Monoubiquitylation of H2A.Z Distinguishes Its Association with Euchromatin or Facultative Heterochromatin

ABSTRACT H2A.Z is a histone H2A variant that is essential for viability in organisms such as Tetrahymena thermophila, Drosophila melanogaster, and mice. In Saccharomyces cerevisiae, loss of H2A.Z is tolerated, but proper regulation of gene expression is affected. Genetics and genome-wide localization studies show that yeast H2A.Z physically localizes to the promoters of genes and functions in part to protect active genes in euchromatin from being silenced by heterochromatin spreading. To date, the function of H2A.Z in mammalian cells is less clear, and evidence so far suggests that it has a role in chromatin compaction and heterochromatin silencing. In this study, we found that the bulk of H2A.Z is excluded from constitutive heterochromatin in differentiated human and mouse cells. Consistent with this observation, analyses of H2A.Z- or H2A-containing mononucleosomes show that the H3 associated with H2A.Z has lower levels of K9 methylation but higher levels of K4 methylation than those associated with H2A. We also found that a fraction of mammalian H2A.Z is monoubiquitylated and that, on the inactive X chromosomes of female cells, the majority of this histone variant is modified by ubiquitin. Finally, ubiquitylation of H2A.Z is mediated by the RING1b E3 ligase of the human polycomb complex, further supporting a silencing role of ubiquitylated H2A.Z. These new findings suggest that mammalian H2A.Z is associated with both euchromatin and facultative heterochromatin and that monoubiquitylation is a specific mark that distinguishes the H2A.Z associated with these different chromatin states.

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Abo1 is required for the H3K9me2 to H3K9me3 transition in heterochromatin

Scientific Reports ◽

10.1038/s41598-020-63209-y ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Wenbo Dong ◽

Eriko Oya ◽

Yasaman Zahedi ◽

Punit Prasad ◽

J. Peter Svensson ◽

...

Keyword(s):

Constitutive Heterochromatin ◽

Genomic Stability ◽

Wild Type ◽

Key Factors ◽

Facultative Heterochromatin ◽

Heterochromatin Formation ◽

Aaa Atpase ◽

Genome Wide ◽

Heterochromatin Silencing

AbstractHeterochromatin regulation is critical for genomic stability. Different H3K9 methylation states have been discovered, with distinct roles in heterochromatin formation and silencing. However, how the transition from H3K9me2 to H3K9me3 is controlled is still unclear. Here, we investigate the role of the conserved bromodomain AAA-ATPase, Abo1, involved in maintaining global nucleosome organisation in fission yeast. We identified several key factors involved in heterochromatin silencing that interact genetically with Abo1: histone deacetylase Clr3, H3K9 methyltransferase Clr4, and HP1 homolog Swi6. Cells lacking Abo1 cultivated at 30 °C exhibit an imbalance of H3K9me2 and H3K9me3 in heterochromatin. In abo1∆ cells, the centromeric constitutive heterochromatin has increased H3K9me2 but decreased H3K9me3 levels compared to wild-type. In contrast, facultative heterochromatin regions exhibit reduced H3K9me2 and H3K9me3 levels in abo1∆. Genome-wide analysis showed that abo1∆ cells have silencing defects in both the centromeres and subtelomeres, but not in a subset of heterochromatin islands in our condition. Thus, our work uncovers a role of Abo1 in stabilising directly or indirectly Clr4 recruitment to allow the H3K9me2 to H3K9me3 transition in heterochromatin.

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The genome-wide role of HSF-1 in the regulation of gene expression in Caenorhabditis elegans

BMC Genomics ◽

10.1186/s12864-016-2837-5 ◽

2016 ◽

Vol 17 (1) ◽

Cited By ~ 66

Author(s):

Jessica Brunquell ◽

Stephanie Morris ◽

Yin Lu ◽

Feng Cheng ◽

Sandy D. Westerheide

Keyword(s):

Gene Expression ◽

Caenorhabditis Elegans ◽

Regulation Of Gene Expression ◽

Genome Wide

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Functional correlation of H3K9me2 and nuclear compartment formation

10.1101/2020.08.28.271221 ◽

2020 ◽

Author(s):

Kei Fukuda ◽

Chikako Shimura ◽

Hisashi Miura ◽

Akie Tanigawa ◽

Takehiro Suzuki ◽

...

Keyword(s):

Transcriptional Activation ◽

Genome Organization ◽

Mammalian Cells ◽

Embryonic Stem ◽

Epigenetic Mark ◽

3 Dimensional ◽

3D Genome ◽

Functional Correlation ◽

Genome Wide

AbstractBackgroundHistone H3 lysine 9 dimethylation (H3K9me2) is a highly conserved silencing epigenetic mark. Chromatin marked with H3K9me2 forms large domains in mammalian cells and correlates well with lamina-associated domains and the B compartment. However, the role of H3K9me2 in 3-dimensional (3D) genome organization remains unclear.ResultsWe investigated the genome-wide H3K9me2 distribution, the transcriptome and 3D genome organization in mouse embryonic stem cells (mESCs) upon the inhibition or depletion of H3K9 methyltransferases (MTases) G9a/GLP, SETDB1, and SUV39H1/2. We found that H3K9me2 is regulated by these five MTases; however, H3K9me2 and transcription in the A and B compartments were largely regulated by different sets of the MTases: H3K9me2 in the A compartments were mainly regulated by G9a/GLP and SETDB1, while H3K9me2 in the B compartments were regulated by all five H3K9 MTases. Furthermore, decreased H3K9me2 correlated with the changes to the more active compartmental state that accompanied transcriptional activation.ConclusionOur data showed that H3K9me2 domain formation is functionally linked to 3D genome organization.

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Multivariable regulation of gene expression plasticity in metazoans

10.1101/692863 ◽

2019 ◽

Author(s):

Long Xiao ◽

Zhiguang Zhao ◽

Fei He ◽

Zhuo Du

Keyword(s):

Gene Expression ◽

Histone Modifications ◽

Regulation Of Gene Expression ◽

Rapid Change ◽

High Plasticity ◽

Expression Levels ◽

Genome Wide ◽

Expression Potential ◽

Highly Correlated

ABSTRACTAn important capacity of genes is the rapid change of expression levels to cope with environment, known as expression plasticity. Elucidating the genomic mechanisms determining expression plasticity is critical for understanding the molecular basis of phenotypic plasticity, fitness, and adaptation. In this study, we systematically quantified genome-wide gene expression plasticity in four metazoan species by integrating changes of expression levels under a large number of genetic and environmental conditions. From this, we demonstrated that expression plasticity measures a distinct feature of gene expression that is orthogonal to other well-studies features including gene expression potential and tissue specificity/broadness. Expression plasticity is conserved across species with important physiological implications. The magnitude of expression plasticity is highly correlated with gene function and genes with high plasticity are implicated in disease susceptibility. Genome-wide analysis identified many conserved promoter cis-elements, trans-acting factors (such as CFCF), and gene body histone modifications (H3K36me3, H3K79me2, and H4K20me1) that are significantly associated with expression plasticity. Analysis of expression changes in perturbation experiments further validated a causal role of specific transcription factors and histone modifications. Collectively, this work reveals general properties, physiological implications, and multivariable regulation of gene expression plasticity in metazoans, extending the mechanistic understanding of gene regulation.

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Directed Evolution of an Enhanced POU Reprogramming Factor for Cell Fate Engineering

Molecular Biology and Evolution ◽

10.1093/molbev/msab075 ◽

2021 ◽

Author(s):

Daisylyn Senna Tan ◽

Yanpu Chen ◽

Ya Gao ◽

Anastasia Bednarz ◽

Yuanjie Wei ◽

...

Keyword(s):

Directed Evolution ◽

Cell Fate ◽

Mammalian Cells ◽

Phenotypic Selection ◽

Biochemical Assays ◽

Genome Wide ◽

Dna Elements ◽

Pioneer Factor ◽

Selection Of

Abstract Transcription factor-driven cell fate engineering in pluripotency induction, transdifferentiation, and forward reprogramming requires efficiency, speed, and maturity for widespread adoption and clinical translation. Here, we used Oct4, Sox2, Klf4, and c-Myc driven pluripotency reprogramming to evaluate methods for enhancing and tailoring cell fate transitions, through directed evolution with iterative screening of pooled mutant libraries and phenotypic selection. We identified an artificially evolved and enhanced POU factor (ePOU) that substantially outperforms wild-type Oct4 in terms of reprogramming speed and efficiency. In contrast to Oct4, not only can ePOU induce pluripotency with Sox2 alone, but it can also do so in the absence of Sox2 in a three-factor ePOU/Klf4/c-Myc cocktail. Biochemical assays combined with genome-wide analyses showed that ePOU possesses a new preference to dimerize on palindromic DNA elements. Yet, the moderate capacity of Oct4 to function as a pioneer factor, its preference to bind octamer DNA and its capability to dimerize with Sox2 and Sox17 proteins remain unchanged in ePOU. Compared with Oct4, ePOU is thermodynamically stabilized and persists longer in reprogramming cells. In consequence, ePOU: 1) differentially activates several genes hitherto not implicated in reprogramming, 2) reveals an unappreciated role of thyrotropin-releasing hormone signaling, and 3) binds a distinct class of retrotransposons. Collectively, these features enable ePOU to accelerate the establishment of the pluripotency network. This demonstrates that the phenotypic selection of novel factor variants from mammalian cells with desired properties is key to advancing cell fate conversions with artificially evolved biomolecules.

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High Throughput Sequencing Following Cross-Linked Immune Precipitation (HITS-CLIP) of Argonaute (AGO) Identifies Mir-9 As a Regulator of MMP2 in the Marrow Microenvironment (ME)

Blood ◽

10.1182/blood.v118.21.2392.2392 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2392-2392 ◽

Cited By ~ 1

Author(s):

Ilango Balakrishnan ◽

Xiaodong Yang ◽

Beverly Torok-Storb ◽

Jay Hesselberth ◽

Manoj M Pillai

Keyword(s):

Gene Expression ◽

High Throughput ◽

Stromal Cells ◽

False Positive ◽

High Throughput Sequencing ◽

Regulation Of Gene Expression ◽

Negative Results ◽

Genome Wide ◽

Direct Binding

Abstract Abstract 2392 There is increasing recognition of the role of small noncoding RNAs in post-transcriptional regulation of gene expression in diverse tissues of eukaryotic organisms including vertebrates. MicroRNAs (miRNAs) are the best studied amongst these small RNAs and are thought to act by binding to the 3' untranslated regions (3' UTRs) of mature mRNAs in a sequence-specific fashion and preventing the initiation of peptide translation and/ or initiating mRNA degradation. Recent evidence suggests that miRNA-based regulation might involve binding to regions other than 3' UTRs including coding regions. Current approaches to defining miRNA-mRNA interactions are mostly restricted to those based on bio-informatic prediction, protein down-regulation following in-vitro transfection of miRNA precursors and luciferase assays to determine binding to 3' UTRs. None of these methods however show direct interaction between a specific miRNA and its purported target RNA. Bio-informatics-based approaches are also prone to false positive and negative results given the short length of sequence matching, and reliance on heuristics and cross-species conservation. Newer genome-wide approaches like HITS-CLIP (High Throughput Sequencing following Cross Linked Immuno Precipitation, or CLIP-Seq) overcome some of these limitations by directly isolating the miRNA-mRNA interactome bound to argonaute (AGO), a critical component of the rna-induced silencing complex (RISC)1. HITS-CLIP utilizes the ability of ultraviolet (UV) light to cross-link RNAs to proteins in their close proximity. The crosslinked miRNA-mRNA-Ago complexes are then isolated and the RNA reverse transcribed to cDNA libraries and sequenced by next generation sequencing (NGS). Given the widespread role of miRNAs in several vertebrate tissues, we hypothesized that miRNA-regulation of gene expression is operant in the hematopoietic microenvironment (ME) and thus contributes to regulation of hematopoiesis. We hence used HITS-CLIP to analyze the miRNA-mRNA interactome of three key cellular components of the ME: stromal cells, endothelium and macrophages. We have previously reported on the use of the stromal cell lines Hs27a and Hs5 to define specific functional niches within the ME. Hs27a can functionally support primitive hematopoietic stem and progenitor cells (HSPC) in cobblestone areas (CSAs) and express high levels of factors known to support HSPC such as SDF1, Jagged1 and Angiopoietin1. In contrast, Hs5 drives HSPC to mature lineages and secretes high levels of cytokines like IL1, IL6 and GCSF. Human umbilical vein endothelial cells (HUVECs) and MCSF-treated CD14+ cells were utilized for the endothelial and macrophage cultures respectively. The HITS-CLIP datasets from each of these populations were enriched for a putative binding site for miR-9 in the coding region of Matrix Metalloproteinase 2 (MMP2) mRNA. MMP2 belongs to a family of endopeptidases critical in the remodeling of extracellular matrix in several tissues and in the egress/ homing of HSPC to their functional niches in the ME. Functional binding of miR-9 to MMP2 was validated by Western-blotting of stromal cells transfected with miR-9 which revealed > 50% reduction of protein levels when compared to control-transfected cells. This was also confirmed by gelatin zymography which showed significantly reduced MMP2 activity in stromal cells transfected with miR-9. Finally, to confirm direct binding of miR-9 to the putative binding region on the MMP2 transcript, we cloned this microRNA responsive region (MRE) downstream of the Renilla luciferase gene and assayed its activity by luciferase assays. MiR-9 transfection down-regulated luciferase activity > 50% confirming direct binding to the MRE. Our results show that genome-wide approaches such as HITS-CLIP can be used to define in vivo miRNA-mRNA interactions in the ME and should be considered in studies that define such interactions given the significant false-positive and false negative results associated with approaches based on bio-informatics alone. The approach can also define specific interactions between miRNAs and mRNAs such as MMP2, of relevance to regulation of the hematopoietic ME. Disclosures: No relevant conflicts of interest to declare.

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Regulation of Gene Transcription by the Histone H2A N-Terminal Domain

Molecular and Cellular Biology ◽

10.1128/mcb.00742-07 ◽

2007 ◽

Vol 27 (21) ◽

pp. 7641-7648 ◽

Cited By ~ 22

Author(s):

Michael A. Parra ◽

John J. Wyrick

Keyword(s):

Gene Transcription ◽

Microarray Data ◽

Transcriptional Repression ◽

Dna Microarrays ◽

Reporter Genes ◽

Histone H2a ◽

Terminal Domain ◽

Genome Wide ◽

Genome Wide Expression

ABSTRACT Histone N-terminal domains play critical roles in regulating chromatin structure and gene transcription. Relatively little is known, however, about the role of the histone H2A N-terminal domain in transcription regulation. We have used DNA microarrays to characterize the changes in genome-wide expression caused by mutations in the N-terminal domain of histone H2A. Our results indicate that the N-terminal domain of histone H2A functions primarily to repress the transcription of a large subset of the Saccharomyces cerevisiae genome and that most of the H2A-repressed genes are also repressed by the histone H2B N-terminal domain. Using the histone H2A microarray data, we selected three reporter genes (BNA1, BNA2, and GCY1), which we subsequently used to map regions in the H2A N-terminal domain responsible for this transcriptional repression. These studies revealed that a small subdomain in the H2A N-terminal tail, comprised of residues 16 to 20, is required for the transcriptional repression of these reporter genes. Deletion of either the entire histone H2A N-terminal domain or just this small subdomain imparts sensitivity to UV irradiation. Finally, we show that two residues in this H2A subdomain, serine-17 and arginine-18, are specifically required for the transcriptional repression of the BNA2 reporter gene.

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The frequency natural antisense transcript first promotes, then represses, frequency gene expression via facultative heterochromatin

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1406130112 ◽

2015 ◽

Vol 112 (14) ◽

pp. 4357-4362 ◽

Cited By ~ 27

Author(s):

Na Li ◽

Tammy M. Joska ◽

Catherine E. Ruesch ◽

Samuel J. Coster ◽

William J. Belden

Keyword(s):

Gene Expression ◽

Antisense Transcript ◽

Inducible Promoter ◽

Facultative Heterochromatin ◽

Heterochromatin Formation ◽

Circadian Phase ◽

Parallel Pathway ◽

Hygromycin Resistance Gene ◽

Heterochromatin Silencing

The circadian clock is controlled by a network of interconnected feedback loops that require histone modifications and chromatin remodeling. Long noncoding natural antisense transcripts (NATs) originate from Period in mammals and frequency (frq) in Neurospora. To understand the role of NATs in the clock, we put the frq antisense transcript qrf (frq spelled backwards) under the control of an inducible promoter. Replacing the endogenous qrf promoter altered heterochromatin formation and DNA methylation at frq. In addition, constitutive, low-level induction of qrf caused a dramatic effect on the endogenous rhythm and elevated circadian output. Surprisingly, even though qrf is needed for heterochromatic silencing, induction of qrf initially promoted frq gene expression by creating a more permissible local chromatin environment. The observation that antisense expression can initially promote sense gene expression before silencing via heterochromatin formation at convergent loci is also found when a NAT to hygromycin resistance gene is driven off the endogenous vivid (vvd) promoter in the Δvvd strain. Facultative heterochromatin silencing at frq functions in a parallel pathway to previously characterized VVD-dependent silencing and is needed to establish the appropriate circadian phase. Thus, repression via dicer-independent siRNA-mediated facultative heterochromatin is largely independent of, and occurs alongside, other feedback processes.

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Chromatin is an ancient innovation conserved between Archaea and Eukarya

eLife ◽

10.7554/elife.00078 ◽

2012 ◽

Vol 1 ◽

Cited By ~ 48

Author(s):

Ron Ammar ◽

Dax Torti ◽

Kyle Tsui ◽

Marinella Gebbia ◽

Tanja Durbic ◽

...

Keyword(s):

Gene Expression ◽

Chromatin Structure ◽

Nucleosome Occupancy ◽

Regulation Of Gene Expression ◽

Haloferax Volcanii ◽

Dna Compaction ◽

Genome Wide ◽

Eukaryotic Chromatin ◽

Transcriptional Start Sites

The eukaryotic nucleosome is the fundamental unit of chromatin, comprising a protein octamer that wraps ∼147 bp of DNA and has essential roles in DNA compaction, replication and gene expression. Nucleosomes and chromatin have historically been considered to be unique to eukaryotes, yet studies of select archaea have identified homologs of histone proteins that assemble into tetrameric nucleosomes. Here we report the first archaeal genome-wide nucleosome occupancy map, as observed in the halophile Haloferax volcanii. Nucleosome occupancy was compared with gene expression by compiling a comprehensive transcriptome of Hfx. volcanii. We found that archaeal transcripts possess hallmarks of eukaryotic chromatin structure: nucleosome-depleted regions at transcriptional start sites and conserved −1 and +1 promoter nucleosomes. Our observations demonstrate that histones and chromatin architecture evolved before the divergence of Archaea and Eukarya, suggesting that the fundamental role of chromatin in the regulation of gene expression is ancient.

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Natural Compounds as Modulators of NADPH Oxidases

Oxidative Medicine and Cellular Longevity ◽

10.1155/2013/271602 ◽

2013 ◽

Vol 2013 ◽

pp. 1-10 ◽

Cited By ~ 58

Author(s):

Tullia Maraldi

Keyword(s):

Mammalian Cells ◽

Redox State ◽

Regulation Of Gene Expression ◽

Natural Compounds ◽

Oxygen Species ◽

Nadph Oxidases ◽

Natural Extracts ◽

Current State ◽

Wide Range

Reactive oxygen species (ROS) are cellular signals generated ubiquitously by all mammalian cells, but their relative unbalance triggers also diseases through intracellular damage to DNA, RNA, proteins, and lipids. NADPH oxidases (NOX) are the only known enzyme family with the sole function to produce ROS. The NOX physiological functions concern host defence, cellular signaling, regulation of gene expression, and cell differentiation. On the other hand, increased NOX activity contributes to a wide range of pathological processes, including cardiovascular diseases, neurodegeneration, organ failure, and cancer. Therefore targeting these enzymatic ROS sources by natural compounds, without affecting the physiological redox state, may be an important tool. This review summarizes the current state of knowledge of the role of NOX enzymes in physiology and pathology and provides an overview of the currently available NADPH oxidase inhibitors derived from natural extracts such as polyphenols.

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