The histone chaperone FACT facilitates heterochromatin spreading through regulation of histone turnover and H3K9 methylation states

2021 ◽  
Author(s):  
Magdalena Murawska ◽  
R. A. Greenstein ◽  
Tamas Schauer ◽  
Karl C.F. Olsen ◽  
Henry Ng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Histone Chaperone ◽  
Replication Cycle ◽  
H3k9 Methylation ◽  
Spreading Process ◽  
Epigenetic Marks ◽  
Heterochromatin Formation ◽  
Heterochromatin Silencing

Heterochromatin formation requires three distinct steps: nucleation, self-propagation (spreading) along the chromosome, and faithful maintenance after each replication cycle. Impeding any of those steps induces heterochromatin defects and improper gene expression. The essential histone chaperone FACT has been implicated in heterochromatin silencing, however, the mechanisms by which FACT engages in this process remain opaque. Here, we pin-pointed its function to the heterochromatin spreading process. FACT impairment reduces nucleation-distal H3K9me3 and HP1/Swi6 accumulation at subtelomeres and de-represses genes in the vicinity of heterochromatin boundaries. FACT promotes spreading by repressing heterochromatic histone turnover, which is crucial for the H3K9me2 to me3 transition that enables spreading. FACT mutant spreading defects are suppressed by removal of the H3K9 methylation antagonist Epe1 via nucleosome stabilization. Together, our study identifies FACT as a histone chaperone that specifically promotes heterochromatin spreading and lends support to the model that regulated histone turnover controls the propagation of epigenetic marks.

scholarly journals SUV39 SET domains mediate crosstalk of heterochromatic histone marks

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Alessandro Stirpe ◽  
Nora Guidotti ◽  
Sarah J Northall ◽  
Sinan Kilic ◽  
Alexandre Hainard ◽  
...  
Keyword(s):  
Structure Function ◽  
Fission Yeast ◽  
Catalytic Domain ◽  
Constitutive Heterochromatin ◽  
Histone H3 ◽  
H3k9 Methylation ◽  
Set Domain ◽  
Heterochromatin Formation ◽  
Heterochromatin Silencing ◽  
Genomic Regions

The SUV39 class of methyltransferase enzymes deposits histone H3 lysine 9 di- and trimethylation (H3K9me2/3), the hallmark of constitutive heterochromatin. How these enzymes are regulated to mark specific genomic regions as heterochromatic is poorly understood. Clr4 is the sole H3K9me2/3 methyltransferase in the fission yeast Schizosaccharomyces pombe, and recent evidence suggests that ubiquitination of lysine 14 on histone H3 (H3K14ub) plays a key role in H3K9 methylation. However, the molecular mechanism of this regulation and its role in heterochromatin formation remain to be determined. Our structure-function approach shows that the H3K14ub substrate binds specifically and tightly to the catalytic domain of Clr4, and thereby stimulates the enzyme by over 250-fold. Mutations that disrupt this mechanism lead to a loss of H3K9me2/3 and abolish heterochromatin silencing similar to clr4 deletion. Comparison with mammalian SET domain proteins suggests that the Clr4 SET domain harbors a conserved sensor for H3K14ub, which mediates licensing of heterochromatin formation.

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

2015 ◽  
Vol 112 (14) ◽  
pp. 4357-4362 ◽  
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.

scholarly journals SUV39 SET domains mediate crosstalk of heterochromatic histone marks

2020 ◽  
Author(s):  
Alessandro Stirpe ◽  
Nora Guidotti ◽  
Sarah Northall ◽  
Sinan Kilic ◽  
Alexandre Hainard ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Constitutive Heterochromatin ◽  
H3k9 Methylation ◽  
Set Domain ◽  
Heterochromatin Formation ◽  
Histone Lysine ◽  
Heterochromatin Silencing ◽  
Central Recruitment ◽  
Genomic Regions ◽  
In Cis

AbstractThe SUV39 class of methyltransferase enzymes deposits histone lysine 9 di- and trimethylation (H3K9me2/3), the epigenetic hallmark of constitutive heterochromatin, which serves as the central recruitment platform for the heterochromatic silencing machinery. How these enzymes are regulated to mark specific genomic regions as heterochromatic is not well understood. Clr4 is the sole H3K9me2/3 methyltransferase in the fission yeast S.pombe and recent evidence suggests that ubiquitination of lysine 14 on H3 tail (H3K14) plays a key role in H3K9 methylation. However, the molecular mechanism of this regulation and its role in heterochromatin formation remains to be determined. Here we present a structure-function approach to understanding how the H3K14ub mark stimulates Clr4 activity in cis. These results show that the H3K14ub substrate binds specifically and tightly to the catalytic domain of Clr4, and thereby activates the enzyme by 250-fold. Mutations that disrupt this mechanism lead to a loss of H3K9me2/3 and abolish heterochromatin silencing similar to a clr4 deletion. Our work reveals a sensor for H3K14 ubiquitylation in the SET domain of Clr4, which mediates the licensing of heterochromatin formation by an epigenetic cross-talk. This sensor is also active in the human SUV39H2 enzyme.

scholarly journals Coordinated regulation of heterochromatin inheritance by Dpb3–Dpb4 complex

2017 ◽  
Vol 114 (47) ◽  
pp. 12524-12529 ◽  
Author(s):  
Haijin He ◽  
Yang Li ◽  
Qianhua Dong ◽  
An-Yun Chang ◽  
Feng Gao ◽  
...  
Keyword(s):  
Dna Replication ◽  
Histone Deacetylases ◽  
Histone Deacetylation ◽  
H3k9 Methylation ◽  
Epigenetic Marks ◽  
Chromatin Remodelers ◽  
Dna Polymerase Epsilon ◽  
Histone Fold ◽  
Heterochromatin Silencing ◽  
Histone Hypoacetylation

During DNA replication, chromatin is disrupted ahead of the replication fork, and epigenetic information must be restored behind the fork. How epigenetic marks are inherited through DNA replication remains poorly understood. Histone H3 lysine 9 (H3K9) methylation and histone hypoacetylation are conserved hallmarks of heterochromatin. We previously showed that the inheritance of H3K9 methylation during DNA replication depends on the catalytic subunit of DNA polymerase epsilon, Cdc20. Here we show that the histone-fold subunit of Pol epsilon, Dpb4, interacts an uncharacterized small histone-fold protein, SPCC16C4.22, to form a heterodimer in fission yeast. We demonstrate that SPCC16C4.22 is nonessential for viability and corresponds to the true ortholog of Dpb3. We further show that the Dpb3–Dpb4 dimer associates with histone deacetylases, chromatin remodelers, and histones and plays a crucial role in the inheritance of histone hypoacetylation in heterochromatin. We solve the 1.9-Å crystal structure of Dpb3–Dpb4 and reveal that they form the H2A–H2B-like dimer. Disruption of Dpb3–Dpb4 dimerization results in loss of heterochromatin silencing. Our findings reveal a link between histone deacetylation and H3K9 methylation and suggest a mechanism for how two processes are coordinated during replication. We propose that the Dpb3–Dpb4 heterodimer together with Cdc20 serves as a platform for the recruitment of chromatin modifiers and remodelers that mediate heterochromatin assembly during DNA replication, and ensure the faithful inheritance of epigenetic marks in heterochromatin.

scholarly journals Pterostilbene Changes Epigenetic Marks at Enhancer Regions of Oncogenes in Breast Cancer Cells

Antioxidants ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1232
Author(s):  
Sadaf Harandi-Zadeh ◽  
Cayla Boycott ◽  
Megan Beetch ◽  
Tony Yang ◽  
Benjamin J. E. Martin ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Dna Methyltransferase ◽  
Epigenetic Marks ◽  
Chip Sequencing ◽  
Dietary Polyphenols ◽  
A Genome ◽  
Epigenetic Patterns

Epigenetic aberrations are linked to sporadic breast cancer. Interestingly, certain dietary polyphenols with anti-cancer effects, such as pterostilbene (PTS), have been shown to regulate gene expression by altering epigenetic patterns. Our group has proposed the involvement of DNA methylation and DNA methyltransferase 3B (DNMT3B) as vital players in PTS-mediated suppression of candidate oncogenes and suggested a role of enhancers as target regions. In the present study, we assess a genome-wide impact of PTS on epigenetic marks at enhancers in highly invasive MCF10CA1a breast cancer cells. Following chromatin immunoprecipitation (ChIP)-sequencing in MCF10CA1a cells treated with 7 μM PTS for 9 days, we discovered that PTS leads to increased binding of DNMT3B at enhancers of 77 genes, and 17 of those genes display an overlapping decrease in the occupancy of trimethylation at lysine 36 of histone 3 (H3K36me3), a mark of active enhancers. We selected two genes, PITPNC1 and LINC00910, and found that their enhancers are hypermethylated in response to PTS. These changes coincided with the downregulation of gene expression. Of importance, we showed that 6 out of 17 target enhancers, including PITPNC1 and LINC00910, are bound by an oncogenic transcription factor OCT1 in MCF10CA1a cells. Indeed, the six enhancers corresponded to genes with established or putative cancer-driving functions. PTS led to a decrease in OCT1 binding at those enhancers, and OCT1 depletion resulted in PITPNC1 and LINC00910 downregulation, further demonstrating a role for OCT1 in transcriptional regulation. Our findings provide novel evidence for the epigenetic regulation of enhancer regions by dietary polyphenols in breast cancer cells.

scholarly journals Tax induces the recruitment of NF-kB to unintegrated HIV-1 DNA to rescue viral gene expression and replication

2021 ◽  
Author(s):  
Ishak D. Irwan ◽  
Bryan R. Cullen
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Lentiviral Vectors ◽  
Expression Vectors ◽  
Viral Gene ◽  
Epigenetic Marks ◽  
Tax Protein ◽  
Human T Cell ◽  
Activation Of Transcription ◽  
Hiv 1

AbstractWe have previously reported that the normally essential step of integration of the HIV-1 proviral DNA intermediate into the host cell genome becomes dispensable in T cells that express the Human T cell leukemia virus 1 (HTLV-1) Tax protein. The rescue of integrase (IN) deficient HIV-1 replication by Tax results from the strong activation of transcription from the long terminal repeat (LTR) promoter on episomal HIV-1 DNA, an effect that is closely correlated with the recruitment of activating epigenetic marks, such as H3Ac, and depletion of repressive epigenetic marks, such as H3K9me3, from chromatinized unintegrated proviruses. In addition, activation of transcription from unintegrated HIV-1 DNA coincides with the recruitment of NF-kB to the two NF-kB binding sites found in the HIV-1 LTR enhancer. Here we report that the recruitment of NF-kB to unintegrated viral DNA precedes, and is a prerequisite for, Tax-induced changes in epigenetic marks, so that an IN-HIV-1 mutant lacking both LTR NF-kB sites is entirely non-responsive to Tax and fails to undergo the epigenetic changes listed above. We also report that heterologous promoters introduced into IN-HIV-1-based vectors are transcriptionally active even in the absence of Tax. Finally, we failed to reproduce a recent report arguing that heterologous promoters introduced into IN-vectors based on HIV-1 are more active if the HIV-1 promoter and enhancer, located in the LTR U3 region, are deleted, in a so-called self inactivating or SIN lentivector design.ImportanceIntegrase-deficient expression vectors based on HIV-1 are becoming increasingly popular as tools for gene therapy in vivo due to their inability to cause insertional mutagenesis. However, many IN-lentiviral vectors are able to achieve only low levels of gene expression and methods to increase this low level have not been extensively explored. Here we analyze how the HTLV-1 Tax protein is able to rescue the replication of IN-HIV-1 in T cells and describe IN-lentiviral vectors that are able to express a heterologous gene effectively.

Swi6/HP1 binding to RNA-DNA hybrids initiates heterochromatin assembly

2020 ◽  
Author(s):  
Jagmohan Singh ◽  
Jyotsna Kumar ◽  
Swati Haldar ◽  
Neelima Gupta ◽  
Viney Kumar ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Rnai Pathway ◽  
Affinity Binding ◽  
Heterochromatin Formation ◽  
Independent Mechanism ◽  
Alternate Model ◽  
Self Association ◽  
Heterochromatin Silencing ◽  
Heterochromatin Structure ◽  
Heterochromatin Assembly

Abstract Heterochromatin formation in fission yeast and metazoans involves di/trimethylation of histone H3 at lysine 9 position (me2/me3-K9-H3) by the histone methyltransferase (HMT) Suv39/Clr4, followed by binding of Swi6/HP1 to me2/me3-K9-H3 via its chromodomain1. Subsequent self-association of Swi6/HP1 on adjacent nucleosomes leads to folded heterochromatin structure1-3. An alternate model suggests a concerted participation of Clr4 and Swi6/HP12,3. HP1 binding to RNA has been invoked for heterochromatin silencing in metazoans4,5. Swi6/HP1 also binds and channels RNA to exosome pathway in fission yeast6. Recruitment of Swi6/HP1 to centromere is also dependent on the RNAi pathway7. Here we show that Swi6/HP1 exhibits binding to RNAs, ranging from promiscuous, low-affinity binding to mRNAs, to moderate-affinity binding to the RNAi-generated siRNAs corresponding to the repeats present in heterochromatin regions7, to high affinity binding to the RNA-DNA hybrids cognate to the repeats. Together with sensitivity of Swi6 localization and silencing to RNaseH, our results suggest a dynamic distribution of Swi6/HP1 among the heterochromatin and euchromatic transcripts and binding to RNA-DNA hybrid as an RNAi-dependent and Me2/me3-K9-H3-independent mechanism of recruitment, leading to heterochromatin formation and silencing.

scholarly journals Host Cell Gene Expression during Human Immunodeficiency Virus Type 1 Latency and Reactivation and Effects of Targeting Genes That Are Differentially Expressed in Viral Latency

2004 ◽  
Vol 78 (17) ◽  
pp. 9458-9473 ◽  
Author(s):  
Vyjayanthi Krishnan ◽  
Steven L. Zeichner
Keyword(s):  
Gene Expression ◽  
Host Cell ◽  
Cell Lines ◽  
Lytic Replication ◽  
Replication Cycle ◽  
Differentially Expressed ◽  
Cellular Genes ◽  
Latently Infected ◽  
Cell Gene Expression ◽  
Infected Cells

ABSTRACT The existence of reservoirs of cells latently infected with human immunodeficiency virus (HIV) is a major obstacle to the elimination of HIV infection. We studied the changes in cellular gene expression that accompany the reactivation and completion of the lytic viral cycle in cell lines chronically infected with HIV-1. We found that several genes exhibited altered expression in the chronically infected cells compared to the uninfected parental cells prior to induction into lytic replication. A number of gene classes showed increased expression in the chronically infected cells, notably including genes encoding proteasomes, histone deacetylases, and many transcription factors. Following induction of the lytic replication cycle, we observed ordered, time-dependent changes in the cellular gene expression pattern. Approximately 1,740 genes, many of which fall into 385 known pathways, were differentially expressed (P < 0.001), indicating that completion of the HIV replication cycle is associated with distinct, temporally ordered changes in host cell gene expression. Maximum changes were observed in the early and intermediate phases of the lytic replication cycle. Since the changes in gene expression in chronically infected cells suggested that cells latently infected with HIV have a different gene expression profile than corresponding uninfected cells, we studied the expression profiles of three different chronically infected cell lines to determine whether they showed similar changes in common cellular genes and pathways. Thirty-two genes showed significant differential expression in all cell lines studied compared to their uninfected parental cell lines. Notable among them were cdc42 and lyn, which were downregulated and are required for HIV Nef binding and viral replication. Other genes previously unrelated to HIV latency or pathogenesis were also differentially expressed. To determine the effects of targeting products of the genes that were differentially expressed in latently infected cells, we treated the latently infected cells with a proteasome inhibitor, clastolactacystin-beta-lactone (CLBL), and an Egr1 activator, resveratrol. We found that treatment with CLBL and resveratrol stimulated lytic viral replication, suggesting that treatment of cells with agents that target cellular genes differentially expressed in latently infected cells can stimulate lytic replication. These findings may offer new insights into the interaction of the latently infected host cell and HIV and suggest therapeutic approaches for inhibiting HIV infection and for manipulating cells latently infected with HIV so as to trigger lytic replication.

500. THE MANIPULATION OF THE EPIGENETIC MARK HISTONE 3 LYSINE 9 TRIMETHYLATION IN DONOR CELLS AND ITS EFFECTS ON THE DEVELOPMENT OF CLONED MOUSE EMBRYOS

2009 ◽  
Vol 21 (9) ◽  
pp. 101
Author(s):  
J. Antony ◽  
F. Oback ◽  
R. Broadhurst ◽  
S. Cole ◽  
C. Graham ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Transfer ◽  
Expression Profiles ◽  
Es Cells ◽  
Embryonic Stem ◽  
Histone Demethylase ◽  
Specific Gene ◽  
Epigenetic Mark ◽  
Epigenetic Marks ◽  
Donor Cells

To produce live cloned mammals from adult somatic cells the nuclei of these cells must be first reprogrammed from a very restricted, cell lineage-specific gene expression profile to an embryo-like expression pattern, compatible with embryonic development. Although this has been achieved in a number of species the efficiency of cloning remains very low. Inadequate reprogramming of epigenetic marks in the donor cells correlated with aberrant embryonic gene expression profiles has been identified as a key cause of this inefficiency. Some of the most common epigenetic marks are chemical modifications of histones, the main structural proteins of chromatin. A range of different histone modifications, including acetylation and methylation, exists and can be attributed to either repression or activation of genes. One epigenetic mark which is known to be very stable and difficult to remove during reprogramming is the trimethylation of lysine 9 in histone H3 (H3K9Me3). To test the hypothesis that H3K9Me3 marks are a major stumbling block for successful cloning we are attempting to remove these marks by overexpression of the H3K9Me3 specific histone demethylase, jmjd2b, in donor cells, prior to their use for nuclear transfer. We have engineered mouse embryonic stem (ES) cells for the tet inducible expression of a fusion protein with a functional jmjd2b or non-functional mutant jmjd2b histone demethylase. Approximately 94% and 88% of the cells can be induced for the expression of functional and mutant jmjd2b-EGFP in the respective ES cell lines. Immunofluorescence analyses have shown that induction of functional jmjd2b-EGFP results in an approximately 50% reduction of H3K9Me3 levels compared to non-induced cells and induced mutant jmjd2b-EGFP cells. The comparison of the in-vitro embryo development following nuclear transfer with induced and non-induced donor cells show significantly better overall development to blastocysts and morulae from induced donor cells with reduced H3K9Me3 levels.

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