Single-genome analysis reveals a heterogeneous association of the herpes simplex virus genome with H3K27me2 and the reader PHF20L1 following infection of human fibroblasts.

The fate of herpesvirus genomes following entry into different cell types is thought to regulate the outcome of infection. For the Herpes simplex virus 1 (HSV-1), latent infection of neurons is characterized by association with repressive heterochromatin marked with Polycomb silencing-associated lysine 27 methylation on histone H3 (H3K27me). However, whether H3K27 methylation plays a role in repressing lytic gene expression in non-neuronal cells is unclear. To address this gap in knowledge, and with consideration that the fate of the viral genome and outcome of HSV-1 infection could be heterogeneous, we developed an assay to quantify the abundance of histone modifications within single viral genome foci of infected fibroblasts. Using this approach, combined with bulk epigenetic techniques, we were unable to detect any role for H3K27me3 during HSV-1 lytic infection of fibroblasts. By contrast, we could detect the lesser studied H3K27me2 on a subpopulation of viral genomes, which was consistent with a role for H3K27 demethylases in promoting lytic gene expression. In addition, viral genomes co-localized with the H3K27me2 reader protein PHF20L1, and this association was enhanced by inhibition of the H3K27 demethylases UTX and JMJD3. Notably, targeting of H3K27me2 to viral genomes was enhanced following infection with a transcriptionally defective virus in the absence of Promyelocytic leukemia nuclear bodies. Collectively, these studies implicate a role for H3K27me2 in fibroblast-associated HSV genome silencing in a manner dependent on genome sub-nuclear localization and transcriptional activity. IMPORTANCE: Investigating the potential mechanisms of gene silencing for DNA viruses in different cell types is important to understand the differential outcomes of infection, particularly for viruses like herpesviruses that can undergo distinct types of infection in different cell types. In addition, investigating chromatin association with viral genomes informs on the mechanisms of epigenetic regulation of DNA processes. However, there is a growing appreciation for heterogeneity in the outcome of infection at the single cell, and even single viral genome, level. Here we describe a novel assay for quantifying viral genome foci with chromatin proteins and show that a portion of genomes are targeted for silencing by H3K27me2 and associate with the reader protein PHF20L1. This study raises important questions regarding the mechanism of H3K27me2-specific targeting to viral genomes, the contribution of epigenetic heterogeneity to herpesvirus infection, and the role of PHF20L1 in regulating the outcome of DNA virus infection.

UHRF1 ubiquitin ligase activity supports the maintenance of low-density CpG methylation.

The RING E3 ubiquitin ligase UHRF1 is an established cofactor for DNA methylation inheritance. Nucleosomal engagement through histone and DNA interactions directs UHRF1 ubiquitin ligase activity toward lysines on histone H3 tails, creating binding sites for DNMT1 through ubiquitin interacting motifs (UIM1 and UIM2). Here, we profile contributions of UHRF1 and DNMT1 to genome-wide DNA methylation inheritance and dissect specific roles for ubiquitin signaling in this process. We reveal DNA methylation maintenance at low-density CpGs is vulnerable to disruption of UHRF1 ubiquitin ligase activity and DNMT1 ubiquitin reading activity through UIM1. Hypomethylation of low-density CpGs in this manner induces formation of partially methylated domains (PMD), a methylation signature observed across human cancers. Furthermore, disrupting DNMT1 UIM2 function abolishes DNA methylation maintenance. Collectively, we show DNMT1-dependent DNA methylation inheritance is a ubiquitin-regulated process and suggest a disrupted UHRF1-DNMT1 ubiquitin signaling axis contributes to the development of PMDs in human cancers.

Single-genome analysis reveals heterogeneous association of the Herpes Simplex Virus genome with H3K27me2 and the reader PHF20L1 following infection of human fibroblasts.

The fate of herpesvirus genomes following entry into different cell types is thought to regulate the outcome of infection. For the Herpes simplex virus 1 (HSV-1), latent infection of neurons is characterized by association with repressive heterochromatin marked with Polycomb silencing-associated lysine 27 methylation on histone H3 (H3K27me). However, whether H3K27 methylation plays a role in repressing lytic gene expression in non-neuronal cells is unclear. To address this gap in knowledge, and with consideration that the fate of the viral genome and outcome of HSV-1 infection could be heterogeneous, we developed an assay to quantify the abundance of histone modifications within single viral genome foci of infected fibroblasts. Using this approach, combined with bulk epigenetic techniques, we were unable to detect any role for H3K27me3 during HSV-1 lytic infection of fibroblasts. In contrast, we could detect the lesser studied H3K27me2 on a subpopulation of viral genomes, which was consistent with a role for H3K27 demethylases in promoting lytic gene expression. This was consistent with a role for H3K27 demethylases in promoting lytic gene expression. In addition, viral genomes co-localized with the H3K27me2 reader protein PHF20L1, and this association was enhanced by inhibition of the H3K27 demethylases UTX and JMJD3. Notably, targeting of H3K27me2 to viral genomes was enhanced following infection with a transcriptionally defective virus in the absence of Promyelocytic leukemia nuclear bodies. Collectively, these studies implicate a role for H3K27me2 in fibroblast-associated HSV genome silencing in a manner dependent on genome sub-nuclear localization and transcriptional activity.

ARID1A-dependent maintenance of H3.3 is required for repressive CHD4-ZMYND8 chromatin interactions at super-enhancers.

BACKGROUND: SWI/SNF (BAF) chromatin remodeling complexes regulate lineage-specific enhancer activity by promoting accessibility for diverse DNA-binding factors and chromatin regulators. Additionally, they are known to modulate the function of the epigenome through regulation of histone post-translational modifications and nucleosome composition, although the way SWI/SNF complexes govern the epigenome remains poorly understood. Here, we investigate the function of ARID1A, a subunit of certain mammalian SWI/SNF chromatin remodeling complexes associated with malignancies and benign diseases originating from the uterine endometrium. RESULTS: Through genome-wide analysis of human endometriotic epithelial cells, we show that more than half of ARID1A binding sites are marked by the variant histone H3.3, including active regulatory elements such as super-enhancers. ARID1A knockdown leads to H3.3 depletion and gain of canonical H3.1/3.2 at ARID1A-bound active regulatory elements, and a concomitant redistribution of H3.3 toward genic elements. ARID1A interactions with the repressive chromatin remodeler CHD4 (NuRD) are associated with H3.3, and ARID1A is required for CHD4 recruitment to H3.3. ZMYND8 interacts with CHD4 to suppress a subset of ARID1A, CHD4, and ZMYND8 co-bound, H3.3+ H4K16ac+ super-enhancers near genes governing extracellular matrix, motility, adhesion, and epithelial-to-mesenchymal transition. Moreover, these gene expression alterations are observed in human endometriomas. CONCLUSIONS: These studies demonstrate that ARID1A-containing BAF complexes are required for maintenance of the histone variant H3.3 at active regulatory elements, such as super-enhancers, and this function is required for the physiologically relevant activities of alternative chromatin remodelers.

Histone H3.3 phosphorylation amplifies stimulation-induced transcription.

Complex organisms can rapidly induce select genes in response to diverse environmental cues. This regulation occurs in the context of large genomes condensed by histone proteins into chromatin. The sensing of pathogens by macrophages engages conserved signalling pathways and transcription factors to coordinate the induction of inflammatory genes1-3. Enriched integration of histone H3.3, the ancestral histone H3 variant, is a general feature of dynamically regulated chromatin and transcription4-7. However, how chromatin is regulated at induced genes, and what features of H3.3 might enable rapid and high-level transcription, are unknown. The amino terminus of H3.3 contains a unique serine residue (Ser31) that is absent in 'canonical' H3.1 and H3.2. Here we show that this residue, H3.3S31, is phosphorylated (H3.3S31ph) in a stimulation-dependent manner along rapidly induced genes in mouse macrophages. This selective mark of stimulation-responsive genes directly engages the histone methyltransferase SETD2, a component of the active transcription machinery, and 'ejects' the elongation corepressor ZMYND118,9. We propose that features of H3.3 at stimulation-induced genes, including H3.3S31ph, provide preferential access to the transcription apparatus. Our results indicate dedicated mechanisms that enable rapid transcription involving the histone variant H3.3, its phosphorylation, and both the recruitment and the ejection of chromatin regulators.

A Read/Write Mechanism Connects p300 Bromodomain Function to H2A.Z Acetylation.

Acetylation of the histone variant H2A.Z (H2A.Zac) occurs at active regulatory regions associated with gene expression. Although the Tip60 complex is proposed to acetylate H2A.Z, functional studies suggest additional enzymes are involved. Here, we show that p300 acetylates H2A.Z at multiple lysines. In contrast, we found that although Tip60 does not efficiently acetylate H2A.Z in vitro, genetic inhibition of Tip60 reduces H2A.Zac in cells. Importantly, we found that interaction between the p300-bromodomain and H4 acetylation (H4ac) enhances p300-driven H2A.Zac. Indeed, H2A.Zac and H4ac show high genomic overlap, especially at active promoters. We also reveal unique chromatin features and transcriptional states at enhancers correlating with co-occurrence or exclusivity of H4ac and H2A.Zac. We propose that differential H4 and H2A.Z acetylation signatures can also define the enhancer state. In conclusion, we show both Tip60 and p300 contribute to H2A.Zac and reveal molecular mechanisms of writer/reader crosstalk between H2A.Z and H4 acetylation through p300.

OGT binds a conserved C-terminal domain of TET1 to regulate TET1 activity and function in development.

TET enzymes convert 5-methylcytosine to 5-hydroxymethylcytosine and higher oxidized derivatives. TETs stably associate with and are post-translationally modified by the nutrient-sensing enzyme OGT, suggesting a connection between metabolism and the epigenome. Here, we show for the first time that modification by OGT enhances TET1 activity in vitro. We identify a TET1 domain that is necessary and sufficient for binding to OGT and report a point mutation that disrupts the TET1-OGT interaction. We show that this interaction is necessary for TET1 to rescue hematopoetic stem cell production in tet mutant zebrafish embryos, suggesting that OGT promotes TET1's function during development. Finally, we show that disrupting the TET1-OGT interaction in mouse embryonic stem cells changes the abundance of TET2 and 5-methylcytosine, which is accompanied by alterations in gene expression. These results link metabolism and epigenetic control, which may be relevant to the developmental and disease processes regulated by these two enzymes.

In vitro analysis of RNA degradation catalyzed by deadenylase enzymes.

In this chapter, we describe a method for purification and analysis of the enzymatic activity of deadenylase enzymes. Nearly all eukaryotic messenger RNAs are modified at the 3' end by the addition of an adenosine polymer: the poly-adenosine tail. The poly(A) tail plays a central role in protein expression and mRNA fate. The poly(A) tail promotes translation of the mRNA. Shortening of the poly(A) tail, referred to as deadenylation, reduces protein synthesis and initiates destruction of the mRNA. A specialized class of exoribonucleases, called deadenylase enzymes, carries out this process. Deadenylases are found throughout eukarya, but their functions remain largely unexplored. We present a detailed protocol to analyze deadenylase activity in vitro. First, recombinant deadenylase enzyme is over-expressed and purified from bacteria. Next, labeled RNA substrate is prepared. Deadenylation reactions are performed, and reaction products are analyzed by denaturing gel electrophoresis. Reaction rates are then determined quantitatively. Crucial controls and experimental parameters are described along with practical tips that promote success.

Human Pumilio proteins recruit multiple deadenylases to efficiently repress messenger RNAs.

PUF proteins are a conserved family of eukaryotic RNA-binding proteins that regulate specific mRNAs: they control many processes including stem cell proliferation, fertility, and memory formation. PUFs repress protein expression from their target mRNAs but the mechanism by which they do so remains unclear, especially for humans. Humans possess two PUF proteins, PUM1 and PUM2, which exhibit similar RNA binding specificities. Here we report new insights into their regulatory activities and mechanisms of action. We developed functional assays to measure sequence-specific repression by PUM1 and PUM2. Both robustly inhibit translation and promote mRNA degradation. Purified PUM complexes were found to contain subunits of the CCR4-NOT (CNOT) complex, which contains multiple enzymes that catalyze mRNA deadenylation. PUMs interact with the CNOT deadenylase subunits in vitro. We used three approaches to determine the importance of deadenylases for PUM repression. First, dominant-negative mutants of CNOT7 and CNOT8 reduced PUM repression. Second, RNA interference depletion of the deadenylases alleviated PUM repression. Third, the poly(A) tail was necessary for maximal PUM repression. These findings demonstrate a conserved mechanism of PUF-mediated repression via direct recruitment of the CCR4-POP2-NOT deadenylase leading to translational inhibition and mRNA degradation. A second, deadenylation independent mechanism was revealed by the finding that PUMs repress an mRNA that lacks a poly(A) tail. Thus, human PUMs are repressors capable of deadenylation-dependent and -independent modes of repression.