3C technologies have enabled studies of the dynamics of high-order chromatin structures and long-range chromatin interactions (26-30). However, how these long-range interactions contribute to biological processes and human diseases is not well understood. Our research has focused on an enhancer containing the PCa risk-associated rs55958994 SNP, integrating higher-order chromatin structure data, gene expression profiling, and functional assays. Our results suggest that the rs55958994-containing enhancer regulates PCa progression through long-range interactions with multiple genes. Among these genes, ITGA5, CDH23, and CNTN1 were verified to be both regulated by the rs55958994-containing enhancer and associated with PCa progression; deletion of the enhancer region in PCa cells induced down-regulation of these target genes and led to defects in tumor initiation and migration and loss of CSCs.. Previous eQTL (expression quantitative trait locus) analyses suggested that rs55958994 is linked to KRT8 ...
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
Chromatin fibers have been observed and measured in frozen hydrated sections of three types of cell (chicken erythrocytes and sperm of Patiria miniata and Thyone briareus) representing an approximately 20-bp range of nucleosomal repeat lengths. For sperm of the starfish P. miniata, it was possible to obtain images of chromatin fibers from cells that were swimming in seawater up to the moment of cryo-immobilization, thus providing a record of the native morphology of the chromatin of these cells. Glutaraldehyde fixation produced no significant changes in the ultrastructure or diameter of chromatin fibers, and fiber diameters observed in cryosections were similar to those recorded after low temperature embedding in Lowicryl K11M. Chromatin fiber diameters measured from cryosections of the three types of nuclei were similar, a striking contrast to the situation for chromatin isolated from these cell types, where a strong positive correlation between diameter and nucleosomal repeat length has been ...
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. ...
Search and download thousands of Swedish university essays. Full text. Free. Essay: Moving Away from Proximal Ligation to Study Higher Order Chromatin Complexes at High Resolution.
Author(s): Chen, Song; Lake, Blue B; Zhang, Kun | Abstract: Single-cell RNA sequencing can reveal the transcriptional state of cells, yet provides little insight into the upstream regulatory landscape associated with open or accessible chromatin regions. Joint profiling of accessible chromatin and RNA within the same cells would permit direct matching of transcriptional regulation to its outputs. Here, we describe droplet-based single-nucleus chromatin accessibility and mRNA expression sequencing (SNARE-seq), a method that can link a cells transcriptome with its accessible chromatin for sequencing at scale. Specifically, accessible sites are captured by Tn5 transposase in permeabilized nuclei to permit, within many droplets in parallel, DNA barcode tagging together with the mRNA molecules from the same cells. To demonstrate the utility of SNARE-seq, we generated joint profiles of 5,081 and 10,309 cells from neonatal and adult mouse cerebral cortices, respectively. We reconstructed the transcriptome and
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 epigenetics landscape of cells plays a key role in the establishment of cell-type specific gene expression programs characteristic of different cellular phenotypes. Different experimental procedures have been developed to obtain insights into the accessible chromatin landscape including DNase-seq, FAIRE-seq and ATAC-seq. However, current downstream computational tools fail to reliably determine regulatory region accessibility from the analysis of these experimental data. In particular, currently available peak calling algorithms are very sensitive to their parameter settings and show highly heterogeneous results, which hampers a trustworthy identification of accessible chromatin regions. Here, we present a novel method that predicts accessible and, more importantly, inaccessible gene-regulatory chromatin regions solely relying on transcriptomics data, which complements and improves the results of currently available computational methods for chromatin accessibility assays. We trained a hierarchical
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
TY - JOUR. T1 - The Hsp90 Molecular Chaperone Regulates the Transcription Factor Network Controlling Chromatin Accessibility. AU - Gvozdenov, Zlata. AU - Bendix, Lindsey D.. AU - Kolhe, Janhavi. AU - Freeman, Brian C.. PY - 2019/12/6. Y1 - 2019/12/6. N2 - Genomic events including gene regulation and chromatin status are controlled by transcription factors. Here we report that the Hsp90 molecular chaperone broadly regulates the transcription factor protein family. Our studies identified a biphasic use of Hsp90 in which early inactivation (15 min) of the chaperone triggered a wide reduction of DNA binding events along the genome with concurrent changes to chromatin structure. Long-term loss (6 h) of Hsp90 resulted in a decline of a divergent yet overlaying pool of transcription factors that produced a distinct chromatin pattern. Although both phases involve protein folding, the early point correlated with Hsp90 acting in a late folding step that is critical for DNA binding function, whereas ...
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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
Background Chromatin organisation affects gene expression and regional mutation frequencies and contributes to carcinogenesis. Aberrant organisation of DNA has been correlated with cancer prognosis in analyses of the chromatin component of tumour cell nuclei using image texture analysis. As yet, the methodology has not been sufficiently validated to permit its clinical application. We aimed to define and validate a novel prognostic biomarker for the automatic detection of heterogeneous chromatin organisation. Methods Machine learning algorithms analysed the chromatin organisation in 461 000 images of tumour cell nuclei stained for DNA from 390 patients (discovery cohort) treated for stage I or II colorectal cancer at the Aker University Hospital (Oslo, Norway). The resulting marker of chromatin heterogeneity, termed Nucleotyping, was subsequently independently validated in six patient cohorts: 442 patients with stage I or II colorectal cancer in the Gloucester Colorectal Cancer Study (UK); 391 ...
Eukaryotic centromeres and telomeres are specialized chromosomal regions that share one common characteristic: their underlying DNA sequences are assembled into heritably repressed chromatin. Silent chromatin in budding and fission yeast is composed of fundamentally divergent proteins tat assemble very different chromatin structures. However, the ultimate behaviour of silent chromatin and the pathways that assemble it seem strikingly similar among Saccharomyces cerevisiae (S. cerevisiae), Schizosaccharomyces pombe (S. pombe) and other eukaryotes. Thus, studies in both yeasts have been instrumental in dissecting the mechanisms that establish and maintain silent chromatin in eukaryotes, contributing substantially to our understanding of epigenetic processes. In this review, we discuss current models for the generation of heterochromatic domains at centromeres and telomeres in the two yeast species.. ...
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 ...
The Chromatin, Replication and Chromosomal Stability Conference took place on June 20-21 in Stockholm, Sweden. In this article, I outline the broad scientific program of the meeting which reflected the wide diversity in epigenetics research. Distinct histone modifications are linked with specific chromatin structures and intranuclear positioning, thereby impacting replication timing and replication initiation, which in turn are related to gene expression and cell differentiation. Interference in any of these interconnected mechanisms can result in DNA breakage and lead to the activation of repair pathways. The DNA repair mechanisms again are influenced by the chromatin structure. In summary, the conference highlighted the functional implication of epigenetics in chromatin compaction, transcription regulation, replication control and DNA repair. The tight control of all these mechanisms defines the final cellular character.
In this report, we show that p53 is organized into two epigenetic domains with distinct chromatin structures, CpG methylation, histone compositions, and modifications, as well as associated regulatory factors. The open chromatin conformation, unmethylated CpG, and histone modifications associated the first domain are characteristic of an active chromatin, whereas the characteristics of the second domain, with full CpG methylation, nuclease resistance, and unacetylated histones, are reminiscent of inactive chromatin.. The promoter region has been known to be organized into a nucleosome-free structure (32), but whether this structure is due to the absence of histones or to a novel organization of the histone-DNA complex has not been investigated. The ChIP technique allows us to address this question, and we show that nuclease hypersensitivity and the absence of a nucleosomal DNA ladder in epigenetic domain I are associated with the depletion of the H2a/H2b dimer. Previous studies have shown that ...
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 ...
Methylation of DNA is one of the earliest described epigenetic modifications. Hypermethylation is associated with gene silencing, while the inhibition of methylation is generally associated with reactivating silenced genes. The packaging of DNA in the nucleus into chromatin also plays a role in regulating gene expression. We sought to understand the crosstalk between changes in methylation status of the genome and changes in chromatin structure. 5-azacytidine (5-azaC), a potent DNA methytransferase inhibitor, has recently generated interest as a potential anti-cancer drug, possibly functioning by reactivating silenced tumor suppressor genes. We treated the hematologic cancer cell lines U-937 and T-HP1 with 5-aza for varying lengths of time. We then harvested DNA for methylation studies, RNA for gene expression studies and chromatin for nuclease accessibility studies. The chromatin accessibility was further measured at two different levels of resolution; the domain level (10s of kb) and ...
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 ...
TY - JOUR. T1 - Effects of chromatin structure on the enzymatic and DNA binding functions of DNA methyltransferases DNMT1 and Dnmt3a in vitro. AU - Robertson, Andrea K.. AU - Geiman, Theresa M.. AU - Sankpal, Umesh Tanaji. AU - Hager, Gordon L.. AU - Robertson, Keith D.. PY - 2004/9/10. Y1 - 2004/9/10. N2 - DNA methylation is an epigenetic modification of the genome critical for numerous processes, including transcriptional repression and maintenance of chromatin structure. Recent studies have revealed connections between DNA methylation and other epigenetic modifications such as ATP-dependent chromatin remodeling. It remains unclear, however, exactly how chromatin and epigenetic chromatin modifications affect the biological properties of the DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B). Using a highly purified system and the 5S rDNA gene as free DNA or assembled into a mononucleosome, we have compared the effects of chromatin structure on DNMT1 and Dnmt3a. The catalytic efficiency for ...
TY - JOUR. T1 - Modeling apoptotic chromatin condensation in normal cell nuclei. Requirement for intranuclear mobility and actin involvement. AU - Widlak, Piotr. AU - Palyvoda, Olena. AU - Kumala, Slawomir. AU - Garrard, William T.. N1 - Copyright: Copyright 2008 Elsevier B.V., All rights reserved.. PY - 2002/6/14. Y1 - 2002/6/14. N2 - Hallmarks of the terminal stages of apoptosis are genomic DNA fragmentation and chromatin condensation. Here, we have studied the mechanism of condensation both in vitro and in vivo. We found that DNA fragmentation per se of isolated nuclei from non-apoptotic cells induced chromatin condensation that closely resembles the morphology seen in apoptotic cells, independent of ATP utilization, at physiological ionic strengths. Interestingly, chromatin condensation was accompanied by release of nuclear actin, and both condensation and actin release could be blocked by reversibly pretreating nuclei with Ca2+, Cu2+, diamide, or low pH, procedures shown to stabilize ...
BACKGROUND: ATP-dependent chromatin remodelling complexes are responsible for establishing and maintaining the positions of nucleosomes. Chromatin remodellers are targeted to chromatin by transcription factors and non-coding RNA to remodel the chromatin into functional states. However, the influence of chromatin remodelling on shaping the functional epigenome is not well understood. Moreover, chromatin remodellers have not been extensively explored as a collective group across two-dimensional and three-dimensional epigenomic layers. RESULTS: Here, we have integrated the genome-wide binding profiles of eight chromatin remodellers together with DNA methylation, nucleosome positioning, histone modification and Hi-C chromosomal contacts to reveal that chromatin remodellers can be stratified into two functional groups. Group 1 (BRG1, SNF2H, CHD3 and CHD4) has a clear preference for binding at actively marked chromatin and Group 2 (BRM, INO80, SNF2L and CHD1) for repressively marked chromatin. We find
DNA repair in the eukaryotic cell disrupts local chromatin organization. To investigate whether the resetting of nucleosomal arrays can be linked to the repair process, we developed model systems, with both Xenopus egg extract and human cell extracts, to follow repair and chromatin assembly in parallel on circular DNA templates. Both systems were able to carry out nucleotide excision repair of DNA lesions. We observed that UV-dependent DNA synthesis occurs simultaneously with chromatin assembly, strongly indicating a mechanistic coupling between the two processes. A complementation assay established that chromatin assembly factor I (CAF1) is necessary for this repair associated chromatin formation.. ...
Even relatively minor errors in chromatin remodeling during spermiogenesis are associated with sperm DNA damage and infertility, yet little is known about the etiology. Mice with severe NPYq deletions are infertile due to severe sperm differentiation defects (Ward and Burgoyne, 2006; Yamauchi et al., 2009). We have recently observed that sperm from these mice presented abnormal chromatin packaging and DNA damage. Moreover, when these sperm were injected into the oocytes, a significant increase of oocyte arrest at pronuclei stage and of chromosome aberrations in the fertilized eggs were noted (Yamauchi et al., 2010). Here we provide evidence that the deficiency of NPYq encoded gene Sly is associated with sperm DNA damage and poor sperm chromatin condensation, and propose that SLY plays a role in spermatid-specific chromatin remodeling.. How can Sly/SLY be involved in sperm DNA damage phenotype? SLY protein has been shown to control the postmeiotic expression of ,100 genes, the majority of which ...
Covalent modifications of histones and histone variants have great influence on chromatin structure, which is involved in the transcriptional regulation of gene expression. Chromatin immunoprecipitation (ChIP) is a powerful tool for studying in vivo DNA-histone interactions. Strawberry is a model for Rosaceae and non-climacteric fruits, in which histone modifications have been implicated to affect fruit development and ripening. However, a validated ChIP method has not been reported in strawberry, probably due to its high levels of polysaccharides which affect the quality of prepared chromatin and the efficiency of immunoprecipitation. We describe a native chromatin immunoprecipitation (N-ChIP) protocol suitable for strawberry by optimizing the parameters for nuclei isolation, chromatin extraction, DNA fragmentation and validation analysis using quantitative real-time PCR (qRT-PCR). The qRT-PCR results show that both the active mark H3K36me3 and the silent mark H3K9me2 are efficiently immunoprecipitated
The chromatin remodeling complexes alter chromatin structures. They remodel nucleosomes in ATP-dependent manner and have essential roles in DNA damage repair, recombination, replication and transcriptional control. Increasing evidences indicate that subunits of chromatin remodelers are mutated and/or deregulated in a number of human cancers, and how they influence the cancer gene expression program during cancer initiation and progression is becomming clearer. Therefore, chromatin remodeling complexes arose as promising new targets for the treatment of human cancers. In this review, chromatin remodeling complexes, their epigenetic reader domains and available inhibitors are described. The insights into the misregulated chromatin remodelers pathways in human malignancies and the novel approach targeting deregulated chromatin remodelers to improve chemotherapy efficiency are discussed. ...
The activity of a single gene is influenced by the composition of the chromatin in which it is embedded. Nucleosome turnover, conformational dynamics, and covalent histone modifications each induce changes in the structure of chromatin and its affinity for regulatory proteins. The dynamics of histone modifications and the persistence of modification patterns for long periods are still largely unknown. In this study, we present a stochastic mathematical model that describes the molecular mechanisms of histone modification pattern formation along a single gene, with non-phenomenological, physical parameters. We find that diffusion and recruitment properties of histone modifying enzymes together with chromatin connectivity allow for a rich repertoire of stochastic histone modification dynamics and pattern formation. We demonstrate that histone modification patterns at a single gene can be established or removed within a few minutes through diffusion and weak recruitment mechanisms of histone modification
TY - JOUR. T1 - Alleviation of historic H1-mediated transcriptional repression and chromatin compaction by the acidic activation region in chromosomal protein HMG-14. AU - Ding, Hanfei. AU - Bustin, Michael. AU - Hansen, Ulla. PY - 1997/10/1. Y1 - 1997/10/1. N2 - Histone H1 promotes the generation of a condensed, transcriptionally inactive, higher-order chromatin structure. Consequently, historic H1 activity must be antagonized in order to convert chromatin to a transcriptionally competent, more extended structure. Using simian virus 40 minichromosomes as a model system, we now demonstrate that the nonhistoric chromosomal protein HMG-14, which is known to preferentially associate with active chromatin, completely alleviates historic H1-mediated inhibition of transcription by RNA polymerase II. HMG-14 also partially disrupts histone H1-dependent compaction of chromatin. Both the transcriptional enhancement and chromatin-unfolding activities of HMG-14 are mediated through its acidic, C-terminal ...
De novo chromatin assembly maintains histone density on the daughter strands in the wake of the replication fork. The heterotrimer chromatin assembly factor 1 (CAF-1) couples DNA replication to histone deposition in vitro, but is not essential for yeast cell proliferation. Depletion of CAF-1 in human cell lines demonstrated that CAF-1 was required for efficient progression through S-phase. Cells lacking CAF-1 accumulated in early and mid S-phase and replicated DNA slowly. The checkpoint kinase Chk1, but not Chk2, was phosphorylated in response to CAF-1 depletion, consistent with a DNA replication defect. CAF-1-depleted cell extracts completely lacked DNA replication-coupled chromatin assembly activity, suggesting that CAF-1 is required for efficient S-phase progression in human cells. These results indicate that, in contrast to yeast, human CAF-1 is necessary for coupling chromatin assembly with DNA replication.. ...
TY - JOUR. T1 - Analysis of chromatin structure by in vivo formaldehyde cross-linking. AU - Orlando, Valerio. AU - Strutt, Helen. AU - Paro, Renato. PY - 1997/2. Y1 - 1997/2. N2 - Recent advances leave no doubt that higher order chromatin structures play a fundamental role in many developmentally important mechanisms of gene regulation. In particular analyses in genetic model systems like yeast and Drosophila uncovered novel proteins that are involved in the regulation of chromatin structures. Many of these proteins do not bind directly to DNA but interact in large multimeric complexes. To identify the DNA elements regulated by these multiprotein complexes, alternative approaches to the standard methods of DNA-protein analysis had to be devised. Here we present a method that preserves the architecture of the higher order chromatin structures by crosslinking cells in vivo with formaldehyde. An immunoprecipitation strategy is then used to identify the DNA targets of chromosomal proteins of ...
Current models suggest that tissue-specific genes are arranged in discrete, independently controlled segments of chromatin referred to as regulatory domains. Transition from a closed to open chromatin structure may be an important step in the regulation of gene expression. To determine whether the human alpha-globin cluster, like the beta-globin cluster, lies within a discrete, erythroid-specific domain, we have examined the long-range genomic organization and chromatin structure around this region. The alpha genes lie adjacent to at least four widely expressed genes. The major alpha-globin regulatory element lies 40 kb away from the cluster within an intron of one of these genes. Therefore, unlike the beta cluster, cis-acting sequences controlling alpha gene expression are dispersed within a region of chromatin that is open in both erythroid and nonerythroid cells. This implies a difference in the hierarchical control of alpha- and beta-globin expression.
P-266. Study question: What is the impact of selecting spermatozoa with the highest chromatin integrity on ICSI outcomes? Summary answer: We selected spermatozoa with the highest progressive motility and chromatin integrity by microfluidic sperm selection (MFSS) and achieved superior implantation and delivery rates. What is known already: Sperm preparation methods aim at providing specimens for insemination with the highest progressive motility independent of phenotypic and genomic integrity. It has recently been recognized that a microfluidics device yielded spermatozoa with the highest progressive motility as well as superior chromatin integrity. Here we compared two sperm selection methods: density gradient centrifugation (DGC) and MFSS. Study design, size, duration: From October 2016 to January 2020, ejaculates that were processed by DGC and MFSS for ICSI treatment from 8 consenting men were screening for DNA fragmentation by TUNEL. In addition, ejaculates from 22 men were processed solely ...
A fraction of rat-liver chromatin that is transcriptionally active in vivo has been purified 6- to 7-fold over whole chromatin. This was accomplished by selectively shearing chromatin with DNase II followed by fractionating the released portion on the basis of its solubility properties in 2 mM MgCl2. The resulting soluble material comprises 11% of the total chromatin DNA and is impoverished in histone and enriched in nonhistone protein. Compared with unsheared chromatin, this minor fraction exhibits marked differences in chromosomal protein species. DNA renaturation studies indicate that this fraction is composed of a specific subset of whole genomal DNA sequences. Furthermore, DNA·RNA hybridization experiments suggest that almost 60% of the nonrepetitious DNA sequences of this minor fraction could code for cellular RNA. ...
DNase I hypersensitive site assay is perhaps the most widely used assay for detection of changes in chromatin structure. Studies of many spatially or temporally regulated genes, such as P-globin, have revealed the correlation of formation of DNase I hypersensitive sites with the status of transcription (22). This assay has also been used extensively in analyses of chromatin structure in injected Xenopus oocytes (24). Although the mechanism for the formation of DNase I hypersensitive sites is still not fully understood, the detection of DNase I hypersensitive sites are widely interpreted as results of chromatin remodeling induced by the binding of transcription factors.. 1. Inject 15-20 oocytes with DNA and with or without mRNA encoding TR/RXR (100 ng/ pL, 27.6 nL/oocyte) and treated with or without 50 nM T3. Incubate the oocytes overnight at 18°C incubator.. 2. Collect healthy oocytes and wash the oocytes once with 500 pL of MBSH buffer.. 3. To 15-20 oocytes, add 240 pL of DNase I buffer. ...
The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at entry sites that contain a consensus sequence motif (MSL recognition element or MRE). However, this motif is only similar to 2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex) and female Kc cells (which lack the complex), we find that the presence of active chromatin modifications, together with an elevated local GC ...
Retroviral integration depends on the interaction between intasomes, host chromatin and cellular targeting cofactors as LEDGF/p75 or BET proteins. Previous studies indicated that the retroviral integrase, by itself, may play a role in the local integration site selection within nucleosomal target DNA. We focused our study on this local association by analyzing the intrinsic properties of various retroviral intasomes to functionally accommodate different chromatin structures in the lack of other cofactors. Using in vitro conditions allowing the efficient catalysis of full site integration without these cofactors, we show that distinct retroviral integrases are not equally affected by chromatin compactness. Indeed, while PFV and MLV integration reactions are favored into dense and stable nucleosomes, HIV-1 and ASV concerted integration reactions are preferred into poorly dense chromatin regions of our nucleosomal acceptor templates. Predicted nucleosome occupancy around integration sites identified in
The DNase I accessibility and chromatin organization of genes within the nucleus do correlate to their transcriptional activity. Here, we show that both serum starvation and overexpression of Tip5, a key regulator of ribosomal RNA gene (rDNA) repression, dictate DNase I accessibility, facilitate the association of rDNA with the nuclear matrix and thus regulate large-scale rDNA chromatin organization. Tip5 contains four AT-hooks and a TAM (Tip5/ARBP/MBD) domain, which were proposed to bind matrix-attachment regions (MARs) of the genome. Remarkably, the TAM domain of Tip5 functions as nucleolar localization and nuclear matrix targeting module, whereas AT-hooks do not mediate association with the nuclear matrix, but they are required for nucleolar targeting. These findings suggest a dual role for Tip5s AT-hooks and TAM domain, targeting the nucleolus and anchoring to the nuclear matrix, and suggest a function for Tip5 in the regulation of higher-order rDNA chromatin structure. ...
Thyroid hormone (T3) plays diverse roles in adult organ function and during vertebrate development. The most important stage of mammalian development affected by T3 is the perinatal period when plasma T3 level peaks. Amphibian metamorphosis resembles this mammalian postembryonic period and is absolutely dependent on T3. The ability to easily manipulate this process makes it an ideal model to study the molecular mechanisms governing T3 action during vertebrate development. T3 functions mostly by regulating gene expression through T3 receptors (TRs). Studies in vitro, in cell cultures and reconstituted frog oocyte transcription system have revealed that TRs can both activate and repress gene transcription in a T3-dependent manner and involve chromatin disruption and histone modifications. These changes are accompanied by the recruitment of diverse cofactor complexes. More recently, genetic studies in mouse and frog have provided strong evidence for a role of cofactor complexes in T3 signaling in vivo.
Thyroid hormone (T3) plays diverse roles in adult organ function and during vertebrate development. The most important stage of mammalian development affected by T3 is the perinatal period when plasma T3 level peaks. Amphibian metamorphosis resembles this mammalian postembryonic period and is absolutely dependent on T3. The ability to easily manipulate this process makes it an ideal model to study the molecular mechanisms governing T3 action during vertebrate development. T3 functions mostly by regulating gene expression through T3 receptors (TRs). Studies in vitro, in cell cultures and reconstituted frog oocyte transcription system have revealed that TRs can both activate and repress gene transcription in a T3-dependent manner and involve chromatin disruption and histone modifications. These changes are accompanied by the recruitment of diverse cofactor complexes. More recently, genetic studies in mouse and frog have provided strong evidence for a role of cofactor complexes in T3 signaling in vivo.
The acidic patch is a highly electronegative cleft on the histone H2A-H2B dimer in the nucleosome. It is a fundamental motif for protein binding and chromatin dynamics, but the cellular impact of targeting this potentially therapeutic site with exogenous molecules remains unclear. Here, we characterize a family of binuclear ruthenium compounds that selectively target the nucleosome acidic patch, generating intra-nucleosomal H2A-H2B cross-links as well as inter-nucleosomal cross-links. In contrast to cisplatin or the progenitor RAPTA-C anticancer drugs, the binuclear agents neither arrest specific cell cycle phases nor elicit DNA damage response, but rather induce an irreversible, anomalous state of condensed chromatin that ultimately results in apoptosis. In vitro, the compounds induce misfolding of chromatin fibre and block the binding of the regulator of chromatin condensation 1 (RCC1) acidic patch-binding protein. This family of chromatin-modifying molecules has potential for applications ...
ABSTRACT DNA in the eukaryotic nucleus is complexed with histone and non-histone proteins into chromatin. Nucleosomes, the basic repeating unit of chromatin, not only package DNA but are also intimately involved the regulation of gene expression. All DNA transactions including replication, transcription, recombination and repair take place in such a chromatin environment. Access to packaged nucleosomal DNA in vivo is mediated at least in part by protein complexes that modify or remodel chromatin. Buried sequences in nucleosomes can also transiently become accessible to DNA binding proteins during cycles of partial, reversible unwrapping of nucleosomal DNA from the histone octamer. We have investigated the ability of the human, bifunctional DNA glycosylase, endonuclease III (hNTH1), to initiate base excision repair (BER) of discretely positioned oxidative lesions in model nucleosomes. hNTH1 was able to process a thymine glycol (Tg) lesion almost as efficiently as naked DNA, when the minor groove of the
Recent genome-wide analyses of yeast and human chromatin revealed the widespread prevalence of DNase I hypersensitive sites (DNase I HSs) at gene regulatory regions with possible roles in eukaryotic gene regulation. The presence of DNase I HSs in plants has been described for only a few genes, and we analyzed the chromatin structure of an 80 kb genomic region containing 30 variably expressed genes by DNase I sensitivity assay at 500 bp resolution in Arabidopsis. Distinct DNase I HSs were found at the 5′ and/or 3′ ends of most genes irrespective of their expression levels. Further analysis of well-characterized genes showed that the DNase I HSs occurred near cis-regulatory elements in the promoters of these genes. Upon transcriptional activation of a heat-inducible gene, the DNase I HS was extended into the vicinity of a cis-element and adjacent TATA element in the promoter. Concomitant with this change in DNase I HS, histones were acetylated, removed from the promoter, and a transcription ...
T. kodakarensis MNase digestion. T. kodakarensis strain KOD1 [22] was cultivated under anaerobic conditions at 85 °C and an insoluble unfixed chromatin fraction prepared from cells at the late‐log/stationary‐phase transition [10]. Chromatin was digested with 1 unit/ml of MNase, or 0.1 units/ml of DNase I for 1 h at 37 °C in the presence of 10 μg/μl RNase A. De‐proteinized genomic DNA was digested with 0.03 units/ml of MNase [10].. S. cerevisiae MNase digestion. EUROSCARF wild‐type reference strain BY4742 was grown and chromatin digestion (pooled triplicate samples) performed as described [11], with chromatin in unfixed detergent‐permeabilised yeast spheroplasts incubated with 600 units/ml of MNase for 3 min at 37 °C. Illumina DNA sequencing. NEBNext DNA sample prep master mix set 1 was used for Illumina adaptor ligation. Adaptor ligates were size selected on polyacrylamide gels to preserve the size distribution of the fragments before sequencing in 100 nucleotide paired end mode ...
Since their discovery in the mid-1990s, nuclear actin-related proteins (ARPs) have gained attention for their roles as structural components of ATP-dependent chromatin-remodeling complexes. These remodelers can move nucleosomes along the DNA, evict them from chromatin, and exchange histone variants to alter chromatin states locally. Chromatin-remodeling facilitates DNA-templated processes such as transcription regulation, DNA replication, and repair. Consistent with a role for ARPs in shaping chromatin structure, recent genetic studies show that they affect developmental and cell-type specific transcriptional programming. Here, we focus on recent results that suggest a specific contribution of ARPs to long-range interactions in the nucleus, and review evidence indicating that some ARPs may act independently of chromatin-remodeling machines.. ...
It has been a puzzle how decondensed interphase chromosomes remain essentially unknotted. The natural expectation is that in the presence of type II DNA topoisomerases that permit passages of double-stranded DNA regions through each other, all chromosomes should reach the state of topological equilibrium. The topological equilibrium in highly crowded interphase chromosomes forming chromosome territories would result in formation of highly knotted chromatin fibres. However, Chromosome Conformation Capture (3C) methods revealed that the decay of contact probabilities with the genomic distance in interphase chromosomes is practically the same as in the crumpled globule state that is formed when long polymers condense without formation of any knots. To remove knots from highly crowded chromatin, one would need an active process that should not only provide the energy to move the system from the state of topological equilibrium but also guide topoisomerase-mediated passages in such a way th
B138 Background: Treatment of breast cancer cells with histone deacetylase (HDAC) inhibitors results in chromatin decondensation and the sensitization of cancer cells to DNA damaging agents such as topoisomerase II inhibitors. We previously reported that the HDAC inhibitors induced chromatin decondensation through the down-regulation of heterochromatin maintenance proteins such as heterochromatin protein 1 (HP1), structural maintenance of chromatin proteins (SMC) 1-5 and DNA methyltransferase 1 (DNMT1). Here we report the role of HDAC2 in the expression of heterochromatin maintenance proteins, chromatin decondensation and DNA damage induced by topoisomerase inhibition. Methods: HDAC2 was selectively depleted using siRNA transfection. HDAC2 depleted cells were evaluated by microarray and Western blot analysis for changes in HP1, DNMT1 and SMC mRNA and protein expression. Chromatin decondensation was evaluated by electron microscopy. DNA damage and cell death induced by the topoisomerase ...
Component of the gypsy chromatin insulator complex which is required for the function of the gypsy chromatin insulator and other endogenous chromatin insulators. Chromatin insulators are regulatory elements which establish independent domains of transcriptional activity within eukaryotic genomes. Insulators have two defining properties; they can block the communication between an enhancer and a promoter when placed between them and can also buffer transgenes from position effect variegation (PEV). Insulators are proposed to structure the chromatin fiber into independent domains of differing transcriptional potential by promoting the formation of distinct chromatin loops. This chromatin looping may involve the formation of insulator bodies, where homotypic interactions between individual subunits of the insulator complex could promote the clustering of widely spaced insulators at the nuclear periphery. Within the gypsy insulator complex, this protein may directly bind to insulator DNA at sites distinct
Gene expression is tightly regulated at the level of transcription through cooperation between cis-regulatory elements and trans-factors that bind to the regulatory elements. Together, these factors regulate the higher order chromatin structure which establishes domains that organize the genome and coordinate gene expression. However, the molecular mechanisms controlling transcription of individual loci within a topological domain (TAD) are not fully understood. The cystic fibrosis transmembrane conductance regulator (CFTR) gene provides a paradigm for investigating these mechanisms. CFTR occupies a TAD bordered by CTCF/cohesin binding sites within which are cell-type-selective cis-regulatory elements for the locus. We showed previously that intronic and extragenic enhancers, when occupied by specific transcription factors, are recruited to the CFTR promoter by a looping mechanism to drive gene expression. Here we use a combination of CRISPR/Cas9 editing of cis-regulatory elements and ...
We have shown for the first time that the function of CAF-1 is important for viability following double-strand DNA repair. Furthermore, CAF-1 functions genetically in both homologous recombination and NHEJ, and its function during double-strand DNA repair is dependent on the interaction between CAF-1 and PCNA. Given the biochemical role of CAF-1 in DNA synthesis-coupled chromatin assembly, we propose that CAF-1 mediates chromatin assembly during double-strand DNA repair in vivo.. We have observed sensitivity of CAF-1 mutants to a variety of double-strand DNA-damaging agents. A systematic analysis of the yeast deletion collection also isolated the cac2Δ as a mutant sensitive to MMS (Chang et al. 2002). Similarly, mutants of the genes encoding Arabidopsis CAF-1, fas1, and fas2 are sensitive to MMS (Takeda et al. 2004). Given the role of CAF-1 in chromatin assembly during NER of single-strand lesions and the sensitivity of CAF-1 mutants to UV irradiation (Gaillard et al. 1996; Kaufman et al. 1997; ...
TY - JOUR. T1 - Chromatin immunoprecipitation for studying transcriptional regulation in Xenopus oocytes and tadpoles.. AU - Stewart, David. AU - Tomita, Akihiro. AU - Shi, Yun Bo. AU - Wong, Jiemin. N1 - Copyright: This record is sourced from MEDLINE/PubMed, a database of the U.S. National Library of Medicine. PY - 2006. Y1 - 2006. N2 - Understanding the accurate temporal and spatial regulation of gene expression during development requires knowledge of the spectrum of transcription factors and cofactors involved and their functional interplay with chromatin. Chromatin immunoprecipitation (ChIP) has become a powerful technique that allows us to do so. A typical ChIP assay involves (1) treating cells or tissues with formaldehyde to rapidly crosslink chromatin-associated proteins to DNA, (2) shearing chromatin by sonication into small fragments, (3) immunoprecipitation of the proteins of interest, (4) reversal of crosslinking, and (5) quantitating the specific associated DNA sequences by PCR. ...
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Eukaryotic organisms package DNA into chromatin for compact storage in the cell nucleus, but this packaging process results in transcriptional repression of genes. Chromatin remodeling complexes have evolved to overcome the transcriptional repression caused by chromatin packaging of DNA into nucleosomes by histones. One example of a chromatin remodeling complex is the SWI/SNF complex in yeast which uses ATP to drive the chromatin apart and make DNA accessible to transcription factors. The yeast SWI2 protein was discovered as the catalytic subunit of the yeast SWI/SNF chromatin remodeling complex and is required for the complex to counteract the repressive nature of chromatin. BRG1 and BRM, SWI2 homologs, are part of human chromatin remodeling complexes and have been shown to play a redundant role in the regulation of certain cell cycle and cellular adhesion genes, as well as cellular pathways. Recent studies showing loss of BRG1 in human tumor cell lines and primary tissue samples, BRG1 ...
And demembranated X. laevis sperm,chromatin decondensation and nuclear membrane assembly had been observed. Demembranation was obtained in the identical manner as in the study described above. In each instances,micrococcal nuclease digestion was utilised to confirm nucleosome formation.plant D-3263 (hydrochloride) web cytoplasm and Animal chromatinBecause Xenopus sperm is deficient in H histones,exposure to micrococcal nuclease results in heterogeneous distribution of DNA fragment sizes. When Xenopus sperm nuclei had been incubated with Nicotiana ovule extracts,the chromatin proteins could be replaced by histones derived from Nicotiana ovules,resulting in remodeling on the chromatin structure. In both situations,nuclear remodeling and nucleosome assembly have been observed,suggesting that transcription variables andor cyclincdk complexes originating in the plant cytoplasm may well contribute towards the induction of nuclear reconstitution and chromatin formation. Having said that,complex ...
RNA Polymerase II (RNAP II) is recruited to core promoters by the pre-initiation complex (PIC) of general transcription factors. Within the PIC, transcription factor for RNA polymerase IIB (TFIIB) determines the start site of transcription. TFIIB binding has not been localized, genome-wide, in metazoans. Serial analysis of chromatin occupancy (SACO) is an unbiased methodology used to empirically identify transcription factor binding regions. In this report, we use TFIIB and SACO to localize TFIIB binding regions across the rat genome. A sample of the TFIIB SACO library was sequenced and 12,968 TFIIB genomic signature tags (GSTs) were assigned to the rat genome. GSTs are 20-22 base pair fragments that are derived from TFIIB bound chromatin. TFIIB localized to both non-protein coding and protein-coding loci. For 21% of the 1783 protein-coding genes in this sample of the SACO library, TFIIB binding mapped near the characterized 5 promoter that is upstream of the transcription start site (TSS). However,
The 3D structure of chromatin in the nucleus is important for gene expression and regulation. Chromosomal conformation capture techniques, such as Hi-C, generate large amounts of data showing interaction points on the genome but these are hard to interpret using standard tools. We have developed CSynth, a high performance 3D genome browser and real time chromatin restraint-based modeller to visualise dynamic and interactive models of chromatin capture data. CSynth does its calculations in the GPU hence is much faster than existing modelling software to infer and visualise the chromatin structure which also allow real-time interaction with the modelling parameters. It also allows straightforward comparison of interaction data and the results of third party 3D modelling outputs. In addition we include an option to view and manipulate these complicated structures using Virtual Reality (VR) allowing scientists to immerse themselves in the models for further understanding. This VR component has also proven
Oligodendrocyte precursor cells (OPCs) constitute the main proliferative cells in the adult brain, and deregulation of OPC proliferation-differentiation balance results in either glioma formation or defective adaptive (re)myelination. OPC differentiation requires significant genetic reprogramming implicating chromatin remodeling. Mounting evidence indicates that chromatin remodelers play important roles during normal development and their mutations are associated with neurodevelopmental defects, with CHD7 haploinsuficiency being the cause of CHARGE syndrome and CHD8 being one of the strongest Autism Spectrum Disorder (ASD) high-risk associated genes. Here, we report on uncharacterized functions of the chromatin remodelers Chd7 and Chd8 in OPCs. Their OPC-chromatin-binding profile combined with transcriptome and chromatin accessibility analyses of Chd7-deleted OPCs, demonstrates that Chd7 protects non-proliferative OPCs from apoptosis by chromatin-closing and transcriptional repression of p53.
It is becoming increasingly evident that the cellular response towards DNA damage is affected by the structure of the chromatin region surrounding the damage site [1], while at the same time the chromatin structure is affected by the damage response [2]. DNA double-strand breaks (DSBs) elicit a response in an Mbp-large chromatin region surrounding the break that involves alterations in several post-translational modifications (PTMs). Phosphorylation of histone variant H2AX at serine 139 (S139) to yield γ-H2AX is a hallmark step in the cellular response to DSB. The γ-H2AX chromatin domains, which can be visualized as ionizing radiation induced foci (IRIF), delineate regions where a large variety of signalling and repair proteins accumulate [3].. Immunofluorescence detection of PTMs demonstrated alterations in several modifications in the γ-H2AX domain following DSB induction that are associated with regulation of chromatin accessibility, recruitment of DNA damage response factors, and ...
In eukaryotic cells DNA is associated with histones and other proteins to form chromatin. The basic unit of chromatin is the nucleosome consisting of 140 bp of DNA wrapped around an octameric core of the four conserved histones H2A, H2B, H3 and H4.The relatively unstructured and highly charged N-terminal tail domains of histones, are central to the processes that modulate chromatin structure. A diverse and elaborate array of post-translational modifications that include acetylation, phosphorylation, methylation, ubiquitination and ADP-ribosylation, occur on the N-terminal tail domains of histones. Acetylation of lysine residues within these N-terminal domains by histone acetyl-transferases (HATs), is associated with transcriptional activation. This modification results in remodeling of the nucleosome structure into an open conformation more accessible to transcription complexes. In most species, histone H3 is primarily acetylated at lysines 9, 14, 18 and 23. Acetylation at lysine 9 appears to
Chromatin is the complex combination of DNA and proteins that makes up chromosomes.[1][2] It is found inside the nuclei of eukaryotic cells. Chromatin is divided into heterochromatin (condensed) and euchromatin (extended) forms.[3][4] Heterochromatin is composed mostly of satellite DNA tandem repeats. The active components of chromatin are DNA and histone proteins, although other proteins also occur.[5] The functions of chromatin are: ...
Abstract. NETosis, a form of cell death that manifests by the release of decondensed chromatin to the extracellular space, provides valuable insights into mechanisms and consequences of cellular demise. Because extracellular chromatin can immobilize microbes, the extended nucleohistone network was called a neutrophil extracellular trap (NET), and the process of chromatin release was proposed to serve an innate immune defense function. Extracellular chromatin NETs were initially observed in studies of neutrophils and are most prominent in these types of granulocytes. Subsequent studies showed that other granulocytes and, in a limited way, other cells of the innate immune response may also release nuclear chromatin following certain kinds of stimulation. Variations of NETosis were noted with cells that remain temporarily motile after the release of chromatin. Numerous stimuli for NETosis were discovered, including bacterial breakdown products, inflammatory stimuli, particulate matter, certain ...
Antisense transcription is widespread in genomes. Despite large differences in gene size and architecture, we find that yeast and human genes share a unique, antisense transcription-associated chromatin signature. We asked whether this signature is related to a biological function for antisense transcription. Using quantitative RNA-FISH, we observed changes in sense transcript distributions in nuclei and cytoplasm as antisense transcript levels were altered. To determine the mechanistic differences underlying these distributions, we developed a mathematical framework describing transcription from initiation to transcript degradation. At GAL1, high levels of antisense transcription alter sense transcription dynamics, reducing rates of transcript production and processing, while increasing transcript stability. This relationship with transcript stability is also observed as a genome-wide association. Establishing the antisense transcription-associated chromatin signature through disruption of the Set3C
ARONOW. Stepwise chromatin restructuring and unexpected rules for interaction of distributed cis-elements that form a locus control region in vivo. Catherine Ley-Ebert, Carolyn Florio, John Maier, Chris Cost and Bruce Aronow. Childrens Hospital Research Foundation and the University of Cincinnati, Cincinnati, Ohio, USA. We have performed a mutational analysis of the human adenosine deaminase (ADA) gene thymic locus control region in transgenic mice using CAT reporter genes. We examined in detail gene expression, chromatin structure based on low and high resolution MNase and DNAse hypersensitivity, restriction enzyme accessibility, and histone acetylation state analyses using chromatin immune precipitations. Mutations and perturbations of LCR cis-elements led to variably compromised LCR function and the formation of several common distinguishable chromatin structures.. Our results suggest that the ADA thymic LCR is formed by the interaction of distributed cis-elements that consist of a 200 97bp ...
This organism was chosen because it has epigenetically defined regional centromeres whose chromatin and protein compositions are similar to those of their human counterparts, to identify factors responsible for the replacement of histone H3 with CENP-A at centromeres.. In this report, the KAIST research group systematically analyzed the roles of the ATP-dependent chromatin-remodelers in the centromeric chromatin assembly of fission yeast as they serve as strong candidates for such factors ...
Chromatin, Chromatid, Chromsome Terminology - posted in General Biology Discussion: Some Questions to which I have found different/conflicting answers depending on what I have read or who I have talked to. Thanks in Advance. 1-Chromatin, chromosomes, and chromatid all consist of DNA AND some sort of proteins? 2-Is the following statement accurate: ALL types/forms of chromatin material and chromatids consist of chromosomes BUT not all types of chromosomes are chromatin or chromatids? 3-An...