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Steven Henikoff - One of the best experts on this subject based on the ideXlab platform.
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Nucleosomes remember where they were
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Steven Henikoff, Kami AhmadAbstract:A central postulate in chromatin biology is that Nucleosomes are inherited through replication, and evidence for the recycling of Nucleosomes from ahead of the replication fork to behind goes back more than 40 y (1, 2). Early electron microscopic observations of chromatin fibers revealed that Nucleosomes form directly behind the replication fork (3), confirmed by later kinetic studies (4). However, it has remained uncertain as to whether histones from a nucleosome ahead of the fork return to the same position on a daughter strand after the fork has passed through. This is a critical question to resolve, because any dispersion of histones behind the fork disperses histone features such as posttranslational modifications that have been causally implicated in the propagation of gene expression states (5). The restoration of nucleosome positions may also be important for transcriptional regulation, given that Nucleosomes act as barriers to transcriptional elongation but are disrupted when RNA polymerase passes through (6). Thus both replication and transcription can potentially disperse Nucleosomes. To address this uncertainty, Schlissel and Rine (7) devise an elegant strategy to permanently mark histones within a 4-nucleosome region of the budding yeast genome, which allows them to precisely determine whether or not those Nucleosomes shift positions after replication fork passage. By engineering the marked region within the repressible and inducible GAL10 gene, this system also allows them to separate the effects of replication fork passage and transcription on nucleosome positioning. Biochemical studies have examined the process of nucleosome redeposition postreplication, but the question of positional memory has not been resolved. Unwinding of a nucleosome in vitro by the action of a helicase and a DNA polymerase resulted in transfer of the histone core to the leading-strand DNA duplex (8). As the leading strand is replicated before the lagging strand in vivo, a similar … [↵][1]1To whom correspondence may be addressed. Email: steveh{at}fhcrc.org. [1]: #xref-corresp-1-1
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precise genome wide mapping of single Nucleosomes and linkers in vivo
Genome Biology, 2018Co-Authors: Răzvan V Chereji, Srinivas Ramachandran, Terri D Bryson, Steven HenikoffAbstract:We developed a chemical cleavage method that releases single nucleosome dyad-containing fragments, allowing us to precisely map both single Nucleosomes and linkers with high accuracy genome-wide in yeast. Our single nucleosome positioning data reveal that Nucleosomes occupy preferred positions that differ by integral multiples of the DNA helical repeat. By comparing nucleosome dyad positioning maps to existing genomic and transcriptomic data, we evaluated the contributions of sequence, transcription, and histones H1 and H2A.Z in defining the chromatin landscape. We present a biophysical model that neglects DNA sequence and shows that steric occlusion suffices to explain the salient features of nucleosome positioning.
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Replicating Nucleosomes.
Science advances, 2015Co-Authors: Srinivas Ramachandran, Steven HenikoffAbstract:Eukaryotic replication disrupts each nucleosome as the fork passes, followed by re-assembly of disrupted Nucleosomes and incorporation of newly synthesized histones into Nucleosomes in the daughter genomes. In this review, we examine this process of replication-coupled nucleosome assembly to understand how characteristic steady state nucleosome landscapes are attained. Recent studies have begun to elucidate mechanisms involved in histone transfer during replication and maturation of the nucleosome landscape after disruption by replication. A fuller understanding of replication-coupled nucleosome assembly will be needed to explain how epigenetic information is replicated at every cell division.
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asymmetric Nucleosomes flank promoters in the budding yeast genome
Genome Research, 2015Co-Authors: Srinivas Ramachandran, Gabriel E Zentner, Steven HenikoffAbstract:Nucleosomes in active chromatin are dynamic, but whether they have distinct structural conformations is unknown. To identify Nucleosomes with alternative structures genome-wide, we used H4S47C-anchored cleavage mapping, which revealed that 5% of budding yeast (Saccharomyces cerevisiae) nucleosome positions have asymmetric histone-DNA interactions. These asymmetric interactions are enriched at nucleosome positions that flank promoters. Micrococcal nuclease (MNase) sequence-based profiles of asymmetric nucleosome positions revealed a corresponding asymmetry in MNase protection near the dyad axis, suggesting that the loss of DNA contacts around H4S47 is accompanied by protection of the DNA from MNase. Chromatin immunoprecipitation mapping of selected nucleosome remodelers indicated that asymmetric Nucleosomes are bound by the RSC chromatin remodeling complex, which is required for maintaining Nucleosomes at asymmetric positions. These results imply that the asymmetric nucleosome-RSC complex is a metastable intermediate representing partial unwrapping and protection of nucleosomal DNA on one side of the dyad axis during chromatin remodeling.
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Nucleosomes are context specific h2a z modulated barriers to rna polymerase
Molecular Cell, 2014Co-Authors: Srinivas Ramachandran, Christopher M Weber, Steven HenikoffAbstract:Summary Nucleosomes are barriers to transcription in vitro; however, their effects on RNA polymerase in vivo are unknown. Here we describe a simple and general strategy to comprehensively map the positions of elongating and arrested RNA polymerase II (RNAPII) at nucleotide resolution. We find that the entry site of the first (+1) nucleosome is a barrier to RNAPII for essentially all genes, including those undergoing regulated pausing farther upstream. In contrast to the +1 nucleosome, gene body Nucleosomes are low barriers and cause RNAPII stalling both at the entry site and near the dyad axis. The extent of the +1 nucleosome barrier correlates with nucleosome occupancy but anticorrelates with enrichment of histone variant H2A.Z. Importantly, depletion of H2A.Z from a nucleosome position results in a higher barrier to RNAPII. Our results suggest that Nucleosomes present significant, context-specific barriers to RNAPII in vivo that can be tuned by the incorporation of H2A.Z.
Alan G Hinnebusch - One of the best experts on this subject based on the ideXlab platform.
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chromatin remodeler ino80c acts independently of h2a z to evict promoter Nucleosomes and stimulate transcription of highly expressed genes in yeast
Nucleic Acids Research, 2020Co-Authors: Emily Biernat, Răzvan V Chereji, Chhabi K Govind, Yashpal Rawal, David J Clark, Alan G HinnebuschAbstract:The chromatin remodelers SWI/SNF and RSC function in evicting promoter Nucleosomes at highly expressed yeast genes, particularly those activated by transcription factor Gcn4. Ino80 remodeling complex (Ino80C) can establish nucleosome-depleted regions (NDRs) in reconstituted chromatin, and was implicated in removing histone variant H2A.Z from the -1 and +1 Nucleosomes flanking NDRs; however, Ino80C's function in transcriptional activation in vivo is not well understood. Analyzing the cohort of Gcn4-induced genes in ino80Δ mutants has uncovered a role for Ino80C on par with SWI/SNF in evicting promoter Nucleosomes and transcriptional activation. Compared to SWI/SNF, Ino80C generally functions over a wider region, spanning the -1 and +1 Nucleosomes, NDR and proximal genic Nucleosomes, at genes highly dependent on its function. Defects in nucleosome eviction in ino80Δ cells are frequently accompanied by reduced promoter occupancies of TBP, and diminished transcription; and Ino80 is enriched at genes requiring its remodeler activity. Importantly, nuclear depletion of Ino80 impairs promoter nucleosome eviction even in a mutant lacking H2A.Z. Thus, Ino80C acts widely in the yeast genome together with RSC and SWI/SNF in evicting promoter Nucleosomes and enhancing transcription, all in a manner at least partly independent of H2A.Z editing.
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swi snf and rsc cooperate to reposition and evict promoter Nucleosomes at highly expressed genes in yeast
Genes & Development, 2018Co-Authors: Yashpal Rawal, Răzvan V Chereji, Chhabi K Govind, David J Clark, Sudha Ananthakrishnan, Alan G HinnebuschAbstract:: The nucleosome remodeling complex RSC functions throughout the yeast genome to set the positions of -1 and +1 Nucleosomes and thereby determines the widths of nucleosome-depleted regions (NDRs). The related complex SWI/SNF participates in nucleosome remodeling/eviction and promoter activation at certain yeast genes, including those activated by transcription factor Gcn4, but did not appear to function broadly in establishing NDRs. By analyzing the large cohort of Gcn4-induced genes in mutants lacking the catalytic subunits of SWI/SNF or RSC, we uncovered cooperation between these remodelers in evicting Nucleosomes from different locations in the promoter and repositioning the +1 nucleosome downstream to produce wider NDRs-highly depleted of Nucleosomes-during transcriptional activation. SWI/SNF also functions on a par with RSC at the most highly transcribed constitutively expressed genes, suggesting general cooperation by these remodelers for maximal transcription. SWI/SNF and RSC occupancies are greatest at the most highly expressed genes, consistent with their cooperative functions in nucleosome remodeling and transcriptional activation. Thus, SWI/SNF acts comparably with RSC in forming wide nucleosome-free NDRs to achieve high-level transcription but only at the most highly expressed genes exhibiting the greatest SWI/SNF occupancies.
Timothy R Hughes - One of the best experts on this subject based on the ideXlab platform.
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Poly-dA:dT tracts form an in vivo nucleosomal turnstile
PLoS ONE, 2014Co-Authors: Carl G. De Boer, Timothy R HughesAbstract:Nucleosomes regulate many DNA-dependent processes by controlling the accessibility of DNA, and DNA sequences such as the poly-dA:dT element are known to affect nucleosome binding. We demonstrate that poly-dA:dT tracts form an asymmetric barrier to nucleosome movement in vivo, mediated by ATP-dependent chromatin remodelers. We theorize that nucleosome transit over poly-A elements is more energetically favourable in one direction, leading to an asymmetric arrangement of Nucleosomes around these sequences. We demonstrate that different arrangements of poly-A and poly-T tracts result in very different outcomes for nucleosome occupancy in yeast, mouse, and human, and show that yeast takes advantage of this phenomenon in its promoter architecture.
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The DNA-encoded nucleosome organization of a eukaryotic genome
Nature, 2008Co-Authors: Noam Kaplan, Timothy R Hughes, Irene K. Moore, Yvonne N. Fondufe-mittendorf, Andrea J. Gossett, Desiree Tillo, Yair Field, Emily M. Leproust, Jason D. Lieb, Jonathan WidomAbstract:The Nucleosomes are the basic repeating units of eukaryotic chromatin, and nucleosome organization is critically important for gene regulation. Kaplan et al. tested the importance of the intrinsic DNA sequence preferences of Nucleosomes by measuring the genome-wide occupancy of Nucleosomes assembled on purified yeast genomic DNA. The resulting map is remarkably similar to in vivo nucleosome maps, indicating that the organization of Nucleosomes in vivo is largely governed by the underlying genomic DNA sequence. This study tests the importance of the intrinsic DNA sequence preferences of Nucleosomes by measuring the genome-wide occupancy of Nucleosomes assembled on purified yeast genomic DNA. The resulting map is similar to in vivo nucleosome maps, indicating that the organization of Nucleosomes in vivo is largely governed by the underlying genomic DNA sequence. Nucleosome organization is critical for gene regulation1. In living cells this organization is determined by multiple factors, including the action of chromatin remodellers2, competition with site-specific DNA-binding proteins3, and the DNA sequence preferences of the Nucleosomes themselves4,5,6,7,8. However, it has been difficult to estimate the relative importance of each of these mechanisms in vivo7,9,10,11, because in vivo nucleosome maps reflect the combined action of all influencing factors. Here we determine the importance of nucleosome DNA sequence preferences experimentally by measuring the genome-wide occupancy of Nucleosomes assembled on purified yeast genomic DNA. The resulting map, in which nucleosome occupancy is governed only by the intrinsic sequence preferences of Nucleosomes, is similar to in vivo nucleosome maps generated in three different growth conditions. In vitro, nucleosome depletion is evident at many transcription factor binding sites and around gene start and end sites, indicating that nucleosome depletion at these sites in vivo is partly encoded in the genome. We confirm these results with a micrococcal nuclease-independent experiment that measures the relative affinity of Nucleosomes for ∼40,000 double-stranded 150-base-pair oligonucleotides. Using our in vitro data, we devise a computational model of nucleosome sequence preferences that is significantly correlated with in vivo nucleosome occupancy in Caenorhabditis elegans. Our results indicate that the intrinsic DNA sequence preferences of Nucleosomes have a central role in determining the organization of Nucleosomes in vivo.
David J Clark - One of the best experts on this subject based on the ideXlab platform.
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chromatin remodeler ino80c acts independently of h2a z to evict promoter Nucleosomes and stimulate transcription of highly expressed genes in yeast
Nucleic Acids Research, 2020Co-Authors: Emily Biernat, Răzvan V Chereji, Chhabi K Govind, Yashpal Rawal, David J Clark, Alan G HinnebuschAbstract:The chromatin remodelers SWI/SNF and RSC function in evicting promoter Nucleosomes at highly expressed yeast genes, particularly those activated by transcription factor Gcn4. Ino80 remodeling complex (Ino80C) can establish nucleosome-depleted regions (NDRs) in reconstituted chromatin, and was implicated in removing histone variant H2A.Z from the -1 and +1 Nucleosomes flanking NDRs; however, Ino80C's function in transcriptional activation in vivo is not well understood. Analyzing the cohort of Gcn4-induced genes in ino80Δ mutants has uncovered a role for Ino80C on par with SWI/SNF in evicting promoter Nucleosomes and transcriptional activation. Compared to SWI/SNF, Ino80C generally functions over a wider region, spanning the -1 and +1 Nucleosomes, NDR and proximal genic Nucleosomes, at genes highly dependent on its function. Defects in nucleosome eviction in ino80Δ cells are frequently accompanied by reduced promoter occupancies of TBP, and diminished transcription; and Ino80 is enriched at genes requiring its remodeler activity. Importantly, nuclear depletion of Ino80 impairs promoter nucleosome eviction even in a mutant lacking H2A.Z. Thus, Ino80C acts widely in the yeast genome together with RSC and SWI/SNF in evicting promoter Nucleosomes and enhancing transcription, all in a manner at least partly independent of H2A.Z editing.
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swi snf and rsc cooperate to reposition and evict promoter Nucleosomes at highly expressed genes in yeast
Genes & Development, 2018Co-Authors: Yashpal Rawal, Răzvan V Chereji, Chhabi K Govind, David J Clark, Sudha Ananthakrishnan, Alan G HinnebuschAbstract:: The nucleosome remodeling complex RSC functions throughout the yeast genome to set the positions of -1 and +1 Nucleosomes and thereby determines the widths of nucleosome-depleted regions (NDRs). The related complex SWI/SNF participates in nucleosome remodeling/eviction and promoter activation at certain yeast genes, including those activated by transcription factor Gcn4, but did not appear to function broadly in establishing NDRs. By analyzing the large cohort of Gcn4-induced genes in mutants lacking the catalytic subunits of SWI/SNF or RSC, we uncovered cooperation between these remodelers in evicting Nucleosomes from different locations in the promoter and repositioning the +1 nucleosome downstream to produce wider NDRs-highly depleted of Nucleosomes-during transcriptional activation. SWI/SNF also functions on a par with RSC at the most highly transcribed constitutively expressed genes, suggesting general cooperation by these remodelers for maximal transcription. SWI/SNF and RSC occupancies are greatest at the most highly expressed genes, consistent with their cooperative functions in nucleosome remodeling and transcriptional activation. Thus, SWI/SNF acts comparably with RSC in forming wide nucleosome-free NDRs to achieve high-level transcription but only at the most highly expressed genes exhibiting the greatest SWI/SNF occupancies.
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mnase sensitive complexes in yeast Nucleosomes and non histone barriers
Molecular Cell, 2017Co-Authors: Răzvan V Chereji, Josefina Ocampo, David J ClarkAbstract:Summary Micrococcal nuclease (MNase) is commonly used to map Nucleosomes genome-wide, but nucleosome maps are affected by the degree of digestion. It has been proposed that many yeast promoters are not nucleosome-free but instead occupied by easily digested, unstable, "fragile" Nucleosomes. We analyzed the histone content of all MNase-sensitive complexes by MNase-ChIP-seq and sonication-ChIP-seq. We find that yeast promoters are predominantly bound by non-histone protein complexes, with little evidence for fragile Nucleosomes. We do detect MNase-sensitive Nucleosomes elsewhere in the genome, including at transcription termination sites. However, they have high A/T content, suggesting that MNase sensitivity does not indicate instability, but rather the preference of MNase for A/T-rich DNA, such that A/T-rich Nucleosomes are digested faster than G/C-rich Nucleosomes. We confirm our observations by analyzing ChIP-exo, chemical mapping, and ATAC-seq data from other laboratories. Thus, histone ChIP-seq experiments are essential to distinguish Nucleosomes from other DNA-binding proteins that protect against MNase.
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Transcription Through the Nucleosome
The Nucleosome, 2007Co-Authors: David J ClarkAbstract:Publisher Summary This chapter focuses on transcription in chromatin, in particular, and on the question of how RNA polymerase transcribes through Nucleosomes. The compact nature of the nucleosome is compatible with its fundamental role in packaging DNA into chromosomes. The mechanism of transcription through Nucleosomes in vivo and in vitro is addressed in the chapter. The nucleosome core is a highly compact structure containing nearly two coils of DNA tightly wrapped around a central core histone octamer. It is sterically impossible for a bulky RNA polymerase molecule to transcribe through such a structure. Electron micrographs suggest that transcription is accompanied by a further decondensation of the chromatin filament containing the gene to an extent that depends primarily on the polymerase density. The passage of RNA polymerase disrupts the nucleosomal organization of a gene. There is evidence both for displacement/transfer of Nucleosomes and for transient or stable unfolding of Nucleosomes because of transcription in vivo. In vitro, prokaryotic RNA polymerases have been used extensively as models for transcription through the nucleosome core. Most of the evidence is consistent with the displacement/transfer model.
Răzvan V Chereji - One of the best experts on this subject based on the ideXlab platform.
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chromatin remodeler ino80c acts independently of h2a z to evict promoter Nucleosomes and stimulate transcription of highly expressed genes in yeast
Nucleic Acids Research, 2020Co-Authors: Emily Biernat, Răzvan V Chereji, Chhabi K Govind, Yashpal Rawal, David J Clark, Alan G HinnebuschAbstract:The chromatin remodelers SWI/SNF and RSC function in evicting promoter Nucleosomes at highly expressed yeast genes, particularly those activated by transcription factor Gcn4. Ino80 remodeling complex (Ino80C) can establish nucleosome-depleted regions (NDRs) in reconstituted chromatin, and was implicated in removing histone variant H2A.Z from the -1 and +1 Nucleosomes flanking NDRs; however, Ino80C's function in transcriptional activation in vivo is not well understood. Analyzing the cohort of Gcn4-induced genes in ino80Δ mutants has uncovered a role for Ino80C on par with SWI/SNF in evicting promoter Nucleosomes and transcriptional activation. Compared to SWI/SNF, Ino80C generally functions over a wider region, spanning the -1 and +1 Nucleosomes, NDR and proximal genic Nucleosomes, at genes highly dependent on its function. Defects in nucleosome eviction in ino80Δ cells are frequently accompanied by reduced promoter occupancies of TBP, and diminished transcription; and Ino80 is enriched at genes requiring its remodeler activity. Importantly, nuclear depletion of Ino80 impairs promoter nucleosome eviction even in a mutant lacking H2A.Z. Thus, Ino80C acts widely in the yeast genome together with RSC and SWI/SNF in evicting promoter Nucleosomes and enhancing transcription, all in a manner at least partly independent of H2A.Z editing.
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swi snf and rsc cooperate to reposition and evict promoter Nucleosomes at highly expressed genes in yeast
Genes & Development, 2018Co-Authors: Yashpal Rawal, Răzvan V Chereji, Chhabi K Govind, David J Clark, Sudha Ananthakrishnan, Alan G HinnebuschAbstract:: The nucleosome remodeling complex RSC functions throughout the yeast genome to set the positions of -1 and +1 Nucleosomes and thereby determines the widths of nucleosome-depleted regions (NDRs). The related complex SWI/SNF participates in nucleosome remodeling/eviction and promoter activation at certain yeast genes, including those activated by transcription factor Gcn4, but did not appear to function broadly in establishing NDRs. By analyzing the large cohort of Gcn4-induced genes in mutants lacking the catalytic subunits of SWI/SNF or RSC, we uncovered cooperation between these remodelers in evicting Nucleosomes from different locations in the promoter and repositioning the +1 nucleosome downstream to produce wider NDRs-highly depleted of Nucleosomes-during transcriptional activation. SWI/SNF also functions on a par with RSC at the most highly transcribed constitutively expressed genes, suggesting general cooperation by these remodelers for maximal transcription. SWI/SNF and RSC occupancies are greatest at the most highly expressed genes, consistent with their cooperative functions in nucleosome remodeling and transcriptional activation. Thus, SWI/SNF acts comparably with RSC in forming wide nucleosome-free NDRs to achieve high-level transcription but only at the most highly expressed genes exhibiting the greatest SWI/SNF occupancies.
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precise genome wide mapping of single Nucleosomes and linkers in vivo
Genome Biology, 2018Co-Authors: Răzvan V Chereji, Srinivas Ramachandran, Terri D Bryson, Steven HenikoffAbstract:We developed a chemical cleavage method that releases single nucleosome dyad-containing fragments, allowing us to precisely map both single Nucleosomes and linkers with high accuracy genome-wide in yeast. Our single nucleosome positioning data reveal that Nucleosomes occupy preferred positions that differ by integral multiples of the DNA helical repeat. By comparing nucleosome dyad positioning maps to existing genomic and transcriptomic data, we evaluated the contributions of sequence, transcription, and histones H1 and H2A.Z in defining the chromatin landscape. We present a biophysical model that neglects DNA sequence and shows that steric occlusion suffices to explain the salient features of nucleosome positioning.
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mnase sensitive complexes in yeast Nucleosomes and non histone barriers
Molecular Cell, 2017Co-Authors: Răzvan V Chereji, Josefina Ocampo, David J ClarkAbstract:Summary Micrococcal nuclease (MNase) is commonly used to map Nucleosomes genome-wide, but nucleosome maps are affected by the degree of digestion. It has been proposed that many yeast promoters are not nucleosome-free but instead occupied by easily digested, unstable, "fragile" Nucleosomes. We analyzed the histone content of all MNase-sensitive complexes by MNase-ChIP-seq and sonication-ChIP-seq. We find that yeast promoters are predominantly bound by non-histone protein complexes, with little evidence for fragile Nucleosomes. We do detect MNase-sensitive Nucleosomes elsewhere in the genome, including at transcription termination sites. However, they have high A/T content, suggesting that MNase sensitivity does not indicate instability, but rather the preference of MNase for A/T-rich DNA, such that A/T-rich Nucleosomes are digested faster than G/C-rich Nucleosomes. We confirm our observations by analyzing ChIP-exo, chemical mapping, and ATAC-seq data from other laboratories. Thus, histone ChIP-seq experiments are essential to distinguish Nucleosomes from other DNA-binding proteins that protect against MNase.
