DNA is wrapped around a histone octamer to form the basic unit of chromatin structure. During embryogenesis, dynamic changes of chromatin structure and chromatin modification occur after fertilization; subsequently, the epigenetic information is inherited through many rounds of the cell cycle. Thus, chromatin is essential for the determination of cell identity. Two strategies are used to modulate a chromatin environment: the covalent modification of histone tails and energy-dependent chromatin remodeling. The acetylation, methylation or phosphorylation of histone tails can have profound effects on chromatin structure and transcription (Jenuwein and Allis, 2001). Chromatin remodeling reactions are catalyzed by large protein complexes that use the energy of ATP hydrolysis to alter the structure or positioning of nucleosomes (Becker and Hörz, 2002; Clapier and Cairns, 2009). In addition to these events, histone variants play important roles in modulating chromatin structure (Henikoff and Ahmad, ...
Preparation of Chromatin Assembly Extracts from Preblastoderm Drosophila Embryos -- Analysis of Reconstituted Chromatin Using a Solid-Phase Approach -- In Vivo Chromatin Decondensation Assays: Molecular Genetic Analysis of Chromatin Unfolding Characteristics of Selected Proteins -- DNA Methyltransferase Probing of Chromatin Structure Within Populations and on Single Molecules -- Visualization of the Expression of HMGN Nucleosomal Binding Proteins in the Developing Mouse Embryo and in Adult Mouse Tissues -- Drug-Induced Premature Chromosome Condensation (PCC) Protocols: Cytogenetic Approaches in Mitotic Chromosome and Interphase Chromatin -- Analysis of DNA Topology in Yeast Chromatin -- Preparation and Analysis of Uniquely Positioned Mononucleosomes -- Monitoring DNA Breaks in Optically Highlighted Chromatin in Living Cells by Laser Scanning Confocal Microscopy -- Methods to Study Transcription-Coupled Repair in Chromatin -- Cytometric Analysis of DNA Damage: Phosphorylation of Histone H2AX as a ...
One of the longest standing problems in DNA repair is how cells relax chromatin in order to make DNA lesions accessible for global nucleotide excision repair (NER). Since chromatin has to be relaxed for efficient lesion detection, the key question is whether chromatin relaxation precedes lesion detection or vice versa. Chromatin accessibility factors have been proposed but not yet identified. Here we show that p53 acts as a chromatin accessibility factor, mediating UV-induced global chromatin relaxation. Using localized subnuclear UV irradiation, we demonstrate that chromatin relaxation is extended over the whole nucleus and that this process requires p53. We show that the sequence for initiation of global NER is as follows: transcription-associated lesion detection; p53-mediated global chromatin relaxation; and global lesion detection. The tumour suppressor p53 is crucial for genomic stability, a role partially explained by its pro-apoptotic capacity. We demonstrate here that p53 is also a ...
The THO complex is involved in transcription, genome stability, and messenger ribonucleoprotein (mRNP) formation, but its precise molecular function remains enigmatic. Under heat shock conditions, THO mutants accumulate large protein-DNA complexes that alter the chromatin density of target genes (heavy chromatin), defining a specific biochemical facet of THO function and a powerful tool of analysis. Here, we show that heavy chromatin distribution is dictated by gene boundaries and that the gene promoter is necessary and sufficient to convey THO sensitivity in these conditions. Single-molecule fluorescence insitu hybridization measurements show that heavy chromatin formation correlates with an unusually high firing pace of the promoter with more than 20 transcription events per minute. Heavy chromatin formation closely follows the modulation of promoter firing and strongly correlates with polymerase occupancy genome wide. We propose that the THO complex is required for tuning the dynamic of ...
Loss of function of CDKN2A/B, also known as INK4/ARF [encoding p16INK4A, p15INK4B, and p14ARF (mouse p19Arf)], confers susceptibility to cancers, whereas its up-regulation during organismal aging provokes cellular senescence and tissue degenerative disorders. To better understand the transcriptional regulation of p16INK4A, a CRISPR screen targeting open, noncoding chromatin regions adjacent to p16INK4A was performed in a human p16INK4A-P2A-mCherry reporter cell line. We identified a repressive element located in the 3′ region adjacent to the ARF promoter that controls p16INK4A expression via long-distance chromatin interactions. Coinfection of lentiviral dCas9-KRAB with selected single-guide RNAs against the repressive element abrogated the ARF/p16INK4A chromatin contacts, thus reactivating p16INK4A expression. Genetic CRISPR screening identified candidate transcription factors inhibiting p16INK4A regulation, including ZNF217, which was confirmed to bind the ARF/p16INK4A interaction loop. In ...
TY - JOUR. T1 - CAME. T2 - Identification of chromatin accessibility from nucleosome occupancy and methylome sequencing. AU - Piao, Yongjun. AU - Lee, Seong Keon. AU - Lee, Eun Joon. AU - Robertson, Keith D. AU - Shi, Huidong. AU - Ryu, Keun Ho. AU - Choi, Jeong Hyeon. PY - 2017/4/15. Y1 - 2017/4/15. N2 - Motivation: Chromatin accessibility plays a key role in epigenetic regulation of gene activation and silencing. Open chromatin regions allow regulatory elements such as transcription factors and polymerases to bind for gene expression while closed chromatin regions prevent the activity of transcriptional machinery. Recently, Methyltransferase Accessibility Protocol for individual templates-Bisulfite Genome Sequencing (MAPit-BGS) and nucleosome occupancy and methylome sequencing (NOMe-seq) have been developed for simultaneously profiling chromatin accessibility and DNA methylation on single molecules. Therefore, there is a great demand in developing computational methods to identify chromatin ...
Here, we introduce the 3D Genome Browser, http://3dgenome.org , which allows users to conveniently explore both their own and over 300 publicly available chromatin interaction data of different types. We design a new binary data format for Hi-C data that reduces the file size by at least a magnitude and allows users to visualize chromatin interactions over millions of base pairs within seconds. Our browser provides multiple methods linking distal cis-regulatory elements with their potential target genes. Users can seamlessly integrate thousands of other omics data to gain a comprehensive view of both regulatory landscape and 3D genome structure.
Two main chromatin assembly pathways ensure the proper transmission of chromatin organization and chromatin-based information throughout the cell cycle. A replication-dependent (RD) pathway that couples chromatin assembly to DNA synthesis and a replication-independent (RI) pathway. Whether these pathways contribute to the establishment of chromatin domains like heterochromatin or euchromatin by introducing modifications on histones or modulating chromatin structure remains unknown. Using Xenopus laevis egg extracts we monitored RD and RI chromatin assembly on single-stranded and double-stranded DNA templates. Even though RD assembly proceeded faster than RI assembly the histone content and saturation level with nucleosomes were similar. Despite these comparable topological features, the hydrodynamic behavior of both chromatin species in sucrose gradient centrifugation clearly differed. The RD assembled chromatin ran at lower sucrose concentrations than the RI created chromatin suggesting ...
Histones are responsible for packaging the genomes of almost all eukaryotes into fundamental repeating nucleosome units. The packaging must facilitate compaction into the cell nucleus but also enable dynamic access to the genome. A variety of mechanisms exist for targeting enzymes to undertake local opening of chromatin such as at active genes or for DNA repair. However, larger scale transitions in chromatin also occur where extended genome regions have altered chromatin organisation. This often involves abundant non-histone chromatin proteins that switch chromatin between states that are not well understood at the structural level. The contribution of highly basic non-histone chromatin proteins in vitro has been investigated using the HMGA2 protein implicated in human stem cell chromatin opening, and the Hematodinium DVNP protein which is suggested to replace histones as the dominant packaging protein in this dinoflagellate. These two proteins are compared to histone H1 which stabilises ...
1. Li X-Y, Thomas S, Sabo PJ, Eisen MB, Stamatoyannopoulos JA, Biggin MD. The role of chromatin accessibility in directing the widespread, overlapping patterns of Drosophila transcription factor binding. Genome Biol. 2011;12: R34. doi: 10.1186/gb-2011-12-4-r34 21473766. 2. Thurman RE, Rynes E, Humbert R, Vierstra J, Maurano MT, Haugen E, et al. The accessible chromatin landscape of the human genome. Nature. 2012;489: 75-82. doi: 10.1038/nature11232 22955617. 3. Klemm SL, Shipony Z, Greenleaf WJ. Chromatin accessibility and the regulatory epigenome. Nat Rev Genet. 2019;20: 207-220. doi: 10.1038/s41576-018-0089-8 30675018. 4. Wu J, Huang B, Chen H, Yin Q, Liu Y, Xiang Y, et al. The landscape of accessible chromatin in mammalian preimplantation embryos. Nature. 2016;534: 652-657. doi: 10.1038/nature18606 27309802. 5. Clark SJ, Argelaguet R, Kapourani C-A, Stubbs TM, Lee HJ, Alda-Catalinas C, et al. scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in ...
The different chromatin features display distinct spatial patterns. It is thus worthwhile to explore the relationship between these patterns and the level of gene expression. Making use of RNA-seq data obtained from the different stages of C. elegans, we quantified the expression level of each gene. For each bin, we then calculated the correlation between the gene expression levels and the average signals of each chromatin feature of the bin. Figure 2b shows the spatial variation of these correlation coefficients around TSSs and TTSs. According to the correlation patterns, there are two main types of chromatin features: ones that are positively correlated with gene expression (such as H3K79me1, H3K79me2 and H3K79me3); and ones that are negatively correlated with gene expression (such as H3K9me2 and H3K9me3). While some features show largely uniform correlations across the 16-kb regions, some others are more variable across the regions. For example, H3K79me2 has a high correlation coefficient ...
Biomedical applications of high-throughput sequencing methods generate a vast amount of data in which numerous chromatin features are mapped along the genome. The results are frequently analysed by creating binary data sets that link the presence/absence of a given feature to specific genomic loci. However, the nucleosome occupancy or chromatin accessibility landscape is essentially continuous. It is currently a challenge in the field to cope with continuous distributions of deep sequencing chromatin readouts and to integrate the different types of discrete chromatin features to reveal linkages between them. Here we introduce the NucTools suite of Perl scripts as well as MATLAB- and R-based visualization programs for a nucleosome-centred downstream analysis of deep sequencing data. NucTools accounts for the continuous distribution of nucleosome occupancy. It allows calculations of nucleosome occupancy profiles averaged over several replicates, comparisons of nucleosome occupancy landscapes between
Due to advances in molecular biology techniques, chromatin structure and function has re-emerged as a key research area in the investigation of gene regulation and expression. This indispensable new book provides the busy researcher with an overview of all the latest research in this important area. Topicality and breadth of coverage is assured by the contributions of an international group of over 30 leading scientists in this field. Contents list: Elements of chromatin structure: histones, nucleosomes, fibers; DNA structure: implications for chromatin structure and function; Replication and assembly; Promoter potentiation and activation: chromatin structure and transcriptional induction of heat shock genes; Initiation of expression: remodelling genes; Transcription on chromatin templates; Chromatin structure and epigenetic regulation in yeast; Epigenetic regulation in Drosophilia: a conspiracy of silence; Boundaries and domains; Epigenetic regulation in mammalian cells.Elgin, Sarah C. is the ...
There are variety of models and techniques to observe the presence of the 30nm fibers but it has been more observed that highly compacted chromatin fiber like 30nm fibers are not necessarily present for any gene regulation such as folding of DNA. Instead, 10 nm chromatin can be condensed enough into compacted domains through frequent bending and making 10nm fibers close to each other. In other words, it does not require to have 30nm fibers but is sufficient to have 10 nm chromatin fibers that is organized in genome to explain the complexities of nuclear organization and gene regulation. ...
Chromatin compacts DNA to an extreme extend and allows eukaryotic genome fit the size of the nucleus. On the other hand, however, it must process the ability to untighten DNA and to permit the cellular machinery access to genome. Chromatin consists of nucleosomes in which a protein core is constituted by four canonical histones H2A, H2B, H3, H4 and wrapped around by 147 bp of DNA. Histone variants, and the chromatin remodelling machinery, can reorganize the compaction of chromatin and thus be important for epigenetic regulation of gene expression.. Histone variant H2A.Z is a universal mark of dynamic nucleosomes. H2A.Z is essential for growth, development and viability of a number of species including mammals. H2A.Z plays critical roles in multiple biological processes including gene transcription and replication, DNA repair, and genome integrity. The chromatin incorporation of H2A.Z is catalysed by SRCAP, an ATP-dependent, multi-component chromatin remodelling complex. The YL1 subunit of SRCAP ...
The genetic information encoded by the DNA sequence, can be expressed in different ways. Genomic imprinting is an epigenetic phenomenon that results in monoallelic expression of imprinted genes in a parent of origin-dependent manner. Imprinted genes are frequently found in clusters and can share common regulatory elements. Most of the imprinted genes are regulated by Imprinting Control Regions (ICRs). H19/Igf2 region is a well known imprinted cluster, which is regulated by insulator function of ICR located upstream of the H19 gene. It has been proposed that the epigenetic control of the insulator function of H19 ICR involves organization of higher order chromatin interactions.. In this study we have investigated the role of post-translational modification in regulating insulator protein CTCF (CCCTC-binding factor). The results indicated novel links between poly(ADP-ribosyl)ation and CTCF, which are essential for regulating insulators function.. We also studied the higher order chromatin ...
TY - JOUR. T1 - Sequence and chromatin determinants of transcription factor binding and the establishment of cell type-specific binding patterns. AU - Srivastava, Divyanshi. AU - Mahony, Shaun. PY - 2019/1/1. Y1 - 2019/1/1. N2 - Transcription factors (TFs) selectively bind distinct sets of sites in different cell types. Such cell type-specific binding specificity is expected to result from interplay between the TFs intrinsic sequence preferences, cooperative interactions with other regulatory proteins, and cell type-specific chromatin landscapes. Cell type-specific TF binding events are highly correlated with patterns of chromatin accessibility and active histone modifications in the same cell type. However, since concurrent chromatin may itself be a consequence of TF binding, chromatin landscapes measured prior to TF activation provide more useful insights into how cell type-specific TF binding events became established in the first place. Here, we review the various sequence and chromatin ...
Abstract: To study the relation between chromatin structure and DNA function in detail it is necessary to have an in vitro procedure for assembling nucleosomes on a naked DNA template with properties similar to native chromatin. Such procedures exist for yeast and animal model systems but have not been developed for plants. The goal of this project was to lay the groundwork for developing a chromatin assembly extract from plants. Extracts from various plant materials were tested to determine their suitability for chromatin reconstitution. Tissues from plants are thought to have much higher levels of protease and nuclease activities than those of animals or yeast. Therefore, methods to determine the relative activity of proteases and nucleases had to be developed to determine if the template DNA, histones, and chromatin assembly proteins could survive the chromatin assembly reaction. Additionally, methods to streamline the isolation of maize nuclei and purification of histones were developed. ...
HI-TECH SOLUTIONS - Exporter, Importer, Manufacturer, Distributor & Supplier of Nuclear Chromatin Decondensation (N.C.D.) based in New Delhi, India
Employing a new algorithm for identifying differentially methylated regions (DMRs) from reduced representation bisulfite sequencing profiles, we identified 1972 hypermethylated and 3250 hypomethylated myogenic DMRs in a comparison of myoblasts (Mb) and myotubes (Mt) with 16 types of nonmuscle cell cultures. DMRs co-localized with a variety of chromatin structures, as deduced from ENCODE whole-genome profiles. Myogenic hypomethylation was highly associated with both weak and strong enhancer-type chromatin, while hypermethylation was infrequently associated with enhancer-type chromatin. Both myogenic hypermethylation and hypomethylation often overlapped weak transcription-type chromatin and Polycomb-repressed-type chromatin. For representative genes, we illustrate relationships between DNA methylation, the local chromatin state, DNaseI hypersensitivity, and gene expression. For example, MARVELD2 exhibited myogenic hypermethylation in transcription-type chromatin that overlapped a silenced promoter in Mb
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Bysani M, Agren R, Davegårdh C, Volkov P, Rönn T, Unneberg P, Bacos K, Ling C Sci Rep 9 (1) - [2019-12-00; online 2019-05-23] Impaired insulin secretion from pancreatic islets is a hallmark of type 2 diabetes (T2D). Altered chromatin structure may contribute to the disease. We therefore studied the impact of T2D on open chromatin in human pancreatic islets. We used assay for transposase-accessible chromatin using sequencing (ATAC-seq) to profile open chromatin in islets from T2D and non-diabetic donors. We identified 57,105 and 53,284 ATAC-seq peaks representing open chromatin regions in islets of non-diabetic and diabetic donors, respectively. The majority of ATAC-seq peaks mapped near transcription start sites. Additionally, peaks were enriched in enhancer regions and in regions where islet-specific transcription factors (TFs), e.g. FOXA2, MAFB, NKX2.2, NKX6.1 and PDX1, bind. Islet ATAC-seq peaks overlap with 13 SNPs associated with T2D (e.g. rs7903146, rs2237897, rs757209, rs11708067 and ...
Chromatin is the template on which DNA-associated transactions take place in eukaryotic organisms. Nucleosomes consisting of the four histones H2A, H2B, H3 and H4 each organize 150bp of DNA and constitute a first layer of chromatin. The three-dimensional organization of chromatin as well as histone post-translational modifications (PTMs) regulate recruitment of chromatin-associated effector proteins (effectors). Heterochromatin protein 1 (HP1) is an effector associated with silenced genome regions. HP1 recognizes histone H3 trimethylated at lysine 9 (H3 K9me3) and can dimerize. This results in a protein with two binding domains allowing multivalent engagement of target chromatin. HP1 can further promote chromatin condensation and inter-fiber contacts. The effector p53 binding protein (53BP1) is a key regulator in the DNA damage repair pathway. It is known to target a trio of PTMs; H4 dimethylated at K20 (H4 K20me2), H2A(.X) ubiquitylated at K15 (H2A.X K15ub) and H2A.X phosphorylated at S139 ...
TY - JOUR. T1 - Transcription of isolated mouse liver chromatin. AU - Bacheler, Lee T.. AU - Smith, Kirby D.. PY - 1976. Y1 - 1976. N2 - Analysis of RNA transcription from isolated mouse liver chromatin has been undertaken by means of RN A-excess hybridizations with small amounts of radioactive DNA. This analysis indicates that mouse liver chromatin is a restricted template for the in vitro synthesis of RNA complements to repetitive DNA, but more RNA species are synthesized than are found in the RNA isolated from mouse liver nuclei. Extraction with 0.5 M NaCl destroys the template restriction of isolated chromatin. RNA synthesized in vitro from DNA or chromatin templates by Escherichia coli RNA polymerase, as well as in vivo mouse liver nuclear RNA, were each hybridized to 125I-labeled DNA of high, intermediate, or low reiteration frequency. Chromatin-primed and nuclear RNA saturate a smaller portion of each DNA fraction than does DNA-primed RNA. However, chromatin-primed RNA saturates more high ...
Principal Investigator: Oliver Bell. In metazoans, packaging of genomic DNA into the nucleosomal protein scaffold of chromatin provides an opportunity to tightly regulate accessibility and readout of the genetic information. In particular, chemical modifications of nucleosomes and DNA have emerged as important determinants of genome accessibility. However, the dynamic regulation of chromatin state and its contribution to epigenetic inheritance of gene expression has remained enigmatic and a key challenge in the field of chromatin biology.. We have developed a novel technology that allows for rapid addition and removal of chromatin regulatory activities to a gene locus in any murine cell type. The Chromatin in vivo Assay (CiA) employs small molecules, which simultaneously bind two distinct peptide domains to induce dimerization between a chromatin modifier and a DNA binding protein. The CiA approach provides high temporal control allowing us to study the kinetics and epigenetic memory of histone ...
Chromatin structure is influenced by multiples factors, such as pH, temperature, nature and concentration of counterions, post-translational modifications of histones and binding of structural non-histone proteins. RNA is also known to contribute to the regulation of chromatin structure as chromatin-induced gene silencing was shown to depend on the RNAi machinery in S. pombe, plants and Drosophila. Moreover, both in Drosophila and mammals, dosage compensation requires the contribution of specific non-coding RNAs. However, whether RNA itself plays a direct structural role in chromatin is not known. Here, we report results that indicate a general structural role for RNA in eukaryotic chromatin. RNA is found associated to purified chromatin prepared from chicken liver, or cultured Drosophila S2 cells, and treatment with RNase A alters the structural properties of chromatin. Our results indicate that chromatin-associated RNAs, which account for 2%-5% of total chromatin-associated nucleic acids, are polyA−
Author Summary Histones are the main protein components of chromatin. The N-terminal tails of histones stick out from the nucleosomes, the building blocks of chromatin, and are involved in the regulation of all DNA-dependent processes. Only Histone H2A has an additional C-terminal tail and currently very little is known about the function of this tail. The H2A C-terminus protrudes from the nucleosome and is located where the DNA enters and leaves the nucleosome. We show here that it can interact with the linker histone H1 that is important for higher order chromatin structure. We also find that this tail is involved in regulating nucleosome dynamics and mobility of H2A itself. The C-terminal H2A tail has also an important function in regulating the activity of chromatin remodelers, enzymes that can reposition nucleosomes. Furthermore we find that cells expressing C-terminally truncated H2A are more sensitive to stress, demonstrating that this tail is important for cellular homeostasis. Together our
The arrangement of compact chromatin of G0 lymphocytes was studied in three-dimensional reconstructions of the ensemble of the chromatin and of individual compact chromatin bodies. Rat spleen was seri
Scientists in Canada and the United States have used three-dimensional imaging techniques to settle a long-standing debate about how DNA and structural proteins are packaged into chromatin fibers. The researchers, whose findings are published in EMBO reports, reveal that the mouse genome consists of 10-nm chromatin fibers but did not find evidence for the wider 30-nm fibers that were previously thought to be important components of the DNA architecture.. "DNA is an exceptionally long molecule that can reach several meters in length. This means it needs to be packaged into a highly compact state to fit within the limited space of the cell nucleus," said David Bazett-Jones, Senior Scientist at the Hospital for Sick Children, Toronto, and Professor at the University of Toronto, Canada. "For the past few decades, scientists have favored structural models for chromatin organization where DNA is first wrapped around proteins in nucleosomes. In one possible model, the strand of repeating nucleosomes is ...
We describe an assay for transposase-accessible chromatin using sequencing (ATAC-seq), based on direct in vitro transposition of sequencing adaptors into native chromatin, as a rapid and sensitive method for integrative epigenomic analysis. ATAC-seq captures open chromatin sites using a simple two-s …
Posttranslational modifications play a key role in recruiting chromatin remodeling and modifying enzymes to specific regions of chromosomes to modulate chromatin structure. Alc1 (amplified in liver cancer 1), a member of the SNF2 ATPase superfamily with a carboxy-terminal macrodomain, is encoded by an oncogene implicated in the pathogenesis of hepatocellular carcinoma. Here we show that Alc1 interacts transiently with chromatin-associated proteins, including histones and the poly(ADP-ribose) polymerase Parp1. Alc1 ATPase and chromatin remodeling activities are strongly activated by Parp1 and its substrate NAD and require an intact macrodomain capable of binding poly(ADP-ribose). Alc1 is rapidly recruited to nucleosomes in vitro and to chromatin in cells when Parp1 catalyzes PAR synthesis. We propose that poly(ADP-ribosyl)ation of chromatin-associated Parp1 serves as a mechanism for targeting a SNF2 family remodeler to chromatin. ...
Chromatin is a complex polymer molecule in eukaryotic cells, primarily consisting of DNA and histones. Many works have shown that the 3D folding of chromatin structure plays an important role in DNA expression. The recently proposed Chro- mosome Conformation Capture technologies, especially the Hi-C assays, provide us an opportunity to study how the 3D structures of the chromatin are organized. Based on the data from Hi-C experiments, many chromatin 3D structure modeling methods have been proposed. However, there is limited ground truth to validate these methods and no robust chromatin structure alignment algorithms to evaluate the performance of these methods. In our work, we first made a thorough literature review of 25 publicly available population Hi-C-based chromatin 3D structure modeling methods. Furthermore, to evaluate and to compare the performance of these methods, we proposed a novel data simulation method, which combined the population Hi-C data and single-cell Hi-C data without ad ...
Beijing, China - Chromatin remodeling proteins (chromatin remodelers) are essential and powerful regulators for critical DNA-templated cellular processes, such as DNA replication, recombination, gene transcription/repression, and DNA damage repair. These molecular and genetic processes are important for a wide spectrum of cellular functions, including cell cycle, death, differentiation, pluripotency, and genome integrity. Recently, many scientific reports have shown that chromatin remodeling proteins could be promising new targets for the treatment of human malignancy.. "This is a hot and exciting research topic for cancer researchers, and our article provides an updated understanding on the functions and mechanisms of chromatin remodelers in human cancers," says Dr. Chun Zhang, the principle investigator of the Department of Nuclear Medicine of Beijing Chao-Yang Hospital and Capital Medical University of China.. Chromatin remodeling is an energy-driven process in which chromatin remodelers use ...
TY - JOUR. T1 - ArchAlign. T2 - Coordinate-free chromatin alignment reveals novel architectures. AU - Lai, William K.M.. AU - Buck, Michael J.. PY - 2010/12/23. Y1 - 2010/12/23. N2 - To facilitate identification and characterization of genomic functional elements, we have developed a chromatin architecture alignment algorithm (ArchAlign). ArchAlign identifies shared chromatin structural patterns from high-resolution chromatin structural datasets derived from next-generation sequencing or tiled microarray approaches for user defined regions of interest. We validated ArchAlign using well characterized functional elements, and used it to explore the chromatin structural architecture at CTCF binding sites in the human genome. ArchAlign is freely available at http://www.acsu.buffalo.edu/~mjbuck/ArchAlign.html.. AB - To facilitate identification and characterization of genomic functional elements, we have developed a chromatin architecture alignment algorithm (ArchAlign). ArchAlign identifies shared ...
Transposable elements (TEs) are major structural components of eukaryotic genomes; however, mobilization of TEs generally has negative effects on the host genome. To counteract this threat, host cells have evolved genetic and epigenetic mechanisms that keep TEs silenced. One such mechanism involves the Piwi-piRNA complex, which represses TEs in animal gonads either by cleaving TE transcripts in the cytoplasm or by directing specific chromatin modifications at TE loci in the nucleus. Most Piwi-interacting RNAs (piRNAs) are derived from genomic piRNA clusters. There has been remarkable progress in our understanding of the mechanisms underlying piRNA biogenesis. However, little is known about how a specific locus in the genome is converted into a piRNA-producing site. In this review, we will discuss a possible link between chromatin boundaries and piRNA cluster formation.
Methyl-CpG-binding protein 2 (MeCP2) is generally considered to act as a transcriptional repressor, whereas recent studies suggest that MeCP2 is also involved in transcription activation. To gain insight into this dual function of MeCP2, we assessed the impact of MeCP2 on higher-order chromatin structure in living cells using mammalian cell systems harbouring a lactose operator and reporter gene-containing chromosomal domain to assess the effect of lactose repressor-tagged MeCP2 (and separate MeCP2 domains) binding in living cells. Our data reveal that targeted binding of MeCP2 elicits extensive chromatin unfolding. MeCP2-induced chromatin unfolding is triggered independently of the methyl-cytosine-binding domain. Interestingly, MeCP2 binding triggers the loss of HP1gamma at the chromosomal domain and an increased HP1gamma mobility, which is not observed for HP1alpha and HP1beta. Surprisingly, MeCP2-induced chromatin unfolding is not associated with transcriptional activation. Our study suggests ...
A primary challenge in the H3K27M field has been to resolve the contradictory observations indicating that PRC2 has a high affinity for H3K27M peptides, yet PRC2 and H3K27M are often mutually excluded from chromatin in H3K27M DIPG (4, 6, 9, 10). Our studies revealed that interaction of H3K27M with PRC2 is a dynamic process that cannot be captured by static, steady-state approaches. Namely, there is an initial phase after H3K27M is expressed and incorporated into chromatin, followed by PRC2 recruitment to H3K27M-containing chromatin, presumably due to its higher affinity toward H3K27M (Fig. 2C). However, in the next phase, PRC2 is released from H3K27M, as they do not colocalize at steady-state conditions in both isogenic 293 T-REx systems (e.g., this study, Figs. 2 and 3) and the H3K27M DIPG themselves (6). This dynamic model therefore accommodates both the finding of high H3K27M and PRC2 affinity in select assays and their failure to be stably colocalized on chromatin in cells. In line with the ...
The assembly of eukaryotic genomes into chromatin is a highly complex and delicate task; the cell must efficiently package and condense the DNA into the eukaryotic nucleus while maintaining specific regions of accessible chromatin to enable important functions with chromatin substrates. While the chromatin structure must remain highly dynamic in order to accommodate changes in the expression of some genes, it also serves to stably maintain the functional states of other genes through epigenetic mechanisms (45, 51). Recently, genetic and biochemical analyses have identified a broad class of multisubunit chromatin remodeling complexes which are likely to play important roles both in the process of chromatin opening and in the maintenance of chromatin in a dynamic or flexible state (26, 48, 53). These complexes remodel or reorganize nucleosomes in a wide range of in vitro assays which test for altered accessibility of nucleosomal DNA.. Nucleosome remodeling complexes are modular entities. The ...
In vivo studies of gene activation have revealed that changes in chromatin are often associated with transcriptional activation (reviewed in references 21,30, and 44). For example, in erythroid cells where each globin gene is sequentially expressed during development, the human β-globin locus is hypersensitive to nucleases (19, 22). In contrast, the entire β-globin locus is condensed into a nuclease-resistant chromatin structure in nonerythroid tissues, where the genes are not transcribed. Thus, transcriptionally active chromatin domains differ from the bulk of the genome in their susceptibility to digestion by nucleases. Detailed studies of nuclease-hypersensitive sites have suggested that interactions of sequence-specific DNA binding factors with chromatin alter the canonical nucleosomal structure (for example, see references2, 7, and 59). These conformational changes generate transcriptionally competent chromatin templates that are accessible to general transcription factors and the RNA ...
Global changes in chromatin accessibility may drive cancer progression by reprogramming transcription factor (TF) binding. In addition, histone acetylation readers such as bromodomain-containing protein 4 (BRD4) have been shown to associate with these TFs and contribute to aggressive cancers including prostate cancer (PC). Here, we show that chromatin accessibility defines castration-resistant prostate cancer (CRPC). We show that the deregulation of androgen receptor (AR) expression is a driver of chromatin relaxation and that AR/androgen-regulated bromodomain-containing proteins (BRDs) mediate this effect. We also report that BRDs are overexpressed in CRPCs and that ATAD2 and BRD2 have prognostic value. Finally, we developed gene stratification signature (BROMO-10) for bromodomain response and PC prognostication, to inform current and future trials with drugs targeting these processes. Our findings provide a compelling rational for combination therapy targeting bromodomains in selected patients in
The re-expression of fetal cardiac genes during disease requires coordinated chromatin remodeling. Local chromatin packing around the nucleosome protein complex is understood with atomic resolution; however, higher level properties of chromatin packing in the cardiovascular system are unknown. We hypothesized that nucleosome occupancy (i.e. exact positioning of nucleosomes at given loci) is a feature that regulates altered gene expression in hypertrophy. To investigate mechanisms that establish chromatin structure, genome-wide nucleosome location in heart was determined by Micrococcal Nuclease treatment followed by next-generation sequencing (MNase-Seq). A total of 137.4M Illumina reads were acquired from normal and hypertrophic myocardium, and Novoalign used to align them to the mouse genome mm9 with an average mapping percentage of 59%. Resulting occupancy profiles from normal and diseased cardiac myocytes exhibit global (i.e. chromosome scale) similarities, but local (i.e. gene/promoter ...
Nucleosomes are the core units of cellular chromatin and are comprised of 147 base pairs (bp) of DNA wrapped around an octamer of histone proteins. Proteins such as chromatin remodelers, transcription factors, and DNA repair proteins interact dynamically with chromatin to regulate access to DNA, control gene transcription, and maintain genome integrity. The extent of association with chromatin changes rapidly in response to stresses, such as immune activation, oxidative stress, or viral infection, resulting in downstream effects on chromatin conformation and transcription of target genes. To elucidate changes in the composition of proteins associated with chromatin under different conditions, we adapted existing protocols to isolate nuclei and fractionate cellular chromatin using a gradient of salt concentrations. The presence of specific proteins in different salt fractions can be assessed by Western blotting or mass spectrometry, providing insight into the degree to which they are associated with
This protocol describes the chromatin preparation from fresh or frozen tissues. The isolated chromatin can be used for chromatin immunoprecipitation assays using Diagenode&rsquo...
Head: Falk Martin, RNDr., Ph.D. ([email protected]; +420-728084060). Research Keywords. Chromatin Structure & Function in physiological and pathological processes. - higher-order chromatin structure in regulation of fundamental nuclear processes like transcription, replication, differentiation, DNA repair etc.; chromatin dynamics and epigenetic modifications; histone code. - higher-order chromatin structure in development of chromosomal aberrations and carcinogenesis (currently mostly focused on):. · molecular pathogenesis of leukemia, e.g. acute promyelocytic leukemia (APL). · mechanism of recurrent chromosomal aberrations in myelodysplastic syndromes (MDS). - telomere biology. - HMG proteins. DNA damage and repair, maintenance of the genome stability- mechanisms of DNA double strand break (DSB) induction, repair, and misrepair; influence of higher-order chromatin on DSB induction and repair efficiency; DSB repair during cell cycle, development, and pathological processes; unstable repeat ...
Epigenetic regulation of gene expression is a developing field of study with many potential therapeutic applications. Chromatin remodeling is necessary for proper mammalian development, and misregulation of this process is associated with many human diseases. Three mechanisms by which chromatin structure is modified include methylation of the DNA, covalent modification of histone tails, and repositioning of nucleosomes by ATP dependant chromatin remodeling enzymes. To further increase our knowledge of the mechanisms and proteins involved in the modification of chromatin structure I undertook two projects; the role of DNA methylation in the regulation of human β-globin gene expression, and analysis of the Chd6 ATPase-/- mouse. Methylation status of the human β-globin gene promoters correlates with the expression pattern of the individual genes. However an extensive locus wide analysis of the methylation pattern in primary human tissue has not been performed. We used bisulfite sequencing to ...
ChromHMM is software for learning and characterizing chromatin states. ChromHMM can integrate multiple chromatin datasets such as ChIP-seq data of various histone modifications to discover de novo the major re-occuring combinatorial and spatial patterns of marks. ChromHMM is based on a multivariate Hidden Markov Model that explicitly models the presence or absence of each chromatin mark. The resulting model can then be used to systematically annotate a genome in one or more cell types. By automatically computing state enrichments for large-scale functional and annotation datasets ChromHMM facilitates the biological characterization of each state. ChromHMM also produces files with genome-wide maps of chromatin state annotations that can be directly visualized in a genome browser. ...
Chromatin influences Human Immunodeficiency Virus (HIV) integration and replication. This review highlights critical host factors that influence chromatin structure and organization and that also impact HIV integration, transcriptional regulation and latency. Furthermore, recent attempts to target chromatin associated factors to reduce the HIV proviral load are discussed.
A key control point in inducible gene expression involves reorganization of chromatin structure across regulatory regions of genes to allow access for the transcriptional machinery. Here we have mapped chromatin accessibility across the promoter region of the GM-CSF gene in unstimulated and stimulated EL-4 T cells and several important points have emerged. First, the entire region that was mapped from −633 to +164 showed intrinsic accessibility to both MNase and restriction enzymes. This is in stark contrast to a recent study on the IL-2 promoter in the same cell type where we showed that there is no accessibility across a large region of the IL-2 gene implying a more "closed" chromatin configuration for this gene (30). Second, two regions of the GM-CSF upstream sequence were relatively inaccessible to digestion in these assays and such regions may represent preferred nucleosome positions. The possibility that nucleosomes are preferentially located in these positions (centered on −100 and ...
How does chromatin structure determine the fate of transcripts? How do transcripts direct changes in chromatin? Our lab is investigating the role of chromatin and RNA in controlling transcriptional and post-transcriptional gene regulatory processes. We are focusing on gaining mechanistic insights into how small non-coding RNAs induce changes in chromatin and transcription in animals. We are also aiming to identify the role of chromatin on post-transcriptional gene regulatory processes. We use an interdisciplinary approach combining biochemistry, genetics, cell, molecular and computational biology and functional genomics as key tools to tackle these exciting questions.. ...
The three-dimensional (3D) organization of the genome (chromatin) plays an important role in key cellular processes such as DNA replication, repair, transcription [1], and epigenetic inheritance [2]. Links between chromatin architecture and diseases such as cancer are being established [3]. Unlike most proteins that adopt the same unique 3D shapes in all cells, the conformational states of the chromatin fiber are not nearly as compact or ordered and are stochastic to some degree. Remarkably, several features of chromatin folding appear to be universal. Chromosomal territories, in which each chromosome occupies a distinct region of the nucleus, have been observed in numerous organisms and cell types, such as yeast [4], human [5], D. melanogaster (fruit fly) [6-8], mouse [9], and Arabidopsis [10]. Chromosome interactions, both within (intra) chromosomes and between (inter) chromosomes, have been observed microscopically [6, 8] and inferred using cross-linking techniques [11] such as the Hi-C ...