase-positive tumor cell lines. Lack of expression of hTR and hTERT in ALT cell lines is associated with histone H3 and H4 hypoacetylation and methylation of Lys9 histone H3. Conversely, hTR and hTERT expression in telomerase-positive cell lines is associated with hyperacetylation of H3 and H4 and methylation of Lys4 H3. Methylation of Lys20 H4 was not linked to gene expression but instead was specific to the hTR and hTERT promoters of ALT cells. This may provide an insight into the differences between ALT and telomerase-positive cells as well as a novel marker for the ALT phenotype. Treatment of normal and ALT cells with 5-azadeoxycytidine in combination with Trichostatin A caused chromatin remodeling of both promoters and reactivation of hTR and hTERT expression in ALT and normal cell lines. This data establishes a definite link between the chromatin environment of the telomerase gene promoters and transcriptional activity.

  15. Chromatin Remodeling Factor LSH is Upregulated by the LRP6-GSK3β-E2F1 Axis Linking Reversely with Survival in Gliomas

    PubMed Central

    Xiao, Desheng; Huang, Jun; Pan, Yu; Li, Hao; Fu, Chunyan; Mao, Chao; Cheng, Yan; Shi, Ying; Chen, Ling; Jiang, Yiqun; Yang, Rui; Liu, Yating; Zhou, Jianhua; Cao, Ya; Liu, Shuang; Tao, Yongguang

    2017-01-01

    The signaling pathway-based stratification in chromatin modification could predict clinical outcome more reliably than morphology-alone-based classification schemes in gliomas. Here we reported a role of the chromatin-remodeling factor lymphoid-specific helicase (LSH) in gliomas. Among astrocytomas of grade I to III and glioblastoma of grade IV, LSH were almost completely expressed in all cases, and strongly correlated with astrocytomas progression and poor prognosis of patients with astrocytomas and glioblastoma. Ectopic expression of LSH promoted tumor formation. Up-regulation of transcription factor E2F1 in astrocytomas and glioblastoma was associated with the progression of gliomas and correlated with LSH expression. Chromatin immunoprecipitation (ChIP) analysis showed transcription factor E2F1 were recruited to the promoter region of LSH, and depletion of E2F1 decreased LSH expression and cell growth. Moreover, glycogen synthase kinase-3β (GSK-3β), an intact complex of E2F1, were also highly expressed in astrocytomas and linked with astrocytomas progression and poor prognosis of patients with astrocytomas and glioblastoma. Inhibition of GSK3β increased the enrichment of E2F1 to the LSH promoter, in turn, increased LSH expression. Lipoprotein receptor-related protein 6 (LRP6), an upstream regulator of GSK3β signaling pathway, was highly expressed in gliomas. Knockdown of LRP6 decreased LSH expression through decrease of recruitment of E2F1 to the LSH promoter leading to inhibition of cell growth. Taken together, this study reveals evidence demonstrating a mechanism by which upregulated promoted gliomas. A mechanistic link between LSH expression and activation of the LPR6/ GSK3β/E2F1 axis in gliomas illustrates a novel role of LSH in malignant astrocytomas and glioblastoma. PMID:28042322

  16. Chromium Cross-Links Histone Deacetylase 1-DNA Methyltransferase 1 Complexes to Chromatin, Inhibiting Histone-Remodeling Marks Critical for Transcriptional Activation▿

    PubMed Central

    Schnekenburger, Michael; Talaska, Glenn; Puga, Alvaro

    2007-01-01

    Transcriptional regulation of gene expression requires posttranslational modification of histone proteins, which, in concert with chromatin-remodeling factors, modulate chromatin structure. Exposure to environmental agents may interfere with specific histone modifications and derail normal patterns of gene expression. To test this hypothesis, we coexposed cells to binary mixtures of benzo[a]pyrene (B[a]P), an environmental procarcinogen that activates Cyp1a1 transcriptional responses mediated by the aryl hydrocarbon receptor (AHR), and chromium, a carcinogenic heavy metal that represses B[a]P-inducible AHR-mediated gene expression. We show that chromium cross-links histone deacetylase 1-DNA methyltransferase 1 (HDAC1-DNMT1) complexes to Cyp1a1 promoter chromatin and inhibits histone marks induced by AHR-mediated gene transactivation, including phosphorylation of histone H3 Ser-10, trimethylation of H3 Lys-4, and various acetylation marks in histones H3 and H4. These changes inhibit RNA polymerase II recruitment without affecting the kinetics of AHR DNA binding. HDAC1 and DNMT1 inhibitors or depletion of HDAC1 or DNMT1 with siRNAs blocks chromium-induced transcriptional repression by decreasing the interaction of these proteins with the Cyp1a1 promoter and allowing histone acetylation to proceed. By inhibiting Cyp1a1 expression, chromium stimulates the formation of B[a]P DNA adducts. Epigenetic modification of gene expression patterns may be a key element of the developmental and carcinogenic outcomes of exposure to chromium and to other environmental agents. PMID:17682057

  17. p63 regulates Satb1 to control tissue-specific chromatin remodeling during development of the epidermis

    PubMed Central

    Fessing, Michael Y.; Mardaryev, Andrei N.; Gdula, Michal R.; Sharov, Andrey A.; Sharova, Tatyana Y.; Rapisarda, Valentina; Gordon, Konstantin B.; Smorodchenko, Anna D.; Poterlowicz, Krzysztof; Ferone, Giustina; Kohwi, Yoshinori; Missero, Caterina

    2011-01-01

    During development, multipotent progenitor cells establish tissue-specific programs of gene expression. In this paper, we show that p63 transcription factor, a master regulator of epidermal morphogenesis, executes its function in part by directly regulating expression of the genome organizer Satb1 in progenitor cells. p63 binds to a proximal regulatory region of the Satb1 gene, and p63 ablation results in marked reduction in the Satb1 expression levels in the epidermis. Satb1−/− mice show impaired epidermal morphology. In Satb1-null epidermis, chromatin architecture of the epidermal differentiation complex locus containing genes associated with epidermal differentiation is altered primarily at its central domain, where Satb1 binding was confirmed by chromatin immunoprecipitation–on-chip analysis. Furthermore, genes within this domain fail to be properly activated upon terminal differentiation. Satb1 expression in p63+/− skin explants treated with p63 small interfering ribonucleic acid partially restored the epidermal phenotype of p63-deficient mice. These data provide a novel mechanism by which Satb1, a direct downstream target of p63, contributes in epidermal morphogenesis via establishing tissue-specific chromatin organization and gene expression in epidermal progenitor cells. PMID:21930775

  18. The role of chromatin-remodeling factor PKL in balancing osmotic stress responses during Arabidopsis seed germination.

    PubMed

    Perruc, Elian; Kinoshita, Natsuko; Lopez-Molina, Luis

    2007-12-01

    Embryogenesis in Arabidopsis results in mature, osmotolerant embryos within dry seeds. Late-embryogenesis programs involve the transcription factors ABI3 and ABI5, which are necessary for osmotolerance. ABI3 and ABI5 are degraded in seeds initiating germination, abolishing their protected state. However, during an early stage of germination, strong osmotic stresses, or ABA exposure maintain ABI3 and ABI5 expression, leading to growth arrest, and osmotolerance. Mild stress stimuli delay ABI3 and ABI5 disappearance, retarding germination but not preventing eventual closure of embryogenesis programs. PICKLE (PKL), a putative chromatin modifier, is necessary to repress ABI3 and ABI5 expression during germination in response to ABA. We show that pkl mutants display persistent high expression of ABI3 and ABI5 upon ABA stimulation. In turn, maintenance of ABI5 expression leads to hypersensitive germination responses to ABA in pkl seeds. We provide evidence that ABI3 and ABI5 are less associated with repressed chromatin in pkl mutants. Our results provide evidence that PKL-dependent repression of embryonic gene expression extends to late-embryogenesis genes and is associated with changes in chromatin. We suggest that effective closure of embryogenesis omostolerance programs during germination prevents excessive plant reactions to stress.

  19. Distinct modes of SMAD2 chromatin binding and remodeling shape the transcriptional response to NODAL/Activin signaling

    PubMed Central

    Coda, Davide M; Gaarenstroom, Tessa; East, Philip; Patel, Harshil; Miller, Daniel S J; Lobley, Anna; Matthews, Nik; Stewart, Aengus; Hill, Caroline S

    2017-01-01

    NODAL/Activin signaling orchestrates key processes during embryonic development via SMAD2. How SMAD2 activates programs of gene expression that are modulated over time however, is not known. Here we delineate the sequence of events that occur from SMAD2 binding to transcriptional activation, and the mechanisms underlying them. NODAL/Activin signaling induces dramatic chromatin landscape changes, and a dynamic transcriptional network regulated by SMAD2, acting via multiple mechanisms. Crucially we have discovered two modes of SMAD2 binding. SMAD2 can bind pre-acetylated nucleosome-depleted sites. However, it also binds to unacetylated, closed chromatin, independently of pioneer factors, where it induces nucleosome displacement and histone acetylation. For a subset of genes, this requires SMARCA4. We find that long term modulation of the transcriptional responses requires continued NODAL/Activin signaling. Thus SMAD2 binding does not linearly equate with transcriptional kinetics, and our data suggest that SMAD2 recruits multiple co-factors during sustained signaling to shape the downstream transcriptional program. DOI: http://dx.doi.org/10.7554/eLife.22474.001 PMID:28191871

  20. Sustained activation of STAT5 is essential for chromatin remodeling and maintenance of mammary-specific function

    SciTech Connect

    Xu, Ren; Nelson, Celeste M.; Muschler, John L.; Veiseh, Mandana; Vonderhaar, Barbara K.; Bissell, Mina J.

    2009-06-03

    Epithelial cells, once dissociated and placed in two-dimensional (2D) cultures, rapidly lose tissue-specific functions. We showed previously that in addition to prolactin, signaling by laminin-111 was necessary to restore functional differentiation of mammary epithelia. Here, we elucidate two additional aspects of laminin-111 action. We show that in 2D cultures, the prolactin receptor is basolaterally localized and physically segregated from its apically placed ligand. Detachment of the cells exposes the receptor to ligation by prolactin leading to signal transducers and activators of transcription protein 5 (STAT5) activation, but only transiently and not sufficiently for induction of milk protein expression. We show that laminin-111 reorganizes mammary cells into polarized acini, allowing both the exposure of the prolactin receptor and sustained activation of STAT5. The use of constitutively active STAT5 constructs showed that the latter is necessary and sufficient for chromatin reorganization and {beta}-casein transcription. These results underscore the crucial role of continuous laminin signaling and polarized tissue architecture in maintenance of transcription factor activation, chromatin organization, and tissue-specific gene expression.

  1. Chromatin-dependent transcription factor accessibility rather than nucleosome remodeling predominates during global transcriptional restructuring in Saccharomyces cerevisiae.

    PubMed

    Zawadzki, Karl A; Morozov, Alexandre V; Broach, James R

    2009-08-01

    Several well-studied promoters in yeast lose nucleosomes upon transcriptional activation and gain them upon repression, an observation that has prompted the model that transcriptional activation and repression requires nucleosome remodeling of regulated promoters. We have examined global nucleosome positioning before and after glucose-induced transcriptional reprogramming, a condition under which more than half of all yeast genes significantly change expression. The majority of induced and repressed genes exhibit no change in promoter nucleosome arrangement, although promoters that do undergo nucleosome remodeling tend to contain a TATA box. Rather, we found multiple examples where the pre-existing accessibility of putative transcription factor binding sites before glucose addition determined whether the corresponding gene would change expression in response to glucose addition. These results suggest that selection of appropriate transcription factor binding sites may be dictated to a large extent by nucleosome prepositioning but that regulation of expression through these sites is dictated not by nucleosome repositioning but by changes in transcription factor activity.

  2. Human INO80/YY1 chromatin remodeling complex transcriptionally regulates the BRCA2- and CDKN1A-interacting protein (BCCIP) in cells.

    PubMed

    Su, Jiaming; Sui, Yi; Ding, Jian; Li, Fuqiang; Shen, Shuang; Yang, Yang; Lu, Zeming; Wang, Fei; Cao, Lingling; Liu, Xiaoxia; Jin, Jingji; Cai, Yong

    2016-10-01

    The BCCIP (BRCA2- and CDKN1A-interacting protein) is an important cofactor for BRCA2 in tumor suppression. Although the low expression of BCCIP is observed in multiple clinically diagnosed primary tumor tissues such as ovarian cancer, renal cell carcinoma and colorectal carcinoma, the mechanism of how BCCIP is regulated in cells is still unclear. The human INO80/YY1 chromatin remodeling complex composed of 15 subunits catalyzes ATP-dependent sliding of nucleosomes along DNA. Here, we first report that BCCIP is a novel target gene of the INO80/YY1 complex by presenting a series of experimental evidence. Gene expression studies combined with siRNA knockdown data locked candidate genes including BCCIP of the INO80/YY1 complex. Silencing or over-expressing the subunits of the INO80/YY1 complex regulates the expression level of BCCIP both in mRNA and proteins in cells. Also, the functions of INO80/YY1 complex in regulating the transactivation of BCCIP were confirmed by luciferase reporter assays. Chromatin immunoprecipitation (ChIP) experiments clarify the enrichment of INO80 and YY1 at +0.17 kb downstream of the BCCIP transcriptional start site. However, this enrichment is significantly inhibited by either knocking down INO80 or YY1, suggesting the existence of both INO80 and YY1 is required for recruiting the INO80/YY1 complex to BCCIP promoter region. Our findings strongly indicate that BCCIP is a potential target gene of the INO80/YY1 complex.

  3. TOPOISOMERASE 6B is involved in chromatin remodelling associated with control of carbon partitioning into secondary metabolites and cell walls, and epidermal morphogenesis in Arabidopsis

    PubMed Central

    Mittal, Amandeep; Balasubramanian, Rajagopal; Cao, Jin; Singh, Prabhjeet; Subramanian, Senthil; Hicks, Glenn; Nothnagel, Eugene A.; Abidi, Noureddine; Janda, Jaroslav; Galbraith, David W.; Rock, Christopher D.

    2014-01-01

    Plant growth is continuous and modular, a combination that allows morphogenesis by cell division and elongation and serves to facilitate adaptation to changing environments. The pleiotropic phenotypes of the harlequin (hlq) mutant, isolated on the basis of ectopic expression of the abscisic acid (ABA)- and auxin-inducible proDc3:GUS reporter gene, were previously characterized. Mutants are skotomorphogenic, have deformed and collapsed epidermal cells which accumulate callose and starch, cell walls abundant in pectins and cell wall proteins, and abnormal and reduced root hairs and leaf trichomes. hlq and two additional alleles that vary in their phenotypic severity of starch accumulation in the light and dark have been isolated, and it is shown that they are alleles of bin3/hyp6/rhl3/Topoisomerase6B. Mutants and inhibitors affecting the cell wall phenocopy several of the traits displayed in hlq. A microarray analysis was performed, and coordinated expression of physically adjacent pairs/sets of genes was observed in hlq, suggesting a direct effect on chromatin. Histones, WRKY and IAA/AUX transcription factors, aquaporins, and components of ubiquitin-E3-ligase-mediated proteolysis, and ABA or biotic stress response markers as well as proteins involved in cellular processes affecting carbon partitioning into secondary metabolites were also identified. A comparative analysis was performed of the hlq transcriptome with other previously published TopoVI mutant transcriptomes, namely bin3, bin5, and caa39 mutants, and limited concordance between data sets was found, suggesting indirect or genotype-specific effects. The results shed light on the molecular mechanisms underlying the det/cop/fus-like pleiotropic phenotypes of hlq and support a broader role for TopoVI regulation of chromatin remodelling to mediate development in response to environmental and hormonal signals. PMID:24821950

  4. Reduced expression of the chromatin remodeling gene ARID1A enhances gastric cancer cell migration and invasion via downregulation of E-cadherin transcription.

    PubMed

    Yan, Hai-Bo; Wang, Xue-Fei; Zhang, Qian; Tang, Zhao-Qing; Jiang, Ying-Hua; Fan, Hui-Zhi; Sun, Yi-hong; Yang, Peng-Yuan; Liu, Feng

    2014-04-01

    The chromatin remodeling gene AT-rich interactive domain-containing protein 1A (ARID1A) encodes the protein BAF250a, a subunit of human SWI/SNF-related complexes. Recent studies have identified ARID1A as a tumor suppressor. Here, we show that ARID1A expression is reduced in gastric cancer (GC) tissues, which are significantly associated with local lymph node metastasis, tumor infiltration and poor patient prognosis. ARID1A silencing enforces the migration and invasion of GC cells, whereas ectopic expression of ARID1A inhibits migration. The adhesive protein E-cadherin is remarkably downregulated in response to ARID1A silencing, but it is upregulated by ARID1A overexpression. E-cadherin overexpression significantly inhibits GC cell migration and invasion, whereas CDH1 (coded E-cadherin) silencing promotes migration. Restored expression of CDH1 in ARID1A-silenced cell lines restores the inhibition of cell migration. Luciferase reporter assays and chromatin immunoprecipitation indicate that the ARID1A-associated SWI/SNF complex binds to the CDH1 promoter and modulates CDH1 transcription. ARID1A knockdown induces evident morphological changes of GC cells with increased expression of mesenchymal markers, indicating an epithelial-mesenchymal transition. ARID1A silencing does not alter the level of β-catenin but induces a subcellular redistribution of β-catenin from the plasma membrane to the cytoplasm and nucleus. Immunohistochemical studies demonstrate that reduced expression of E-cadherin is associated with local lymph node metastasis, tumor infiltration and poor clinical prognosis. ARID1A and E-cadherin expression show a strong correlation in 75.4% of the analyzed GC tissues. They are synergistically downregulated in 23.5% of analyzed GC tissues. In conclusion, ARID1A targets E-cadherin during the modulation of GC cell migration and invasion.

  5. The Arabidopsis SWI2/SNF2 Chromatin Remodeler BRAHMA Regulates Polycomb Function during Vegetative Development and Directly Activates the Flowering Repressor Gene SVP

    PubMed Central

    Li, Chenlong; Chen, Chen; Gao, Lei; Yang, Songguang; Nguyen, Vi; Shi, Xuejiang; Siminovitch, Katherine; Kohalmi, Susanne E.; Huang, Shangzhi; Wu, Keqiang; Chen, Xuemei; Cui, Yuhai

    2015-01-01

    The chromatin remodeler BRAHMA (BRM) is a Trithorax Group (TrxG) protein that antagonizes the functions of Polycomb Group (PcG) proteins in fly and mammals. Recent studies also implicate such a role for Arabidopsis (Arabidopsis thaliana) BRM but the molecular mechanisms underlying the antagonism are unclear. To understand the interplay between BRM and PcG during plant development, we performed a genome-wide analysis of trimethylated histone H3 lysine 27 (H3K27me3) in brm mutant seedlings by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Increased H3K27me3 deposition at several hundred genes was observed in brm mutants and this increase was partially supressed by removal of the H3K27 methyltransferase CURLY LEAF (CLF) or SWINGER (SWN). ChIP experiments demonstrated that BRM directly binds to a subset of the genes and prevents the inappropriate association and/or activity of PcG proteins at these loci. Together, these results indicate a crucial role of BRM in restricting the inappropriate activity of PcG during plant development. The key flowering repressor gene SHORT VEGETATIVE PHASE (SVP) is such a BRM target. In brm mutants, elevated PcG occupancy at SVP accompanies a dramatic increase in H3K27me3 levels at this locus and a concomitant reduction of SVP expression. Further, our gain- and loss-of-function genetic evidence establishes that BRM controls flowering time by directly activating SVP expression. This work reveals a genome-wide functional interplay between BRM and PcG and provides new insights into the impacts of these proteins in plant growth and development. PMID:25615622

  6. The Arabidopsis SWI2/SNF2 chromatin Remodeler BRAHMA regulates polycomb function during vegetative development and directly activates the flowering repressor gene SVP.

    PubMed

    Li, Chenlong; Chen, Chen; Gao, Lei; Yang, Songguang; Nguyen, Vi; Shi, Xuejiang; Siminovitch, Katherine; Kohalmi, Susanne E; Huang, Shangzhi; Wu, Keqiang; Chen, Xuemei; Cui, Yuhai

    2015-01-01

    The chromatin remodeler BRAHMA (BRM) is a Trithorax Group (TrxG) protein that antagonizes the functions of Polycomb Group (PcG) proteins in fly and mammals. Recent studies also implicate such a role for Arabidopsis (Arabidopsis thaliana) BRM but the molecular mechanisms underlying the antagonism are unclear. To understand the interplay between BRM and PcG during plant development, we performed a genome-wide analysis of trimethylated histone H3 lysine 27 (H3K27me3) in brm mutant seedlings by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Increased H3K27me3 deposition at several hundred genes was observed in brm mutants and this increase was partially supressed by removal of the H3K27 methyltransferase CURLY LEAF (CLF) or SWINGER (SWN). ChIP experiments demonstrated that BRM directly binds to a subset of the genes and prevents the inappropriate association and/or activity of PcG proteins at these loci. Together, these results indicate a crucial role of BRM in restricting the inappropriate activity of PcG during plant development. The key flowering repressor gene SHORT VEGETATIVE PHASE (SVP) is such a BRM target. In brm mutants, elevated PcG occupancy at SVP accompanies a dramatic increase in H3K27me3 levels at this locus and a concomitant reduction of SVP expression. Further, our gain- and loss-of-function genetic evidence establishes that BRM controls flowering time by directly activating SVP expression. This work reveals a genome-wide functional interplay between BRM and PcG and provides new insights into the impacts of these proteins in plant growth and development.

  7. Functional Proteomics Establishes the Interaction of SIRT7 with Chromatin Remodeling Complexes and Expands Its Role in Regulation of RNA Polymerase I Transcription*

    PubMed Central

    Tsai, Yuan-Chin; Greco, Todd M.; Boonmee, Apaporn; Miteva, Yana; Cristea, Ileana M.

    2012-01-01

    Among mammalian sirtuins, SIRT7 is the only enzyme residing in nucleoli where ribosomal DNA is transcribed. Recent reports established that SIRT7 associates with RNA Pol I machinery and is required for rDNA transcription. Although defined by its homology to the yeast histone deacetylase Sir2, current knowledge suggests that SIRT7 itself has little to no deacetylase activity. Because only two SIRT7 interactions have been thus far described: RNA Pol I and upstream binding factor, identification of proteins and complexes associating with SIRT7 is critical to understanding its functions. Here, we present the first characterization of SIRT7 interaction networks. We have systematically investigated protein interactions of three EGFP-tagged SIRT7 constructs: wild type, a point mutation affecting rDNA transcription, and a deletion mutant lacking the predicted coiled-coil domain. A combinatorial proteomics and bioinformatics approach was used to integrate gene ontology classifications, functional protein networks, and normalized abundances of proteins co-isolated with SIRT7. The resulting refined proteomic data set confirmed SIRT7 interactions with RNA Pol I and upstream binding factor and highlighted association with factors involved in RNA Pol I- and II-dependent transcriptional processes and several nucleolus-localized chromatin remodeling complexes. Particularly enriched were members of the B-WICH complex, such as Mybbp1a, WSTF, and SNF2h. Prominent interactions were validated by a selected reaction monitoring-like approach using metabolic labeling with stable isotopes, confocal microscopy, reciprocal immunoaffinity precipitation, and co-isolation with endogenous SIRT7. To extend the current knowledge of mechanisms involved in SIRT7-dependent regulation of rDNA transcription, we showed that small interfering RNA-mediated SIRT7 knockdown leads to reduced levels of RNA Pol I protein, but not messenger RNA, which was confirmed in diverse cell types. The down-regulation of

  8. Functional Proteomics Establishes the Interaction of SIRT7 with Chromatin Remodeling Complexes and Expands Its Role in Regulation of RNA Polymerase I Transcription*

    PubMed Central

    Tsai, Yuan-Chin; Greco, Todd M.; Boonmee, Apaporn; Miteva, Yana; Cristea, Ileana M.

    2012-01-01

    Among mammalian sirtuins, SIRT7 is the only enzyme residing in nucleoli where ribosomal DNA is transcribed. Recent reports established that SIRT7 associates with RNA Pol I machinery and is required for rDNA transcription. Although defined by its homology to the yeast histone deacetylase Sir2, current knowledge suggests that SIRT7 itself has little to no deacetylase activity. Because only two SIRT7 interactions have been thus far described: RNA Pol I and upstream binding factor, identification of proteins and complexes associating with SIRT7 is critical to understanding its functions. Here, we present the first characterization of SIRT7 interaction networks. We have systematically investigated protein interactions of three EGFP-tagged SIRT7 constructs: wild type, a point mutation affecting rDNA transcription, and a deletion mutant lacking the predicted coiled-coil domain. A combinatorial proteomics and bioinformatics approach was used to integrate gene ontology classifications, functional protein networks, and normalized abundances of proteins co-isolated with SIRT7. The resulting refined proteomic data set confirmed SIRT7 interactions with RNA Pol I and upstream binding factor and highlighted association with factors involved in RNA Pol I- and II-dependent transcriptional processes and several nucleolus-localized chromatin remodeling complexes. Particularly enriched were members of the B-WICH complex, such as Mybbp1a, WSTF, and SNF2h. Prominent interactions were validated by a selected reaction monitoring-like approach using metabolic labeling with stable isotopes, confocal microscopy, reciprocal immunoaffinity precipitation, and co-isolation with endogenous SIRT7. To extend the current knowledge of mechanisms involved in SIRT7-dependent regulation of rDNA transcription, we showed that small interfering RNA-mediated SIRT7 knockdown leads to reduced levels of RNA Pol I protein, but not messenger RNA, which was confirmed in diverse cell types. The down-regulation of

  9. Autism-Associated Chromatin Regulator Brg1/SmarcA4 Is Required for Synapse Development and Myocyte Enhancer Factor 2-Mediated Synapse Remodeling

    PubMed Central

    Zhang, Zilai; Cao, Mou; Chang, Chia-Wei; Wang, Cindy; Shi, Xuanming; Zhan, Xiaoming; Birnbaum, Shari G.; Bezprozvanny, Ilya; Huber, Kimberly M.

    2015-01-01

    Synapse development requires normal neuronal activities and the precise expression of synapse-related genes. Dysregulation of synaptic genes results in neurological diseases such as autism spectrum disorders (ASD). Mutations in genes encoding chromatin-remodeling factor Brg1/SmarcA4 and its associated proteins are the genetic causes of several developmental diseases with neurological defects and autistic symptoms. Recent large-scale genomic studies predicted Brg1/SmarcA4 as one of the key nodes of the ASD gene network. We report that Brg1 deletion in early postnatal hippocampal neurons led to reduced dendritic spine density and maturation and impaired synapse activities. In developing mice, neuronal Brg1 deletion caused severe neurological defects. Gene expression analyses indicated that Brg1 regulates a significant number of genes known to be involved in synapse function and implicated in ASD. We found that Brg1 is required for dendritic spine/synapse elimination mediated by the ASD-associated transcription factor myocyte enhancer factor 2 (MEF2) and that Brg1 regulates the activity-induced expression of a specific subset of genes that overlap significantly with the targets of MEF2. Our analyses showed that Brg1 interacts with MEF2 and that MEF2 is required for Brg1 recruitment to target genes in response to neuron activation. Thus, Brg1 plays important roles in both synapse development/maturation and MEF2-mediated synapse remodeling. Our study reveals specific functions of the epigenetic regulator Brg1 in synapse development and provides insights into its role in neurological diseases such as ASD. PMID:26459759

  10. Hsf1 and Hsp90 orchestrate temperature-dependent global transcriptional remodelling and chromatin architecture in Candida albicans

    PubMed Central

    Leach, Michelle D.; Farrer, Rhys A.; Tan, Kaeling; Miao, Zhengqiang; Walker, Louise A.; Cuomo, Christina A.; Wheeler, Robert T.; Brown, Alistair J. P.; Wong, Koon Ho; Cowen, Leah E.

    2016-01-01

    Fever is a universal response to infection, and opportunistic pathogens such as Candida albicans have evolved complex circuitry to sense and respond to heat. Here we harness RNA-seq and ChIP-seq to discover that the heat shock transcription factor, Hsf1, binds distinct motifs in nucleosome-depleted promoter regions to regulate heat shock genes and genes involved in virulence in C. albicans. Consequently, heat shock increases C. albicans host cell adhesion, damage and virulence. Hsf1 activation depends upon the molecular chaperone Hsp90 under basal and heat shock conditions, but the effects are opposite and in part controlled at the level of Hsf1 expression and DNA binding. Finally, we demonstrate that Hsp90 regulates global transcription programs by modulating nucleosome levels at promoters of stress-responsive genes. Thus, we describe a mechanism by which C. albicans responds to temperature via Hsf1 and Hsp90 to orchestrate gene expression and chromatin architecture, thereby enabling thermal adaptation and virulence. PMID:27226156

  11. CRTC1 Nuclear Translocation Following Learning Modulates Memory Strength via Exchange of Chromatin Remodeling Complexes on the Fgf1 Gene.

    PubMed

    Uchida, Shusaku; Teubner, Brett J W; Hevi, Charles; Hara, Kumiko; Kobayashi, Ayumi; Dave, Rutu M; Shintaku, Tatsushi; Jaikhan, Pattaporn; Yamagata, Hirotaka; Suzuki, Takayoshi; Watanabe, Yoshifumi; Zakharenko, Stanislav S; Shumyatsky, Gleb P

    2017-01-10

    Memory is formed by synapse-to-nucleus communication that leads to regulation of gene transcription, but the identity and organizational logic of signaling pathways involved in this communication remain unclear. Here we find that the transcription cofactor CRTC1 is a critical determinant of sustained gene transcription and memory strength in the hippocampus. Following associative learning, synaptically localized CRTC1 is translocated to the nucleus and regulates Fgf1b transcription in an activity-dependent manner. After both weak and strong training, the HDAC3-N-CoR corepressor complex leaves the Fgf1b promoter and a complex involving the translocated CRTC1, phosphorylated CREB, and histone acetyltransferase CBP induces transient transcription. Strong training later substitutes KAT5 for CBP, a process that is dependent on CRTC1, but not on CREB phosphorylation. This in turn leads to long-lasting Fgf1b transcription and memory enhancement. Thus, memory strength relies on activity-dependent changes in chromatin and temporal regulation of gene transcription on specific CREB/CRTC1 gene targets.

  12. Deficiency of zebrafish fgf20a results in aberrant skull remodeling that mimics both human cranial disease and evolutionarily important fish skull morphologies.

    PubMed

    Cooper, W James; Wirgau, Rachel M; Sweet, Elly M; Albertson, R Craig

    2013-01-01

    The processes that direct skull remodeling are of interest to both human-oriented studies of cranial dysplasia and evolutionary studies of skull divergence. There is increasing awareness that these two fields can be mutually informative when natural variation mimics pathology. Here we describe a zebrafish mutant line, devoid of blastema (dob), which does not have a functional fgf20a protein, and which also presents cranial defects similar to both adaptive and clinical variation. We used geometric morphometric methods to provide quantitative descriptions of the effects of the dob mutation on skull morphogenesis. In combination with "whole-mount in situ hybridization" labeling of normal fgf20a expression and assays for osteoblast and osteoclast activity, the results of these analyses indicate that cranial dysmorphologies in dob zebrafish are generated by aberrations in post-embryonic skull remodeling via decreased osteoblasotgenesis and increased osteoclastogenesis. Mutational effects include altered skull vault geometries and midfacial hypoplasia that are consistent with key diagnostic signs for multiple human craniofacial syndromes. These phenotypic shifts also mimic changes in the functional morphology of fish skulls that have arisen repeatedly in several highly successful radiations (e.g., damselfishes and East-African rift-lake cichlids). Our results offer the dob/fgf20a mutant as an experimentally tractable model with which to examine post-embryonic skull development as it relates to human disease and vertebrate evolution.

  13. Deficiency of zebrafish fgf20a results in aberrant skull remodeling that mimics both human cranial disease and evolutionarily important fish skull morphologies

    PubMed Central

    Cooper, W. James; Wirgau, Rachel M.; Sweet, Elly M.; Albertson, R. Craig

    2013-01-01

    The processes that direct skull remodeling are of interest to both human-oriented studies of cranial dysplasia and evolutionary studies of skull divergence. There is increasing awareness that these two fields can be mutually informative when natural variation mimics pathology. Here we describe a zebrafish mutant line, devoid of blastema(dob), which does not have a functional fgf20a protein, and which also presents cranial defects similar to both adaptive and clinical variation. We used geometric morphometric methods to provide quantitative descriptions of the effects of the dob mutation on skull morphogenesis. In combination with whole-mount in situ hybridization labeling of normal fgf20a expression and assays for osteoblast and osteoclast activity, the results of these analyses indicate that cranial dysmorphologies in dob zebrafish are generated by aberrations in post-embryonic skull remodeling via decreased osteoblasotgenesis and increased osteoclastogenesis. Mutational effects include altered skull vault geometries and midfacial hypoplasia that are consistent with key diagnostic signs for multiple human craniofacial syndromes. These phenotypic shifts also mimic changes in the functional morphology of fish skulls that have arisen repeatedly in several highly successful radiations (e.g., damselfishes and East-African rift-lake cichlids). Our results offer the dob/fgf20a mutant as an experimentally tractable model with which to examine post-embryonic skull development as it relates to human disease and evolution. PMID:24261444

  14. A unique missense allele of BAF155, a core BAF chromatin remodeling complex protein, causes neural tube closure defects in mice.

    PubMed

    Harmacek, Laura; Watkins-Chow, Dawn E; Chen, Jianfu; Jones, Kenneth L; Pavan, William J; Salbaum, J Michael; Niswander, Lee

    2014-05-01

    Failure of embryonic neural tube closure results in the second most common class of birth defects known as neural tube defects (NTDs). While NTDs are likely the result of complex multigenic dysfunction, it is not known whether polymorphisms in epigenetic regulators may be risk factors for NTDs. Here we characterized Baf155(msp3) , a unique ENU-induced allele in mice. Homozygous Baf155(mps3) embryos exhibit highly penetrant exencephaly, allowing us to investigate the roles of an assembled, but malfunctional BAF chromatin remodeling complex in vivo at the time of neural tube closure. Evidence of defects in proliferation and apoptosis were found within the neural tube. RNA-Seq analysis revealed that surprisingly few genes showed altered expression in Baf155 mutant neural tissue, given the broad epigenetic role of the BAF complex, but included genes involved in neural development and cell survival. Moreover, gene expression changes between individual mutants were variable even though the NTD was consistently observed. This suggests that inconsistent gene regulation contributes to failed neural tube closure. These results shed light on the role of the BAF complex in the process of neural tube closure and highlight the importance of studying missense alleles to understand epigenetic regulation during critical phases of development.

  15. SNF2 chromatin remodeler-family proteins FRG1 and -2 are required for RNA-directed DNA methylation.

    PubMed

    Groth, Martin; Stroud, Hume; Feng, Suhua; Greenberg, Maxim V C; Vashisht, Ajay A; Wohlschlegel, James A; Jacobsen, Steven E; Ausin, Israel

    2014-12-09

    DNA methylation in Arabidopsis thaliana is maintained by at least four different enzymes: DNA methyltransferase1 (MET1), chromomethylase3 (CMT3), domains rearranged methyltransferase2 (DRM2), and chromomethylase2 (CMT2). However, DNA methylation is established exclusively by the enzyme DRM2, which acts in the RNA-directed DNA methylation (RdDM) pathway. Some RdDM components belong to gene families and have partially redundant functions, such as the endoribonucleases dicer-like 2, 3, and 4, and involved in de novo2 (IDN2) interactors IDN2-like 1 and 2. Traditional mutagenesis screens usually fail to detect genes if they are redundant, as the loss of one gene can be compensated by a related gene. In an effort to circumvent this issue, we used coexpression data to identify closely related genes that are coregulated with genes in the RdDM pathway. Here we report the discovery of two redundant proteins, SNF2-ring-helicase-like1 and -2 (FRG1 and -2) that are putative chromatin modifiers belonging to the SNF2 family of helicase-like proteins. Analysis of genome-wide bisulfite sequencing shows that simultaneous mutations of FRG1 and -2 cause defects in methylation at specific RdDM targeted loci. We also show that FRG1 physically associates with Su(var)3-9-related SUVR2, a known RdDM component, in vivo. Combined, our results identify FRG1 and FRG2 as previously unidentified components of the RdDM machinery.

  16. SNF2 chromatin remodeler-family proteins FRG1 and -2 are required for RNA-directed DNA methylation

    PubMed Central

    Groth, Martin; Stroud, Hume; Feng, Suhua; Greenberg, Maxim V. C.; Vashisht, Ajay A.; Wohlschlegel, James A.; Jacobsen, Steven E.; Ausin, Israel

    2014-01-01

    DNA methylation in Arabidopsis thaliana is maintained by at least four different enzymes: DNA METHYLTRANSFERASE1 (MET1), CHROMOMETHYLASE3 (CMT3), DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2), and CHROMOMETHYLASE2 (CMT2). However, DNA methylation is established exclusively by the enzyme DRM2, which acts in the RNA-directed DNA methylation (RdDM) pathway. Some RdDM components belong to gene families and have partially redundant functions, such as the endoribonucleases DICER-LIKE 2, 3, and 4, and INVOLVED IN DE NOVO2 (IDN2) interactors IDN2-LIKE 1 and 2. Traditional mutagenesis screens usually fail to detect genes if they are redundant, as the loss of one gene can be compensated by a related gene. In an effort to circumvent this issue, we used coexpression data to identify closely related genes that are coregulated with genes in the RdDM pathway. Here we report the discovery of two redundant proteins, SNF2-RING-HELICASE–LIKE1 and -2 (FRG1 and -2) that are putative chromatin modifiers belonging to the SNF2 family of helicase-like proteins. Analysis of genome-wide bisulfite sequencing shows that simultaneous mutations of FRG1 and -2 cause defects in methylation at specific RdDM targeted loci. We also show that FRG1 physically associates with Su(var)3-9–related SUVR2, a known RdDM component, in vivo. Combined, our results identify FRG1 and FRG2 as previously unidentified components of the RdDM machinery. PMID:25425661

  17. Recruitment by the Repressor Freud-1 of Histone Deacetylase-Brg1 Chromatin Remodeling Complexes to Strengthen HTR1A Gene Repression.

    PubMed

    Souslova, Tatiana; Mirédin, Kim; Millar, Anne M; Albert, Paul R

    2016-12-02

    Five-prime repressor element under dual repression binding protein-1 (Freud-1)/CC2D1A is genetically linked to intellectual disability and implicated in neuronal development. Freud-1 represses the serotonin-1A (5-HT1A) receptor gene HTR1A by histone deacetylase (HDAC)-dependent or HDAC-independent mechanisms in 5-HT1A-negative (e.g., HEK-293) or 5-HT1A-expressing cells (SK-N-SH), respectively. To identify the underlying mechanisms, Freud-1-associated proteins were affinity-purified from HEK-293 nuclear extracts and members of the Brg1/SMARCCA chromatin remodeling and Sin3A-HDAC corepressor complexes were identified. Pull-down assays using recombinant proteins showed that Freud-1 interacts directly with the Brg1 carboxyl-terminal domain; interaction with Brg1 required the carboxyl-terminal of Freud-1. Freud-1 complexes in HEK-293 and SK-N-SH cells differed, with low levels of BAF170/SMARCC2 and BAF57/SMARCE1 in HEK-293 cells and low-undetectable BAF155/SMARCC1, Sin3A, and HDAC1/2 in SK-N-SH cells. Similarly, by quantitative chromatin immunoprecipitation, Brg1-BAF170/57 and Sin3A-HDAC complexes were observed at the HTR1A promoter in HEK-293 cells, whereas in SK-N-SH cells, Sin3A-HDAC proteins were not detected. Quantifying 5-HT1A receptor mRNA levels in cells treated with siRNA to Freud-1, Brg1, or both RNAs addressed the functional role of the Freud-1-Brg1 complex. In HEK-293 cells, 5-HT1A receptor mRNA levels were increased only when both Freud-1 and Brg1 were depleted, but in SK-N-SH cells, depletion of either protein upregulated 5-HT1A receptor RNA. Thus, recruitment by Freud-1 of Brg1, BAF155, and Sin3A-HDAC complexes appears to strengthen repression of the HTR1A gene to prevent its expression inappropriate cell types, while recruitment of the Brg1-BAF170/57 complex is permissive to 5-HT1A receptor expression. Alterations in Freud-1-Brg1 interactions in mutants associated with intellectual disability could impair gene repression leading to altered neuronal

  18. Versatile reporter systems show that transactivation by human T-cell leukemia virus type 1 Tax occurs independently of chromatin remodeling factor BRG1.

    PubMed

    Zhang, Ling; Liu, Meihong; Merling, Randall; Giam, Chou-Zen

    2006-08-01

    Potent activation of human T-cell leukemia virus type 1 (HTLV-1) gene expression is mediated by the virus-encoded transactivator protein Tax and three imperfect 21-bp repeats in the viral long terminal repeats. Each 21-bp repeat contains a cAMP-responsive-element core flanked by 5' G-rich and 3' C-rich sequences. Tax alone does not bind DNA. Rather, it interacts with basic domain-leucine zipper transcription factors CREB and ATF-1 to form ternary complexes with the 21-bp repeats. In the context of the ternary complexes, Tax contacts the G/C-rich sequences and recruits transcriptional coactivators CREB-binding protein (CBP)/p300 to effect potent transcriptional activation. Using an easily transduced and chromosomally integrated reporter system derived from a self-inactivating lentivirus vector, we showed in a BRG1- and BRM1-deficient adrenal carcinoma cell line, SW-13, that Tax- and 21-bp repeat-mediated transactivation does not require BRG1 or BRM1 and is not enhanced by BRG1. With a similar reporter system, we further demonstrated that Tax- and tumor necrosis factor alpha-induced NF-kappaB activation occurs readily in SW-13 cells in the absence of BRG1 and BRM1. These results suggest that the assembly of stable multiprotein complexes containing Tax, CREB/ATF-1, and CBP/p300 on the 21-bp repeats is the principal mechanism employed by Tax to preclude nucleosome formation at the HTLV-1 enhancer/promoter. This most likely bypasses the need for BRG1-containing chromatin-remodeling complexes. Likewise, recruitment of CBP/p300 by NF-kappaB may be sufficient to disrupt histone-DNA interaction for the initiation of transcription.

  19. SWR1 Chromatin-Remodeling Complex Subunits and H2A.Z Have Non-overlapping Functions in Immunity and Gene Regulation in Arabidopsis.

    PubMed

    Berriri, Souha; Gangappa, Sreeramaiah N; Kumar, S Vinod

    2016-07-06

    Incorporation of the histone variant H2A.Z into nucleosomes by the SWR1 chromatin remodeling complex is a critical step in eukaryotic gene regulation. In Arabidopsis, SWR1c and H2A.Z have been shown to control gene expression underlying development and environmental responses. Although they have been implicated in defense, the specific roles of the complex subunits and H2A.Z in immunity are not well understood. In this study, we analyzed the roles of the SWR1c subunits, PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1 (PIE1), ACTIN-RELATED PROTEIN6 (ARP6), and SWR1 COMPLEX 6 (SWC6), as well as H2A.Z, in defense and gene regulation. We found that SWR1c components play different roles in resistance to different pathogens. Loss of PIE1 and SWC6 function as well as depletion of H2A.Z led to reduced basal resistance, while loss of ARP6 fucntion resulted in enhanced resistance. We found that mutations in PIE1 and SWC6 resulted in impaired effector-triggered immunity. Mutation in SWR1c components and H2A.Z also resulted in compromised jasmonic acid/ethylene-mediated immunity. Genome-wide expression analyses similarly reveal distinct roles for H2A.Z and SWR1c components in gene regulation, and suggest a potential role for PIE1 in the regulation of the cross talk between defense signaling pathways. Our data show that although they are part of the same complex, Arabidopsis SWR1c components could have non-redundant functions in plant immunity and gene regulation.

  20. The chromatin remodelling component SMARCB1/INI1 influences the metastatic behavior of colorectal cancer through a gene signature mapping to chromosome 22

    PubMed Central

    2013-01-01

    Background INI1 (Integrase interactor 1), also known as SMARCB1, is the most studied subunit of chromatin remodelling complexes. Its role in colorectal tumorigenesis is not known. Methods We examined SMARCB1/INI1 protein expression in 134 cases of colorectal cancer (CRC) and 60 matched normal mucosa by using tissue microarrays and western blot and categorized the results according to mismatch repair status (MMR), CpG island methylator phenotype, biomarkers of tumor differentiation CDX2, CK20, vimentin and p53. We validated results in two independent data sets and in cultured CRC cell lines. Results Herein, we show that negative SMARCB1/INI1 expression (11% of CRCs) associates with loss of CDX2, poor differentiation, liver metastasis and shorter patients’ survival regardless of the MMR status or tumor stage. Unexpectedly, even CRCs displaying diffuse nuclear INI1 staining (33%) show an adverse prognosis and vimentin over-expression, in comparison with the low expressing group (56%). The negative association of SMARCB1/INI1-lack of expression with a metastatic behavior is enhanced by the TP53 status. By interrogating global gene expression from two independent cohorts of 226 and 146 patients, we confirm the prognostic results and identify a gene signature characterized by SMARCB1/INI1 deregulation. Notably, the top genes of the signature (BCR, COMT, MIF) map on the long arm of chromosome 22 and are closely associated with SMARCB1/INI1. Conclusion Our findings suggest that SMARCB1/INI1-dysregulation and genetic hot-spots on the long arm of chromosome 22 might play an important role in the CRC metastatic behavior and be clinically relevant as novel biomarkers. PMID:24286138

  1. Aberrant Epicardial Adipose Tissue Extracellular Matrix Remodeling in Patients with Severe Ischemic Cardiomyopathy: Insight from Comparative Quantitative Proteomics

    PubMed Central

    Jiang, Ding-Sheng; Zeng, Hao-Long; Li, Rui; Huo, Bo; Su, Yun-Shu; Fang, Jing; Yang, Qing; Liu, Li-Gang; Hu, Min; Cheng, Cai; Zhu, Xue-Hai; Yi, Xin; Wei, Xiang

    2017-01-01

    There is ample evidence indicating that epicardial adipose tissue (EAT) volume and thickness is positively associated with coronary artery disease (CAD). However, the exact pathological changes in the human EAT after myocardial ischemia remains largely unclear. In the current study, we applied a comparative quantitative proteomics to elucidate the altered biological processes in the EAT of ischemic cardiomyopathy (ICM) patients. A total of 1649 proteins were successfully quantified in our study, among which 165 proteins were significantly changed (ratio <0.8 or >1.2 fold and p < 0.05 in both repetitions) in EAT of ICM individuals. Gene ontology (GO) enrichment analysis revealed that cardiac structure and cellular metabolism were over-represented among these regulated proteins. The hypertrophic cardiomyopathy, adrenergic signaling in cardiomyocytes, extracellular matrix (ECM)-receptor interaction, phagosome, Glycolysis/Gluconeogenesis, and PPAR signaling pathway were highlighted by the KEGG PATHWAY analysis. More importantly, we found that the proteins responsible for extracellular matrix organization were dramatically increased in EAT of ICM patients. In addition, the picrosirius red (PSR) staining results showed that the collagen fiber content was prominently increased, which indicated the EAT of ICM individuals underwent extracellular matrix remodeling and ERK1/2 activation maybe responsible for these pathological changes partially. PMID:28256566

  2. Physical and Functional Interactions between Drosophila Homologue of Swc6/p18Hamlet Subunit of the SWR1/SRCAP Chromatin-remodeling Complex with the DNA Repair/Transcription Factor TFIIH*

    PubMed Central

    Herrera-Cruz, Mariana; Cruz, Grisel; Valadez-Graham, Viviana; Fregoso-Lomas, Mariana; Villicaña, Claudia; Vázquez, Martha; Reynaud, Enrique; Zurita, Mario

    2012-01-01

    The multisubunit DNA repair and transcription factor TFIIH maintains an intricate cross-talk with different factors to achieve its functions. The p8 subunit of TFIIH maintains the basal levels of the complex by interacting with the p52 subunit. Here, we report that in Drosophila, the homolog of the p8 subunit (Dmp8) is encoded in a bicistronic transcript with the homolog of the Swc6/p18Hamlet subunit (Dmp18) of the SWR1/SRCAP chromatin remodeling complex. The SWR1 and SRCAP complexes catalyze the exchange of the canonical histone H2A with the H2AZ histone variant. In eukaryotic cells, bicistronic transcripts are not common, and in some cases, the two encoded proteins are functionally related. We found that Dmp18 physically interacts with the Dmp52 subunit of TFIIH and co-localizes with TFIIH in the chromatin. We also demonstrated that Dmp18 genetically interacts with Dmp8, suggesting that a cross-talk might exist between TFIIH and a component of a chromatin remodeler complex involved in histone exchange. Interestingly, our results also show that when the level of one of the two proteins is decreased and the other maintained, a specific defect in the fly is observed, suggesting that the organization of these two genes in a bicistronic locus has been selected during evolution to allow co-regulation of both genes. PMID:22865882

  3. The analysis of chromatin remodeling and the staining for DNA methylation and histone acetylation do not provide definitive indicators of the developmental ability of inter-species cloned embryos.

    PubMed

    Lee, Eugine; Kim, Ji Hye; Park, Seon Mi; Jeong, Yeon Ik; Lee, Jong Yun; Park, Sun Woo; Choi, Jiho; Kim, Huen Suk; Jeong, Yeon Woo; Kim, Sue; Hyun, Sang Hwan; Hwang, Woo Suk

    2008-05-01

    The restricted supply of oocytes in the domestic dog limits the development of reproductive biotechnologies in this species. Inter-species somatic cell nuclear transfer could be an alternative for cloning animals whose oocytes are difficult to obtain. In this study, the possibility of cloning dog embryos using pig oocytes was investigated by evaluating nuclear remodeling. Chromatin remodeling, assessed by premature chromosome condensation, pseudo-pronuclei formation, DNA methylation and histone acetylation, along with the developmental ability was compared between intra- and inter-species cloned embryos. The incidence of premature chromosome condensation was significantly higher in intra-species cloned embryos relative to inter-species cloned embryos (87.2% vs. 61.7%; P<0.05), but comparable pseudo-pronuclei formation was observed in both (85.3% vs. 75.8%). None of the inter-species cloned embryos developed beyond the 8-cell stage while 18.3% of intra-species cloned embryos developed to the blastocyst stage. The relative level of both DNA methylation and histone acetylation was similar between intra- and inter-species cloned embryos at all times examined. These results suggest that although partial chromatin remodeling occurs, further investigation is needed to be able to use pig oocytes as recipient oocytes in dog cloning.

  4. Connecting the dots: chromatin and alternative splicing in EMT

    PubMed Central

    Warns, Jessica A.; Davie, James R.; Dhasarathy, Archana

    2015-01-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process. PMID:26291837

  5. Connecting the dots: chromatin and alternative splicing in EMT.

    PubMed

    Warns, Jessica A; Davie, James R; Dhasarathy, Archana

    2016-02-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases, and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process.

  6. Single Molecule Studies of Chromatin

    SciTech Connect

    Jeans, C; Colvin, M E; Thelen, M P; Noy, A

    2004-01-06

    The DNA in eukaryotic cells is tightly packaged as chromatin through interactions with histone proteins to form nucleosomes. These nucleosomes are themselves packed together through interactions with linker histone and non-histone proteins. In order for processes such as DNA replication, DNA repair, and transcription to occur, the chromatin fiber must be remodeled such that the necessary enzymes can access the DNA. The structure of the chromatin fiber beyond the level of the single nucleosome and the structural changes which accompany the remodeling process are poorly understood. We are studying the structures and forces behind the remodeling process through the use of atomic force microscopy (AFM). This allows both high-resolution imaging of the chromatin, and manipulation of individual fibers. Pulling a single chromatin fiber apart using the AFM tip yields information on the forces which hold the structure together. We have isolated chromatin fibers from chicken erythrocytes and Chinese hamster ovary cell lines. AFM images of these fibers will be presented, along with preliminary data from the manipulation of these fibers using the AFM tip. The implications of these data for the structure of chromatin undergoing the remodeling process are discussed.

  7. Negative Regulation of p21Waf1/Cip1 by Human INO80 Chromatin Remodeling Complex Is Implicated in Cell Cycle Phase G2/M Arrest and Abnormal Chromosome Stability

    PubMed Central

    Cao, Lingling; Ding, Jian; Dong, Liguo; Zhao, Jiayao; Su, Jiaming; Wang, Lingyao; Sui, Yi; Zhao, Tong; Wang, Fei; Jin, Jingji; Cai, Yong

    2015-01-01

    We previously identified an ATP-dependent human Ino80 (INO80) chromatin remodeling complex which shares a set of core subunits with yeast Ino80 complex. Although research evidence has suggested that INO80 complex functions in gene transcription and genome stability, the precise mechanism remains unclear. Herein, based on gene expression profiles from the INO80 complex-knockdown in HeLa cells, we first demonstrate that INO80 complex negatively regulates the p21Waf1/Cip1 (p21) expression in a p53-mediated mechanism. In chromatin immunoprecipitation (ChIP) and a sequential ChIP (Re-ChIP) assays, we determined that the INO80 complex and p53 can bind to the same promoter region of p21 gene (-2.2kb and -1.0kb upstream of the p21 promoter region), and p53 is required for the recruitment of the INO80 complex to the p21 promoter. RNAi knockdown strategies of INO80 not only led to prolonged progression of cell cycle phase G2/M to G1, but it also resulted in abnormal chromosome stability. Interestingly, high expression of p21 was observed in most morphologically-changed cells, suggesting that negative regulation of p21 by INO80 complex might be implicated in maintaining the cell cycle process and chromosome stability. Together, our findings will provide a theoretical basis to further elucidate the cellular mechanisms of the INO80 complex. PMID:26340092

  8. Negative Regulation of p21Waf1/Cip1 by Human INO80 Chromatin Remodeling Complex Is Implicated in Cell Cycle Phase G2/M Arrest and Abnormal Chromosome Stability.

    PubMed

    Cao, Lingling; Ding, Jian; Dong, Liguo; Zhao, Jiayao; Su, Jiaming; Wang, Lingyao; Sui, Yi; Zhao, Tong; Wang, Fei; Jin, Jingji; Cai, Yong

    2015-01-01

    We previously identified an ATP-dependent human Ino80 (INO80) chromatin remodeling complex which shares a set of core subunits with yeast Ino80 complex. Although research evidence has suggested that INO80 complex functions in gene transcription and genome stability, the precise mechanism remains unclear. Herein, based on gene expression profiles from the INO80 complex-knockdown in HeLa cells, we first demonstrate that INO80 complex negatively regulates the p21Waf1/Cip1 (p21) expression in a p53-mediated mechanism. In chromatin immunoprecipitation (ChIP) and a sequential ChIP (Re-ChIP) assays, we determined that the INO80 complex and p53 can bind to the same promoter region of p21 gene (-2.2 kb and -1.0 kb upstream of the p21 promoter region), and p53 is required for the recruitment of the INO80 complex to the p21 promoter. RNAi knockdown strategies of INO80 not only led to prolonged progression of cell cycle phase G2/M to G1, but it also resulted in abnormal chromosome stability. Interestingly, high expression of p21 was observed in most morphologically-changed cells, suggesting that negative regulation of p21 by INO80 complex might be implicated in maintaining the cell cycle process and chromosome stability. Together, our findings will provide a theoretical basis to further elucidate the cellular mechanisms of the INO80 complex.

  9. Telomere dysfunction drives aberrant hematopoietic differentiation and myelodysplastic syndrome.

    PubMed

    Colla, Simona; Ong, Derrick Sek Tong; Ogoti, Yamini; Marchesini, Matteo; Mistry, Nipun A; Clise-Dwyer, Karen; Ang, Sonny A; Storti, Paola; Viale, Andrea; Giuliani, Nicola; Ruisaard, Kathryn; Ganan Gomez, Irene; Bristow, Christopher A; Estecio, Marcos; Weksberg, David C; Ho, Yan Wing; Hu, Baoli; Genovese, Giannicola; Pettazzoni, Piergiorgio; Multani, Asha S; Jiang, Shan; Hua, Sujun; Ryan, Michael C; Carugo, Alessandro; Nezi, Luigi; Wei, Yue; Yang, Hui; D'Anca, Marianna; Zhang, Li; Gaddis, Sarah; Gong, Ting; Horner, James W; Heffernan, Timothy P; Jones, Philip; Cooper, Laurence J N; Liang, Han; Kantarjian, Hagop; Wang, Y Alan; Chin, Lynda; Bueso-Ramos, Carlos; Garcia-Manero, Guillermo; DePinho, Ronald A

    2015-05-11

    Myelodysplastic syndrome (MDS) risk correlates with advancing age, therapy-induced DNA damage, and/or shorter telomeres, but whether telomere erosion directly induces MDS is unknown. Here, we provide the genetic evidence that telomere dysfunction-induced DNA damage drives classical MDS phenotypes and alters common myeloid progenitor (CMP) differentiation by repressing the expression of mRNA splicing/processing genes, including SRSF2. RNA-seq analyses of telomere dysfunctional CMP identified aberrantly spliced transcripts linked to pathways relevant to MDS pathogenesis such as genome stability, DNA repair, chromatin remodeling, and histone modification, which are also enriched in mouse CMP haploinsufficient for SRSF2 and in CD34(+) CMML patient cells harboring SRSF2 mutation. Together, our studies establish an intimate link across telomere biology, aberrant RNA splicing, and myeloid progenitor differentiation.

  10. Chromatin and Transcription in Yeast

    PubMed Central

    Rando, Oliver J.; Winston, Fred

    2012-01-01

    Understanding the mechanisms by which chromatin structure controls eukaryotic transcription has been an intense area of investigation for the past 25 years. Many of the key discoveries that created the foundation for this field came from studies of Saccharomyces cerevisiae, including the discovery of the role of chromatin in transcriptional silencing, as well as the discovery of chromatin-remodeling factors and histone modification activities. Since that time, studies in yeast have continued to contribute in leading ways. This review article summarizes the large body of yeast studies in this field. PMID:22345607

  11. C/EBPα and PU.1 are involved in distinct differentiation responses of acute promyelocytic leukemia HL-60 and NB4 cells via chromatin remodeling.

    PubMed

    Savickiene, Jurate; Treigyte, Grazina; Vistartaite, Giedre; Tunaitis, Virginijus; Magnusson, Karl-Eric; Navakauskiene, Ruta

    2011-01-01

    C/EBPα and PU.1 are the basic transcription factors that control differentiation-related genes, including granulocyte- colony-stimulating factor (G-CSFR) and human neutrophil elastase (HNE). Here, we analyzed a role of C/EBPα and PU.1 in human acute leukemia cell lines, HL-60 and NB4, in association with a modified chromatin structure by histone deacetylase inhibitors, FK228, sodium phenyl butyrate and vitamin B3. We found that sodium phenyl butyrate alone and 6h-pretreatment with phenyl butyrate or FK228 before the induction of differentiation with all-trans-retinoic acid in the presence of vitamin B3 effectively accelerated and enhanced differentiation to granulocytes in HL-60 but not in NB4 cells as detected by NBT test and the expression of CD11b and CD114 (G-CSFR) using flow cytometric analysis. HDACIs induced a time- and dose-dependent accumulation of hyper-acetylated histone H4 in both cell lines with the delay in NB4 cells. Time-dependent different induction of HL-60 and NB4 cell differentiation was paralleled by the activation of C/EBPα and PU.1 binding to the G-CSFR and the HNE promoters in electrophoretic mobility shift assay. Chromatin immunoprecipitation analysis revealed histone H4 acetylation in the G-CSF receptor promoter at the C/EBPα binding site in HL-60 but not in NB4 cells under the combined treatment. The results indicate that epigenetic events, such as histone acetylation, are involved in the activity modulation of the key transcription factors responsible for the induction of granulocytic differentiation in promyelocytic leukemia cells.

  12. Chromatin roadblocks to reprogramming 50 years on.

    PubMed

    Skene, Peter J; Henikoff, Steven

    2012-10-29

    A half century after John Gurdon demonstrated nuclear reprogramming, for which he was awarded the 2012 Nobel Prize in Physiology or Medicine, his group provides insights into the molecular mechanisms whereby chromatin remodeling is required for nuclear reprogramming. Among the issues addressed in Gurdon's latest work are the chromatin impediments to artificially induced reprogramming, discovered by Shinya Yamanaka, who shared the award with Gurdon.

  13. Recruitment of the SWI-SNF Chromatin Remodeling Complex as a Mechanism of Gene Activation by the Glucocorticoid Receptor τ1 Activation Domain

    PubMed Central

    Wallberg, Annika E.; Neely, Kristen E.; Hassan, Ahmed H.; Gustafsson, Jan-Åke; Workman, Jerry L.; Wright, Anthony P. H.

    2000-01-01

    The SWI-SNF complex has been shown to alter nucleosome conformation in an ATP-dependent manner, leading to increased accessibility of nucleosomal DNA to transcription factors. In this study, we show that the SWI-SNF complex can potentiate the activity of the glucocorticoid receptor (GR) through the N-terminal transactivation domain, τ1, in both yeast and mammalian cells. GR-τ1 can directly interact with purified SWI-SNF complex, and mutations in τ1 that affect the transactivation activity in vivo also directly affect τ1 interaction with SWI-SNF. Furthermore, the SWI-SNF complex can stimulate τ1-driven transcription from chromatin templates in vitro. Taken together, these results support a model in which the GR can directly recruit the SWI-SNF complex to target promoters during glucocorticoid-dependent gene activation. We also provide evidence that the SWI-SNF and SAGA complexes represent independent pathways of τ1-mediated activation but play overlapping roles that are able to compensate for one another under some conditions. PMID:10688647

  14. Single Molecule Studies of Chromatin

    SciTech Connect

    Jeans, C; Thelen, M P; Noy, A

    2006-02-06

    In eukaryotic cells, DNA is packaged as chromatin, a highly ordered structure formed through the wrapping of the DNA around histone proteins, and further packed through interactions with a number of other proteins. In order for processes such as DNA replication, DNA repair, and transcription to occur, the structure of chromatin must be remodeled such that the necessary enzymes can access the DNA. A number of remodeling enzymes have been described, but our understanding of the remodeling process is hindered by a lack of knowledge of the fine structure of chromatin, and how this structure is modulated in the living cell. We have carried out single molecule experiments using atomic force microscopy (AFM) to study the packaging arrangements in chromatin from a variety of cell types. Comparison of the structures observed reveals differences which can be explained in terms of the cell type and its transcriptional activity. During the course of this project, sample preparation and AFM techniques were developed and optimized. Several opportunities for follow-up work are outlined which could provide further insight into the dynamic structural rearrangements of chromatin.

  15. Swi/SNF-GCN5-dependent chromatin remodelling determines induced expression of GDH3, one of the paralogous genes responsible for ammonium assimilation and glutamate biosynthesis in Saccharomyces cerevisiae.

    PubMed

    Avendaño, Amaranta; Riego, Lina; DeLuna, Alexander; Aranda, Cristina; Romero, Guillermo; Ishida, Cecilia; Vázquez-Acevedo, Miriam; Rodarte, Beatriz; Recillas-Targa, Félix; Valenzuela, Lourdes; Zonszein, Sergio; González, Alicia

    2005-07-01

    It is accepted that Saccharomyces cerevisiae genome arose from complete duplication of eight ancestral chromosomes; functionally normal ploidy was recovered because of the massive loss of 90% of duplicated genes. There is evidence that indicates that part of this selective conservation of gene pairs is compelling to yeast facultative metabolism. As an example, the duplicated NADP-glutamate dehydrogenase pathway has been maintained because of the differential expression of the paralogous GDH1 and GDH3 genes, and the biochemical specialization of the enzymes they encode. The present work has been aimed to the understanding of the regulatory mechanisms that modulate GDH3 transcriptional activation. Our results show that GDH3 expression is repressed in glucose-grown cultures, as opposed to what has been observed for GDH1, and induced under respiratory conditions, or under stationary phase. Although GDH3 pertains to the nitrogen metabolic network, and its expression is Gln3p-regulated, complete derepression is ultimately determined by the carbon source through the action of the SAGA and SWI/SNF chromatin remodelling complexes. GDH3 carbon-mediated regulation is over-imposed to that exerted by the nitrogen source, highlighting the fact that operation of facultative metabolism requires strict control of enzymes, like Gdh3p, involved in biosynthetic pathways that use tricarboxylic acid cycle intermediates.

  16. Long-term hydroxytamoxifen treatment of an MCF-7-derived breast cancer cell line irreversibly inhibits the expression of estrogenic genes through chromatin remodeling.

    PubMed

    Badia, E; Duchesne, M J; Semlali, A; Fuentes, M; Giamarchi, C; Richard-Foy, H; Nicolas, J C; Pons, M

    2000-08-01

    Antiestrogen resistance is frequently observed in patients after longterm treatment with tamoxifen, a nonsteroidal antiestrogen widely used for endocrine therapy of breast cancer. In vitro studies in resistant cells showed that the expression of natural estrogen-responsive genes is frequently altered. Using MVLN cells, an MCF-7-derived cell model, we previously demonstrated that 4-hydroxytamoxifen (OHT) treatment irreversibly inactivated an estrogen-regulated chimeric luciferase response by a direct effect of the drug and not through a cell selection process (E. Badia et al., Cancer Res., 54: 5860-5866, 1994). In the present study, we present tamoxifen-resistant but still estrogen-dependent clones isolated after long-term treatment of MVLN cells with OHT and show that progesterone receptor (PR) expression was irreversibly decreased in some of these clones, whereas the PRA:PRB ratio of residual PR remained unchanged. The irreversible inactivation of both chimeric luciferase gene and PR gene expression was associated with the disappearance of DNase 1-hypersensitive sites. In the case of the chimeric gene, at least one of these sites was close to the estrogen responsive element. Genomic sequencing analysis of a clone with very low PR content did not reveal any methylation on CpG dinucleotides or any mutation in the PR gene promoter region. In all of the resistant clones tested and independently of their PR content, estrogen receptor expression was only lowered by half and remained functional, whereas pS2 expression was not modified. We also observed that the residual luciferase activity level (1-2%) of the MVLN clones, the luciferase expression of which had been irreversibly inactivated, was raised 4-fold by trichostatin A treatment. We conclude that long-term OHT treatment may modify the chromatin structure and thus could contribute to differentially silencing natural target genes.

  17. Aging by epigenetics-A consequence of chromatin damage?

    SciTech Connect

    Sedivy, John M. Banumathy, Gowrishankar; Adams, Peter D.

    2008-06-10

    Chromatin structure is not fixed. Instead, chromatin is dynamic and is subject to extensive developmental and age-associated remodeling. In some cases, this remodeling appears to counter the aging and age-associated diseases, such as cancer, and extend organismal lifespan. However, stochastic non-deterministic changes in chromatin structure might, over time, also contribute to the break down of nuclear, cell and tissue function, and consequently aging and age-associated diseases.

  18. Meta-analysis of gene expression patterns in animal models of prenatal alcohol exposure suggests role for protein synthesis inhibition and chromatin remodeling

    PubMed Central

    Rogic, Sanja; Wong, Albertina; Pavlidis, Paul

    2017-01-01

    Background Prenatal alcohol exposure (PAE) can result in an array of morphological, behavioural and neurobiological deficits that can range in their severity. Despite extensive research in the field and a significant progress made, especially in understanding the range of possible malformations and neurobehavioral abnormalities, the molecular mechanisms of alcohol responses in development are still not well understood. There have been multiple transcriptomic studies looking at the changes in gene expression after PAE in animal models, however there is a limited apparent consensus among the reported findings. In an effort to address this issue, we performed a comprehensive re-analysis and meta-analysis of all suitable, publically available expression data sets. Methods We assembled ten microarray data sets of gene expression after PAE in mouse and rat models consisting of samples from a total of 63 ethanol-exposed and 80 control animals. We re-analyzed each data set for differential expression and then used the results to perform meta-analyses considering all data sets together or grouping them by time or duration of exposure (pre- and post-natal, acute and chronic, respectively). We performed network and Gene Ontology enrichment analysis to further characterize the identified signatures. Results For each sub-analysis we identified signatures of differential expressed genes that show support from multiple studies. Overall, the changes in gene expression were more extensive after acute ethanol treatment during prenatal development than in other models. Considering the analysis of all the data together, we identified a robust core signature of 104 genes down-regulated after PAE, with no up-regulated genes. Functional analysis reveals over-representation of genes involved in protein synthesis, mRNA splicing and chromatin organization. Conclusions Our meta-analysis shows that existing studies, despite superficial dissimilarity in findings, share features that allow us

  19. Chromatin remodeling gene AT-rich interactive domain-containing protein 1A suppresses gastric cancer cell proliferation by targeting PIK3CA and PDK1

    PubMed Central

    Wang, Jie; Cui, Shu-Jian; Wang, Xiao-Qing; Jiang, Ying-Hua; Feng, Li; Yang, Peng-Yuan; Liu, Feng

    2016-01-01

    The tumor suppressor gene AT-rich interactive domain-containing protein 1A (ARID1A) was frequently mutated in cancers. The modulation mechanism of ARID1A for PI3K/AKT signaling in gastric cancer (GC) remains elusive. Here, we found that depletion of endogenous ARID1A enhanced the in vitro proliferation, colony formation, cellular growth, nutrient uptake and in vivo xenograft tumor growth of GC cells. PI3K/AKT activation by ARID1A-silencing was profiled using a phospho-protein antibody array. The phosphorylation of PDK1, AKT, GSK3β and 70S6K, and the protein and mRNA expressions of PI3K and PDK1, were upregulated by ARID1A-silencing. Chromatin immunoprecipitation and luciferase reporter assay revealed that ARID1A-involved SWI/SNF complex inhibited PIK3CA and PDK1 transcription by direct binding to their promoters. Serial deletion mutation analyses revealed that the ARID1A central region containing the HIC1-binding domain, but not the ARID DNA-binding domain and the C-terminal domain, was essential for the inhibition of GC cell growth, PI3K/AKT pathway phosphorylation and its transcriptional modulation activity of PIK3CA and PDK1. The proliferation, cellular growth and glucose consumption of ARID1A-deficient GC cells were efficiently prohibited by allosteric inhibitors mk2206 and LY294002, which targeting AKT and PI3K, respectively. Both inhibitors also downregulated the phosphorylation of PI3K/AKT pathway in ARID1A-deficient GC cells. Such cells were sensitized to the treatment of LY294002, and AT7867, another inhibitor of AKT and p70S6K. The administration of LY294002 alone inhibited the in vivo growth of ARID1A- deficient GC cells in mouse xenograft model. Our study provides a novel insight into the modulatory function and mechanism of ARID1A in PI3K/AKT signaling in GC. PMID:27323812

  20. Expression of Tau Produces Aberrant Plasma Membrane Blebbing in Glial Cells Through RhoA-ROCK-Dependent F-Actin Remodeling.

    PubMed

    Torres-Cruz, Francisco M; Rodríguez-Cruz, Fanny; Escobar-Herrera, Jaime; Barragán-Andrade, Norma; Basurto-Islas, Gustavo; Ripova, Daniela; Ávila, Jesús; Garcia-Sierra, Francisco

    2016-03-21

    Abnormal aggregation of Tau in glial cells has been reported in Alzheimer's disease (AD) and other tauopathies; however, the pathological significance of these aggregates remains unsolved to date. In this study, we evaluated whether full-length Tau (Tau441) and its aspartic acid421-truncated Tau variant (Tau421) produce alterations in the normal organization of the cytoskeleton and plasma membrane (PM) when transiently expressed in cultured C6-glial cells. Forty-eight hours post-transfection, abnormal microtubule bundling was observed in the majority of the cells, which expressed either Tau441 or Tau421. Moreover, both variants of Tau produced extensive PM blebbing associated with cortical redistribution of filamentous actin (F-Actin). These effects were reverted when Tau-expressing cells were incubated with drugs that depolymerize F-Actin. In addition, when glial cells showing Tau-induced PM blebbing were incubated with inhibitors of the Rho-associated protein kinase (ROCK) signaling pathway, both formation of abnormal PM blebs and F-Actin remodeling were avoided. All of these effects were initiated upstream by abnormal Tau-induced microtubule bundling, which may release the microtubule-bound guanine nucleotide exchange factor-H1 (GEF-H1) into the cytoplasm in order to activate its major effector RhoA-GTPase. These results may represent a new mechanism of Tau toxicity in which Tau-induced microtubule bundling produces activation of the Rho-GTPase-ROCK pathway that in turn mediates the remodeling of cortical Actin and PM blebbing. In AD and other tauopathies, these Tau-induced abnormalities may occur and contribute to the impairment of glial activity.

  1. Chromatin Dynamics during Lytic Infection with Herpes Simplex Virus 1

    PubMed Central

    Conn, Kristen L.; Schang, Luis M.

    2013-01-01

    Latent HSV-1 genomes are chromatinized with silencing marks. Since 2004, however, there has been an apparent inconsistency in the studies of the chromatinization of the HSV-1 genomes in lytically infected cells. Nuclease protection and chromatin immunoprecipitation assays suggested that the genomes were not regularly chromatinized, having only low histone occupancy. However, the chromatin modifications associated with transcribed and non-transcribed HSV-1 genes were those associated with active or repressed transcription, respectively. Moreover, the three critical HSV-1 transcriptional activators all had the capability to induce chromatin remodelling, and interacted with critical chromatin modifying enzymes. Depletion or overexpression of some, but not all, chromatin modifying proteins affected HSV-1 transcription, but often in unexpected manners. Since 2010, it has become clear that both cellular and HSV-1 chromatins are highly dynamic in infected cells. These dynamics reconcile the weak interactions between HSV-1 genomes and chromatin proteins, detected by nuclease protection and chromatin immunoprecipitation, with the proposed regulation of HSV-1 gene expression by chromatin, supported by the marks in the chromatin in the viral genomes and the abilities of the HSV-1 transcription activators to modulate chromatin. It also explains the sometimes unexpected results of interventions to modulate chromatin remodelling activities in infected cells. PMID:23863878

  2. Chromatin hydrodynamics.

    PubMed

    Bruinsma, Robijn; Grosberg, Alexander Y; Rabin, Yitzhak; Zidovska, Alexandra

    2014-05-06

    Following recent observations of large scale correlated motion of chromatin inside the nuclei of live differentiated cells, we present a hydrodynamic theory-the two-fluid model-in which the content of a nucleus is described as a chromatin solution with the nucleoplasm playing the role of the solvent and the chromatin fiber that of a solute. This system is subject to both passive thermal fluctuations and active scalar and vector events that are associated with free energy consumption, such as ATP hydrolysis. Scalar events drive the longitudinal viscoelastic modes (where the chromatin fiber moves relative to the solvent) while vector events generate the transverse modes (where the chromatin fiber moves together with the solvent). Using linear response methods, we derive explicit expressions for the response functions that connect the chromatin density and velocity correlation functions to the corresponding correlation functions of the active sources and the complex viscoelastic moduli of the chromatin solution. We then derive general expressions for the flow spectral density of the chromatin velocity field. We use the theory to analyze experimental results recently obtained by one of the present authors and her co-workers. We find that the time dependence of the experimental data for both native and ATP-depleted chromatin can be well-fitted using a simple model-the Maxwell fluid-for the complex modulus, although there is some discrepancy in terms of the wavevector dependence. Thermal fluctuations of ATP-depleted cells are predominantly longitudinal. ATP-active cells exhibit intense transverse long wavelength velocity fluctuations driven by force dipoles. Fluctuations with wavenumbers larger than a few inverse microns are dominated by concentration fluctuations with the same spectrum as thermal fluctuations but with increased intensity.

  3. Reshaping chromatin after DNA damage: the choreography of histone proteins.

    PubMed

    Polo, Sophie E

    2015-02-13

    DNA damage signaling and repair machineries operate in a nuclear environment where DNA is wrapped around histone proteins and packaged into chromatin. Understanding how chromatin structure is restored together with the DNA sequence during DNA damage repair has been a topic of intense research. Indeed, chromatin integrity is central to cell functions and identity. However, chromatin shows remarkable plasticity in response to DNA damage. This review presents our current knowledge of chromatin dynamics in the mammalian cell nucleus in response to DNA double strand breaks and UV lesions. I provide an overview of the key players involved in regulating histone dynamics in damaged chromatin regions, focusing on histone chaperones and their concerted action with histone modifiers, chromatin remodelers and repair factors. I also discuss how these dynamics contribute to reshaping chromatin and, by altering the chromatin landscape, may affect the maintenance of epigenetic information.

  4. Chromatin Dynamics

    PubMed Central

    Hübner, Michael R.; Spector, David L.

    2010-01-01

    The expression patterns of many protein-coding genes are orchestrated in response to exogenous stimuli, as well as cell-type-specific developmental programs. In recent years, researchers have shown that dynamic chromatin movements and interactions in the nucleus play a crucial role in gene regulation. In this review, we highlight our current understanding of the organization of chromatin in the interphase nucleus and the impact of chromatin dynamics on gene expression. We also discuss the current state of knowledge with regard to the localization of active and inactive genes within the three-dimensional nuclear space. Furthermore, we address recent findings that demonstrate the movements of chromosomal regions and genomic loci in association with changes in transcriptional activity. Finally, we discuss the role of intra-and interchromosomal interactions in the control of coregulated genes. PMID:20462379

  5. Lung neuroendocrine tumours: deep sequencing of the four World Health Organization histotypes reveals chromatin-remodelling genes as major players and a prognostic role for TERT, RB1, MEN1 and KMT2D.

    PubMed

    Simbolo, Michele; Mafficini, Andrea; Sikora, Katarzyna O; Fassan, Matteo; Barbi, Stefano; Corbo, Vincenzo; Mastracci, Luca; Rusev, Borislav; Grillo, Federica; Vicentini, Caterina; Ferrara, Roberto; Pilotto, Sara; Davini, Federico; Pelosi, Giuseppe; Lawlor, Rita T; Chilosi, Marco; Tortora, Giampaolo; Bria, Emilio; Fontanini, Gabriella; Volante, Marco; Scarpa, Aldo

    2017-03-01

    Next-generation sequencing (NGS) was applied to 148 lung neuroendocrine tumours (LNETs) comprising the four World Health Organization classification categories: 53 typical carcinoid (TCs), 35 atypical carcinoid (ACs), 27 large-cell neuroendocrine carcinomas, and 33 small-cell lung carcinomas. A discovery screen was conducted on 46 samples by the use of whole-exome sequencing and high-coverage targeted sequencing of 418 genes. Eighty-eight recurrently mutated genes from both the discovery screen and current literature were verified in the 46 cases of the discovery screen, and validated on additional 102 LNETs by targeted NGS; their prevalence was then evaluated on the whole series. Thirteen of these 88 genes were also evaluated for copy number alterations (CNAs). Carcinoids and carcinomas shared most of the altered genes but with different prevalence rates. When mutations and copy number changes were combined, MEN1 alterations were almost exclusive to carcinoids, whereas alterations of TP53 and RB1 cell cycle regulation genes and PI3K/AKT/mTOR pathway genes were significantly enriched in carcinomas. Conversely, mutations in chromatin-remodelling genes, including those encoding histone modifiers and members of SWI-SNF complexes, were found at similar rates in carcinoids (45.5%) and carcinomas (55.0%), suggesting a major role in LNET pathogenesis. One AC and one TC showed a hypermutated profile associated with a POLQ damaging mutation. There were fewer CNAs in carcinoids than in carcinomas; however ACs showed a hybrid pattern, whereby gains of TERT, SDHA, RICTOR, PIK3CA, MYCL and SRC were found at rates similar to those in carcinomas, whereas the MEN1 loss rate mirrored that of TCs. Multivariate survival analysis revealed RB1 mutation (p = 0.0005) and TERT copy gain (p = 0.016) as independent predictors of poorer prognosis. MEN1 mutation was associated with poor prognosis in AC (p = 0.0045), whereas KMT2D mutation correlated with longer survival in SCLC

  6. Ubiquitous Over-Expression of Chromatin Remodeling Factor SRG3 Ameliorates the T Cell-Mediated Exacerbation of EAE by Modulating the Phenotypes of both Dendritic Cells and Macrophages.

    PubMed

    Lee, Sung Won; Park, Hyun Jung; Jeon, Sung Ho; Lee, Changjin; Seong, Rho Hyun; Park, Se-Ho; Hong, Seokmann

    2015-01-01

    Although SWI3-related gene (SRG3), a chromatin remodeling factor, is critical for various biological processes including early embryogenesis and thymocyte development, it is unclear whether SRG3 is involved in the differentiation of CD4+ T cells, the key mediator of adaptive immune responses. Because it is known that experimental autoimmune encephalomyelitis (EAE) development is determined by the activation of CD4+ T helper cells, here, we investigated the role of SRG3 in EAE development using SRG3 transgenic mouse models exhibiting two distinct SRG3 expression patterns: SRG3 expression driven by either the CD2 or β-actin promoter. We found that the outcome of EAE development was completely different depending on the expression pattern of SRG3. The specific over-expression of SRG3 using the CD2 promoter facilitated EAE via the induction of Th1 and Th17 cells, whereas the ubiquitous over-expression of SRG3 using the β-actin promoter inhibited EAE by promoting Th2 differentiation and suppressing Th1 and Th17 differentiation. In addition, the ubiquitous over-expression of SRG3 polarized CD4+ T cell differentiation towards the Th2 phenotype by converting dendritic cells (DCs) or macrophages to Th2 types. SRG3 over-expression not only reduced pro-inflammatory cytokine production by DCs but also shifted macrophages from the inducible nitric oxide synthase (iNOS)-expressing M1 phenotype to the arginase-1-expressing M2 phenotype during EAE. In addition, Th2 differentiation in β-actin-SRG3 Tg mice during EAE was associated with an increase in the basophil and mast cell populations and in IL4 production. Furthermore, the increased frequency of Treg cells in the spinal cord of β-actin-SRG3 Tg mice might induce the suppression of and accelerate the recovery from EAE symptoms. Taken together, our results provide the first evidence supporting the development of a new therapeutic strategy for EAE involving the modulation of SRG3 expression to induce M2 and Th2 polarization

  7. Chromatin signatures of the Drosophila replication program.

    PubMed

    Eaton, Matthew L; Prinz, Joseph A; MacAlpine, Heather K; Tretyakov, George; Kharchenko, Peter V; MacAlpine, David M

    2011-02-01

    DNA replication initiates from thousands of start sites throughout the Drosophila genome and must be coordinated with other ongoing nuclear processes such as transcription to ensure genetic and epigenetic inheritance. Considerable progress has been made toward understanding how chromatin modifications regulate the transcription program; in contrast, we know relatively little about the role of the chromatin landscape in defining how start sites of DNA replication are selected and regulated. Here, we describe the Drosophila replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. We find that while the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. We identify chromatin features that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. We also generate accurate and predictive models from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events.

  8. reSETting chromatin during transcription elongation

    PubMed Central

    Smolle, Michaela; Workman, Jerry L.; Venkatesh, Swaminathan

    2013-01-01

    Maintenance of ordered chromatin structure over the body of genes is vital for the regulation of transcription. Increased access to the underlying DNA sequence results in the recruitment of RNA polymerase II to inappropriate, promoter-like sites within genes, resulting in unfettered transcription. Two new papers show how the Set2-mediated methylation of histone H3 on Lys36 (H3K36me) maintains chromatin structure by limiting histone dynamics over gene bodies, either by recruiting chromatin remodelers that preserve ordered nucleosomal distribution or by lowering the binding affinity of histone chaperones for histones, preventing their removal. PMID:23257840

  9. Interactions of transcription factors with chromatin.

    PubMed

    van Bakel, Harm

    2011-01-01

    Sequence-specific transcription factors (TFs) play a central role in regulating transcription initiation by directing the recruitment and activity of the general transcription machinery and accessory factors. It is now well established that many of the effects exerted by TFs in eukaryotes are mediated through interactions with a host of coregulators that modify the chromatin state, resulting in a more open (in case of activation) or closed conformation (in case of repression). The relationship between TFs and chromatin is a two-way street, however, as chromatin can in turn influence the recognition and binding of target sequences by TFs. The aim of this chapter is to highlight how this dynamic interplay between TF-directed remodelling of chromatin and chromatin-adjusted targeting of TF binding determines where and how transcription is initiated, and to what degree it is productive.

  10. Mechanism of Transcriptional Regulation by Androgen Receptor and its Coactivators in the Context of Chromatin

    DTIC Science & Technology

    2002-07-01

    acetylation over the promoter region was also observed. This histone acetylation is correlated with the recruitment of CBP/p300. Taken together, our...data suggest that hormone-dependent activation by AR is associated with two types of chromatin remodeling, histone acetylation and chromatin remodeling...number of transcriptional cofactors as histone acetyltransferaseases (HAT), deacetylases, or methyltransferases. Acetylation of histone tails is

  11. Nucleosome Remodeling and Epigenetics

    PubMed Central

    Becker, Peter B.; Workman, Jerry L.

    2013-01-01

    Eukaryotic chromatin is kept flexible and dynamic to respond to environmental, metabolic, and developmental cues through the action of a family of so-called “nucleosome remodeling” ATPases. Consistent with their helicase ancestry, these enzymes experience conformation changes as they bind and hydrolyze ATP. At the same time they interact with DNA and histones, which alters histone–DNA interactions in target nucleosomes. Their action may lead to complete or partial disassembly of nucleosomes, the exchange of histones for variants, the assembly of nucleosomes, or the movement of histone octamers on DNA. “Remodeling” may render DNA sequences accessible to interacting proteins or, conversely, promote packing into tightly folded structures. Remodeling processes participate in every aspect of genome function. Remodeling activities are commonly integrated with other mechanisms such as histone modifications or RNA metabolism to assemble stable, epigenetic states. PMID:24003213

  12. The great repression: chromatin and cryptic transcription.

    PubMed

    Hennig, Bianca P; Fischer, Tamás

    2013-01-01

    The eukaryotic chromatin structure is essential in correctly defining transcription units. Impairing this structure can activate cryptic promoters, and lead to the accumulation of aberrant RNA transcripts. Here we discuss critical pathways that are responsible for the repression of cryptic transcription and the maintenance of genome integrity.

  13. Plant chromatin warms up in Madrid: meeting summary of the 3rd European Workshop on Plant Chromatin 2013, Madrid, Spain.

    PubMed

    Jarillo, José A; Gaudin, Valérie; Hennig, Lars; Köhler, Claudia; Piñeiro, Manuel

    2014-04-01

    The 3rd European Workshop on Plant Chromatin (EWPC) was held on August 2013 in Madrid, Spain. A number of different topics on plant chromatin were presented during the meeting, including new factors mediating Polycomb Group protein function in plants, chromatin-mediated reprogramming in plant developmental transitions, the role of histone variants, and newly identified chromatin remodeling factors. The function of interactions between chromatin and transcription factors in the modulation of gene expression, the role of chromatin dynamics in the control of nuclear processes and the influence of environmental factors on chromatin organization were also reported. In this report, we highlight some of the new insights emerging in this growing area of research, presented at the 3rd EWPC.

  14. Regulation of chromatin structure in the cardiovascular system.

    PubMed

    Rosa-Garrido, Manuel; Karbassi, Elaheh; Monte, Emma; Vondriska, Thomas M

    2013-01-01

    It has been appreciated for some time that cardiovascular disease involves large-scale transcriptional changes in various cell types. What has become increasingly clear only in the past few years, however, is the role of chromatin remodeling in cardiovascular phenotypes in normal physiology, as well as in development and disease. This review summarizes the state of the chromatin field in terms of distinct mechanisms to regulate chromatin structure in vivo, identifying when these modes of regulation have been demonstrated in cardiovascular tissues. We describe areas in which a better understanding of chromatin structure is leading to new insights into the fundamental biology of cardiovascular disease. 

  15. Colorectal Carcinomas With CpG Island Methylator Phenotype 1 Frequently Contain Mutations in Chromatin Regulators

    PubMed Central

    Tahara, Tomomitsu; Yamamoto, Eiichiro; Madireddi, Priyanka; Suzuki, Hiromu; Maruyama, Reo; Chung, Woonbok; Garriga, Judith; Jelinek, Jaroslav; Yamano, Hiro-o; Sugai, Tamotsu; Kondo, Yutaka; Toyota, Minoru; Issa, Jean-Pierre J.; Estécio, Marcos R. H.

    2014-01-01

    BACKGROUND & AIMS Subgroups of colorectal carcinomas (CRCs) characterized by DNA methylation anomalies are termed CpG island methylator phenotype (CIMP)1, CIMP2, or CIMP-negative. The pathogenesis of CIMP1 colorectal carcinomas, and their effects on patients’ prognoses and responses to treatment, differ from those of other CRCs. We sought to identify genetic somatic alterations associated with CIMP1 CRCs. METHODS We examined genomic DNA samples from 100 primary CRCs, 10 adenomas, and adjacent normal-appearing mucosae from patients undergoing surgery or colonoscopy at 3 tertiary medical centers. We performed exome sequencing of 16 colorectal tumors and their adjacent normal tissues. Extensive comparison with known somatic alterations in CRCs allowed segregation of CIMP1-exclusive alterations. The prevalence of mutations in selected genes was determined from an independent cohort. RESULTS We found that genes that regulate chromatin were mutated in CIMP1 CRCs; the highest rates of mutation were observed in CHD7 and CHD8, which encode members of the chromodomain helicase/adenosine triphosphate—dependent chromatin remodeling family. Somaticmutations in these 2 genes were detected in 5 of 9 CIMP1 CRCs. A prevalence screen showed that nonsilencing mutations in CHD7 and CHD8 occurred significantly more frequently in CIMP1 tumors (18 of 42 [43%]) than in CIMP2 (3 of 34 [9%]; P < .01) or CIMP-negative tumors (2 of 34 [6%]; P < .001). CIMP1 markers had increased binding by CHD7, compared with all genes. Genes altered in patients with CHARGE syndrome (congenital malformations involving the central nervous system, eye, ear, nose, and mediastinal organs) who had CHD7 mutations were also altered in CRCs with mutations in CHD7. CONCLUSIONS Aberrations in chromatin remodeling could contribute to the development of CIMP1 CRCs. A better understanding of the biological determinants of CRCs can be achieved when these tumors are categorized according to their epigenetic status. PMID

  16. Polarization Aberrations

    NASA Technical Reports Server (NTRS)

    Mcguire, James P., Jr.; Chipman, Russell A.

    1990-01-01

    The analysis of the polarization characteristics displayed by optical systems can be divided into two categories: geometrical and physical. Geometrical analysis calculates the change in polarization of a wavefront between pupils in an optical instrument. Physical analysis propagates the polarized fields wherever the geometrical analysis is not valid, i.e., near the edges of stops, near images, in anisotropic media, etc. Polarization aberration theory provides a starting point for geometrical design and facilitates subsequent optimization. The polarization aberrations described arise from differences in the transmitted (or reflected) amplitudes and phases at interfaces. The polarization aberration matrix (PAM) is calculated for isotropic rotationally symmetric systems through fourth order and includes the interface phase, amplitude, linear diattenuation, and linear retardance aberrations. The exponential form of Jones matrices used are discussed. The PAM in Jones matrix is introduced. The exact calculation of polarization aberrations through polarization ray tracing is described. The report is divided into three sections: I. Rotationally Symmetric Optical Systems; II. Tilted and Decentered Optical Systems; and Polarization Analysis of LIDARs.

  17. Three-dimensional structure of human chromatin accessibility complex hCHRAC by electron microscopy

    SciTech Connect

    Hu, M.; Hainfeld, J.; Zhang, Y.-B.; Qian, L.; Brinas, R. P.; Kuznetsova, L.

    2008-12-01

    ATP-dependent chromatin remodeling complexes modulate the dynamic assembly and remodeling of chromatin involved in DNA transcription, replication, and repair. There is little structural detail known about these important multiple-subunit enzymes that catalyze chromatin remodeling processes. Here we report a three-dimensional structure of the human chromatin accessibility complex, hCHRAC, using single particle reconstruction by negative stain electron microscopy. This structure shows an asymmetric 15 x 10 x 12 nm disk shape with several lobes protruding out of its surfaces. Based on the factors of larger contact area, smaller steric hindrance, and direct involvement of hCHRAC in interactions with the nucleosome, we propose that four lobes on one side form a multiple-site contact surface 10 nm in diameter for nucleosome binding. This work provides the first determination of the three-dimensional structure of the ISWI-family of chromatin remodeling complexes.

  18. HDACi--targets beyond chromatin.

    PubMed

    Buchwald, Marc; Krämer, Oliver H; Heinzel, Thorsten

    2009-08-08

    Histone deacetylases (HDACs) play an important role in gene regulation. Inhibitors of HDACs (HDACi) are novel anti-cancer drugs, which induce histone (hyper-) acetylation and counteract aberrant gene repression. On the other hand, HDACi treatment can also result in decreased gene expression, and targeting HDACs affects more than chromatin. Recently, HDACi were shown to evoke non-histone protein acetylation, which can alter signaling networks relevant for tumorgenesis. Furthermore, HDACi can promote the degradation of (proto-) oncoproteins. Here, we summarize these findings and discuss how these substances could be beneficial for the treatment and prevention of human ailments, such as cancer and unbalanced immune functions.

  19. Impaired TIP60-mediated H4K16 acetylation accounts for the aberrant chromatin accumulation of 53BP1 and RAP80 in Fanconi anemia pathway-deficient cells.

    PubMed

    Renaud, Emilie; Barascu, Aurelia; Rosselli, Filippo

    2016-01-29

    To rescue collapsed replication forks cells utilize homologous recombination (HR)-mediated mechanisms to avoid the induction of gross chromosomal abnormalities that would be generated by non-homologous end joining (NHEJ). Using DNA interstrand crosslinks as a replication barrier, we investigated how the Fanconi anemia (FA) pathway promotes HR at stalled replication forks. FA pathway inactivation results in Fanconi anemia, which is associated with a predisposition to cancer. FANCD2 monoubiquitination and assembly in subnuclear foci appear to be involved in TIP60 relocalization to the chromatin to acetylates histone H4K16 and prevents the binding of 53BP1 to its docking site, H4K20Me2. Thus, FA pathway loss-of-function results in accumulation of 53BP1, RIF1 and RAP80 at damaged chromatin, which impair DNA resection at stalled replication fork-associated DNA breaks and impede HR. Consequently, DNA repair in FA cells proceeds through the NHEJ pathway, which is likely responsible for the accumulation of chromosome abnormalities. We demonstrate that the inhibition of NHEJ or deacetylase activity rescue HR in FA cells.

  20. Changing chromatin fiber conformation by nucleosome repositioning.

    PubMed

    Müller, Oliver; Kepper, Nick; Schöpflin, Robert; Ettig, Ramona; Rippe, Karsten; Wedemann, Gero

    2014-11-04

    Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ?10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell.

  1. Changing Chromatin Fiber Conformation by Nucleosome Repositioning

    PubMed Central

    Müller, Oliver; Kepper, Nick; Schöpflin, Robert; Ettig, Ramona; Rippe, Karsten; Wedemann, Gero

    2014-01-01

    Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ∼10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell. PMID:25418099

  2. An Overview of Chromatin-Regulating Proteins in Cells

    PubMed Central

    Zhang, Pingyu; Torres, Keila; Liu, Xiuping; Liu, Chang-gong; Pollock, Raphael E.

    2016-01-01

    In eukaryotic cells, gene expressions on chromosome DNA are orchestrated by a dynamic chromosome structure state that is largely controlled by chromatin-regulating proteins, which regulate chromatin structures, release DNA from the nucleosome, and activate or suppress gene expression by modifying nucleosome histones or mobilizing DNA-histone structure. The two classes of chromatin- regulating proteins are 1) enzymes that modify histones through methylation, acetylation, phosphorylation, adenosine diphosphate–ribosylation, glycosylation, sumoylation, or ubiquitylation and 2) enzymes that remodel DNA-histone structure with energy from ATP hydrolysis. Chromatin-regulating proteins, which modulate DNA-histone interaction, change chromatin conformation, and increase or decrease the binding of functional DNA-regulating protein complexes, have major functions in nuclear processes, including gene transcription and DNA replication, repair, and recombination. This review provides a general overview of chromatin-regulating proteins, including their classification, molecular functions, and interactions with the nucleosome in eukaryotic cells. PMID:26796306

  3. Revealing remodeler function: Varied and unique

    NASA Astrophysics Data System (ADS)

    Eastlund, Allen

    Chromatin remodelers perform a necessary and required function for the successful expression of our genetic code. By modifying, shifting, or ejecting nucleosomes from the chromatin structure they allow access to the underlying DNA to the rest of the cell's machinery. This research has focused on two major remodeler motors from major families of chromatin remodelers: the trimeric motor domain of RSC and the motor domain of the ISWI family, ISWI. Using primarily stopped-flow spectrofluorometry, I have categorized the time-dependent motions of these motor domains along their preferred substrate, double-stranded DNA. Combined with collected ATP utilization data, I present the subsequent analysis and associated conclusions that stem from the underlying assumptions and models. Interestingly, there is little in common between the investigated proteins aside from their favored medium. While RSC exhibits modest translocation characteristics and highly effective motion with the ability for large molecular forces, ISWI is not only structurally different but highly inefficient in its motion leading to difficulties in determining its specific translocation mechanics. While chromatin remodeling is a ubiquitous facet of eukaryotic life, there remains much to be understood about their general mechanisms.

  4. Histone variants and chromatin assembly in plant abiotic stress responses.

    PubMed

    Zhu, Yan; Dong, Aiwu; Shen, Wen-Hui

    2013-01-01

    Genome organization into nucleosomes and higher-order chromatin structures has profound implications for the regulation of gene expression, DNA replication and repair. The structure of chromatin can be remodeled by several mechanisms; among others, nucleosome assembly/disassembly and replacement of canonical histones with histone variants constitute important ones. In this review, we provide a brief description on the current knowledge about histone chaperones involved in nucleosome assembly/disassembly and histone variants in Arabidopsis thaliana. We discuss recent advances in revealing crucial functions of histone chaperones, nucleosome assembly/disassembly and histone variants in plant response to abiotic stresses. It appears that chromatin structure remodeling may provide a flexible, global and stable means for the regulation of gene transcription to help plants more effectively cope with environmental stresses. This article is part of a Special Issue entitled: Histone chaperones and chromatin assembly.

  5. Chromatin enrichment for proteomics

    PubMed Central

    Kustatscher, Georg; Wills, Karen L. H.; Furlan, Cristina; Rappsilber, Juri

    2015-01-01

    During interphase, chromatin hosts fundamental cellular processes, such as gene expression, DNA replication and DNA damage repair. To analyze chromatin on a proteomic scale, we have developed chromatin enrichment for proteomics (ChEP), which is a simple biochemical procedure that enriches interphase chromatin in all its complexity. It enables researchers to take a ‘snapshot’ of chromatin and to isolate and identify even transiently bound factors. In ChEP, cells are fixed with formaldehyde; subsequently, DNA together with all cross-linked proteins is isolated by centrifugation under denaturing conditions. This approach enables the analysis of global chromatin composition and its changes, which is in contrast with existing chromatin enrichment procedures, which either focus on specific chromatin loci (e.g., affinity purification) or are limited in specificity, such as the analysis of the chromatin pellet (i.e., analysis of all insoluble nuclear material). ChEP takes half a day to complete and requires no specialized laboratory skills or equipment. ChEP enables the characterization of chromatin response to drug treatment or physiological processes. Beyond proteomics, ChEP may preclear chromatin for chromatin immunoprecipitation (ChIP) analyses. PMID:25101823

  6. Retinal remodeling.

    PubMed

    Jones, B W; Kondo, M; Terasaki, H; Lin, Y; McCall, M; Marc, R E

    2012-07-01

    Retinal photoreceptor degeneration takes many forms. Mutations in rhodopsin genes or disorders of the retinal pigment epithelium, defects in the adenosine triphosphate binding cassette transporter, ABCR gene defects, receptor tyrosine kinase defects, ciliopathies and transport defects, defects in both transducin and arrestin, defects in rod cyclic guanosine 3',5'-monophosphate phosphodiesterase, peripherin defects, defects in metabotropic glutamate receptors, synthetic enzymatic defects, defects in genes associated with signaling, and many more can all result in retinal degenerative disease like retinitis pigmentosa (RP) or RP-like disorders. Age-related macular degeneration (AMD) and AMD-like disorders are possibly due to a constellation of potential gene targets and gene/gene interactions, while other defects result in diabetic retinopathy or glaucoma. However, all of these insults as well as traumatic insults to the retina result in retinal remodeling. Retinal remodeling is a universal finding subsequent to retinal degenerative disease that results in deafferentation of the neural retina from photoreceptor input as downstream neuronal elements respond to loss of input with negative plasticity. This negative plasticity is not passive in the face of photoreceptor degeneration, with a phased revision of retinal structure and function found at the molecular, synaptic, cell, and tissue levels involving all cell classes in the retina, including neurons and glia. Retinal remodeling has direct implications for the rescue of vision loss through bionic or biological approaches, as circuit revision in the retina corrupts any potential surrogate photoreceptor input to a remnant neural retina. However, there are a number of potential opportunities for intervention that are revealed through the study of retinal remodeling, including therapies that are designed to slow down photoreceptor loss, interventions that are designed to limit or arrest remodeling events, and

  7. Determinants of Sir2-Mediated, Silent Chromatin Cohesion.

    PubMed

    Chen, Yu-Fan; Chou, Chia-Ching; Gartenberg, Marc R

    2016-08-01

    Cohesin associates with distinct sites on chromosomes to mediate sister chromatid cohesion. Single cohesin complexes are thought to bind by encircling both sister chromatids in a topological embrace. Transcriptionally repressed chromosomal domains in the yeast Saccharomyces cerevisiae represent specialized sites of cohesion where cohesin binds silent chromatin in a Sir2-dependent fashion. In this study, we investigated the molecular basis for Sir2-mediated cohesion. We identified a cluster of charged surface residues of Sir2, collectively termed the EKDK motif, that are required for cohesin function. In addition, we demonstrated that Esc8, a Sir2-interacting factor, is also required for silent chromatin cohesion. Esc8 was previously shown to associate with Isw1, the enzymatic core of ISW1 chromatin remodelers, to form a variant of the ISW1a chromatin remodeling complex. When ESC8 was deleted or the EKDK motif was mutated, cohesin binding at silenced chromatin domains persisted but cohesion of the domains was abolished. The data are not consistent with cohesin embracing both sister chromatids within silent chromatin domains. Transcriptional silencing remains largely intact in strains lacking ESC8 or bearing EKDK mutations, indicating that silencing and cohesion are separable functions of Sir2 and silent chromatin.

  8. DNA Damage Repair in the Context of Plant Chromatin1

    PubMed Central

    2015-01-01

    The integrity of DNA molecules is constantly challenged. All organisms have developed mechanisms to detect and repair multiple types of DNA lesions. The basic principles of DNA damage repair (DDR) in prokaryotes and unicellular and multicellular eukaryotes are similar, but the association of DNA with nucleosomes in eukaryotic chromatin requires mechanisms that allow access of repair enzymes to the lesions. This is achieved by chromatin-remodeling factors, and their necessity for efficient DDR has recently been demonstrated for several organisms and repair pathways. Plants share many features of chromatin organization and DNA repair with fungi and animals, but they differ in other, important details, which are both interesting and relevant for our understanding of genome stability and genetic diversity. In this Update, we compare the knowledge of the role of chromatin and chromatin-modifying factors during DDR in plants with equivalent systems in yeast and humans. We emphasize plant-specific elements and discuss possible implications. PMID:26089404

  9. Remodeling of heterochromatin induced by heavy metals in extreme old age.

    PubMed

    Lezhava, Teimuraz; Monaselidze, Jamlet; Jokhadze, Tinatin; Gorgoshidze, Maia; Kiladze, Maia; Gaiozishvili, Maia

    2011-09-01

    The levels of chromosome instability and heat absorption of chromatin have been studied in cultured lymphocytes derived from blood of 80-93- and 18-30-year-old individuals, under the effect of heavy metal Cu(II) and Cd(II) salts. The analysis of the results obtained indicates that 50 μM Cu(II) induced a significantly higher level of cells with chromosome aberrations in old donors (13.8 ± 1.5% vs control, 3.8 ± 1.7%), whereas treatment with 100 μM Cd(II) did not induce any changes in the background index. Analysis of the lymphocyte melting curves showed that Cu(II) ions caused more effective condensation of heterochromatin in old healthy individuals compared with young donors, which was expressed by the increase of the T (m) of elderly chromatin by ~3°C compared with the norm. Treatment of lymphocyte chromatin of old individuals with 100 μM Cd(II) caused decondensation (deheterochromatinization) of both the facultative and constitutive domains of heterochromatin. The deheterochromatinization T (m) was decreased by ~3-3.5°C compared with the T (m) observed for young individuals. Thus, the chromatin of cultured lymphocytes from the old-aged individuals underwent modification under the influence of copper and cadmium salts. Cu(II) caused additional heterochromatinization of heterochromatin, and Cd(II) caused deheterochromatinization of facultative and constitutive heterochromatin. Our data may be important as new information on the remodeling of constitutive and facultative heterochromatin induced by heavy metals in aging, aging pathology, and pathology linked with metal ions.

  10. Nucleosome remodelers in double-strand break repair.

    PubMed

    Seeber, Andrew; Hauer, Michael; Gasser, Susan M

    2013-04-01

    ATP-dependent nucleosome remodelers use ATP hydrolysis to shift, evict and exchange histone dimers or octamers and have well-established roles in transcription. Earlier work has suggested a role for nucleosome remodelers such as INO80 in double-strand break (DSB) repair. This review will begin with an update on recent studies that explore how remodelers are recruited to DSBs. We then examine their impact on various steps of repair, focusing on resection and the formation of the Rad51-ssDNA nucleofilament. Finally, we will explore new studies that implicate remodelers in the physical movement of chromatin in response to damage.

  11. Skew aberration: a form of polarization aberration.

    PubMed

    Yun, Garam; Crabtree, Karlton; Chipman, Russell A

    2011-10-15

    We define a new class of aberration, skew aberration, which is a component of polarization aberration. Skew aberration is an intrinsic rotation of polarization states due to the geometric transformation of local coordinates, independent of coatings and interface polarization. Skew aberration in a radially symmetric system has the form of a circular retardance tilt plus coma aberration. Skew aberration causes undesired polarization distribution in the exit pupil. We demonstrate statistics on skew aberration of 2383 optical systems described in Code V's U.S. patent library [Code V Version 10.3 (Synopsys, 2011), pp. 22-24]; the mean skew aberration is 0.89° and the standard deviation is 1.37°. The maximum skew aberration found is 17.45° and the minimum is -11.33°. U.S. patent 2,896,506, which has ±7.01° of skew aberration, is analyzed in detail. Skew aberration should be of concern in microlithography optics and other high NA and large field of view optical systems.

  12. Long range chromatin organization

    PubMed Central

    Acuña, Luciana I Gómez; Kornblihtt, Alberto R

    2014-01-01

    Splicing is a predominantly co-transcriptional process that has been shown to be tightly coupled to transcription. Chromatin structure is a key factor that mediates this functional coupling. In light of recent evidence that shows the importance of higher order chromatin organization in the coordination and regulation of gene expression, we discuss here the possible roles of long-range chromatin organization in splicing and alternative splicing regulation. PMID:25764333

  13. A Broad Set of Chromatin Factors Influences Splicing

    PubMed Central

    Allemand, Eric; Myers, Michael P.; Garcia-Bernardo, Jose; Harel-Bellan, Annick; Krainer, Adrian R.; Muchardt, Christian

    2016-01-01

    Several studies propose an influence of chromatin on pre-mRNA splicing, but it is still unclear how widespread and how direct this phenomenon is. We find here that when assembled in vivo, the U2 snRNP co-purifies with a subset of chromatin-proteins, including histones and remodeling complexes like SWI/SNF. Yet, an unbiased RNAi screen revealed that the outcome of splicing is influenced by a much larger variety of chromatin factors not all associating with the spliceosome. The availability of this broad range of chromatin factors impacting splicing further unveiled their very context specific effect, resulting in either inclusion or skipping, depending on the exon under scrutiny. Finally, a direct assessment of the impact of chromatin on splicing using an in vitro co-transcriptional splicing assay with pre-mRNAs transcribed from a nucleosomal template, demonstrated that chromatin impacts nascent pre-mRNP in their competence for splicing. Altogether, our data show that numerous chromatin factors associated or not with the spliceosome can affect the outcome of splicing, possibly as a function of the local chromatin environment that by default interferes with the efficiency of splicing. PMID:27662573

  14. Super-resolution microscopy reveals decondensed chromatin structure at transcription sites

    NASA Astrophysics Data System (ADS)

    Wang, Yejun; Maharana, Shovamayee; Wang, Michelle D.; Shivashankar, G. V.

    2014-03-01

    Remodeling of the local chromatin structure is essential for the regulation of gene expression. While a number of biochemical and bioimaging experiments suggest decondensed chromatin structures are associated with transcription, a direct visualization of DNA and transcriptionally active RNA polymerase II (RNA pol II) at super-resolution is still lacking. Here we investigate the structure of chromatin isolated from HeLa cells using binding activatable localization microscopy (BALM). The sample preparation method preserved the structural integrity of chromatin. Interestingly, BALM imaging of the chromatin spreads revealed the presence of decondensed chromatin as gap structures along the spreads. These gaps were enriched with phosphorylated S5 RNA pol II, and were sensitive to the cellular transcriptional state. Taken together, we could visualize the decondensed chromatin regions together with active RNA pol II for the first time using super-resolution microscopy.

  15. Tension-dependent nucleosome remodeling at the pericentromere in yeast.

    PubMed

    Verdaasdonk, Jolien S; Gardner, Ryan; Stephens, Andrew D; Yeh, Elaine; Bloom, Kerry

    2012-07-01

    Nucleosome positioning is important for the structural integrity of chromosomes. During metaphase the mitotic spindle exerts physical force on pericentromeric chromatin. The cell must adjust the pericentromeric chromatin to accommodate the changing tension resulting from microtubule dynamics to maintain a stable metaphase spindle. Here we examine the effects of spindle-based tension on nucleosome dynamics by measuring the histone turnover of the chromosome arm and the pericentromere during metaphase in the budding yeast Saccharomyces cerevisiae. We find that both histones H2B and H4 exhibit greater turnover in the pericentromere during metaphase. Loss of spindle-based tension by treatment with the microtubule-depolymerizing drug nocodazole or compromising kinetochore function results in reduced histone turnover in the pericentromere. Pericentromeric histone dynamics are influenced by the chromatin-remodeling activities of STH1/NPS1 and ISW2. Sth1p is the ATPase component of the Remodels the Structure of Chromatin (RSC) complex, and Isw2p is an ATP-dependent DNA translocase member of the Imitation Switch (ISWI) subfamily of chromatin-remodeling factors. The balance between displacement and insertion of pericentromeric histones provides a mechanism to accommodate spindle-based tension while maintaining proper chromatin packaging during mitosis.

  16. Chromatin Topological Transitions

    NASA Astrophysics Data System (ADS)

    Lavelle, C.; Bancaud, A.; Recouvreux, P.; Barbi, M.; Victor, J.; Viovy, J.

    DNA transaction events occurring during a cell cycle (transcription,repair, replication) are always associated with severe topological constraints on the double helix. However, since nuclear DNA is bound to various proteins (including histones) that control its accessibility and 3D organization, these topological constraints propagate or accumulate on a chromatin substrate. This paper focuses on chromatin fiber response to physiological mechanical constraints expected to occur during transcription elongation. We will show in particular how recent single molecule techniques help us to understand how chromatin conformational dynamics could manage harsh DNA supercoiling changes.

  17. Persistent Chromatin Modifications Induced by High Fat Diet.

    PubMed

    Leung, Amy; Trac, Candi; Du, Juan; Natarajan, Rama; Schones, Dustin E

    2016-05-13

    Obesity is a highly heritable complex disease that results from the interaction of multiple genetic and environmental factors. Formerly obese individuals are susceptible to metabolic disorders later in life, even after lifestyle changes are made to mitigate the obese state. This is reminiscent of the metabolic memory phenomenon originally observed for persistent complications in diabetic patients, despite subsequent glycemic control. Epigenetic modifications represent a potential mediator of this observed memory. We previously demonstrated that a high fat diet leads to changes in chromatin accessibility in the mouse liver. The regions of greatest chromatin changes in accessibility are largely strain-dependent, indicating a genetic component in diet-induced chromatin alterations. We have now examined the persistence of diet-induced chromatin accessibility changes upon diet reversal in two strains of mice. We find that a substantial fraction of loci that undergo chromatin accessibility changes with a high fat diet remains in the remodeled state after diet reversal in C57BL/6J mice. In contrast, the vast majority of diet-induced chromatin accessibility changes in A/J mice are transient. Our data also indicate that the persistent chromatin accessibility changes observed in C57BL/6J mice are associated with specific transcription factors and histone post-translational modifications. The persistent loci identified here are likely to be contributing to the overall phenotype and are attractive targets for therapeutic intervention.

  18. C/EBP maintains chromatin accessibility in liver and facilitates glucocorticoid receptor recruitment to steroid response elements.

    PubMed

    Grøntved, Lars; John, Sam; Baek, Songjoon; Liu, Ying; Buckley, John R; Vinson, Charles; Aguilera, Greti; Hager, Gordon L

    2013-05-29

    Mechanisms regulating transcription factor interaction with chromatin in intact mammalian tissues are poorly understood. Exploiting an adrenalectomized mouse model with depleted endogenous glucocorticoids, we monitor changes of the chromatin landscape in intact liver tissue following glucocorticoid injection. Upon activation of the glucocorticoid receptor (GR), proximal regions of activated and repressed genes are remodelled, and these remodelling events correlate with RNA polymerase II occupancy of regulated genes. GR is exclusively associated with accessible chromatin and 62% percent of GR-binding sites are occupied by C/EBPβ. At the majority of these sites, chromatin is preaccessible suggesting a priming function of C/EBPβ for GR recruitment. Disruption of C/EBPβ binding to chromatin results in attenuation of pre-programmed chromatin accessibility, GR recruitment and GR-induced chromatin remodelling specifically at sites co-occupied by GR and C/EBPβ. Collectively, we demonstrate a highly cooperative mechanism by which C/EBPβ regulates selective GR binding to the genome in liver tissue. We suggest that selective targeting of GR in other tissues is likely mediated by the combined action of cell-specific priming proteins and chromatin remodellers.

  19. Making copies of chromatin: the challenge of nucleosomal organization and epigenetic information.

    PubMed

    Corpet, Armelle; Almouzni, Geneviève

    2009-01-01

    Understanding the basic mechanisms underlying chromatin dynamics during DNA replication in eukaryotic cells is of fundamental importance. Beyond DNA compaction, chromatin organization represents a means to regulate genome function. Thus, the inheritance and maintenance of the DNA sequence, along with its organization into chromatin, is central for eukaryotic life. To orchestrate DNA replication in the context of chromatin is a challenge, both in terms of accessibility to the compact structures and maintenance of chromatin organization. To meet the challenge of maintenance, cells have evolved efficient nucleosome dynamics involving assembly pathways and chromatin maturation mechanisms that restore chromatin organization in the wake of DNA replication. In this review, we describe our current knowledge concerning how these pathways operate at the nucleosomal level and highlight the key players, such as histone chaperones, chromatin remodelers or modifiers, involved in the process of chromatin duplication. Major advances have been made recently concerning de novo nucleosome assembly and our understanding of its coordination with recycling of parental histones is progressing. Insights into the transmission of chromatin-based information during replication have important implications in the field of epigenetics to fully comprehend how the epigenetic landscape might, or at times might not, be stably maintained in the face of dramatic changes in chromatin structure.

  20. Chicago aberration correction work.

    PubMed

    Beck, V D

    2012-12-01

    The author describes from his personal involvement the many improvements to electron microscopy Albert Crewe and his group brought by minimizing the effects of aberrations. The Butler gun was developed to minimize aperture aberrations in a field emission electron gun. In the 1960s, Crewe anticipated using a spherical aberration corrector based on Scherzer's design. Since the tolerances could not be met mechanically, a method of moving the center of the octopoles electrically was developed by adding lower order multipole fields. Because the corrector was located about 15 cm ahead of the objective lens, combination aberrations would arise with the objective lens. This fifth order aberration would then limit the aperture of the microscope. The transformation of the off axis aberration coefficients of a round lens was developed and a means to cancel anisotropic coma was developed. A new method of generating negative spherical aberration was invented using the combination aberrations of hexapoles. Extensions of this technique to higher order aberrations were developed. An electrostatic electron mirror was invented, which allows the cancellation of primary spherical aberration and first order chromatic aberration. A reduction of chromatic aberration by two orders of magnitude was demonstrated using such a system.

  1. DNA Methylation and Chromatin Remodeling: The Blueprint of Cancer Epigenetics

    PubMed Central

    Shenoy, Smita; Bairy, Kurady Laxminarayana

    2016-01-01

    Epigenetics deals with the interactions between genes and the immediate cellular environment. These interactions go a long way in shaping up each and every person's individuality. Further, reversibility of epigenetic interactions may offer a dynamic control over the expression of various critical genes. Thus, tweaking the epigenetic machinery may help cause or cure diseases, especially cancer. Therefore, cancer epigenetics, especially at a molecular level, needs to be scrutinised closely, as it could potentially serve as the future pharmaceutical goldmine against neoplastic diseases. However, in view of its rapidly enlarging scope of application, it has become difficult to keep abreast of scientific information coming out of various epigenetic studies directed against cancer. Using this review, we have attempted to shed light on two of the most important mechanisms implicated in cancer, that is, DNA (deoxyribonucleic acid) methylation and histone modifications, and their place in cancer pathogenesis. Further, we have attempted to take stock of the new epigenetic drugs that have emerged onto the market as well as those in the pipeline that offer hope in mankind's fight against cancer. PMID:27119045

  2. DNA Methylation and Chromatin Remodeling: The Blueprint of Cancer Epigenetics.

    PubMed

    Bhattacharjee, Dipanjan; Shenoy, Smita; Bairy, Kurady Laxminarayana

    2016-01-01

    Epigenetics deals with the interactions between genes and the immediate cellular environment. These interactions go a long way in shaping up each and every person's individuality. Further, reversibility of epigenetic interactions may offer a dynamic control over the expression of various critical genes. Thus, tweaking the epigenetic machinery may help cause or cure diseases, especially cancer. Therefore, cancer epigenetics, especially at a molecular level, needs to be scrutinised closely, as it could potentially serve as the future pharmaceutical goldmine against neoplastic diseases. However, in view of its rapidly enlarging scope of application, it has become difficult to keep abreast of scientific information coming out of various epigenetic studies directed against cancer. Using this review, we have attempted to shed light on two of the most important mechanisms implicated in cancer, that is, DNA (deoxyribonucleic acid) methylation and histone modifications, and their place in cancer pathogenesis. Further, we have attempted to take stock of the new epigenetic drugs that have emerged onto the market as well as those in the pipeline that offer hope in mankind's fight against cancer.

  3. Chromatin Memory in the Development of Human Cancers

    PubMed Central

    Yao, Yixin; Des Marais, Thomas L; Costa, Max

    2014-01-01

    Cancer is a complex disease with acquired genomic and epigenomic alterations that affect cell proliferation, viability and invasiveness. Almost all the epigenetic mechanisms including cytosine methylation and hydroxymethylation, chromatin remodeling and non-coding RNAs have been found associate with carcinogenesis and cancer specific expression profile. Altered histone modification as an epigenetic hallmark is frequently found in tumors. Understanding the epigenetic alterations induced by carcinogens or infectious agents may help us understand early epigenetic changes prior to the development of cancer. In this review, we focus on chromatin remodeling and the associated histone modifiers in the development of cancer; the application of these modifiers as a cancer therapy target in different clinical trial phases is also discussed. PMID:25606572

  4. Dynamic regulation of transcription factors by nucleosome remodeling.

    PubMed

    Li, Ming; Hada, Arjan; Sen, Payel; Olufemi, Lola; Hall, Michael A; Smith, Benjamin Y; Forth, Scott; McKnight, Jeffrey N; Patel, Ashok; Bowman, Gregory D; Bartholomew, Blaine; Wang, Michelle D

    2015-06-05

    The chromatin landscape and promoter architecture are dominated by the interplay of nucleosome and transcription factor (TF) binding to crucial DNA sequence elements. However, it remains unclear whether nucleosomes mobilized by chromatin remodelers can influence TFs that are already present on the DNA template. In this study, we investigated the interplay between nucleosome remodeling, by either yeast ISW1a or SWI/SNF, and a bound TF. We found that a TF serves as a major barrier to ISW1a remodeling, and acts as a boundary for nucleosome repositioning. In contrast, SWI/SNF was able to slide a nucleosome past a TF, with concurrent eviction of the TF from the DNA, and the TF did not significantly impact the nucleosome positioning. Our results provide direct evidence for a novel mechanism for both nucleosome positioning regulation by bound TFs and TF regulation via dynamic repositioning of nucleosomes.

  5. Ectopically tethered CP190 induces large-scale chromatin decondensation

    NASA Astrophysics Data System (ADS)

    Ahanger, Sajad H.; Günther, Katharina; Weth, Oliver; Bartkuhn, Marek; Bhonde, Ramesh R.; Shouche, Yogesh S.; Renkawitz, Rainer

    2014-01-01

    Insulator mediated alteration in higher-order chromatin and/or nucleosome organization is an important aspect of epigenetic gene regulation. Recent studies have suggested a key role for CP190 in such processes. In this study, we analysed the effects of ectopically tethered insulator factors on chromatin structure and found that CP190 induces large-scale decondensation when targeted to a condensed lacO array in mammalian and Drosophila cells. In contrast, dCTCF alone, is unable to cause such a decondensation, however, when CP190 is present, dCTCF recruits it to the lacO array and mediates chromatin unfolding. The CP190 induced opening of chromatin may not be correlated with transcriptional activation, as binding of CP190 does not enhance luciferase activity in reporter assays. We propose that CP190 may mediate histone modification and chromatin remodelling activity to induce an open chromatin state by its direct recruitment or targeting by a DNA binding factor such as dCTCF.

  6. Insulation of the Chicken β-Globin Chromosomal Domain from a Chromatin-Condensing Protein, MENT

    PubMed Central

    Istomina, Natalia E.; Shushanov, Sain S.; Springhetti, Evelyn M.; Karpov, Vadim L.; A. Krasheninnikov, Igor; Stevens, Kimberly; Zaret, Kenneth S.; Singh, Prim B.; Grigoryev, Sergei A.

    2003-01-01

    Active genes are insulated from developmentally regulated chromatin condensation in terminally differentiated cells. We mapped the topography of a terminal stage-specific chromatin-condensing protein, MENT, across the active chicken β-globin domain. We observed two sharp transitions of MENT concentration coinciding with the β-globin boundary elements. The MENT distribution profile was opposite to that of acetylated core histones but correlated with that of histone H3 dimethylated at lysine 9 (H3me2K9). Ectopic MENT expression in NIH 3T3 cells caused a large-scale and specific remodeling of chromatin marked by H3me2K9. MENT colocalized with H3me2K9 both in chicken erythrocytes and NIH 3T3 cells. Mutational analysis of MENT and experiments with deacetylase inhibitors revealed the essential role of the reaction center loop domain and an inhibitory affect of histone hyperacetylation on the MENT-induced chromatin remodeling in vivo. In vitro, the elimination of the histone H3 N-terminal peptide containing lysine 9 by trypsin blocked chromatin self-association by MENT, while reconstitution with dimethylated but not acetylated N-terminal domain of histone H3 specifically restored chromatin self-association by MENT. We suggest that histone H3 modification at lysine 9 directly regulates chromatin condensation by recruiting MENT to chromatin in a fashion that is spatially constrained from active genes by gene boundary elements and histone hyperacetylation. PMID:12944473

  7. Insulation of the chicken beta-globin chromosomal domain from a chromatin-condensing protein, MENT.

    PubMed

    Istomina, Natalia E; Shushanov, Sain S; Springhetti, Evelyn M; Karpov, Vadim L; Krasheninnikov, Igor A; Stevens, Kimberly; Zaret, Kenneth S; Singh, Prim B; Grigoryev, Sergei A

    2003-09-01

    Active genes are insulated from developmentally regulated chromatin condensation in terminally differentiated cells. We mapped the topography of a terminal stage-specific chromatin-condensing protein, MENT, across the active chicken beta-globin domain. We observed two sharp transitions of MENT concentration coinciding with the beta-globin boundary elements. The MENT distribution profile was opposite to that of acetylated core histones but correlated with that of histone H3 dimethylated at lysine 9 (H3me2K9). Ectopic MENT expression in NIH 3T3 cells caused a large-scale and specific remodeling of chromatin marked by H3me2K9. MENT colocalized with H3me2K9 both in chicken erythrocytes and NIH 3T3 cells. Mutational analysis of MENT and experiments with deacetylase inhibitors revealed the essential role of the reaction center loop domain and an inhibitory affect of histone hyperacetylation on the MENT-induced chromatin remodeling in vivo. In vitro, the elimination of the histone H3 N-terminal peptide containing lysine 9 by trypsin blocked chromatin self-association by MENT, while reconstitution with dimethylated but not acetylated N-terminal domain of histone H3 specifically restored chromatin self-association by MENT. We suggest that histone H3 modification at lysine 9 directly regulates chromatin condensation by recruiting MENT to chromatin in a fashion that is spatially constrained from active genes by gene boundary elements and histone hyperacetylation.

  8. Dietary control of chromatin

    PubMed Central

    Huang, Zhiguang; Cai, Ling; Tu, Benjamin P

    2015-01-01

    Organisms must be able to rapidly alter gene expression in response to changes in their nutrient environment. This review summarizes evidence that epigenetic modifications of chromatin depend on particular metabolites of intermediary metabolism, enabling the facile regulation of gene expression in tune with metabolic state. Nutritional or dietary control of chromatin is an often-overlooked, yet fundamental regulatory mechanism directly linked to human physiology. Nutrient-sensitive epigenetic marks are dynamic, suggesting rapid turnover, and may have functions beyond the regulation of gene transcription, including pH regulation and as carbon sources in cancer cells. PMID:26094239

  9. Repression and activation by multiprotein complexes that alter chromatin structure.

    PubMed

    Kingston, R E; Bunker, C A; Imbalzano, A N

    1996-04-15

    Recent studies have provided strong evidence that macromolecular complexes are used in the cell to remodel chromatin structure during activation and to create an inaccessible structure during repression, Although there is not yet any rigorous demonstration that modification of chromatin structure plays a direct, causal role in either activation or repression, there is sufficient smoke to indicate the presence of a blazing inferno nearby. It is clear that complexes that remodel chromatin are tractable in vitro; hopefully this will allow the establishment of systems that provide a direct analysis of the role that remodeling might play in activation. These studies indicate that establishment of functional systems to corroborate the elegant genetic studies on repression might also be tractable. As the mechanistic effects of these complexes are sorted out, it will become important to understand how the complexes are regulated. In many of the instances discussed above, the genes whose products make up these complexes were identified in genetic screens for effects on developmental processes. This implies a regulation of the activity of these complexes in response to developmental cues and further implies that the work to fully understand these complexes will occupy a generation of scientists.

  10. Programming off and on states in chromatin: mechanisms of Polycomb and trithorax group complexes.

    PubMed

    Simon, Jeffrey A; Tamkun, John W

    2002-04-01

    Polycomb and trithorax group proteins are evolutionarily conserved chromatin components that maintain stable states of gene expression. Recent studies have identified and characterized several multiprotein complexes containing these transcriptional regulators. Advances in understanding molecular activities of these complexes in vitro, and functional domains present in their subunits, suggest that they control transcription through multistep mechanisms that involve nucleosome modification, chromatin remodeling, and interaction with general transcription factors.

  11. ERECTA signaling controls Arabidopsis inflorescence architecture through chromatin-mediated activation of PRE1 expression.

    PubMed

    Cai, Hanyang; Zhao, Lihua; Wang, Lulu; Zhang, Man; Su, Zhenxia; Cheng, Yan; Zhao, Heming; Qin, Yuan

    2017-03-13

    Flowering plants display a remarkable diversity in inflorescence architecture, and pedicel length is one of the key contributors to this diversity. In Arabidopsis thaliana, the receptor-like kinase ERECTA (ER) mediated signaling pathway plays important roles in regulating inflorescence architecture by promoting cell proliferation. However, the regulating mechanism remains elusive in the pedicel. Genetic interactions between ERECTA signaling and the chromatin remodeling complex SWR1 in the control of inflorescence architecture were studied. Comparative transcriptome analysis was applied to identify downstream components. Chromatin immunoprecipitation and nucleosome occupancy was further investigated. The results indicated that the chromatin remodeler SWR1 coordinates with ERECTA signaling in regulating inflorescence architecture by activating the expression of PRE1 family genes and promoting pedicel elongation. It was found that SWR1 is required for the incorporation of the H2A.Z histone variant into nucleosomes of the whole PRE1 gene family and the ERECTA controlled expression of PRE1 gene family through regulating nucleosome dynamics. We propose that utilization of a chromatin remodeling complex to regulate gene expression is a common theme in developmental control across kingdoms. These findings shed light on the mechanisms through which chromatin remodelers orchestrate complex transcriptional regulation of gene expression in coordination with a developmental cue.

  12. Lysosome-mediated processing of chromatin in senescence.

    PubMed

    Ivanov, Andre; Pawlikowski, Jeff; Manoharan, Indrani; van Tuyn, John; Nelson, David M; Rai, Taranjit Singh; Shah, Parisha P; Hewitt, Graeme; Korolchuk, Viktor I; Passos, Joao F; Wu, Hong; Berger, Shelley L; Adams, Peter D

    2013-07-08

    Cellular senescence is a stable proliferation arrest, a potent tumor suppressor mechanism, and a likely contributor to tissue aging. Cellular senescence involves extensive cellular remodeling, including of chromatin structure. Autophagy and lysosomes are important for recycling of cellular constituents and cell remodeling. Here we show that an autophagy/lysosomal pathway processes chromatin in senescent cells. In senescent cells, lamin A/C-negative, but strongly γ-H2AX-positive and H3K27me3-positive, cytoplasmic chromatin fragments (CCFs) budded off nuclei, and this was associated with lamin B1 down-regulation and the loss of nuclear envelope integrity. In the cytoplasm, CCFs were targeted by the autophagy machinery. Senescent cells exhibited markers of lysosomal-mediated proteolytic processing of histones and were progressively depleted of total histone content in a lysosome-dependent manner. In vivo, depletion of histones correlated with nevus maturation, an established histopathologic parameter associated with proliferation arrest and clinical benignancy. We conclude that senescent cells process their chromatin via an autophagy/lysosomal pathway and that this might contribute to stability of senescence and tumor suppression.

  13. The yeast PHO5 promoter: from single locus to systems biology of a paradigm for gene regulation through chromatin

    PubMed Central

    Korber, Philipp; Barbaric, Slobodan

    2014-01-01

    Chromatin dynamics crucially contributes to gene regulation. Studies of the yeast PHO5 promoter were key to establish this nowadays accepted view and continuously provide mechanistic insight in chromatin remodeling and promoter regulation, both on single locus as well as on systems level. The PHO5 promoter is a context independent chromatin switch module where in the repressed state positioned nucleosomes occlude transcription factor sites such that nucleosome remodeling is a prerequisite for and not consequence of induced gene transcription. This massive chromatin transition from positioned nucleosomes to an extensive hypersensitive site, together with respective transitions at the co-regulated PHO8 and PHO84 promoters, became a prime model for dissecting how remodelers, histone modifiers and chaperones co-operate in nucleosome remodeling upon gene induction. This revealed a surprisingly complex cofactor network at the PHO5 promoter, including five remodeler ATPases (SWI/SNF, RSC, INO80, Isw1, Chd1), and demonstrated for the first time histone eviction in trans as remodeling mode in vivo. Recently, the PHO5 promoter and the whole PHO regulon were harnessed for quantitative analyses and computational modeling of remodeling, transcription factor binding and promoter input-output relations such that this rewarding single-locus model becomes a paradigm also for theoretical and systems approaches to gene regulatory networks. PMID:25190457

  14. Chromatin and DNA replication.

    PubMed

    MacAlpine, David M; Almouzni, Geneviève

    2013-08-01

    The size of a eukaryotic genome presents a unique challenge to the cell: package and organize the DNA to fit within the confines of the nucleus while at the same time ensuring sufficient dynamics to allow access to specific sequences and features such as genes and regulatory elements. This is achieved via the dynamic nucleoprotein organization of eukaryotic DNA into chromatin. The basic unit of chromatin, the nucleosome, comprises a core particle with 147 bp of DNA wrapped 1.7 times around an octamer of histones. The nucleosome is a highly versatile and modular structure, both in its composition, with the existence of various histone variants, and through the addition of a series of posttranslational modifications on the histones. This versatility allows for both short-term regulatory responses to external signaling, as well as the long-term and multigenerational definition of large functional chromosomal domains within the nucleus, such as the centromere. Chromatin organization and its dynamics participate in essentially all DNA-templated processes, including transcription, replication, recombination, and repair. Here we will focus mainly on nucleosomal organization and describe the pathways and mechanisms that contribute to assembly of this organization and the role of chromatin in regulating the DNA replication program.

  15. Archaeal chromatin proteins.

    PubMed

    Zhang, ZhenFeng; Guo, Li; Huang, Li

    2012-05-01

    Archaea, along with Bacteria and Eukarya, are the three domains of life. In all living cells, chromatin proteins serve a crucial role in maintaining the integrity of the structure and function of the genome. An array of small, abundant and basic DNA-binding proteins, considered candidates for chromatin proteins, has been isolated from the Euryarchaeota and the Crenarchaeota, the two major phyla in Archaea. While most euryarchaea encode proteins resembling eukaryotic histones, crenarchaea appear to synthesize a number of unique DNA-binding proteins likely involved in chromosomal organization. Several of these proteins (e.g., archaeal histones, Sac10b homologs, Sul7d, Cren7, CC1, etc.) have been extensively studied. However, whether they are chromatin proteins and how they function in vivo remain to be fully understood. Future investigation of archaeal chromatin proteins will lead to a better understanding of chromosomal organization and gene expression in Archaea and provide valuable information on the evolution of DNA packaging in cellular life.

  16. Analysis of Chromatin Organisation

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2011-01-01

    Terms to be familiar with before you start to solve the test: chromatin, nucleases, sucrose density gradient centrifugation, melting point, gel electrophoresis, ethidium bromide, autoradiography, Southern blotting, Northern blotting, Sanger sequencing, restriction endonucleases, exonucleases, linker DNA, chloroform extraction, nucleosomes,…

  17. Chromatin Structure Following UV-Induced DNA Damage—Repair or Death?

    PubMed Central

    Farrell, Andrew W.; Halliday, Gary M.; Lyons, James Guy

    2011-01-01

    In eukaryotes, DNA is compacted into a complex structure known as chromatin. The unravelling of DNA is a crucial step in DNA repair, replication, transcription and recombination as this allows access to DNA for these processes. Failure to package DNA into the nucleosome, the individual unit of chromatin, can lead to genomic instability, driving a cell into apoptosis, senescence, or cellular proliferation. Ultraviolet (UV) radiation damage causes destabilisation of chromatin integrity. UV irradiation induces DNA damage such as photolesions and subjects the chromatin to substantial rearrangements, causing the arrest of transcription forks and cell cycle arrest. Highly conserved processes known as nucleotide and base excision repair (NER and BER) then begin to repair these lesions. However, if DNA repair fails, the cell may be forced into apoptosis. The modification of various histones as well as nucleosome remodelling via ATP-dependent chromatin remodelling complexes are required not only to repair these UV-induced DNA lesions, but also for apoptosis signalling. Histone modifications and nucleosome remodelling in response to UV also lead to the recruitment of various repair and pro-apoptotic proteins. Thus, the way in which a cell responds to UV irradiation via these modifications is important in determining its fate. Failure of these DNA damage response steps can lead to cellular proliferation and oncogenic development, causing skin cancer, hence these chromatin changes are critical for a proper response to UV-induced injury. PMID:22174650

  18. Aberrant Expression of Histone Deacetylases 4 in Cognitive Disorders: Molecular Mechanisms and a Potential Target

    PubMed Central

    Wu, Yili; Hou, Fei; Wang, Xin; Kong, Qingsheng; Han, Xiaolin; Bai, Bo

    2016-01-01

    Histone acetylation is a major mechanism of chromatin remodeling, contributing to epigenetic regulation of gene transcription. Histone deacetylases (HDACs) are involved in both physiological and pathological conditions by regulating the status of histone acetylation. Although histone deacetylase 4 (HDAC4), a member of the HDAC family, may lack HDAC activity, it is actively involved in regulating the transcription of genes involved in synaptic plasticity, neuronal survival, and neurodevelopment by interacting with transcription factors, signal transduction molecules and HDAC3, another member of the HDAC family. HDAC4 is highly expressed in brain and its homeostasis is crucial for the maintenance of cognitive function. Accumulated evidence shows that HDAC4 expression is dysregulated in several brain disorders, including neurodegenerative diseases and mental disorders. Moreover, cognitive impairment is a characteristic feature of these diseases. It indicates that aberrant HDAC4 expression plays a pivotal role in cognitive impairment of these disorders. This review aims to describe the current understanding of HDAC4’s role in the maintenance of cognitive function and its dysregulation in neurodegenerative diseases and mental disorders, discuss underlying molecular mechanisms, and provide an outlook into targeting HDAC4 as a potential therapeutic approach to rescue cognitive impairment in these diseases. PMID:27847464

  19. Interpreting Chromosome Aberration Spectra

    NASA Technical Reports Server (NTRS)

    Levy, Dan; Reeder, Christopher; Loucas, Bradford; Hlatky, Lynn; Chen, Allen; Cornforth, Michael; Sachs, Rainer

    2007-01-01

    Ionizing radiation can damage cells by breaking both strands of DNA in multiple locations, essentially cutting chromosomes into pieces. The cell has enzymatic mechanisms to repair such breaks; however, these mechanisms are imperfect and, in an exchange process, may produce a large-scale rearrangement of the genome, called a chromosome aberration. Chromosome aberrations are important in killing cells, during carcinogenesis, in characterizing repair/misrepair pathways, in retrospective radiation biodosimetry, and in a number of other ways. DNA staining techniques such as mFISH ( multicolor fluorescent in situ hybridization) provide a means for analyzing aberration spectra by examining observed final patterns. Unfortunately, an mFISH observed final pattern often does not uniquely determine the underlying exchange process. Further, resolution limitations in the painting protocol sometimes lead to apparently incomplete final patterns. We here describe an algorithm for systematically finding exchange processes consistent with any observed final pattern. This algorithm uses aberration multigraphs, a mathematical formalism that links the various aspects of aberration formation. By applying a measure to the space of consistent multigraphs, we will show how to generate model-specific distributions of aberration processes from mFISH experimental data. The approach is implemented by software freely available over the internet. As a sample application, we apply these algorithms to an aberration data set, obtaining a distribution of exchange cycle sizes, which serves to measure aberration complexity. Estimating complexity, in turn, helps indicate how damaging the aberrations are and may facilitate identification of radiation type in retrospective biodosimetry.

  20. Global methylation status of sperm DNA in carriers of chromosome structural aberrations

    PubMed Central

    Olszewska, Marta; Barciszewska, Miroslawa Z; Fraczek, Monika; Huleyuk, Nataliya; Chernykh, Vyacheslav B; Zastavna, Danuta; Barciszewski, Jan; Kurpisz, Maciej

    2017-01-01

    Male infertility might be clearly associated with aberrant DNA methylation patterns in human spermatozoa. An association between oxidative stress and the global methylation status of the sperm genome has also been suggested. The aim of the present study was to determine whether the global sperm DNA methylation status was affected in the spermatozoa of carriers of chromosome structural aberrations. The relationships between the 5-methylcytosine (m5C) levels in spermatozoa and chromatin integrity status were evaluated. The study patients comprised male carriers of chromosome structural aberrations with reproductive failure (n = 24), and the controls comprised normozoospermic sperm volunteers (n = 23). The global m5C level was measured using thin-layer chromatography (TLC) and immunofluorescence (IF) techniques. The sperm chromatin integrity was assessed using aniline blue (AB) staining and TUNEL assay. The mean m5C levels were similar between the investigated chromosome structural aberrations carriers (P) and controls (K). However, sperm chromatin integrity tests revealed significantly higher values in chromosomal rearrangement carriers than in controls (P < 0.05). Although the potential relationship between sperm chromatin integrity status and sperm DNA fragmentation and the m5C level juxtaposed in both analyzed groups (P vs K) was represented in a clearly opposite manner, the low chromatin integrity might be associated with the high hypomethylation status of the sperm DNA observed in carriers of chromosome structural aberrations. PMID:26908061

  1. Retinal remodeling in inherited photoreceptor degenerations.

    PubMed

    Marc, Robert E; Jones, Bryan W

    2003-10-01

    Photoreceptor degenerations initiated in rods or the retinal pigmented epithelium usually evoke secondary cone death and sensory deafferentation of the surviving neural retina. In the mature central nervous system, deafferentation evokes atrophy and connective re-patterning. It has been assumed that the neural retina does not remodel, and that it is a passive survivor. Screening of advanced stages of human and rodent retinal degenerations with computational molecular phenotyping has exposed a prolonged period of aggressive negative remodeling in which neurons migrate along aberrant glial columns and seals, restructuring the adult neural retina (1). Many neurons die, but survivors rewire the remnant inner plexiform layer (IPL), forming thousands of novel ectopic microneuromas in the remnant inner nuclear layer (INL). Bipolar and amacrine cells engage in new circuits that are most likely corruptive. Remodeling in human and rodent retinas emerges regardless of the molecular defects that initially trigger retinal degenerations. Although remodeling may constrain therapeutic intervals for molecular, cellular, or bionic rescue, the exposure of intrinsic retinal remodeling by the removal of sensory control in retinal degenerations suggests that neuronal organization in the normal retina may be more plastic than previously believed.

  2. Interphase Chromosome Conformation and Chromatin-Chromatin Interactions in Human Epithelial Cells Cultured Under Different Gravity Conditions

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Wong, Michael; Hada, Megumi; Wu, Honglu

    2015-01-01

    Microgravity has been shown to alter global gene expression patterns and protein levels both in cultured cells and animal models. It has been suggested that the packaging of chromatin fibers in the interphase nucleus is closely related to genome function, and the changes in transcriptional activity are tightly correlated with changes in chromatin folding. This study explores the changes of chromatin conformation and chromatin-chromatin interactions in the simulated microgravity environment, and investigates their correlation to the expression of genes located at different regions of the chromosome. To investigate the folding of chromatin in interphase under various culture conditions, human epithelial cells, fibroblasts, and lymphocytes were fixed in the G1 phase. Interphase chromosomes were hybridized with a multicolor banding in situ hybridization (mBAND) probe for chromosome 3 which distinguishes six regions of the chromosome as separate colors. After images were captured with a laser scanning confocal microscope, the 3-dimensional structure of interphase chromosome 3 was reconstructed at multi-mega base pair scale. In order to determine the effects of microgravity on chromosome conformation and orientation, measures such as distance between homologous pairs, relative orientation of chromosome arms about a shared midpoint, and orientation of arms within individual chromosomes were all considered as potentially impacted by simulated microgravity conditions. The studies revealed non-random folding of chromatin in interphase, and suggested an association of interphase chromatin folding with radiation-induced chromosome aberration hotspots. Interestingly, the distributions of genes with expression changes over chromosome 3 in cells cultured under microgravity environment are apparently clustered on specific loci and chromosomes. This data provides important insights into how mammalian cells respond to microgravity at molecular level.

  3. Probing Nucleosome Remodeling by Unzipping Single DNA Molecules

    NASA Astrophysics Data System (ADS)

    Wang, Michelle

    2006-03-01

    At the core of eukaryotic chromatin is the nucleosome, which consists of 147 bp of DNA wrapped 1.65 turns around an octamer of histone proteins. Even this lowest level of genomic compaction presents a strong barrier to DNA-binding cellular factors that are required for essential processes such as transcription, DNA replication, recombination and repair. Chromatin remodeling enzymes use the energy of ATP hydrolysis to regulate accessibility of the genetic code by altering chromatin structure. While remodeling enzymes have been the subject of extensive research in recent years, their precise mechanism remains unclear. In order to probe the structure of individual nucleosomes and their remodeling, we assembled a histone octamer onto a DNA segment containing a strong nucleosome positioning sequence. As the DNA double helix was unzipped through the nucleosome using a feedback-enhanced optical trap, the presence of the nucleosome was detected as a series of dramatic increases in the tension in the DNA, followed by sudden tension reductions. Analysis of the unzipping force throughout the disruption accurately revealed the spatial location and fine structure of the nucleosome to near base pair precision. Using this approach, we investigate how remodeling enzymes may alter the location and structure of a nucleosome.

  4. Impact of Chromatin Structure on PR Signaling: Transition from Local to Global Analysis

    PubMed Central

    Grøntved, Lars; Hager, Gordon L

    2011-01-01

    The progesterone receptor (PR) interacts with chromatin in a highly dynamic manner that requires ongoing chromatin remodeling, interaction with chaparones and activity of the proteasome. Here we discuss dynamic interaction of steroid receptor with chromatin, with special attention not only to PR but also to the glucocorticoid receptor (GR), as these receptors share many similarities regarding interaction with, and remodeling of, chromatin. Both receptors can bind nucleosomal DNA and have accordingly been described as pioneering factors. However recent genomic approaches (ChIP-seq and DHS-seq) show that a large fraction of receptor binding events occur at pre-accessible chromatin. Thus factors which generate and maintain accessible chromatin during development, and in fully differentiated tissue, contribute a major fraction of receptor tissue specificity. In addition, chromosome conformation capture techniques suggest that steroid receptors preferentially sequester within distinct nuclear hubs. We will integrate dynamic studies from single cells and genomic studies from cell populations, and discuss how genomic approaches have reshaped our current understanding of mechanisms that control steroid receptor interaction with chromatin. PMID:21958695

  5. Nucleosome breathing and remodeling constrain CRISPR-Cas9 function

    PubMed Central

    Isaac, R Stefan; Jiang, Fuguo; Doudna, Jennifer A; Lim, Wendell A; Narlikar, Geeta J; Almeida, Ricardo

    2016-01-01

    The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo. DOI: http://dx.doi.org/10.7554/eLife.13450.001 PMID:27130520

  6. Chromatin changes predict recurrence after radical prostatectomy

    PubMed Central

    Hveem, Tarjei S; Kleppe, Andreas; Vlatkovic, Ljiljana; Ersvær, Elin; Wæhre, Håkon; Nielsen, Birgitte; Kjær, Marte Avranden; Pradhan, Manohar; Syvertsen, Rolf Anders; Nesheim, John Arne; Liestøl, Knut; Albregtsen, Fritz; Danielsen, Håvard E

    2016-01-01

    Background: Pathological evaluations give the best prognostic markers for prostate cancer patients after radical prostatectomy, but the observer variance is substantial. These risk assessments should be supported and supplemented by objective methods for identifying patients at increased risk of recurrence. Markers of epigenetic aberrations have shown promising results in several cancer types and can be assessed by automatic analysis of chromatin organisation in tumour cell nuclei. Methods: A consecutive series of 317 prostate cancer patients treated with radical prostatectomy at a national hospital between 1987 and 2005 were followed for a median of 10 years (interquartile range, 7–14). On average three tumour block samples from each patient were included to account for tumour heterogeneity. We developed a novel marker, termed Nucleotyping, based on automatic assessment of disordered chromatin organisation, and validated its ability to predict recurrence after radical prostatectomy. Results: Nucleotyping predicted recurrence with a hazard ratio (HR) of 3.3 (95% confidence interval (CI), 2.1–5.1). With adjustment for clinical and pathological characteristics, the HR was 2.5 (95% CI, 1.5–4.1). An updated stratification into three risk groups significantly improved the concordance with patient outcome compared with a state-of-the-art risk-stratification tool (P<0.001). The prognostic impact was most evident for the patients who were high-risk by clinical and pathological characteristics and for patients with Gleason score 7. Conclusion: A novel assessment of epigenetic aberrations was capable of improving risk stratification after radical prostatectomy. PMID:27124335

  7. The Oncoprotein BRD4-NUT Generates Aberrant Histone Modification Patterns.

    PubMed

    Zee, Barry M; Dibona, Amy B; Alekseyenko, Artyom A; French, Christopher A; Kuroda, Mitzi I

    2016-01-01

    Defects in chromatin proteins frequently manifest in diseases. A striking case of a chromatin-centric disease is NUT-midline carcinoma (NMC), which is characterized by expression of NUT as a fusion partner most frequently with BRD4. ChIP-sequencing studies from NMC patients revealed that BRD4-NUT (B4N) covers large genomic regions and elevates transcription within these domains. To investigate how B4N modulates chromatin, we performed affinity purification of B4N when ectopically expressed in 293-TREx cells and quantified the associated histone posttranslational modifications (PTM) using proteomics. We observed significant enrichment of acetylation particularly on H3 K18 and of combinatorial patterns such as H3 K27 acetylation paired with K36 methylation. We postulate that B4N complexes override the preexisting histone code with new PTM patterns that reflect aberrant transcription and that epigenetically modulate the nucleosome environment toward the NMC state.

  8. The Oncoprotein BRD4-NUT Generates Aberrant Histone Modification Patterns

    PubMed Central

    Zee, Barry M.; Dibona, Amy B.; Alekseyenko, Artyom A.; French, Christopher A.; Kuroda, Mitzi I.

    2016-01-01

    Defects in chromatin proteins frequently manifest in diseases. A striking case of a chromatin-centric disease is NUT-midline carcinoma (NMC), which is characterized by expression of NUT as a fusion partner most frequently with BRD4. ChIP-sequencing studies from NMC patients revealed that BRD4-NUT (B4N) covers large genomic regions and elevates transcription within these domains. To investigate how B4N modulates chromatin, we performed affinity purification of B4N when ectopically expressed in 293-TREx cells and quantified the associated histone posttranslational modifications (PTM) using proteomics. We observed significant enrichment of acetylation particularly on H3 K18 and of combinatorial patterns such as H3 K27 acetylation paired with K36 methylation. We postulate that B4N complexes override the preexisting histone code with new PTM patterns that reflect aberrant transcription and that epigenetically modulate the nucleosome environment toward the NMC state. PMID:27698495

  9. Cas9 Functionally Opens Chromatin

    PubMed Central

    Barkal, Amira A.; Srinivasan, Sharanya; Hashimoto, Tatsunori; Gifford, David K.; Sherwood, Richard I.

    2016-01-01

    Using a nuclease-dead Cas9 mutant, we show that Cas9 reproducibly induces chromatin accessibility at previously inaccessible genomic loci. Cas9 chromatin opening is sufficient to enable adjacent binding and transcriptional activation by the settler transcription factor retinoic acid receptor at previously unbound motifs. Thus, we demonstrate a new use for Cas9 in increasing surrounding chromatin accessibility to alter local transcription factor binding. PMID:27031353

  10. Molecular Toxicology of Chromatin

    DTIC Science & Technology

    1992-01-01

    FINAL 01 Jan 89 TO 31 Dec 91 4. ITL ANO SUS Y, L RE %UMAS MOLECULAR TOXICOLOGY OF CHROMATIN AFOSR-89-0231 PE - 61102F AUT PR - 2312 TA - A5 Dr Ernest Kun...Waterbury, CT), 2-mercaptoethanol, NAD+, NADPH, nucleo- tides, sodium tungstate , hydrogen peroxide, Tris and MES buffers from Sigma (St. Louis, MO...ml) with sodium tungstate (5.93 g, in 20 ml H20) for 1.5 h followed by extraction of the green product into ethyl acetate, washing with 0.1 N HCl, and

  11. RNF20-SNF2H Pathway of Chromatin Relaxation in DNA Double-Strand Break Repair.

    PubMed

    Kato, Akihiro; Komatsu, Kenshi

    2015-07-14

    Rapid progress in the study on the association of histone modifications with chromatin remodeling factors has broadened our understanding of chromatin dynamics in DNA transactions. In DNA double-strand break (DSB) repair, the well-known mark of histones is the phosphorylation of the H2A variant, H2AX, which has been used as a surrogate marker of DSBs. The ubiquitylation of histone H2B by RNF20 E3 ligase was recently found to be a DNA damage-induced histone modification. This modification is required for DSB repair and regulated by a distinctive pathway from that of histone H2AX phosphorylation. Moreover, the connection between H2B ubiquitylation and the chromatin remodeling activity of SNF2H has been elucidated. In this review, we summarize the current knowledge of RNF20-mediated processes and the molecular link to H2AX-mediated processes during DSB repair.

  12. Mapping chromatin modifications in nanochannels

    NASA Astrophysics Data System (ADS)

    Lim, Shuang Fang; Karpusenko, Alena; Riehn, Robert

    2013-03-01

    DNA and chromatin are elongated to a fixed fraction of their contour length when introduced into quasi-1d nanochannels. Because single molecules are analyzed, their hold great potential for the analysis for the genetic analysis of material from single cells. In this study, we have reconstituted chromatin with histones from a variety of sources, and mapped the modification profile of the chromatin. We monitored methylation and acetylation patterns of the histone tail protein residues using fluorescently labelled antibodies. Using those, we distinguished chromatin reconstituted from chicken erythrocytes, calf thymus, and HeLa cells. We discuss prospects for profiling histone modifications for whole chromosomes from single cells.

  13. Chromatin structure in barley nuclei.

    PubMed

    Mithieux, G; Roux, B

    1983-10-03

    In order to study the chromatin structure of a higher plant we used a high-yield method, which allows one to obtain up to 10(9) nuclei/kg fresh barley leaves. Significant amounts of low-ionic-strength-soluble chromatin can be extracted from these nuclei. Physicochemical properties were examined and discussed. Electric birefringence allowed us to observe the same transition in electro-optical properties as has been observed for animal chromatin, and suggested the existence of a symetrical structure occurring for approximately six nucleosomes. Circular dichroism showed that barley oligonucleosomes exhibit a higher molar ellipticity at 282 nm than total soluble chromatin and than their animal counterparts.

  14. Zelda is differentially required for chromatin accessibility, transcription factor binding, and gene expression in the early Drosophila embryo

    PubMed Central

    Schulz, Katharine N.; Bondra, Eliana R.; Moshe, Arbel; Villalta, Jacqueline E.; Lieb, Jason D.; Kaplan, Tommy; McKay, Daniel J.; Harrison, Melissa M.

    2015-01-01

    The transition from a specified germ cell to a population of pluripotent cells occurs rapidly following fertilization. During this developmental transition, the zygotic genome is largely transcriptionally quiescent and undergoes significant chromatin remodeling. In Drosophila, the DNA-binding protein Zelda (also known as Vielfaltig) is required for this transition and for transcriptional activation of the zygotic genome. Open chromatin is associated with Zelda-bound loci, as well as more generally with regions of active transcription. Nonetheless, the extent to which Zelda influences chromatin accessibility across the genome is largely unknown. Here we used formaldehyde-assisted isolation of regulatory elements to determine the role of Zelda in regulating regions of open chromatin in the early embryo. We demonstrate that Zelda is essential for hundreds of regions of open chromatin. This Zelda-mediated chromatin accessibility facilitates transcription-factor recruitment and early gene expression. Thus, Zelda possesses some key characteristics of a pioneer factor. Unexpectedly, chromatin at a large subset of Zelda-bound regions remains open even in the absence of Zelda. The GAGA factor-binding motif and embryonic GAGA factor binding are specifically enriched in these regions. We propose that both Zelda and GAGA factor function to specify sites of open chromatin and together facilitate the remodeling of the early embryonic genome. PMID:26335634

  15. Chromatin fiber polymorphism triggered by variations of DNA linker lengths.

    PubMed

    Collepardo-Guevara, Rosana; Schlick, Tamar

    2014-06-03

    Deciphering the factors that control chromatin fiber structure is key to understanding fundamental chromosomal processes. Although details remain unknown, it is becoming clear that chromatin is polymorphic depending on internal and external factors. In particular, different lengths of the linker DNAs joining successive nucleosomes (measured in nucleosome-repeat lengths or NRLs) that characterize different cell types and cell cycle stages produce different structures. NRL is also nonuniform within single fibers, but how this diversity affects chromatin fiber structure is not clear. Here we perform Monte Carlo simulations of a coarse-grained oligonucleosome model to help interpret fiber structure subject to intrafiber NRL variations, as relevant to proliferating cells of interphase chromatin, fibers subject to remodeling factors, and regulatory DNA sequences. We find that intrafiber NRL variations have a profound impact on chromatin structure, with a wide range of different architectures emerging (highly bent narrow forms, canonical and irregular zigzag fibers, and polymorphic conformations), depending on the NRLs mixed. This stabilization of a wide range of fiber forms might allow NRL variations to regulate both fiber compaction and selective DNA exposure. The polymorphic forms spanning canonical to sharply bent structures, like hairpins and loops, arise from large NRL variations and are surprisingly more compact than uniform NRL structures. They are distinguished by tail-mediated far-nucleosome interactions, in addition to the near-nucleosome interactions of canonical 30-nm fibers. Polymorphism is consistent with chromatin's diverse biological functions and heterogeneous constituents. Intrafiber NRL variations, in particular, may contribute to fiber bending and looping and thus to distant communication in associated regulatory processes.

  16. Differential association of chromatin proteins identifies BAF60a/SMARCD1 as a regulator of embryonic stem cell differentiation.

    PubMed

    Alajem, Adi; Biran, Alva; Harikumar, Arigela; Sailaja, Badi Sri; Aaronson, Yair; Livyatan, Ilana; Nissim-Rafinia, Malka; Sommer, Andreia Gianotti; Mostoslavsky, Gustavo; Gerbasi, Vincent R; Golden, Daniel E; Datta, Arnab; Sze, Siu Kwan; Meshorer, Eran

    2015-03-31

    Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized chromatin proteins. To identify chromatin regulators in ESCs, we developed a simple biochemical assay named D-CAP (differential chromatin-associated proteins), using brief micrococcal nuclease digestion of chromatin, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Using D-CAP, we identified several differentially chromatin-associated proteins between undifferentiated and differentiated ESCs, including the chromatin remodeling protein SMARCD1. SMARCD1 depletion in ESCs led to altered chromatin and enhanced endodermal differentiation. Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses suggested that SMARCD1 is both an activator and a repressor and is enriched at developmental regulators and that its chromatin binding coincides with H3K27me3. SMARCD1 knockdown caused H3K27me3 redistribution and increased H3K4me3 around the transcription start site (TSS). One of the identified SMARCD1 targets was Klf4. In SMARCD1-knockdown clones, KLF4, as well as H3K4me3 at the Klf4 locus, remained high and H3K27me3 was abolished. These results propose a role for SMARCD1 in restricting pluripotency and activating lineage pathways by regulating H3K27 methylation.

  17. FACT Assists Base Excision Repair by Boosting the Remodeling Activity of RSC

    PubMed Central

    Ouararhni, Khalid; Roulland, Yohan; Ben Simon, Elsa; Kundu, Tapas; Hamiche, Ali; Angelov, Dimitar; Dimitrov, Stefan

    2016-01-01

    FACT, in addition to its role in transcription, is likely implicated in both transcription-coupled nucleotide excision repair and DNA double strand break repair. Here, we present evidence that FACT could be directly involved in Base Excision Repair and elucidate the chromatin remodeling mechanisms of FACT during BER. We found that, upon oxidative stress, FACT is released from transcription related protein complexes to get associated with repair proteins and chromatin remodelers from the SWI/SNF family. We also showed the rapid recruitment of FACT to the site of damage, coincident with the glycosylase OGG1, upon the local generation of oxidized DNA. Interestingly, FACT facilitates uracil-DNA glycosylase in the removal of uracil from nucleosomal DNA thanks to an enhancement in the remodeling activity of RSC. This discloses a novel property of FACT wherein it has a co-remodeling activity and strongly enhances the remodeling capacity of the chromatin remodelers. Altogether, our data suggest that FACT may acts in concert with RSC to facilitate excision of DNA lesions during the initial step of BER. PMID:27467129

  18. Chromatin condensation during terminal erythropoiesis.

    PubMed

    Zhao, Baobing; Yang, Jing; Ji, Peng

    2016-09-02

    Mammalian terminal erythropoiesis involves gradual but dramatic chromatin condensation steps that are essential for cell differentiation. Chromatin and nuclear condensation is followed by a unique enucleation process, which is believed to liberate more spaces for hemoglobin enrichment and enable the generation of a physically flexible mature red blood cell. Although these processes have been known for decades, the mechanisms are still unclear. Our recent study reveals an unexpected nuclear opening formation during mouse terminal erythropoiesis that requires caspase-3 activity. Major histones, except H2AZ, are partially released from the opening, which is important for chromatin condensation. Block of the nuclear opening through caspase inhibitor or knockdown of caspase-3 inhibits chromatin condensation and enucleation. We also demonstrate that nuclear opening and histone release are cell cycle regulated. These studies reveal a novel mechanism for chromatin condensation in mammalia terminal erythropoiesis.

  19. Stepwise nucleosome translocation by RSC remodeling complexes.

    PubMed

    Harada, Bryan T; Hwang, William L; Deindl, Sebastian; Chatterjee, Nilanjana; Bartholomew, Blaine; Zhuang, Xiaowei

    2016-02-19

    The SWI/SNF-family remodelers regulate chromatin structure by coupling the free energy from ATP hydrolysis to the repositioning and restructuring of nucleosomes, but how the ATPase activity of these enzymes drives the motion of DNA across the nucleosome remains unclear. Here, we used single-molecule FRET to monitor the remodeling of mononucleosomes by the yeast SWI/SNF remodeler, RSC. We observed that RSC primarily translocates DNA around the nucleosome without substantial displacement of the H2A-H2B dimer. At the sites where DNA enters and exits the nucleosome, the DNA moves largely along or near its canonical wrapping path. The translocation of DNA occurs in a stepwise manner, and at both sites where DNA enters and exits the nucleosome, the step size distributions exhibit a peak at approximately 1-2 bp. These results suggest that the movement of DNA across the nucleosome is likely coupled directly to DNA translocation by the ATPase at its binding site inside the nucleosome.

  20. CCSI: a database providing chromatin-chromatin spatial interaction information.

    PubMed

    Xie, Xiaowei; Ma, Wenbin; Songyang, Zhou; Luo, Zhenhua; Huang, Junfeng; Dai, Zhiming; Xiong, Yuanyan

    2016-01-01

    Distal regulatory elements have been shown to regulate gene transcription through spatial interactions, and single nucleotide polymorphisms (SNPs) are linked with distal gene expression by spatial proximity, which helps to explain the causal role of disease-associated SNPs in non-coding region. Therefore, studies on spatial interactions between chromatin have created a new avenue for elucidating the mechanism of transcriptional regulation in disease pathogenesis. Recently, a growing number of chromatin interactions have been revealed by means of 3C, 4C, 5C, ChIA-PET and Hi-C technologies. To interpret and utilize these interactions, we constructed chromatin-chromatin spatial interaction (CCSI) database by integrating and annotating 91 sets of chromatin interaction data derived from published literature, UCSC database and NCBI GEO database, resulting in a total of 3,017,962 pairwise interactions (false discovery rate < 0.05), covering human, mouse and yeast. A web interface has been designed to provide access to the chromatin interactions. The main features of CCSI are (i) showing chromatin interactions and corresponding genes, enhancers and SNPs within the regions in the search page; (ii) offering complete interaction datasets, enhancer and SNP information in the download page; and (iii) providing analysis pipeline for the annotation of interaction data. In conclusion, CCSI will facilitate exploring transcriptional regulatory mechanism in disease pathogenesis associated with spatial interactions among genes, regulatory regions and SNPs. Database URL: http://songyanglab.sysu.edu.cn/ccsi.

  1. Remodeling of cell-cell junctions in arrhythmogenic cardiomyopathy.

    PubMed

    Asimaki, Angeliki; Saffitz, Jeffrey E

    2014-02-01

    Arrhythmogenic cardiomyopathy (AC) is a primary myocardial disorder characterized by a high incidence of ventricular arrhythmias often preceding the onset of ventricular remodeling and dysfunction. Approximately 50% of patients diagnosed with AC have one or more mutations in genes encoding desmosomal proteins, although non-desmosomal genes have also been associated with the disease. Increasing evidence implicates remodeling of intercalated disk proteins reflecting abnormal responses to mechanical load and aberrant cell signaling pathways in the pathogenesis of AC. This review summarizes recent advances in understanding disease mechanisms in AC that have come from studies of human myocardium and experimental models.

  2. Pre-TCR signaling and CD8 gene bivalent chromatin resolution during thymocyte development.

    PubMed

    Harker, Nicola; Garefalaki, Anna; Menzel, Ursula; Ktistaki, Eleni; Naito, Taku; Georgopoulos, Katia; Kioussis, Dimitris

    2011-06-01

    The CD8 gene is silent in CD4(-)CD8(-) double-negative thymocytes, expressed in CD4(+)CD8(+) double-positive cells, and silenced in cells committing to the CD4(+) single-positive (SP) lineage, remaining active in the CD8(+) SP lineage. In this study, we show that the chromatin of the CD8 locus is remodeled in C57BL/6 and B6/J Rag1(-/-) MOM double-negative thymocytes as indicated by DNaseI hypersensitivity and widespread bivalent chromatin marks. Pre-TCR signaling coincides with chromatin bivalency resolution into monovalent activating modifications in double-positive and CD8 SP cells. Shortly after commitment to CD4 SP cell lineage, monovalent repressive characteristics and chromatin inaccessibility are established. Differential binding of Ikaros, NuRD, and heterochromatin protein 1α on the locus during these processes may participate in the complex regulation of CD8.

  3. Roles of pRB in the Regulation of Nucleosome and Chromatin Structures.

    PubMed

    Uchida, Chiharu

    2016-01-01

    Retinoblastoma protein (pRB) interacts with E2F and other protein factors to play a pivotal role in regulating the expression of target genes that induce cell cycle arrest, apoptosis, and differentiation. pRB controls the local promoter activity and has the ability to change the structure of nucleosomes and/or chromosomes via histone modification, epigenetic changes, chromatin remodeling, and chromosome organization. Functional inactivation of pRB perturbs these cellular events and causes dysregulated cell growth and chromosome instability, which are hallmarks of cancer cells. The role of pRB in regulation of nucleosome/chromatin structures has been shown to link to tumor suppression. This review focuses on the ability of pRB to control nucleosome/chromatin structures via physical interactions with histone modifiers and chromatin factors and describes cancer therapies based on targeting these protein factors.

  4. Roles of pRB in the Regulation of Nucleosome and Chromatin Structures

    PubMed Central

    2016-01-01

    Retinoblastoma protein (pRB) interacts with E2F and other protein factors to play a pivotal role in regulating the expression of target genes that induce cell cycle arrest, apoptosis, and differentiation. pRB controls the local promoter activity and has the ability to change the structure of nucleosomes and/or chromosomes via histone modification, epigenetic changes, chromatin remodeling, and chromosome organization. Functional inactivation of pRB perturbs these cellular events and causes dysregulated cell growth and chromosome instability, which are hallmarks of cancer cells. The role of pRB in regulation of nucleosome/chromatin structures has been shown to link to tumor suppression. This review focuses on the ability of pRB to control nucleosome/chromatin structures via physical interactions with histone modifiers and chromatin factors and describes cancer therapies based on targeting these protein factors. PMID:28101510

  5. Chromatin structure and dynamics in hot environments: architectural proteins and DNA topoisomerases of thermophilic archaea.

    PubMed

    Visone, Valeria; Vettone, Antonella; Serpe, Mario; Valenti, Anna; Perugino, Giuseppe; Rossi, Mosè; Ciaramella, Maria

    2014-09-25

    In all organisms of the three living domains (Bacteria, Archaea, Eucarya) chromosome-associated proteins play a key role in genome functional organization. They not only compact and shape the genome structure, but also regulate its dynamics, which is essential to allow complex genome functions. Elucidation of chromatin composition and regulation is a critical issue in biology, because of the intimate connection of chromatin with all the essential information processes (transcription, replication, recombination, and repair). Chromatin proteins include architectural proteins and DNA topoisomerases, which regulate genome structure and remodelling at two hierarchical levels. This review is focussed on architectural proteins and topoisomerases from hyperthermophilic Archaea. In these organisms, which live at high environmental temperature (>80 °C <113 °C), chromatin proteins and modulation of the DNA secondary structure are concerned with the problem of DNA stabilization against heat denaturation while maintaining its metabolic activity.

  6. Chromatin modification by PSC occurs at one PSC per nucleosome and does not require the acidic patch of histone H2A.

    PubMed

    Lo, Stanley M; McElroy, Kyle A; Francis, Nicole J

    2012-01-01

    Chromatin architecture is regulated through both enzymatic and non-enzymatic activities. For example, the Polycomb Group (PcG) proteins maintain developmental gene silencing using an array of chromatin-based mechanisms. The essential Drosophila PcG protein, Posterior Sex Combs (PSC), compacts chromatin and inhibits chromatin remodeling and transcription through a non-enzymatic mechanism involving nucleosome bridging. Nucleosome bridging is achieved through a combination of nucleosome binding and self-interaction. Precisely how PSC interacts with chromatin to bridge nucleosomes is not known and is the subject of this work. We determine the stoichiometry of PSC-chromatin interactions in compact chromatin (in which nucleosomes are bridged) using Scanning Transmission Electron Microscopy (STEM). We find that full compaction occurs with one PSC per nucleosome. In addition to compacting chromatin, we show that PSC oligomerizes nucleosome arrays. PSC-mediated oligomerization of chromatin occurs at similar stoichiometry as compaction suggesting it may also involve nucleosome bridging. Interactions between the tail of histone H4 and the acidic patch of histone H2A are important for chromatin folding and oligomerization, and several chromatin proteins bind the histone H2A acidic patch. However, mutation of the acidic patch of histone H2A does not affect PSC's ability to inhibit chromatin remodeling or bridge nucleosomes. In fact, PSC does not require nucleosomes for bridging activity but can bridge naked DNA segments. PSC clusters nucleosomes on sparsely assembled templates, suggesting it interacts preferentially with nucleosomes over bare DNA. This may be due to the ability of PSC to bind free histones. Our data are consistent with a model in which each PSC binds a nucleosome and at least one other PSC to directly bridge nucleosomes and compact chromatin, but also suggest that naked DNA can be included in compacted structures. We discuss how our data highlight the diversity

  7. Drosophila Brahma complex remodels nucleosome organizations in multiple aspects.

    PubMed

    Shi, Jiejun; Zheng, Meizhu; Ye, Youqiong; Li, Min; Chen, Xiaolong; Hu, Xinjie; Sun, Jin; Zhang, Xiaobai; Jiang, Cizhong

    2014-09-01

    ATP-dependent chromatin remodeling complexes regulate nucleosome organizations. In Drosophila, gene Brm encodes the core Brahma complex, the ATPase subunit of SWI/SNF class of chromatin remodelers. Its role in modulating the nucleosome landscape in vivo is unclear. In this study, we knocked down Brm in Drosophila third instar larvae to explore the changes in nucleosome profiles and global gene transcription. The results show that Brm knockdown leads to nucleosome occupancy changes throughout the entire genome with a bias in occupancy decrease. In contrast, the knockdown has limited impacts on nucleosome position shift. The knockdown also alters another important physical property of nucleosome positioning, fuzziness. Nucleosome position shift, gain or loss and fuzziness changes are all enriched in promoter regions. Nucleosome arrays around the 5' ends of genes are reorganized in five patterns as a result of Brm knockdown. Intriguingly, the concomitant changes in the genes adjacent to the Brahma-dependent remodeling regions have important roles in development and morphogenesis. Further analyses reveal abundance of AT-rich motifs for transcription factors in the remodeling regions.

  8. Radiation-induced DNA damage and chromatin structure

    NASA Technical Reports Server (NTRS)

    Rydberg, B.; Chatterjee, A. (Principal Investigator)

    2001-01-01

    DNA lesions induced by ionizing radiation in cells are clustered and not randomly distributed. For low linear energy transfer (LET) radiation this clustering occurs mainly on the small scales of DNA molecules and nucleosomes. For example, experimental evidence suggests that both strands of DNA on the nucleosomal surface can be damaged in single events and that this damage occurs with a 10-bp modulation because of protection by histones. For high LET radiation, clustering also occurs on a larger scale and depends on chromatin organization. A particularly significant clustering occurs when an ionizing particle traverses the 30 nm chromatin fiber with generation of heavily damaged DNA regions with an average size of about 2 kbp. On an even larger scale, high LET radiation can produce several DNA double-strand breaks in closer proximity than expected from randomness. It is suggested that this increases the probability of misrejoining of DNA ends and generation of lethal chromosome aberrations.

  9. Structure of chromatin in spermatozoa.

    PubMed

    Björndahl, Lars; Kvist, Ulrik

    2014-01-01

    The specialized structure of the sperm chromatin has a dual function - first to protect the DNA from damage during storage and transport to the oocyte, and then to enable a rapid and complete unpacking of the undamaged paternal genome in the ooplasm. It is evident that zinc has a pivotal role in maintaining the structural stability and in enabling a rapid decondensation at the appropriate time. It is important for the sperm chromatin structure that the spermatozoa are ejaculated together with the zinc-rich prostatic secretion. Early exposure to zinc-binding seminal vesicular fluid can deplete the sperm chromatin of zinc and most likely induce surplus formation of disulfide bridges, likely to cause incomplete and delayed decondensation of the sperm chromatin in the oocyte. A premature decrease in sperm chromatin structure stability is likely to increase the risk for damage to the DNA due to increased access to the genome for DNA damaging compounds. The status of the sperm chromatin structure can vary in vitro depending on the exposure to zinc-depleting conditions when spermatozoa are stored in semen after ejaculation. When sperm DNA damage tests are evaluated and validated, it is therefore essential to also take into account the dynamics of zinc-dependent and zinc-independent sperm chromatin stability.

  10. The interplay among chromatin dynamics, cell cycle checkpoints and repair mechanisms modulates the cellular response to DNA damage.

    PubMed

    Lazzaro, Federico; Giannattasio, Michele; Muzi-Falconi, Marco; Plevani, Paolo

    2007-06-01

    Cells are continuously under the assault of endogenous and exogenous genotoxic stress that challenges the integrity of DNA. To cope with such a formidable task cells have evolved surveillance mechanisms, known as checkpoints, and a variety of DNA repair systems responding to different types of DNA lesions. These lesions occur in the context of the chromatin structure and, as expected for all DNA transactions, the cellular response to DNA damage is going to be influenced by the chromatin enviroment. In this review, we will discuss recent studies implicating chromatin remodelling factors and histone modifications in the response to DNA double-strand breaks (DSBs) and in checkpoint activation in response to UV lesions.

  11. Remodeling A School Shop?

    ERIC Educational Resources Information Center

    Baker, G. E.

    1970-01-01

    Presents guidelines for remodeling a school shop combining major considerations of funds, program changes, class management, and flexibility, with the needs of wiring, painting, and placement of equipment. (Author)

  12. Mesoscale Modeling of Chromatin Folding

    NASA Astrophysics Data System (ADS)

    Schlick, Tamar

    2009-03-01

    Eukaryotic chromatin is the fundamental protein/nucleic acid unit that stores the genetic material. Understanding how chromatin fibers fold and unfold in physiological conditions is important for interpreting fundamental biological processes like DNA replication and transcription regulation. Using a mesoscopic model of oligonucleosome chains and tailored sampling protocols, we elucidate the energetics of oligonucleosome folding/unfolding and the role of each histone tail, linker histones, and divalent ions in regulating chromatin structure. The resulting compact topologies reconcile features of the zigzag model with straight linker DNAs with the solenoid model with bent linker DNAs for optimal fiber organization and reveal dynamic and energetic aspects involved.

  13. Structure and Dynamic Properties of a Glucocorticoid Receptor-Induced Chromatin Transition

    PubMed Central

    Fletcher, Terace M.; Ryu, Byung-Woo; Baumann, Christopher T.; Warren, Barbour S.; Fragoso, Gilberto; John, Sam; Hager, Gordon L.

    2000-01-01

    Activation of the mouse mammary tumor virus (MMTV) promoter by the glucocorticoid receptor (GR) is associated with a chromatin structural transition in the B nucleosome region of the viral long terminal repeat (LTR). Recent evidence indicates that this transition extends upstream of the B nucleosome, encompassing a region larger than a single nucleosome (G. Fragoso, W. D. Pennie, S. John, and G. L. Hager, Mol. Cell. Biol. 18:3633–3644). We have reconstituted MMTV LTR DNA into a polynucleosome array using Drosophila embryo extracts. We show binding of purified GR to specific GR elements within a large, multinucleosome array and describe a GR-induced nucleoprotein transition that is dependent on ATP and a HeLa nuclear extract. Previously uncharacterized GR binding sites in the upstream C nucleosome region are involved in the extended region of chromatin remodeling. We also show that GR-dependent chromatin remodeling is a multistep process; in the absence of ATP, GR binds to multiple sites on the chromatin array and prevents restriction enzyme access to recognition sites. Upon addition of ATP, GR induces remodeling and a large increase in access to enzymes sites within the transition region. These findings suggest a dynamic model in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, and is then lost from the template. This model is consistent with the recent description of a “hit-and-run” mechanism for GR action in living cells (J. G. McNally, W. G. Müller, D. Walker, and G. L. Hager, Science 287:1262–1264, 2000). PMID:10938123

  14. Isolation of active regulatory elements from eukaryotic chromatin using FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements)

    PubMed Central

    Giresi, Paul G.; Lieb, Jason D.

    2009-01-01

    The binding of sequence-specific regulatory factors and the recruitment of chromatin remodeling activities cause nucleosomes to be evicted from chromatin in eukaryotic cells. Traditionally, these active sites have been identified experimentally through their sensitivity to nucleases. Here we describe the details of a simple procedure for the genome-wide isolation of nucleosome-depleted DNA from human chromatin, termed FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements). We also provide protocols for different methods of detecting FAIRE-enriched DNA, including use of PCR, DNA microarrays, and next-generation sequencing. FAIRE works on all eukaryotic chromatin tested to date. To perform FAIRE, chromatin is crosslinked with formaldehyde, sheared by sonication, and phenol-chloroform extracted. Most genomic DNA is crosslinked to nucleosomes and is sequestered to the interphase, whereas DNA recovered in the aqueous phase corresponds to nucleosome-depleted regions of the genome. The isolated regions are largely coincident with the location of DNaseI hypersensitive sites, transcriptional start sites, enhancers, insulators, and active promoters. Given its speed and simplicity, FAIRE has utility in establishing chromatin profiles of diverse cell types in health and disease, isolating DNA regulatory elements en masse for further characterization, and as a screening assay for the effects of small molecules on chromatin organization. PMID:19303047

  15. Unusual chromatin in human telomeres.

    PubMed Central

    Tommerup, H; Dousmanis, A; de Lange, T

    1994-01-01

    We report that human telomeres have an unusual chromatin structure characterized by diffuse micrococcal nuclease patterns. The altered chromatin manifested itself only in human telomeres that are relatively short (2 to 7 kb). In contrast, human and mouse telomeres with telomeric repeat arrays of 14 to 150 kb displayed a more canonical chromatin structure with extensive arrays of tightly packed nucleosomes. All telomeric nucleosomes showed a shorter repeat size than bulk nucleosomes, and telomeric mononucleosomal particles were found to be hypersensitive to micrococcal nuclease. However, telomeric nucleosomes were similar to bulk nucleosomes in the rate at which they sedimented through sucrose gradients. We speculate that mammalian telomeres have a bipartite structure with unusual chromatin near the telomere terminus and a more canonical nucleosomal organization in the proximal part of the telomere. Images PMID:8065312

  16. Regulation of cellular chromatin state

    PubMed Central

    Mishra, Rakesh K; Dhawan, Jyotsna

    2010-01-01

    The identity and functionality of eukaryotic cells is defined not just by their genomic sequence which remains constant between cell types, but by their gene expression profiles governed by epigenetic mechanisms. Epigenetic controls maintain and change the chromatin state throughout development, as exemplified by the setting up of cellular memory for the regulation and maintenance of homeotic genes in proliferating progenitors during embryonic development. Higher order chromatin structure in reversibly arrested adult stem cells also involves epigenetic regulation and in this review we highlight common trends governing chromatin states, focusing on quiescence and differentiation during myogenesis. Together, these diverse developmental modules reveal the dynamic nature of chromatin regulation providing fresh insights into the role of epigenetic mechanisms in potentiating development and differentiation. PMID:20592864

  17. Protective role of RAD50 on chromatin bridges during abnormal cytokinesis.

    PubMed

    Schröder-Heurich, Bianca; Wieland, Britta; Lavin, Martin F; Schindler, Detlev; Dörk, Thilo

    2014-03-01

    Faithful chromosome segregation is required for preserving genomic integrity. Failure of this process may entail chromatin bridges preventing normal cytokinesis. To test whether RAD50, a protein normally involved in DNA double-strand break repair, is involved in abnormal cytokinesis and formation of chromatin bridges, we used immunocytochemical and protein interaction assays. RAD50 localizes to chromatin bridges during aberrant cytokinesis and subsequent stages of the cell cycle, either decorating the entire bridge or focally accumulating at the midbody zone. Ionizing radiation led to an ∼4-fold increase in the rate of chromatin bridges in an ataxia telangiectatica mutated (ATM)-dependent manner in human RAD50-proficient fibroblasts but not in RAD50-deficient cells. Cells with a RAD50-positive chromatin bridge were able to continue cell cycling and to progress through S phase (44%), whereas RAD50 knockdown caused a deficiency in chromatin bridges as well as an ∼4-fold prolonged duration of mitosis. RAD50 colocalized and directly interacted with Aurora B kinase and phospho-histone H3, and Aurora B kinase inhibition led to a deficiency in RAD50-positive bridges. Based on these observations, we propose that RAD50 is a crucial factor for the stabilization and shielding of chromatin bridges. Our study provides evidence for a hitherto unknown role of RAD50 in abnormal cytokinesis.

  18. Linker DNA destabilizes condensed chromatin.

    PubMed

    Green, G R; Ferlita, R R; Walkenhorst, W F; Poccia, D L

    2001-01-01

    The contribution of the linker region to maintenance of condensed chromatin was examined in two model systems, namely sea urchin sperm nuclei and chicken red blood cell nuclei. Linkerless nuclei, prepared by extensive digestion with micrococcal nuclease, were compared with Native nuclei using several assays, including microscopic appearance, nuclear turbidity, salt stability, and trypsin resistance. Chromatin in the Linkerless nuclei was highly condensed, resembling pyknotic chromatin in apoptotic cells. Linkerless nuclei were more stable in low ionic strength buffers and more resistant to trypsin than Native nuclei. Analysis of histones from the trypsinized nuclei by polyacrylamide gel electrophoresis showed that specific histone H1, H2B, and H3 tail regions stabilized linker DNA in condensed nuclei. Thermal denaturation of soluble chromatin preparations from differentially trypsinized sperm nuclei demonstrated that the N-terminal regions of histones Sp H1, Sp H2B, and H3 bind tightly to linker DNA, causing it to denature at a high temperature. We conclude that linker DNA exerts a disruptive force on condensed chromatin structure which is counteracted by binding of specific histone tail regions to the linker DNA. The inherent instability of the linker region may be significant in all eukaryotic chromatins and may promote gene activation in living cells.

  19. Relationships between chromosome structure and chromosomal aberrations

    NASA Astrophysics Data System (ADS)

    Eidelman, Yuri; Andreev, Sergey

    An interphase nucleus of human lymphocyte was simulated by the novel Monte Carlo tech-nique. The main features of interphase chromosome structure and packaging were taken into account: different levels of chromatin organisation; nonrandom localisation of chromosomes within a nucleus; chromosome loci dynamics. All chromosomes in a nucleus were modelled as polymer globules. A dynamic pattern of intra/interchromosomal contacts was simulated. The detailed information about chromosomal contacts, such as distribution of intrachromoso-mal contacts over the length of each chromosome and dependence of contact probability on genomic separation between chromosome loci, were calculated and compared to the new exper-imental data obtained by the Hi-C technique. Types and frequencies of simple and complex radiation-induced chromosomal exchange aberrations (CA) induced by X-rays were predicted with taking formation and decay of chromosomal contacts into account. Distance dependence of exchange formation probability was calculated directly. mFISH data for human lymphocytes were analysed. The calculated frequencies of simple CA agreed with the experimental data. Complex CA were underestimated despite the dense packaging of chromosome territories within a nucleus. Possible influence of chromosome-nucleus structural organisation on the frequency and spectrum of radiation-induced chromosome aberrations is discussed.

  20. DNA Repair Defects and Chromosomal Aberrations

    NASA Technical Reports Server (NTRS)

    Hada, Megumi; George, K. A.; Huff, J. L.; Pluth, J. M.; Cucinotta, F. A.

    2009-01-01

    Yields of chromosome aberrations were assessed in cells deficient in DNA doublestrand break (DSB) repair, after exposure to acute or to low-dose-rate (0.018 Gy/hr) gamma rays or acute high LET iron nuclei. We studied several cell lines including fibroblasts deficient in ATM (ataxia telangiectasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase (DNA-PK) activity. Chromosomes were analyzed using the fluorescence in situ hybridization (FISH) chromosome painting method in cells at the first division post irradiation, and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma irradiation induced greater yields of both simple and complex exchanges in the DSB repair-defective cells than in the normal cells. The quadratic dose-response terms for both simple and complex chromosome exchanges were significantly higher for the ATM- and NBS-deficient lines than for normal fibroblasts. However, in the NBS cells the linear dose-response term was significantly higher only for simple exchanges. The large increases in the quadratic dose-response terms in these repair-defective cell lines points the importance of the functions of ATM and NBS in chromatin modifications to facilitate correct DSB repair and minimize the formation of aberrations. The differences found between ATM- and NBS-deficient cells at low doses suggest that important questions should with regard to applying observations of radiation sensitivity at high dose to low-dose exposures. For aberrations induced by iron nuclei, regression models preferred purely linear dose responses for simple exchanges and quadratic dose responses for complex exchanges. Relative biological effectiveness (RBE) factors of all of

  1. Synaptic, transcriptional and chromatin genes disrupted in autism.

    PubMed

    De Rubeis, Silvia; He, Xin; Goldberg, Arthur P; Poultney, Christopher S; Samocha, Kaitlin; Cicek, A Erucment; Kou, Yan; Liu, Li; Fromer, Menachem; Walker, Susan; Singh, Tarinder; Klei, Lambertus; Kosmicki, Jack; Shih-Chen, Fu; Aleksic, Branko; Biscaldi, Monica; Bolton, Patrick F; Brownfeld, Jessica M; Cai, Jinlu; Campbell, Nicholas G; Carracedo, Angel; Chahrour, Maria H; Chiocchetti, Andreas G; Coon, Hilary; Crawford, Emily L; Curran, Sarah R; Dawson, Geraldine; Duketis, Eftichia; Fernandez, Bridget A; Gallagher, Louise; Geller, Evan; Guter, Stephen J; Hill, R Sean; Ionita-Laza, Juliana; Jimenz Gonzalez, Patricia; Kilpinen, Helena; Klauck, Sabine M; Kolevzon, Alexander; Lee, Irene; Lei, Irene; Lei, Jing; Lehtimäki, Terho; Lin, Chiao-Feng; Ma'ayan, Avi; Marshall, Christian R; McInnes, Alison L; Neale, Benjamin; Owen, Michael J; Ozaki, Noriio; Parellada, Mara; Parr, Jeremy R; Purcell, Shaun; Puura, Kaija; Rajagopalan, Deepthi; Rehnström, Karola; Reichenberg, Abraham; Sabo, Aniko; Sachse, Michael; Sanders, Stephan J; Schafer, Chad; Schulte-Rüther, Martin; Skuse, David; Stevens, Christine; Szatmari, Peter; Tammimies, Kristiina; Valladares, Otto; Voran, Annette; Li-San, Wang; Weiss, Lauren A; Willsey, A Jeremy; Yu, Timothy W; Yuen, Ryan K C; Cook, Edwin H; Freitag, Christine M; Gill, Michael; Hultman, Christina M; Lehner, Thomas; Palotie, Aaarno; Schellenberg, Gerard D; Sklar, Pamela; State, Matthew W; Sutcliffe, James S; Walsh, Christiopher A; Scherer, Stephen W; Zwick, Michael E; Barett, Jeffrey C; Cutler, David J; Roeder, Kathryn; Devlin, Bernie; Daly, Mark J; Buxbaum, Joseph D

    2014-11-13

    The genetic architecture of autism spectrum disorder involves the interplay of common and rare variants and their impact on hundreds of genes. Using exome sequencing, here we show that analysis of rare coding variation in 3,871 autism cases and 9,937 ancestry-matched or parental controls implicates 22 autosomal genes at a false discovery rate (FDR) < 0.05, plus a set of 107 autosomal genes strongly enriched for those likely to affect risk (FDR < 0.30). These 107 genes, which show unusual evolutionary constraint against mutations, incur de novo loss-of-function mutations in over 5% of autistic subjects. Many of the genes implicated encode proteins for synaptic formation, transcriptional regulation and chromatin-remodelling pathways. These include voltage-gated ion channels regulating the propagation of action potentials, pacemaking and excitability-transcription coupling, as well as histone-modifying enzymes and chromatin remodellers-most prominently those that mediate post-translational lysine methylation/demethylation modifications of histones.

  2. An annotated list of bivalent chromatin regions in human ES cells: a new tool for cancer epigenetic research.

    PubMed

    Court, Franck; Arnaud, Philippe

    2017-01-17

    CpG islands (CGI) marked by bivalent chromatin in stem cells are believed to be more prone to aberrant DNA methylation in tumor cells. The robustness and genome-wide extent of this instructive program in different cancer types remain to be determined. To address this issue we developed a user-friendly approach to integrate the stem cell chromatin signature in customized DNA methylation analyses. We used publicly available ChIP-sequencing datasets of several human embryonic stem cell (hESC) lines to determine the extent of bivalent chromatin genome-wide. We then created annotated lists of high-confidence bivalent, H3K4me3-only and H3K27me3-only chromatin regions. The main features of bivalent regions included localization in CGI/promoters, depletion in retroelements and enrichment in specific histone modifications, including the poorly characterized H3K23me2 mark. Moreover, bivalent promoters could be classified in three clusters based on PRC2 and PolII complexes occupancy. Genes with bivalent promoters of the PRC2-defined cluster displayed the lowest expression upon differentiation. As proof-of-concept, we assessed the DNA methylation pattern of eight types of tumors and confirmed that aberrant cancer-associated DNA hypermethylation preferentially targets CGI characterized by bivalent chromatin in hESCs. We also found that such aberrant DNA hypermethylation affected particularly bivalent CGI/promoters associated with genes that tend to remain repressed upon differentiation. Strikingly, bivalent CGI were the most affected by aberrant DNA hypermethylation in both CpG Island Methylator Phenotype-positive (CIMP+) and CIMP-negative tumors, suggesting that, besides transcriptional silencing in the pre-tumorigenic cells, the bivalent chromatin signature in hESCs is a key determinant of the instructive program for aberrant DNA methylation.

  3. Chromatin Modifying Agents in the In Vitro Production of Bovine Embryos

    PubMed Central

    Monteiro, Fabio Morato; Oliveira, Clara Slade; Oliveira, Letícia Zoccolaro; Saraiva, Naiara Zoccal; Mercadante, Maria Eugênia Zerlotti; Lopes, Flavia Lombardi; Arnold, Daniel Robert; Garcia, Joaquim Mansano

    2011-01-01

    The low efficiency observed in cloning by nuclear transfer is related to an aberrant gene expression following errors in epigenetic reprogramming. Recent studies have focused on further understanding of the modifications that take place in the chromatin of embryos during the preimplantation period, through the use of chromatin modifying agents. The goal of these studies is to identify the factors involved in nuclear reprogramming and to adjust in vitro manipulations in order to better mimic in vivo conditions. Therefore, proper knowledge of epigenetic reprogramming is necessary to prevent possible epigenetic errors and to improve efficiency and the use of in vitro fertilization and cloning technologies in cattle and other species. PMID:20936105

  4. Genome-Wide Analysis of Chromatin Accessibility in Arabidopsis Infected with Pseudomonas syringae.

    PubMed

    Bordiya, Yogendra; Kang, Hong-Gu

    2017-01-01

    Changes in chromatin accessibility are an important aspect of the molecular changes that occur in eukaryotic cells responding to stress, and they appear to play a critical role in stress-induced transcriptional activation/reprogramming and epigenetic changes. In plants, pathogen infection has been shown to induce rapid and drastic transcriptional reprogramming; growing evidence suggests that chromatin remodeling plays an essential role in this phenomenon. The recent development of genomic tools to assess chromatin accessibility presents a significant opportunity to investigate the relationship between chromatin dynamicity and gene expression. In this protocol, we have adopted a popular chromatin accessibility assay, DNase-seq, to measure chromatin accessibility in Arabidopsis infected with the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). DNase-seq provides information on chromatin accessibility through the sequencing of DNA fragments generated by DNase I digestion of open chromatin, followed by mapping these sequences on a reference genome. Of the two popular DNase-seq approaches, we based our method on the Stamatoyannopoulos protocol, which involves two DNase cleavages rather than a single cleavage, followed by size fractionation. Please note that this two-cleavage approach is widely accepted and has been used extensively by ENCODE (Encyclopedia of DNA Elements) project, a public research consortium investigating cis- and trans-elements in the transcriptional regulation in animal cells. To enhance the quality of the chromatin accessibility assay, we modified this protocol by including two centrifugation steps for nuclear enrichment and size fractionation and an extra washing step for removal of chloroplasts and Pst. The outcomes obtained by this approach are also discussed.

  5. Chromatin structure and DNA damage

    SciTech Connect

    Gale, J.M.

    1987-01-01

    This dissertation examines the structure and structural transitions of chromatin in relation to DNA damage. The ability of intact and histone H1 depleted chromatin fibers to fold into higher ordered structures in vitro was examined following DNA photodamage introduced by two different agents. (1) 254-nm UV radiation and (2) trimethylpsoralen (plus near-UV radiation). Both agents are highly specific for DNA and form adducts predicted to cause different degrees of distortion in the DNA helix. The salt-induced structural transitions of intact and histone H1 depleted chromatin fibers were monitored by both analytical ultracentrifugation and light scattering. Our results show that even in the presence of extremely large, nonphysiological amounts of photodamage by either agent the ability of chromatin to fold into higher ordered structures is not affected. The compact, 30 nm fiber must therefore be able to accommodate a large amount of DNA damage without any measurable changes in the overall size or degree of compaction of this structure. The distribution of pyrimidine dimers was mapped at the single nucleotide level in nucleosome core DNA from UV-irradiated mononucleosomes, chromatin fibers, and human cells in culture using the 3' ..-->.. 5' exonuclease activity of T4 DNA polymerase.

  6. Chromatin modification in zebrafish development.

    PubMed

    Cayuso Mas, Jordi; Noël, Emily S; Ober, Elke A

    2011-01-01

    The generation of complex organisms requires that an initial population of cells with identical gene expression profiles can adopt different cell fates during development by progressively diverging transcriptional programs. These programs depend on the binding of transcritional regulators to specific genomic sites, which in turn is controlled by modifications of the chromatin. Chromatin modifications may occur directly upon DNA by methylation of specific nucleotides, or may involve post-translational modification of histones. Local regulation of histone post-translational modifications regionalizes the genome into euchromatic regions, which are more accessible to DNA-binding factors, and condensed heterochromatic regions, inhibiting the binding of such factors. In addition, these modifications may be required in a genome-wide fashion for processes such as DNA replication or chromosome condensation. From an embryologist's point of view chromatin modifications are intensively studied in the context of imprinting and have more recently received increasing attention in understanding the basis of pluripotency and cellular differentiation. Here, we describe recently uncovered roles of chromatin modifications in zebrafish development and regeneration, as well as available resources and commonly used techniques. We provide a general introduction into chromatin modifications and their respective functions with a focus on gene transcription, as well as key aspects of their roles in the early zebrafish embryo, neural development, formation of the digestive system and tissue regeneration.

  7. Chromatin shapes the mitotic spindle.

    PubMed

    Dinarina, Ana; Pugieux, Céline; Corral, Maria Mora; Loose, Martin; Spatz, Joachim; Karsenti, Eric; Nédélec, François

    2009-08-07

    In animal and plant cells, mitotic chromatin locally generates microtubules that self-organize into a mitotic spindle, and its dimensions and bipolar symmetry are essential for accurate chromosome segregation. By immobilizing microscopic chromatin-coated beads on slide surfaces using a microprinting technique, we have examined the effect of chromatin on the dimensions and symmetry of spindles in Xenopus laevis cytoplasmic extracts. While circular spots with diameters around 14-18 microm trigger bipolar spindle formation, larger spots generate an incorrect number of poles. We also examined lines of chromatin with various dimensions. Their length determined the number of poles that formed, with a 6 x 18 microm rectangular patch generating normal spindle morphology. Around longer lines, multiple poles formed and the structures were disorganized. While lines thinner than 10 mum generated symmetric structures, thicker lines induced the formation of asymmetric structures where all microtubules are on the same side of the line. Our results show that chromatin defines spindle shape and orientation. For a video summary of this article, see the PaperFlick file available with the online Supplemental Data.

  8. Extensive Transcriptional Regulation of Chromatin Modifiers during Human Neurodevelopment

    PubMed Central

    Weng, Matthias K.; Zimmer, Bastian; Pöltl, Dominik; Broeg, Marc P.; Ivanova, Violeta; Gaspar, John A.; Sachinidis, Agapios; Wüllner, Ullrich

    2012-01-01

    Epigenetic changes, including histone modifications or chromatin remodeling are regulated by a large number of human genes. We developed a strategy to study the coordinate regulation of such genes, and to compare different cell populations or tissues. A set of 150 genes, comprising different classes of epigenetic modifiers was compiled. This new tool was used initially to characterize changes during the differentiation of human embryonic stem cells (hESC) to central nervous system neuroectoderm progenitors (NEP). qPCR analysis showed that more than 60% of the examined transcripts were regulated, and >10% of them had a >5-fold increased expression. For comparison, we differentiated hESC to neural crest progenitors (NCP), a distinct peripheral nervous system progenitor population. Some epigenetic modifiers were regulated into the same direction in NEP and NCP, but also distinct differences were observed. For instance, the remodeling ATPase SMARCA2 was up-regulated >30-fold in NCP, while it remained unchanged in NEP; up-regulation of the ATP-dependent chromatin remodeler CHD7 was increased in NEP, while it was down-regulated in NCP. To compare the neural precursor profiles with those of mature neurons, we analyzed the epigenetic modifiers in human cortical tissue. This resulted in the identification of 30 regulations shared between all cell types, such as the histone methyltransferase SETD7. We also identified new markers for post-mitotic neurons, like the arginine methyl transferase PRMT8 and the methyl transferase EZH1. Our findings suggest a hitherto unexpected extent of regulation, and a cell type-dependent specificity of epigenetic modifiers in neurodifferentiation. PMID:22590590

  9. The adenovirus E1A N-terminal repression domain represses transcription from a chromatin template in vitro

    PubMed Central

    Loewenstein, Paul M.; Wu, Shwu-Yuan; Chiang, Cheng-Ming

    2013-01-01

    The adenovirus repression domain of E1A 243R at the E1A N-terminus (E1A 1–80) transcriptionally represses genes involved in differentiation and cell cycle progression. E1A 1–80 represses transcription in vitro from naked DNA templates through its interaction with p300 and TFIID. E1A 1–80 can also interact with several chromatin remodeling factors and associates with chromatin in vivo. We show here that E1A 243R and E1A 1–80 can repress transcription from a reconstituted chromatin template in vitro. Temporal analysis reveals strong repression by E1A 1–80 when added at pre-activation, activation and early transcription stages. Interestingly, E1A 1–80 can greatly enhance transcription from chromatin templates, but not from naked DNA, when added at pre-initiation complex (PIC) formation and transcription-initiation stages. These data reveal a new dimension for E1A 1–80's interface with chromatin and may reflect its interaction with key players in PIC formation, p300 and TFIID, and/or possibly a role in chromatin remodeling. PMID:22521914

  10. The adenovirus E1A N-terminal repression domain represses transcription from a chromatin template in vitro.

    PubMed

    Loewenstein, Paul M; Wu, Shwu-Yuan; Chiang, Cheng-Ming; Green, Maurice

    2012-06-20

    The adenovirus repression domain of E1A 243R at the E1A N-terminus (E1A 1-80) transcriptionally represses genes involved in differentiation and cell cycle progression. E1A 1-80 represses transcription in vitro from naked DNA templates through its interaction with p300 and TFIID. E1A 1-80 can also interact with several chromatin remodeling factors and associates with chromatin in vivo. We show here that E1A 243R and E1A 1-80 can repress transcription from a reconstituted chromatin template in vitro. Temporal analysis reveals strong repression by E1A 1-80 when added at pre-activation, activation and early transcription stages. Interestingly, E1A 1-80 can greatly enhance transcription from chromatin templates, but not from naked DNA, when added at pre-initiation complex (PIC) formation and transcription-initiation stages. These data reveal a new dimension for E1A 1-80's interface with chromatin and may reflect its interaction with key players in PIC formation, p300 and TFIID, and/or possibly a role in chromatin remodeling.

  11. Beyond transcription factors: The role of chromatin modifying enzymes in regulating transcription required for memory

    PubMed Central

    Barrett, Ruth M.; Wood, Marcelo A.

    2008-01-01

    One of the alluring aspects of examining chromatin modifications in the role of modulating transcription required for long-term memory processes is that these modifications may provide transient and potentially stable epigenetic marks in the service of activating and/or maintaining transcriptional processes. These, in turn, may ultimately participate in the molecular mechanisms required for neuronal changes subserving long-lasting changes in behavior. As an epigenetic mechanism of transcriptional control, chromatin modification has been shown to participate in maintaining cellular memory (e.g., cell fate) and may underlie the strengthening and maintenance of synaptic connections required for long-term changes in behavior. Epigenetics has become central to several fields of neurobiology, where researchers have found that regulation of chromatin modification has a significant role in epilepsy, drug addiction, depression, neurodegenerative diseases, and memory. In this review, we will discuss the role of chromatin modifying enzymes in memory processes, as well as how recent studies in yeast genetics and cancer biology may impact the way we think about how chromatin modification and chromatin remodeling regulate neuronal function. PMID:18583646

  12. Chromatin modification and NBS1: their relationship in DNA double-strand break repair.

    PubMed

    Saito, Yuichiro; Zhou, Hui; Kobayashi, Junya

    2016-01-01

    The importance of chromatin modification, including histone modification and chromatin remodeling, for DNA double-strand break (DSB) repair, as well as transcription and replication, has been elucidated. Phosphorylation of H2AX to γ-H2AX is one of the first responses following DSB detection, and this histone modification is important for the DSB damage response by triggering several events, including the accumulation of DNA damage response-related proteins and subsequent homologous recombination (HR) repair. The roles of other histone modifications such as acetylation, methylation and ubiquitination have also been recently clarified, particularly in the context of HR repair. NBS1 is a multifunctional protein that is involved in various DNA damage responses. Its recently identified binding partner RNF20 is an E3 ubiquitin ligase that facilitates the monoubiquitination of histone H2B, a process that is crucial for recruitment of the chromatin remodeler SNF2h to DSB damage sites. Evidence suggests that SNF2h functions in HR repair, probably through regulation of end-resection. Moreover, several recent reports have indicated that SNF2h can function in HR repair pathways as a histone remodeler and that other known histone remodelers can also participate in DSB damage responses. On the other hand, information about the roles of such chromatin modifications and NBS1 in non-homologous end joining (NHEJ) repair of DSBs and stalled fork-related damage responses is very limited; therefore, these aspects and processes need to be further studied to advance our understanding of the mechanisms and molecular players involved.

  13. Chromatin Modifications during Repair of Environmental Exposure-Induced DNA Damage: A Potential Mechanism for Stable Epigenetic Alterations

    PubMed Central

    O’Hagan, Heather M.

    2014-01-01

    Exposures to environmental toxicants and toxins cause epigenetic changes that likely play a role in the development of diseases associated with exposure. The mechanism behind these exposure-induced epigenetic changes is currently unknown. One commonality between most environmental exposures is that they cause DNA damage either directly or through causing an increase in reactive oxygen species, which can damage DNA. Like transcription, DNA damage repair must occur in the context of chromatin requiring both histone modifications and ATP-dependent chromatin remodeling. These chromatin changes aid in DNA damage accessibility and signaling. Several proteins and complexes involved in epigenetic silencing during both development and cancer have been found to be localized to sites of DNA damage. The chromatin-based response to DNA damage is considered a transient event, with chromatin being restored to normal as DNA damage repair is completed. However, in individuals chronically exposed to environmental toxicants or with chronic inflammatory disease, repeated DNA damage-induced chromatin rearrangement may ultimately lead to permanent epigenetic alterations. Understanding the mechanism behind exposure-induced epigenetic changes will allow us to develop strategies to prevent or reverse these changes. This review focuses on epigenetic changes and DNA damage induced by environmental exposures, the chromatin changes that occur around sites of DNA damage, and how these transient chromatin changes may lead to heritable epigenetic alterations at sites of chronic exposure. PMID:24259318

  14. Remodeling the Media Center.

    ERIC Educational Resources Information Center

    Baule, Steven M.

    1998-01-01

    Discusses items that need to be considered when remodeling a school media center. Highlights include space and location for various functions, including projections of print versus electronic media; electrical and data wiring needs; lighting; security and supervision; and reuse of existing furniture and equipment. (LRW)

  15. Remodeling of ribosomal genes in somatic cells by Xenopus egg extract

    SciTech Connect

    Ostrup, Olga; Hyttel, Poul; Klaerke, Dan A.; Collas, Philippe

    2011-09-02

    Highlights: {yields} Xenopus egg extract remodels nuclei and alter cell growth characteristics. {yields} Ribosomal genes are reprogrammed within 6 h after extract exposure. {yields} rDNA reprogramming involves promoter targeting of SNF2H remodeling complex. {yields} Xenopus egg extract does not initiate stress-related response in somatic cells. {yields} Aza-cytidine elicits a stress-induced response in reprogrammed cells. -- Abstract: Extracts from Xenopus eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression. This occurs within hours of extract treatment and is distinct from a stress response. Egg extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling complex component SNF2H without affecting occupancy of the transcription factor UBF and the stress silencers SUV39H1 and SIRT1. During this process, nucleolar localization of UBF and SIRT1 is not altered. On contrary, azacytidine pre-treatment has an adverse effect on rDNA remodeling induced by extract and elicits a stress-type nuclear response. Thus, an early event of Xenopus egg extract-mediated nuclear reprogramming is the remodeling of ribosomal genes involving nucleolar remodeling complex. Condition-specific and rapid silencing of ribosomal genes may serve as a sensitive marker for evaluation of various reprogramming methods.

  16. Hotspots of aberrant enhancer activity punctuate the colorectal cancer epigenome

    PubMed Central

    Cohen, Andrea J.; Saiakhova, Alina; Corradin, Olivia; Luppino, Jennifer M.; Lovrenert, Katreya; Bartels, Cynthia F.; Morrow, James J.; Mack, Stephen C.; Dhillon, Gursimran; Beard, Lydia; Myeroff, Lois; Kalady, Matthew F.; Willis, Joseph; Bradner, James E.; Keri, Ruth A.; Berger, Nathan A.; Pruett-Miller, Shondra M.; Markowitz, Sanford D.; Scacheri, Peter C.

    2017-01-01

    In addition to mutations in genes, aberrant enhancer element activity at non-coding regions of the genome is a key driver of tumorigenesis. Here, we perform epigenomic enhancer profiling of a cohort of more than forty genetically diverse human colorectal cancer (CRC) specimens. Using normal colonic crypt epithelium as a comparator, we identify enhancers with recurrently gained or lost activity across CRC specimens. Of the enhancers highly recurrently activated in CRC, most are constituents of super enhancers, are occupied by AP-1 and cohesin complex members, and originate from primed chromatin. Many activate known oncogenes, and CRC growth can be mitigated through pharmacologic inhibition or genome editing of these loci. Nearly half of all GWAS CRC risk loci co-localize to recurrently activated enhancers. These findings indicate that the CRC epigenome is defined by highly recurrent epigenetic alterations at enhancers which activate a common, aberrant transcriptional programme critical for CRC growth and survival. PMID:28169291

  17. Locus-specific epigenetic remodeling controls addiction- and depression-related behaviors.

    PubMed

    Heller, Elizabeth A; Cates, Hannah M; Peña, Catherine J; Sun, Haosheng; Shao, Ningyi; Feng, Jian; Golden, Sam A; Herman, James P; Walsh, Jessica J; Mazei-Robison, Michelle; Ferguson, Deveroux; Knight, Scott; Gerber, Mark A; Nievera, Christian; Han, Ming-Hu; Russo, Scott J; Tamminga, Carol S; Neve, Rachael L; Shen, Li; Zhang, H Steve; Zhang, Feng; Nestler, Eric J

    2014-12-01

    Chronic exposure to drugs of abuse or stress regulates transcription factors, chromatin-modifying enzymes and histone post-translational modifications in discrete brain regions. Given the promiscuity of the enzymes involved, it has not yet been possible to obtain direct causal evidence to implicate the regulation of transcription and consequent behavioral plasticity by chromatin remodeling that occurs at a single gene. We investigated the mechanism linking chromatin dynamics to neurobiological phenomena by applying engineered transcription factors to selectively modify chromatin at a specific mouse gene in vivo. We found that histone methylation or acetylation at the Fosb locus in nucleus accumbens, a brain reward region, was sufficient to control drug- and stress-evoked transcriptional and behavioral responses via interactions with the endogenous transcriptional machinery. This approach allowed us to relate the epigenetic landscape at a given gene directly to regulation of its expression and to its subsequent effects on reward behavior.

  18. POLYTENE CHROMOSOME SQUASH METHODS FOR STUDYING TRANSCRIPTION AND EPIGENETIC CHROMATIN MODIFICATION IN DROSOPHILA USING ANTIBODIES

    PubMed Central

    Johansen, Kristen M.; Cai, Weili; Deng, Huai; Bao, Xiaomin; Zhang, Weiguo; Girton, Jack; Johansen, Jørgen

    2009-01-01

    The giant polytene chromosomes from Drosophila third instar larval salivary glands provide an important model system for studying the architectural changes in chromatin morphology associated with the process of transcription initiation and elongation. Especially, analysis of the heat shock response has proved useful in correlating chromatin structure remodeling with transcriptional activity. An important tool for such studies is the labeling of polytene chromosome squash preparations with antibodies to the enzymes, transcription factors, or histone modifications of interest. However, in any immunohistochemical experiment there will be advantages and disadvantages to different methods of fixation and sample preparation, the relative merits of which must be balanced. Here we provide detailed protocols for polytene chromosome squash preparation and discuss their relative pros and cons in terms of suitability for reliable antibody labeling and preservation of high resolution chromatin structure. PMID:19272452

  19. Alterations of nuclear envelope and chromatin organization in mandibuloacral dysplasia, a rare form of laminopathy.

    PubMed

    Filesi, Ilaria; Gullotta, Francesca; Lattanzi, Giovanna; D'Apice, Maria Rosaria; Capanni, Cristina; Nardone, Anna Maria; Columbaro, Marta; Scarano, Gioacchino; Mattioli, Elisabetta; Sabatelli, Patrizia; Maraldi, Nadir M; Biocca, Silvia; Novelli, Giuseppe

    2005-10-17

    Autosomal recessive mandibuloacral dysplasia [mandibuloacral dysplasia type A (MADA); Online Mendelian Inheritance in Man (OMIM) no. 248370] is caused by a mutation in LMNA encoding lamin A/C. Here we show that this mutation causes accumulation of the lamin A precursor protein, a marked alteration of the nuclear architecture and, hence, chromatin disorganization. Heterochromatin domains are altered or completely lost in MADA nuclei, consistent with the finding that heterochromatin-associated protein HP1beta and histone H3 methylated at lysine 9 and their nuclear envelope partner protein lamin B receptor (LBR) are delocalized and solubilized. Both accumulation of lamin A precursor and chromatin defects become more severe in older patients. These results strongly suggest that altered chromatin remodeling is a key event in the cascade of epigenetic events causing MADA and could be related to the premature-aging phenotype.

  20. Aberrant apoptotic machinery confers melanoma dual resistance to BRAFV600E inhibitor and immune effector cells: immunosensitization by a histone deacetylase inhibitor

    PubMed Central

    Jazirehi, Ali R; Nazarian, Ramin; Torres-Collado, Antoni Xavier; Economou, James S

    2014-01-01

    BRAFV600E-inhibitors (BRAFi; e.g., vemurafenib) and modern immune-based therapies such as PD-1/PD-L1 and CTLA-4 checkpoints blockade and adoptive cell transfer (ACT) have significantly improved the care of melanoma patients. Having these two effective (BRAFi and immunotherapy) therapies raises the question whether there is a rational biological basis for using them in combination. We developed an in vitro model to determine whether tumor resistance mechanisms to a small molecule inhibitor of a driver oncogene, and to cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-delivered apoptotic death signals were exclusive or intersecting. We generated melanoma sublines resistant to BRAFi vemurafenib and to CTL recognizing the MART-1 melanoma antigen. Vemurafenib-resistant (VemR) sublines were cross-resistant to MART CTL and NK cells indicating that a common apoptotic pathway governing tumor response to both modalities was disrupted. Pretreatment of VemR melanomas with a histone deacetylase inhibitor (HDACi) restored sensitivity to MART CTL and NK apoptosis by skewing the apoptotic gene programs towards a proapoptotic phenotype. Our in vitro findings suggest that during the course of acquisition of BRAFi resistance, melanomas develop cross-resistance to CTL- and NK-killing. Further, aberrant apoptotic pathways, amenable by an FDA-approved chromatin remodeling drug, regulate tumor resistance mechanisms to immune effector cells. These results may provide rational molecular basis for further investigations to combine these therapies clinically. PMID:24660121

  1. Aberration correction of unstable resonators

    NASA Technical Reports Server (NTRS)

    Lang, Robert J. (Inventor)

    1994-01-01

    Construction of aspheric reflectors for unstable resonator lasers to provide an arbitrary laser mode inside the resonator to correct aberrations of an output beam by the construction of the shape of an end reflector opposite the output reflector of the resonator cavity, such as aberrations resulting from refraction of a beam exiting the solid of the resonator having an index of refraction greater than 1 or to produce an aberration in the output beam that will precisely compensate for the aberration of an optical train into which the resonator beam is coupled.

  2. A Network of Chromatin Factors Is Regulating the Transition to Postembryonic Development in Caenorhabditis elegans

    PubMed Central

    Erdelyi, Peter; Wang, Xing; Suleski, Marina; Wicky, Chantal

    2016-01-01

    Mi2 proteins are evolutionarily conserved, ATP-dependent chromatin remodelers of the CHD family that play key roles in stem cell differentiation and reprogramming. In Caenorhabditis elegans, the let-418 gene encodes one of the two Mi2 homologs, which is part of at least two chromatin complexes, namely the Nucleosome Remodeling and histone Deacetylase (NuRD) complex and the MEC complex, and functions in larval development, vulval morphogenesis, lifespan regulation, and cell fate determination. To explore the mechanisms involved in the action of LET-418/Mi2, we performed a genome-wide RNA interference (RNAi) screen for suppressors of early larval arrest associated with let-418 mutations. We identified 29 suppressor genes, of which 24 encode chromatin regulators, mostly orthologs of proteins present in transcriptional activator complexes. The remaining five genes vary broadly in their predicted functions. All suppressor genes could suppress multiple aspects of the let-418 phenotype, including developmental arrest and ectopic expression of germline genes in the soma. Analysis of available transcriptomic data and quantitative PCR revealed that LET-418 and the suppressors of early larval arrest are regulating common target genes. These suppressors might represent direct competitors of LET-418 complexes for chromatin regulation of crucial genes involved in the transition to postembryonic development. PMID:28007841

  3. Klotho and Phosphate Are Modulators of Pathologic Uremic Cardiac Remodeling

    PubMed Central

    Shi, Mingjun; Cho, Han Jun; Adams-Huet, Beverley; Paek, Jean; Hill, Kathy; Shelton, John; Amaral, Ansel P.; Faul, Christian; Taniguchi, Masatomo; Wolf, Myles; Brand, Markus; Takahashi, Masaya; Kuro-o, Makoto; Hill, Joseph A.

    2015-01-01

    Cardiac dysfunction in CKD is characterized by aberrant cardiac remodeling with hypertrophy and fibrosis. CKD is a state of severe systemic Klotho deficiency, and restoration of Klotho attenuates vascular calcification associated with CKD. We examined the role of Klotho in cardiac remodeling in models of Klotho deficiency—genetic Klotho hypomorphism, high dietary phosphate intake, aging, and CKD. Klotho-deficient mice exhibited cardiac dysfunction and hypertrophy before 12 weeks of age followed by fibrosis. In wild-type mice, the induction of CKD led to severe cardiovascular changes not observed in control mice. Notably, non-CKD mice fed a high-phosphate diet had lower Klotho levels and greatly accelerated cardiac remodeling associated with normal aging compared with those on a normal diet. Chronic elevation of circulating Klotho because of global overexpression alleviated the cardiac remodeling induced by either high-phosphate diet or CKD. Regardless of the cause of Klotho deficiency, the extent of cardiac hypertrophy and fibrosis correlated tightly with plasma phosphate concentration and inversely with plasma Klotho concentration, even when adjusted for all other covariables. High-fibroblast growth factor–23 concentration positively correlated with cardiac remodeling in a Klotho-deficient state but not a Klotho-replete state. In vitro, Klotho inhibited TGF-β1–, angiotensin II–, or high phosphate–induced fibrosis and abolished TGF-β1– or angiotensin II–induced hypertrophy of cardiomyocytes. In conclusion, Klotho deficiency is a novel intermediate mediator of pathologic cardiac remodeling, and fibroblast growth factor–23 may contribute to cardiac remodeling in concert with Klotho deficiency in CKD, phosphotoxicity, and aging. PMID:25326585

  4. SWI/SNF Protein Component BAF250a Regulates Cardiac Progenitor Cell Differentiation by Modulating Chromatin Accessibility during Second Heart Field Development*

    PubMed Central

    Lei, Ienglam; Gao, Xiaolin; Sham, Mai Har; Wang, Zhong

    2012-01-01

    ATP-dependent SWI/SNF chromatin remodeling complexes alter the structure of chromatin at specific loci and facilitate tissue-specific gene regulation during development. Several SWI/SNF subunits are required for cardiogenesis. However, the function and mechanisms of SWI/SNF in mediating cardiac progenitor cell (CPC) differentiation during cardiogenesis are not well understood. Our studies of the SWI/SNF chromatin remodeling complex identified that BAF250a, a regulatory subunit of the SWI/SNF, plays a key role in CPC differentiation. BAF250a ablation in mouse second heart field (SHF) led to trabeculation defects in the right ventricle, ventricular septal defect, persistent truncus arteriosus, reduced myocardial proliferation, and embryonic lethality around E13. Using an embryonic stem cell culture system that models the formation and differentiation of SHF CPCs in vivo, we have shown that BAF250a ablation in CPCs specifically inhibits cardiomyocyte formation. Moreover, BAF250a selectively regulates the expression of key cardiac factors Mef2c, Nkx2.5, and Bmp10 in SHF CPCs. Chromatin immunoprecipitation and DNase I digestion assays indicate that BAF250a regulates gene expression by binding selectively to its target gene promoters and recruiting Brg1, the catalytic subunit of SWI/SNF, to modulate chromatin accessibility. Our results thus identify BAF250a-mediated chromatin remodeling as an essential epigenetic mechanism mediating CPC differentiation. PMID:22621927

  5. DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus: Mechanism of allele methylation polymorphism

    PubMed Central

    Russo, Giusi; Landi, Rosaria; Pezone, Antonio; Morano, Annalisa; Zuchegna, Candida; Romano, Antonella; Muller, Mark T.; Gottesman, Max E.; Porcellini, Antonio; Avvedimento, Enrico V.

    2016-01-01

    We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR). We find that HR modifies the DNA methylation pattern of the repaired segment. HR also alters local histone H3 methylation as well chromatin structure by inducing DNA-chromatin loops connecting the 5′ and 3′ ends of the repaired gene. During a two-week period after repair, transcription-associated demethylation promoted by Base Excision Repair enzymes further modifies methylation of the repaired DNA. Subsequently, the repaired genes display stable but diverse methylation profiles. These profiles govern the levels of expression in each clone. Our data argue that DNA methylation and chromatin remodelling induced by HR may be a source of permanent variation of gene expression in somatic cells. PMID:27629060

  6. SUN4 is essential for nuclear remodeling during mammalian spermiogenesis.

    PubMed

    Calvi, Alessandra; Wong, Arnette Shi Wei; Wright, Graham; Wong, Esther Sook Miin; Loo, Tsui Han; Stewart, Colin L; Burke, Brian

    2015-11-15

    One of the more dramatic examples of cellular reorganization occurs during spermiogenesis in which a roughly spherical spermatid is transformed into a mature sperm cell. A highlight of this process involves nuclear remodeling whereby the round spermatid nucleus is sculpted into an elongated and polar structure. This transformation in nuclear architecture features chromatin condensation, changes in the composition and organization of the nuclear lamina and redistribution and elimination of nuclear pore complexes. The manchette, a cytoplasmic microtubule-based structure is thought to play a crucial role in the remodeling process. Here we show that SUN4, a spermatid nuclear membrane protein has an essential function in coupling the manchette to the nuclear periphery. In the absence of SUN4, manchette microtubules appear highly disorganized and the nucleus itself fails to elongate. Consequently, mice deficient in SUN4 display globozoospermia with associated infertility.

  7. Engineered Split-TET2 Enzyme for Inducible Epigenetic Remodeling

    PubMed Central

    2017-01-01

    The Ten-eleven translocation (TET) family of 5-methylcytosine (5mC) dioxygenases catalyze the conversion of 5mC into 5-hydroxymethylcytosine (5hmC) and further oxidized species to promote active DNA demethylation. Here we engineered a split-TET2 enzyme to enable temporal control of 5mC oxidation and subsequent remodeling of epigenetic states in mammalian cells. We further demonstrate the use of this chemically inducible system to dissect the correlation between DNA hydroxymethylation and chromatin accessibility in the mammalian genome. This chemical-inducible epigenome remodeling tool will find broad use in interrogating cellular systems without altering the genetic code, as well as in probing the epigenotype–phenotype relations in various biological systems. PMID:28294608

  8. Camera processing with chromatic aberration.

    PubMed

    Korneliussen, Jan Tore; Hirakawa, Keigo

    2014-10-01

    Since the refractive index of materials commonly used for lens depends on the wavelengths of light, practical camera optics fail to converge light to a single point on an image plane. Known as chromatic aberration, this phenomenon distorts image details by introducing magnification error, defocus blur, and color fringes. Though achromatic and apochromatic lens designs reduce chromatic aberration to a degree, they are complex and expensive and they do not offer a perfect correction. In this paper, we propose a new postcapture processing scheme designed to overcome these problems computationally. Specifically, the proposed solution is comprised of chromatic aberration-tolerant demosaicking algorithm and post-demosaicking chromatic aberration correction. Experiments with simulated and real sensor data verify that the chromatic aberration is effectively corrected.

  9. Components of the Human SWI/SNF Complex Are Enriched in Active Chromatin and Are Associated with the Nuclear Matrix

    PubMed Central

    Reyes, Jose C.; Muchardt, Christian; Yaniv, Moshe

    1997-01-01

    Biochemical and genetic evidence suggest that the SWI/SNF complex is involved in the remodeling of chromatin during gene activation. We have used antibodies specific against three human subunits of this complex to study its subnuclear localization, as well as its potential association with active chromatin and the nuclear skeleton. Immunofluorescence studies revealed a punctate nuclear labeling pattern that was excluded from the nucleoli and from regions of condensed chromatin. Dual labeling failed to reveal significant colocalization of BRG1 or hBRM proteins with RNA polymerase II or with nuclear speckles involved in splicing. Chromatin fractionation experiments showed that both soluble and insoluble active chromatin are enriched in the hSWI/SNF proteins as compared with bulk chromatin. hSWI/SNF proteins were also found to be associated with the nuclear matrix or nuclear scaffold, suggesting that a fraction of the hSWI/SNF complex could be involved in the chromatin organization properties associated with matrix attachment regions. PMID:9128241

  10. Histone lysine methylation and chromatin replication.

    PubMed

    Rivera, Carlos; Gurard-Levin, Zachary A; Almouzni, Geneviève; Loyola, Alejandra

    2014-12-01

    In eukaryotic organisms, the replication of the DNA sequence and its organization into chromatin are critical to maintain genome integrity. Chromatin components, such as histone variants and histone post-translational modifications, along with the higher-order chromatin structure, impact several DNA metabolic processes, including replication, transcription, and repair. In this review we focus on lysine methylation and the relationships between this histone mark and chromatin replication. We first describe studies implicating lysine methylation in regulating early steps in the replication process. We then discuss chromatin reassembly following replication fork passage, where the incorporation of a combination of newly synthesized histones and parental histones can impact the inheritance of lysine methylation marks on the daughter strands. Finally, we elaborate on how the inheritance of lysine methylation can impact maintenance of the chromatin landscape, using heterochromatin as a model chromatin domain, and we discuss the potential mechanisms involved in this process.

  11. Nucleolin is a histone chaperone with FACT-like activity and assists remodeling of nucleosomes

    PubMed Central

    Angelov, Dimitar; Bondarenko, Vladimir A; Almagro, Sébastien; Menoni, Hervé; Mongélard, Fabien; Hans, Fabienne; Mietton, Flore; Studitsky, Vasily M; Hamiche, Ali; Dimitrov, Stefan; Bouvet, Philippe

    2006-01-01

    Remodeling machines play an essential role in the control of gene expression, but how their activity is regulated is not known. Here we report that the nuclear protein nucleolin possesses a histone chaperone activity and that this factor greatly enhances the activity of the chromatin remodeling machineries SWI/SNF and ACF. Interestingly, nucleolin is able to induce the remodeling by SWI/SNF of macroH2A, but not of H2ABbd nucleosomes, which are otherwise resistant to remodeling. This new histone chaperone promotes the destabilization of the histone octamer, helping the dissociation of a H2A–H2B dimer, and stimulates the SWI/SNF-mediated transfer of H2A–H2B dimers. Furthermore, nucleolin facilitates transcription through the nucleosome, which is reminiscent of the activity of the FACT complex. This work defines new functions for histone chaperones in chromatin remodeling and regulation of transcription and explains how nucleolin could act on transcription. PMID:16601700

  12. Chromatin Structure in Telomere Dynamics

    PubMed Central

    Galati, Alessandra; Micheli, Emanuela; Cacchione, Stefano

    2013-01-01

    The establishment of a specific nucleoprotein structure, the telomere, is required to ensure the protection of chromosome ends from being recognized as DNA damage sites. Telomere shortening below a critical length triggers a DNA damage response that leads to replicative senescence. In normal human somatic cells, characterized by telomere shortening with each cell division, telomere uncapping is a regulated process associated with cell turnover. Nevertheless, telomere dysfunction has also been associated with genomic instability, cell transformation, and cancer. Despite the essential role telomeres play in chromosome protection and in tumorigenesis, our knowledge of the chromatin structure involved in telomere maintenance is still limited. Here we review the recent findings on chromatin modifications associated with the dynamic changes of telomeres from protected to deprotected state and their role in telomere functions. PMID:23471416

  13. Global genome nucleotide excision repair is organized into domains that promote efficient DNA repair in chromatin

    PubMed Central

    Yu, Shirong; Evans, Katie; Bennett, Mark; Webster, Richard M.; Leadbitter, Matthew; Teng, Yumin; Waters, Raymond

    2016-01-01

    The rates at which lesions are removed by DNA repair can vary widely throughout the genome, with important implications for genomic stability. To study this, we measured the distribution of nucleotide excision repair (NER) rates for UV-induced lesions throughout the budding yeast genome. By plotting these repair rates in relation to genes and their associated flanking sequences, we reveal that, in normal cells, genomic repair rates display a distinctive pattern, suggesting that DNA repair is highly organized within the genome. Furthermore, by comparing genome-wide DNA repair rates in wild-type cells and cells defective in the global genome–NER (GG-NER) subpathway, we establish how this alters the distribution of NER rates throughout the genome. We also examined the genomic locations of GG-NER factor binding to chromatin before and after UV irradiation, revealing that GG-NER is organized and initiated from specific genomic locations. At these sites, chromatin occupancy of the histone acetyl-transferase Gcn5 is controlled by the GG-NER complex, which regulates histone H3 acetylation and chromatin structure, thereby promoting efficient DNA repair of UV-induced lesions. Chromatin remodeling during the GG-NER process is therefore organized into these genomic domains. Importantly, loss of Gcn5 significantly alters the genomic distribution of NER rates; this has implications for the effects of chromatin modifiers on the distribution of mutations that arise throughout the genome. PMID:27470111

  14. Restriction of histone gene transcription to S phase by phosphorylation of a chromatin boundary protein

    PubMed Central

    Kurat, Christoph F.; Lambert, Jean-Philippe; van Dyk, Dewald; Tsui, Kyle; van Bakel, Harm; Kaluarachchi, Supipi; Friesen, Helena; Kainth, Pinay; Nislow, Corey; Figeys, Daniel; Fillingham, Jeffrey; Andrews, Brenda J.

    2011-01-01

    The cell cycle-regulated expression of core histone genes is required for DNA replication and proper cell cycle progression in eukaryotic cells. Although some factors involved in histone gene transcription are known, the molecular mechanisms that ensure proper induction of histone gene expression during S phase remain enigmatic. Here we demonstrate that S-phase transcription of the model histone gene HTA1 in yeast is regulated by a novel attach–release mechanism involving phosphorylation of the conserved chromatin boundary protein Yta7 by both cyclin-dependent kinase 1 (Cdk1) and casein kinase 2 (CK2). Outside S phase, integrity of the AAA-ATPase domain is required for Yta7 boundary function, as defined by correct positioning of the histone chaperone Rtt106 and the chromatin remodeling complex RSC. Conversely, in S phase, Yta7 is hyperphosphorylated, causing its release from HTA1 chromatin and productive transcription. Most importantly, abrogation of Yta7 phosphorylation results in constitutive attachment of Yta7 to HTA1 chromatin, preventing efficient transcription post-recruitment of RNA polymerase II (RNAPII). Our study identified the chromatin boundary protein Yta7 as a key regulator that links S-phase kinases with RNAPII function at cell cycle-regulated histone gene promoters. PMID:22156209

  15. Restriction of histone gene transcription to S phase by phosphorylation of a chromatin boundary protein.

    PubMed

    Kurat, Christoph F; Lambert, Jean-Philippe; van Dyk, Dewald; Tsui, Kyle; van Bakel, Harm; Kaluarachchi, Supipi; Friesen, Helena; Kainth, Pinay; Nislow, Corey; Figeys, Daniel; Fillingham, Jeffrey; Andrews, Brenda J

    2011-12-01

    The cell cycle-regulated expression of core histone genes is required for DNA replication and proper cell cycle progression in eukaryotic cells. Although some factors involved in histone gene transcription are known, the molecular mechanisms that ensure proper induction of histone gene expression during S phase remain enigmatic. Here we demonstrate that S-phase transcription of the model histone gene HTA1 in yeast is regulated by a novel attach-release mechanism involving phosphorylation of the conserved chromatin boundary protein Yta7 by both cyclin-dependent kinase 1 (Cdk1) and casein kinase 2 (CK2). Outside S phase, integrity of the AAA-ATPase domain is required for Yta7 boundary function, as defined by correct positioning of the histone chaperone Rtt106 and the chromatin remodeling complex RSC. Conversely, in S phase, Yta7 is hyperphosphorylated, causing its release from HTA1 chromatin and productive transcription. Most importantly, abrogation of Yta7 phosphorylation results in constitutive attachment of Yta7 to HTA1 chromatin, preventing efficient transcription post-recruitment of RNA polymerase II (RNAPII). Our study identified the chromatin boundary protein Yta7 as a key regulator that links S-phase kinases with RNAPII function at cell cycle-regulated histone gene promoters.

  16. Chromosome Aberrations in Astronauts

    NASA Technical Reports Server (NTRS)

    George, Kerry A.; Durante, M.; Cucinotta, Francis A.

    2007-01-01

    A review of currently available data on in vivo induced chromosome damage in the blood lymphocytes of astronauts proves that, after protracted exposure of a few months or more to space radiation, cytogenetic biodosimetry analyses of blood collected within a week or two of return from space provides a reliable estimate of equivalent radiation dose and risk. Recent studies indicate that biodosimetry estimates from single spaceflights lie within the range expected from physical dosimetry and biophysical models, but very large uncertainties are associated with single individual measurements and the total sample population remains low. Retrospective doses may be more difficult to estimate because of the fairly rapid time-dependent loss of "stable" aberrations in blood lymphocytes. Also, biodosimetry estimates from individuals who participate in multiple missions, or very long (interplanetary) missions, may be complicated by an adaptive response to space radiation and/or changes in lymphocyte survival and repopulation. A discussion of published data is presented and specific issues related to space radiation biodosimetry protocols are discussed.

  17. Correction of Distributed Optical Aberrations

    SciTech Connect

    Baker, K; Olivier, S; Carrano, C; Phillion, D

    2006-02-12

    The objective of this project was to demonstrate the use of multiple distributed deformable mirrors (DMs) to improve the performance of optical systems with distributed aberrations. This concept is expected to provide dramatic improvement in the optical performance of systems in applications where the aberrations are distributed along the optical path or within the instrument itself. Our approach used multiple actuated DMs distributed to match the aberration distribution. The project developed the algorithms necessary to determine the required corrections and simulate the performance of these multiple DM systems.

  18. Remodeling with the sun

    SciTech Connect

    Bodzin, S.

    1997-05-01

    Remodeling is the perfect time to improve daylighting, direct gain heating and shading with passive solar techniques. It can also provide the best opportunity to add solar water heating or even photoboltaics to a home. This article describes addition of such energy efficient plans to a home in terms of what is needed and what the benefits are: adding windows, North glass, east and west glass, south glass, daylighting, the roof, shingles and roofing tiles, walls and floors, solar hot water, photovoltaics. Two side bars discuss the sunplace: a passive solar room and angles and overhangs.

  19. Chromatin boundary elements organize genomic architecture and developmental gene regulation in Drosophila Hox clusters

    PubMed Central

    Ma, Zhibo; Li, Mo; Roy, Sharmila; Liu, Kevin J; Romine, Matthew L; Lane, Derrick C; Patel, Sapna K; Cai, Haini N

    2016-01-01

    The three-dimensional (3D) organization of the eukaryotic genome is critical for its proper function. Evidence suggests that extensive chromatin loops form the building blocks of the genomic architecture, separating genes and gene clusters into distinct functional domains. These loops are anchored in part by a special type of DNA elements called chromatin boundary elements (CBEs). CBEs were originally found to insulate neighboring genes by blocking influences of transcriptional enhancers or the spread of silent chromatin. However, recent results show that chromatin loops can also play a positive role in gene regulation by looping out intervening DNA and “delivering” remote enhancers to gene promoters. In addition, studies from human and model organisms indicate that the configuration of chromatin loops, many of which are tethered by CBEs, is dynamically regulated during cell differentiation. In particular, a recent work by Li et al has shown that the SF1 boundary, located in the Drosophila Hox cluster, regulates local genes by tethering different subsets of chromatin loops: One subset enclose a neighboring gene ftz, limiting its access by the surrounding Scr enhancers and restrict the spread of repressive histones during early embryogenesis; and the other loops subdivide the Scr regulatory region into independent domains of enhancer accessibility. The enhancer-blocking activity of these CBE elements varies greatly in strength and tissue distribution. Further, tandem pairing of SF1 and SF2 facilitate the bypass of distal enhancers in transgenic flies, providing a mechanism for endogenous enhancers to circumvent genomic interruptions resulting from chromosomal rearrangement. This study demonstrates how a network of chromatin boundaries, centrally organized by SF1, can remodel the 3D genome to facilitate gene regulation during development. PMID:27621770

  20. Nucleosome remodeling by the SWI/SNF complex is enhanced by yeast High Mobility Group Box (HMGB) proteins

    PubMed Central

    Hepp, Matias I.; Alarcon, Valentina; Dutta, Arnob; Workman, Jerry L.; Gutiérrez, José L.

    2014-01-01

    Regulation of gene expression at the level of transcription involves the concerted action of several proteins and protein complexes committed to dynamically alter the surrounding chromatin environment of a gene being activated or repressed. ATP-dependent chromatin remodeling complexes are key actors in chromatin remodeling, and the SWI/SNF complex is the founding member. While many studies have linked the action of these complexes to specific transcriptional regulation of a large number of genes and much is known about their catalytic activity, less is known about the nuclear elements that can enhance or modulate their activity. A number of studies have found that certain High Mobility Group (HMG) proteins are able to stimulate ATP-dependent chromatin remodeling activity, but their influence on the different biochemical outcomes of this activity is still unknown. In this work we studied the influence of the yeast Nhp6A, Nhp6B and Hmo1 proteins (HMGB family members) on different biochemical outcomes of yeast SWI/SNF remodeling activity. We found that all these HMG proteins stimulate the sliding activity of ySWI/SNF, while transient exposure of nucleosomal DNA and octamer transfer catalyzed by this complex are only stimulated by Hmo1. Consistently, only Hmo1 stimulates SWI/SNF binding to the nucleosome. Additionally, the sliding activity of another chromatin remodeling complex, ISW1a, is only stimulated by Hmo1. Further analyses show that these differential stimulatory effects of Hmo1 are dependent on the presence of its C-terminal tail, which contains a stretch of acidic and basic residues. PMID:24972368

  1. Transcriptional regulation of bone and joint remodeling by NFAT

    PubMed Central

    Sitara, Despina; Aliprantis, Antonios O.

    2010-01-01

    Summary Osteoporosis and arthritis are highly prevalent diseases and a significant cause of morbidity and mortality worldwide. These diseases result from aberrant tissue remodeling leading to weak, fracture-prone bones or painful, dysfunctional joints. The nuclear factor of activated T cells (NFAT) transcription factor family controls diverse biologic processes in vertebrates. Here, we review the scientific evidence that links NFAT-regulated gene transcription to bone and joint pathology. A particular emphasis is placed on the role of NFATs in bone resorption and formation by osteoclasts and osteoblasts, respectively. In addition, emerging data that connect NFATs with cartilage biology, angiogenesis, nociception, and neurogenic inflammation are explored. The goal of this article is to highlight the importance of tissue remodeling in musculoskeletal disease and situate NFAT-driven cellular responses within this context to inspire future research endeavors. PMID:20193006

  2. Molecular Mechanisms of Pulmonary Vascular Remodeling in Pulmonary Arterial Hypertension

    PubMed Central

    Leopold, Jane A.; Maron, Bradley A.

    2016-01-01

    Pulmonary arterial hypertension (PAH) is a devastating disease that is precipitated by hypertrophic pulmonary vascular remodeling of distal arterioles to increase pulmonary artery pressure and pulmonary vascular resistance in the absence of left heart, lung parenchymal, or thromboembolic disease. Despite available medical therapy, pulmonary artery remodeling and its attendant hemodynamic consequences result in right ventricular dysfunction, failure, and early death. To limit morbidity and mortality, attention has focused on identifying the cellular and molecular mechanisms underlying aberrant pulmonary artery remodeling to identify pathways for intervention. While there is a well-recognized heritable genetic component to PAH, there is also evidence of other genetic perturbations, including pulmonary vascular cell DNA damage, activation of the DNA damage response, and variations in microRNA expression. These findings likely contribute, in part, to dysregulation of proliferation and apoptosis signaling pathways akin to what is observed in cancer; changes in cellular metabolism, metabolic flux, and mitochondrial function; and endothelial-to-mesenchymal transition as key signaling pathways that promote pulmonary vascular remodeling. This review will highlight recent advances in the field with an emphasis on the aforementioned molecular mechanisms as contributors to the pulmonary vascular disease pathophenotype. PMID:27213345

  3. Protein-Remodeling Factors As Potential Therapeutics for Neurodegenerative Disease

    PubMed Central

    Jackrel, Meredith E.; Shorter, James

    2017-01-01

    Protein misfolding is implicated in numerous neurodegenerative disorders including amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease. A unifying feature of patients with these disorders is the accumulation of deposits comprised of misfolded protein. Aberrant protein folding can cause toxicity through a loss or gain of protein function, or both. An intriguing therapeutic approach to counter these disorders is the application of protein-remodeling factors to resolve these misfolded conformers and return the proteins to their native fold and function. Here, we describe the application of protein-remodeling factors to alleviate protein misfolding in neurodegenerative disease. We focus on Hsp104, Hsp110/Hsp70/Hsp40, NMNAT, and HtrA1, which can prevent and reverse protein aggregation. While many of these protein-remodeling systems are highly promising, their activity can be limited. Thus, engineering protein-remodeling factors to enhance their activity could be therapeutically valuable. Indeed, engineered Hsp104 variants suppress neurodegeneration in animal models, which opens the way to novel therapeutics and mechanistic probes to help understand neurodegenerative disease. PMID:28293166

  4. Chromatin endogenous cleavage and psoralen crosslinking assays to analyze rRNA gene chromatin in vivo.

    PubMed

    Griesenbeck, Joachim; Wittner, Manuel; Charton, Romain; Conconi, Antonio

    2012-01-01

    In eukaryotes, multiple copies of ribosomal RNA (rRNA) genes co-exist in two different chromatin states: actively transcribed (nucleosome depleted) chromatin, and nontranscribed (nucleosomal) chromatin. The presence of two rRNA gene populations compromises the interpretation of analyses obtained by the standard biochemical methods that are used to study chromatin structure (e.g., nuclease digestion and chromatin immunoprecipitation). Here, we provide a protocol to investigate the specific association of proteins with the two rRNA gene chromatin populations in vivo, using Saccharomyces cerevisiae as a model eukaryote.

  5. Flightless I (Drosophila) homolog facilitates chromatin accessibility of the estrogen receptor α target genes in MCF-7 breast cancer cells

    SciTech Connect

    Jeong, Kwang Won

    2014-04-04

    Highlights: • H3K4me3 and Pol II binding at TFF1 promoter were reduced in FLII-depleted MCF-7 cells. • FLII is required for chromatin accessibility of the enhancer of ERalpha target genes. • Depletion of FLII causes inhibition of proliferation of MCF-7 cells. - Abstract: The coordinated activities of multiple protein complexes are essential to the remodeling of chromatin structure and for the recruitment of RNA polymerase II (Pol II) to the promoter in order to facilitate the initiation of transcription in nuclear receptor-mediated gene expression. Flightless I (Drosophila) homolog (FLII), a nuclear receptor coactivator, is associated with the SWI/SNF-chromatin remodeling complex during estrogen receptor (ER)α-mediated transcription. However, the function of FLII in estrogen-induced chromatin opening has not been fully explored. Here, we show that FLII plays a critical role in establishing active histone modification marks and generating the open chromatin structure of ERα target genes. We observed that the enhancer regions of ERα target genes are heavily occupied by FLII, and histone H3K4me3 and Pol II binding induced by estrogen are decreased in FLII-depleted MCF-7 cells. Furthermore, formaldehyde-assisted isolation of regulatory elements (FAIRE)-quantitative polymerase chain reaction (qPCR) experiments showed that depletion of FLII resulted in reduced chromatin accessibility of multiple ERα target genes. These data suggest FLII as a key regulator of ERα-mediated transcription through its role in regulating chromatin accessibility for the binding of RNA Polymerase II and possibly other transcriptional coactivators.

  6. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes

    PubMed Central

    Biankin, Andrew V.; Waddell, Nicola; Kassahn, Karin S.; Gingras, Marie-Claude; Muthuswamy, Lakshmi B.; Johns, Amber L.; Miller, David K.; Wilson, Peter J.; Patch, Ann-Marie; Wu, Jianmin; Chang, David K.; Cowley, Mark J.; Gardiner, Brooke B.; Song, Sarah; Harliwong, Ivon; Idrisoglu, Senel; Nourse, Craig; Nourbakhsh, Ehsan; Manning, Suzanne; Wani, Shivangi; Gongora, Milena; Pajic, Marina; Scarlett, Christopher J.; Gill, Anthony J.; Pinho, Andreia V.; Rooman, Ilse; Anderson, Matthew; Holmes, Oliver; Leonard, Conrad; Taylor, Darrin; Wood, Scott; Xu, Qinying; Nones, Katia; Fink, J. Lynn; Christ, Angelika; Bruxner, Tim; Cloonan, Nicole; Kolle, Gabriel; Newell, Felicity; Pinese, Mark; Mead, R. Scott; Humphris, Jeremy L.; Kaplan, Warren; Jones, Marc D.; Colvin, Emily K.; Nagrial, Adnan M.; Humphrey, Emily S.; Chou, Angela; Chin, Venessa T.; Chantrill, Lorraine A.; Mawson, Amanda; Samra, Jaswinder S.; Kench, James G.; Lovell, Jessica A.; Daly, Roger J.; Merrett, Neil D.; Toon, Christopher; Epari, Krishna; Nguyen, Nam Q.; Barbour, Andrew; Zeps, Nikolajs; Kakkar, Nipun; Zhao, Fengmei; Wu, Yuan Qing; Wang, Min; Muzny, Donna M.; Fisher, William E.; Brunicardi, F. Charles; Hodges, Sally E.; Reid, Jeffrey G.; Drummond, Jennifer; Chang, Kyle; Han, Yi; Lewis, Lora R.; Dinh, Huyen; Buhay, Christian J.; Beck, Timothy; Timms, Lee; Sam, Michelle; Begley, Kimberly; Brown, Andrew; Pai, Deepa; Panchal, Ami; Buchner, Nicholas; De Borja, Richard; Denroche, Robert E.; Yung, Christina K.; Serra, Stefano; Onetto, Nicole; Mukhopadhyay, Debabrata; Tsao, Ming-Sound; Shaw, Patricia A.; Petersen, Gloria M.; Gallinger, Steven; Hruban, Ralph H.; Maitra, Anirban; Iacobuzio-Donahue, Christine A.; Schulick, Richard D.; Wolfgang, Christopher L.; Morgan, Richard A.; Lawlor, Rita T.; Capelli, Paola; Corbo, Vincenzo; Scardoni, Maria; Tortora, Giampaolo; Tempero, Margaret A.; Mann, Karen M.; Jenkins, Nancy A.; Perez-Mancera, Pedro A.; Adams, David J.; Largaespada, David A.; Wessels, Lodewyk F. A.; Rust, Alistair G.; Stein, Lincoln D.; Tuveson, David A.; Copeland, Neal G.; Musgrove, Elizabeth A.; Scarpa, Aldo; Eshleman, James R.; Hudson, Thomas J.; Sutherland, Robert L.; Wheeler, David A.; Pearson, John V.; McPherson, John D.; Gibbs, Richard A.; Grimmond, Sean M.

    2012-01-01

    Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis. PMID:23103869

  7. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes.

    PubMed

    Biankin, Andrew V; Waddell, Nicola; Kassahn, Karin S; Gingras, Marie-Claude; Muthuswamy, Lakshmi B; Johns, Amber L; Miller, David K; Wilson, Peter J; Patch, Ann-Marie; Wu, Jianmin; Chang, David K; Cowley, Mark J; Gardiner, Brooke B; Song, Sarah; Harliwong, Ivon; Idrisoglu, Senel; Nourse, Craig; Nourbakhsh, Ehsan; Manning, Suzanne; Wani, Shivangi; Gongora, Milena; Pajic, Marina; Scarlett, Christopher J; Gill, Anthony J; Pinho, Andreia V; Rooman, Ilse; Anderson, Matthew; Holmes, Oliver; Leonard, Conrad; Taylor, Darrin; Wood, Scott; Xu, Qinying; Nones, Katia; Fink, J Lynn; Christ, Angelika; Bruxner, Tim; Cloonan, Nicole; Kolle, Gabriel; Newell, Felicity; Pinese, Mark; Mead, R Scott; Humphris, Jeremy L; Kaplan, Warren; Jones, Marc D; Colvin, Emily K; Nagrial, Adnan M; Humphrey, Emily S; Chou, Angela; Chin, Venessa T; Chantrill, Lorraine A; Mawson, Amanda; Samra, Jaswinder S; Kench, James G; Lovell, Jessica A; Daly, Roger J; Merrett, Neil D; Toon, Christopher; Epari, Krishna; Nguyen, Nam Q; Barbour, Andrew; Zeps, Nikolajs; Kakkar, Nipun; Zhao, Fengmei; Wu, Yuan Qing; Wang, Min; Muzny, Donna M; Fisher, William E; Brunicardi, F Charles; Hodges, Sally E; Reid, Jeffrey G; Drummond, Jennifer; Chang, Kyle; Han, Yi; Lewis, Lora R; Dinh, Huyen; Buhay, Christian J; Beck, Timothy; Timms, Lee; Sam, Michelle; Begley, Kimberly; Brown, Andrew; Pai, Deepa; Panchal, Ami; Buchner, Nicholas; De Borja, Richard; Denroche, Robert E; Yung, Christina K; Serra, Stefano; Onetto, Nicole; Mukhopadhyay, Debabrata; Tsao, Ming-Sound; Shaw, Patricia A; Petersen, Gloria M; Gallinger, Steven; Hruban, Ralph H; Maitra, Anirban; Iacobuzio-Donahue, Christine A; Schulick, Richard D; Wolfgang, Christopher L; Morgan, Richard A; Lawlor, Rita T; Capelli, Paola; Corbo, Vincenzo; Scardoni, Maria; Tortora, Giampaolo; Tempero, Margaret A; Mann, Karen M; Jenkins, Nancy A; Perez-Mancera, Pedro A; Adams, David J; Largaespada, David A; Wessels, Lodewyk F A; Rust, Alistair G; Stein, Lincoln D; Tuveson, David A; Copeland, Neal G; Musgrove, Elizabeth A; Scarpa, Aldo; Eshleman, James R; Hudson, Thomas J; Sutherland, Robert L; Wheeler, David A; Pearson, John V; McPherson, John D; Gibbs, Richard A; Grimmond, Sean M

    2012-11-15

    Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.

  8. Sequence Features and Transcriptional Stalling within Centromere DNA Promote Establishment of CENP-A Chromatin

    PubMed Central

    Catania, Sandra; Pidoux, Alison L.; Allshire, Robin C.

    2015-01-01

    Centromere sequences are not conserved between species, and there is compelling evidence for epigenetic regulation of centromere identity, with location being dictated by the presence of chromatin containing the histone H3 variant CENP-A. Paradoxically, in most organisms CENP-A chromatin generally occurs on particular sequences. To investigate the contribution of primary DNA sequence to establishment of CENP-A chromatin in vivo, we utilised the fission yeast Schizosaccharomyces pombe. CENP-ACnp1 chromatin is normally assembled on ∼10 kb of central domain DNA within these regional centromeres. We demonstrate that overproduction of S. pombe CENP-ACnp1 bypasses the usual requirement for adjacent heterochromatin in establishing CENP-ACnp1 chromatin, and show that central domain DNA is a preferred substrate for de novo establishment of CENP-ACnp1 chromatin. When multimerised, a 2 kb sub-region can establish CENP-ACnp1 chromatin and form functional centromeres. Randomization of the 2 kb sequence to generate a sequence that maintains AT content and predicted nucleosome positioning is unable to establish CENP-ACnp1 chromatin. These analyses indicate that central domain DNA from fission yeast centromeres contains specific information that promotes CENP-ACnp1 incorporation into chromatin. Numerous transcriptional start sites were detected on the forward and reverse strands within the functional 2 kb sub-region and active promoters were identified. RNAPII is enriched on central domain DNA in wild-type cells, but only low levels of transcripts are detected, consistent with RNAPII stalling during transcription of centromeric DNA. Cells lacking factors involved in restarting transcription—TFIIS and Ubp3—assemble CENP-ACnp1 on central domain DNA when CENP-ACnp1 is at wild-type levels, suggesting that persistent stalling of RNAPII on centromere DNA triggers chromatin remodelling events that deposit CENP-ACnp1. Thus, sequence-encoded features of centromeric DNA create an

  9. To Remodel or To Build?

    ERIC Educational Resources Information Center

    Rosenblum, Todd

    2009-01-01

    The question of remodeling an existing house to make it wheelchair accessible or building a new barrier-free house is a difficult decision. This article presents some initial questions and considerations followed by a list of pros and cons for remodeling an existing house vs. building a new house.

  10. Proteomics of a fuzzy organelle: interphase chromatin

    PubMed Central

    Kustatscher, Georg; Hégarat, Nadia; Wills, Karen L H; Furlan, Cristina; Bukowski-Wills, Jimi-Carlo; Hochegger, Helfrid; Rappsilber, Juri

    2014-01-01

    Chromatin proteins mediate replication, regulate expression, and ensure integrity of the genome. So far, a comprehensive inventory of interphase chromatin has not been determined. This is largely due to its heterogeneous and dynamic composition, which makes conclusive biochemical purification difficult, if not impossible. As a fuzzy organelle, it defies classical organellar proteomics and cannot be described by a single and ultimate list of protein components. Instead, we propose a new approach that provides a quantitative assessment of a protein's probability to function in chromatin. We integrate chromatin composition over a range of different biochemical and biological conditions. This resulted in interphase chromatin probabilities for 7635 human proteins, including 1840 previously uncharacterized proteins. We demonstrate the power of our large-scale data-driven annotation during the analysis of cyclin-dependent kinase (CDK) regulation in chromatin. Quantitative protein ontologies may provide a general alternative to list-based investigations of organelles and complement Gene Ontology. PMID:24534090

  11. Roberts syndrome: New evidence supporting an altered metaphase chromatin structure

    SciTech Connect

    Shang, X.M.; Schultz, E.L.; Tonk, V.

    1994-09-01

    Roberts syndrome is a rare autosomal recessive disease clinically manifested in the newborn by mental and growth retardation, tetraphocomelia, and a variety of craniofacial abnormalities. Cell lines derived from RS patients exhibit subtle mutagen hypersensitivity and cytogenetic abnormalities which include random chromosome loss and the splaying of heterochromatic chromosomal regions. The latter, typically detected on C-banded metaphases, has been used prenatally for the diagnosis of RS. To gain further insights into the RS defect, we have examined a number of parameters related to metaphase chromatin structure, with observations as follows. (1) The heterochromatic splaying associated with RS was found to be visible on G- as well as C-banded metaphases. (2) Quantitative evaluations using fluorescence image analysis revealed that RS metaphase chromosomes bind DAPI less efficiently than chromosomes from normal cells. (3) Denaturation of chromosomal DNA with either a C-banding procedure or 70% formamide at 70{degree}C each produced an aberrant hybridization pattern on RS chromosomes in FISH experiments employing biotinylated total human DNA as probe. (4) RS cells exhibited a >3-fold increase in sensitivity to VM-26, a potent inhibitor of topoisomerase II. Collectively, the aforementioned data support the notion that the primary defect in RS results in an altered metaphase chromatin structure.

  12. No-Regrets Remodeling, 2nd Edition

    SciTech Connect

    2013-12-01

    No-Regrets Remodeling, sponsored by Oak Ridge National Laboratory, is an informative publication that walks homeowners and/or remodelers through various home remodeling projects. In addition to remodeling information, the publication provides instruction on how to incorporate energy efficiency into the remodeling process. The goal of the publication is to improve homeowner satisfaction after completing a remodeling project and to provide the homeowner with a home that saves energy and is comfortable and healthy.

  13. Chromatin plasticity as a differentiation index during muscle differentiation of C2C12 myoblasts.

    PubMed

    Watanabe, Tomonobu M; Higuchi, Sayaka; Kawauchi, Keiko; Tsukasaki, Yoshikazu; Ichimura, Taro; Fujita, Hideaki

    2012-02-24

    Skeletal muscle undergoes complicated differentiation steps that include cell-cycle arrest, cell fusion, and maturation, which are controlled through sequential expression of transcription factors. During muscle differentiation, remodeling of the epigenetic landscape is also known to take place on a large scale, determining cell fate. In an attempt to determine the extent of epigenetic remodeling during muscle differentiation, we characterized the plasticity of the chromatin structure using C2C12 myoblasts. Differentiation of C2C12 cells was induced by lowering the serum concentration after they had reached full confluence, resulting in the formation of multi-nucleated myotubes. Upon induction of differentiation, the nucleus size decreased whereas the aspect ratio increased, indicating the presence of force on the nucleus during differentiation. Movement of the nucleus was also suppressed when differentiation was induced, indicating that the plasticity of chromatin changed upon differentiation. To evaluate the histone dynamics during differentiation, FRAP experiment was performed, which showed an increase in the immobile fraction of histone proteins when differentiation was induced. To further evaluate the change in the histone dynamics during differentiation, FCS was performed, which showed a decrease in histone mobility on differentiation. We here show that the plasticity of chromatin decreases upon differentiation, which takes place in a stepwise manner, and that it can be used as an index for the differentiation stage during myogenesis using the state diagram developed with the parameters obtained in this study.

  14. Genome-Wide Views of Chromatin Structure

    PubMed Central

    Rando, Oliver J.; Chang, Howard Y.

    2010-01-01

    Eukaryotic genomes are packaged into a nucleoprotein complex known as chromatin, which affects most processes that occur on DNA. Along with genetic and biochemical studies of resident chromatin proteins and their modifying enzymes, mapping of chromatin structure in vivo is one of the main pillars in our understanding of how chromatin relates to cellular processes. In this review, we discuss the use of genomic technologies to characterize chromatin structure in vivo, with a focus on data from budding yeast and humans. The picture emerging from these studies is the detailed chromatin structure of a typical gene, where the typical behavior gives insight into the mechanisms and deep rules that establish chromatin structure. Important deviation from the archetype is also observed, usually as a consequence of unique regulatory mechanisms at special genomic loci. Chromatin structure shows substantial conservation from yeast to humans, but mammalian chromatin has additional layers of complexity that likely relate to the requirements of multicellularity such as the need to establish faithful gene regulatory mechanisms for cell differentiation. PMID:19317649

  15. Locus-Specific Epigenetic Remodeling Controls Addiction- and Depression-Related Behaviors (NN-A50213-T)

    PubMed Central

    Heller, Elizabeth A.; Cates, Hannah M.; Peña, Catherine J.; Sun, Haosheng; Shao, Ningyi; Feng, Jian; Golden, Sam A.; Herman, James P.; Walsh, Jessica J.; Mazei-Robison, Michelle; Ferguson, Deveroux; Knight, Scott; Gerber, Mark A.; Nievera, Christian; Han, Ming-Hu; Russo, Scott J.; Tamminga, Carol S.; Neve, Rachael L.; Shen, Li; Zhang, H. Steve; Zhang, Feng; Nestler, Eric J.

    2014-01-01

    Chronic exposure to drugs of abuse or stress regulates transcription factors, chromatin modifying enzymes, and histone posttranslational modifications in discrete brain regions. Due to the promiscuity of the enzymes involved, it has not yet been possible to obtain direct causal evidence to implicate the regulation of transcription and consequent behavioral plasticity by chromatin remodeling that occurs at a single gene. Here, we investigate the mechanism linking chromatin dynamics to neurobiological phenomena by applying engineered transcription factors to selectively modify chromatin at a specific gene in vivo. We found that histone methylation or acetylation at the FosB locus in nucleus accumbens—a brain reward region—is sufficient to control drug- and stress-evoked transcriptional and behavioral responses via interactions with the endogenous transcriptional machinery. This approach allows us to relate the epigenetic landscape at a given gene directly to regulation of its expression and to its subsequent effects on reward behavior. PMID:25347353

  16. Nuclear Phosphoinositide Regulation of Chromatin.

    PubMed

    Hamann, Bree L; Blind, Raymond D

    2017-03-03

    Phospholipid signaling has clear connections to a wide array of cellular processes, particularly in gene expression and in controlling the chromatin biology of cells. However, most of the work elucidating how phospholipid signaling pathways contribute to cellular physiology have studied cytoplasmic membranes, while relatively little attention has been paid to the role of phospholipid signaling in the nucleus. Recent work from several labs has shown that nuclear phospholipid signaling can have important roles that are specific to this cellular compartment. This review focuses on the nuclear phospholipid functions and the activities of phospholipid signaling enzymes that regulate metazoan chromatin and gene expression. In particular, we highlight the roles that nuclear phosphoinositides play in several nuclear-driven physiological processes, such as differentiation, proliferation, and gene expression. Taken together, the recent discovery of several specifically nuclear phospholipid functions could have dramatic impact on our understanding of the fundamental mechanisms that enable tight control of cellular physiology. This article is protected by copyright. All rights reserved.

  17. Identification of lamin B–regulated chromatin regions based on chromatin landscapes

    PubMed Central

    Zheng, Xiaobin; Kim, Youngjo; Zheng, Yixian

    2015-01-01

    Lamins, the major structural components of the nuclear lamina (NL) found beneath the nuclear envelope, are known to interact with most of the nuclear peripheral chromatin in metazoan cells. Although NL–chromatin associations correlate with a repressive chromatin state, the role of lamins in tethering chromatin to NL and how such tether influences gene expression have remained challenging to decipher. Studies suggest that NL proteins regulate chromatin in a context-dependent manner. Therefore understanding the context of chromatin states based on genomic features, including chromatin–NL interactions, is important to the study of lamins and other NL proteins. By modeling genome organization based on combinatorial patterns of chromatin association with lamin B1, core histone modification, and core and linker histone occupancy, we report six distinct large chromatin landscapes, referred to as histone lamin landscapes (HiLands)-red (R), -orange (O), -yellow (Y), -green (G), -blue (B), and -purple (P), in mouse embryonic stem cells (mESCs). This HiLands model demarcates the previously mapped lamin-associated chromatin domains (LADs) into two HiLands, HiLands-B and HiLands-P, which are similar to facultative and constitutive heterochromatins, respectively. Deletion of B-type lamins in mESCs caused a reduced interaction between regions of HiLands-B and NL as measured by emerin–chromatin interaction. Our findings reveal the importance of analyzing specific chromatin types when studying the function of NL proteins in chromatin tether and regulation. PMID:25995381

  18. A systematic analysis of factors localized to damaged chromatin reveals PARP-dependent recruitment of transcription factors

    PubMed Central

    Izhar, Lior; Adamson, Britt; Ciccia, Alberto; Lewis, Jedd; Pontano-Vaites, Laura; Leng, Yumei; Liang, Anthony C.; Westbrook, Thomas F.; Harper, J. Wade; Elledge, Stephen J.

    2015-01-01

    Localization to sites of DNA damage is a hallmark of DNA damage response (DDR) proteins. To identify new DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the ALS candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a PARP-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors and >70% of randomly tested transcription factors localized to sites of DNA damage and approximately 90% were PARP-dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding domain-dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP-dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins. PMID:26004182

  19. Control of Genome Integrity by RFC Complexes; Conductors of PCNA Loading onto and Unloading from Chromatin during DNA Replication

    PubMed Central

    Shiomi, Yasushi; Nishitani, Hideo

    2017-01-01

    During cell division, genome integrity is maintained by faithful DNA replication during S phase, followed by accurate segregation in mitosis. Many DNA metabolic events linked with DNA replication are also regulated throughout the cell cycle. In eukaryotes, the DNA sliding clamp, proliferating cell nuclear antigen (PCNA), acts on chromatin as a processivity factor for DNA polymerases. Since its discovery, many other PCNA binding partners have been identified that function during DNA replication, repair, recombination, chromatin remodeling, cohesion, and proteolysis in cell-cycle progression. PCNA not only recruits the proteins involved in such events, but it also actively controls their function as chromatin assembles. Therefore, control of PCNA-loading onto chromatin is fundamental for various replication-coupled reactions. PCNA is loaded onto chromatin by PCNA-loading replication factor C (RFC) complexes. Both RFC1-RFC and Ctf18-RFC fundamentally function as PCNA loaders. On the other hand, after DNA synthesis, PCNA must be removed from chromatin by Elg1-RFC. Functional defects in RFC complexes lead to chromosomal abnormalities. In this review, we summarize the structural and functional relationships among RFC complexes, and describe how the regulation of PCNA loading/unloading by RFC complexes contributes to maintaining genome integrity. PMID:28134787

  20. Control of Genome Integrity by RFC Complexes; Conductors of PCNA Loading onto and Unloading from Chromatin during DNA Replication.

    PubMed