Silent Chromatin Research Articles

R-loops and regulatory changes in chronologically ageing fission yeast cells drive non-random patterns of genome rearrangements.

Aberrant repair of DNA double-strand breaks can recombine distant chromosomal breakpoints. Chromosomal rearrangements compromise genome function and are a hallmark of ageing. Rearrangements are challenging to detect in non-dividing cell populations, because they reflect individually rare, heterogeneous events. The genomic distribution of de novo rearrangements in non-dividing cells, and their dynamics during ageing, remain therefore poorly characterized. Studies of genomic instability during ageing have focussed on mitochondrial DNA, small genetic variants, or proliferating cells. To characterize genome rearrangements during cellular ageing in non-dividing cells, we interrogated a single diagnostic measure, DNA breakpoint junctions, using Schizosaccharomyces pombe as a model system. Aberrant DNA junctions that accumulated with age were associated with microhomology sequences and R-loops. Global hotspots for age-associated breakpoint formation were evident near telomeric genes and linked to remote breakpoints elsewhere in the genome, including the mitochondrial chromosome. Formation of breakpoint junctions at global hotspots was inhibited by the Sir2 histone deacetylase and might be triggered by an age-dependent de-repression of chromatin silencing. An unexpected mechanism of genomic instability may cause more local hotspots: age-associated reduction in an RNA-binding protein triggering R-loops at target loci. This result suggests that biological processes other than transcription or replication can drive genome rearrangements. Notably, we detected similar signatures of genome rearrangements that accumulated in old brain cells of humans. These findings provide insights into the unique patterns and possible mechanisms of genome rearrangements in non-dividing cells, which can be promoted by ageing-related changes in gene-regulatory proteins.

A unique GCN5 histone acetyltransferase complex controls erythrocyte invasion and virulence in the malaria parasite Plasmodium falciparum.

The histone acetyltransferase GCN5-associated SAGA complex is evolutionarily conserved from yeast to human and functions as a general transcription co-activator in global gene regulation. In this study, we identified a divergent GCN5 complex in Plasmodium falciparum, which contains two plant homeodomain (PHD) proteins (PfPHD1 and PfPHD2) and a plant apetela2 (AP2)-domain transcription factor (PfAP2-LT). To dissect the functions of the PfGCN5 complex, we generated parasite lines with either the bromodomain in PfGCN5 or the PHD domain in PfPHD1 deleted. The two deletion mutants closely phenocopied each other, exhibiting significantly reduced merozoite invasion of erythrocytes and elevated sexual conversion. These domain deletions caused dramatic decreases not only in histone H3K9 acetylation but also in H3K4 trimethylation, indicating synergistic crosstalk between the two euchromatin marks. Domain deletion in either PfGCN5 or PfPHD1 profoundly disturbed the global transcription pattern, causing altered expression of more than 60% of the genes. At the schizont stage, these domain deletions were linked to specific down-regulation of merozoite genes involved in erythrocyte invasion, many of which contain the AP2-LT binding motif and are also regulated by AP2-I and BDP1, suggesting targeted recruitment of the PfGCN5 complex to the invasion genes by these specific factors. Conversely, at the ring stage, PfGCN5 or PfPHD1 domain deletions disrupted the mutually exclusive expression pattern of the entire var gene family, which encodes the virulent factor PfEMP1. Correlation analysis between the chromatin state and alteration of gene expression demonstrated that up- and down-regulated genes in these mutants are highly correlated with the silent and active chromatin states in the wild-type parasite, respectively. Collectively, the PfGCN5 complex represents a novel HAT complex with a unique subunit composition including an AP2 transcription factor, which signifies a new paradigm for targeting the co-activator complex to regulate general and parasite-specific cellular processes in this low-branching parasitic protist.

NAT10-Mediated N4-Acetylcytidine of RNA Contributes to Post-transcriptional Regulation of Mouse Oocyte Maturation in vitro.

N4-acetylcytidine (ac4C), a newly identified epigenetic modification within mRNA, has been characterized as a crucial regulator of mRNA stability and translation efficiency. However, the role of ac4C during oocyte maturation, the process mainly controlled via post-transcriptional mechanisms, has not been explored. N-acetyltransferase 10 (NAT10) is the only known enzyme responsible for ac4C production in mammals and ac4C-binding proteins have not been reported yet. In this study, we have documented decreasing trends of both ac4C and NAT10 expression from immature to mature mouse oocytes. With NAT10 knockdown mediated by small interfering RNA (siRNA) in germinal vesicle (GV)-stage oocytes, ac4C modification was reduced and meiotic maturation in vitro was significantly retarded. Specifically, the rate of first polar body extrusion was significantly decreased with NAT10 knockdown (34.6%) compared to control oocytes without transfection (74.6%) and oocytes transfected with negative control siRNA (72.6%) (p < 0.001), while rates of germinal vesicle breakdown (GVBD) were not significantly different (p = 0.6531). RNA immunoprecipitation and high-throughput sequencing using HEK293T cells revealed that the modulated genes were enriched in biological processes associated with nucleosome assembly, chromatin silencing, chromatin modification and cytoskeletal anchoring. In addition, we identified TBL3 as a potential ac4C-binding protein by a bioinformatics algorithm and RNA pulldown with HEK293T cells, which may mediate downstream cellular activities. Taken together, our results suggest that NAT10-mediated ac4C modification is an important regulatory factor during oocyte maturation in vitro and TBL3 is a potential ac4C-binding protein.

Meiotic Silencing in Pigs: A Case Study in a Translocated Azoospermic Boar.

Carriers of balanced constitutional reciprocal translocations usually present a normal phenotype, but often show reproductive disorders. For the first time in pigs, we analyzed the meiotic process of an autosome–autosome translocation associated with azoospermia. Meiotic process analysis revealed the presence of unpaired autosomal segments with histone γH2AX accumulation sometimes associated with the XY body. Additionally, γH2AX signals were observed on apparently synapsed autosomes other than the SSC1 or SSC15, as previously observed in Ataxia with oculomotor apraxia type 2 patients or knock-out mice for the Senataxin gene. Gene expression showed a downregulation of genes selected on chromosomes 1 and 15, but no upregulation of SSCX genes. We hypothesized that the total meiotic arrest observed in this boar might be due to the silencing of crucial autosomal genes by the mechanism referred to as meiotic silencing of unsynapsed chromatin (MSUC).

Abstract 2126: Therapeutic potential of CPI-0209

Abstract Second-generation EZH2 inhibitor CPI-0209 has therapeutic potential for androgen receptor-dependent prostate cancer Rentian Wu, Feng Zhao, Patricia J. Keller, Jennifer A. Mertz, John P. McGrath, Barbara Bryant, Andrew R. Conery, Kaiming Sun, Jing Wang, and Patrick Trojer Post-translational modification of histones plays critical roles in epigenetic regulation of gene expression. Enhancer of Zeste Homolog 2 (EZH2), a subunit of Polycomb Repressive Complex 2 (PRC2), catalyzes trimethylation of histone H3 on lysine 27 (H3K27me3). Chromatin methylation by EHZ2 promotes chromatin architectural changes and silencing of gene expression. EZH2-mediated gene silencing was shown to contribute to tumor initiation and progression. Amplification and overexpression of EZH2 along with down regulation of its target genes correlate with treatment resistance and poor prognosis in various tumor types including metastatic castration resistant prostate cancer (mCRPC). While most mCRPC patients are initially responsive to agents targeting the androgen receptor (AR) signaling pathway, tumors eventually develop resistance and progress. Other than EZH2 overexpression, mechanisms of resistance involve AR alterations, including AR amplification, alteration of AR splice variants which constitutively activate ARS, and AR mutations that affect native ligand-binding specificity. CPI-0209 is an orally bioavailable and selective EZH2 and EZH1 inhibitor with highly enhanced potency and extended residence time compared to first-generation EZH2 inhibitors. CPI-0209 is currently being clinically developed as monotherapy and in combination with other cancer therapeutic agents [NCT04104776]. Here we demonstrated that AR-dependent prostate cancer cell lines including LNCaP, 22Rv1 and VCaP were sensitive to CPI-0209 treatment, while AR-independent cell lines like PC3 and DU145 were insensitive to CPI-0209. Sensitivity to CPI-0209 were also observed in AR-dependent cell line-derived xenografts (CDX) and patient-derived xenografts (PDX) models. In AR-dependent cells, CPI-0209 synergized with enzalutamide and overcame the anti-androgens resistance induced by AR alterations. In prostate cancer CDX and PDX models, the combination treatment of CPI-0209 and enzalutamide showed greater tumor growth inhibition compared to enzalutamide monotherapy. Transcriptomic analysis revealed that CPI-0209 treatment modulated both AR-related and AR-independent pathways, which reveals a potential mechanism to explain the synergy of CPI-0209 and AR inhibitors. Thus, our results indicate that CPI-0209 may have the potential to be effective for AR-dependent mCRPC that are resistant to current AR inhibitors, supporting potential clinical development in mCRPC. Keywords: EZH2, prostate cancer, epigenetics, resistant Citation Format: Rentian Wu, Feng Zhao, Patricia J. Keller, Jennifer A. Mertz, John P. McGrath, Barbara Bryant, Andrew R. Conery, Kaiming Sun, Jing Wang, Patrick Trojer. Therapeutic potential of CPI-0209 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2126.

Abstract 1131: ORIC-944, a potent and selective allosteric PRC2 inhibitor, demonstrates robust in vivo activity in prostate cancer models

Abstract The polycomb repressive complex 2 (PRC2) is responsible for the methylation of histone 3 at lysine 27 (H3K27) which leads to long-term transcriptional silencing. Through this epigenetic chromatin silencing mechanism, PRC2 plays a key role in regulating cellular functions such as cell growth and differentiation. PRC2 comprises three core subunits: the catalytic subunit enhancer of zeste homolog 2 (EZH2), embryonic ectoderm development (EED) and suppressor of zeste 12 (SUZ12). EED directly interacts with histone H3K27me3 and is essential for the histone methyltransferase activity of PRC2. PRC2 dysregulation occurs in multiple solid tumors and hematological malignancies, resulting in elevated levels of PRC2 activity and H3K27 trimethylation, and has been linked to poor prognosis in patients with metastatic prostate cancer. First-generation PRC2 inhibitors which target EZH2 have demonstrated clinical activity in several cancers, yet the pharmacological and ADME properties of these compounds require high doses that only achieve partial target inhibition at clinically active levels and exhibit drug-drug interaction (DDI) liabilities. As an alternative strategy to fully inhibit the PRC2 complex, we developed ORIC-944, a potent and highly selective allosteric inhibitor of PRC2 via binding the EED subunit. This unique EED targeting strategy can more completely inhibit PRC2, for example, in the presence of innate or acquired resistance mutations in EZH2, and by addressing the potential compensatory escape mechanism of EZH1-driven tumor growth. ORIC-944 has potential best-in-class drug properties compared to first generation PRC2 inhibitors, including superior potency and improved DDI liabilities. In diffuse large B-cell lymphoma (DLBCL) xenografts in vivo, ORIC-944 significantly depleted H3K27 trimethylation and induced tumor regressions in a dose-dependent manner. ORIC-944 demonstrated strong tumor growth inhibition as a single agent with once daily oral dosing in both enzalutamide-responsive and enzalutamide-resistant prostate cancer models. ORIC-944 caused a significant reduction in tumor cell proliferation and anti-apoptotic signaling, as measured by Ki67 and survivin, respectively. Moreover, in PK/PD assessments, ORIC-944 strongly depleted H3K27me3 in treated tumors. These in vivo studies established that effective single agent inhibition of PRC2 via EED results in decreased tumor cell growth in PRC2-dependent prostate cancer models. In summary, ORIC-944 is a potent, highly selective, allosteric, orally bioavailable PRC2 inhibitor via the EED subunit that represents a differentiated strategy to block PRC2 activity in selected cancers. We are developing ORIC-944 as a best-in-class PRC2 inhibitor for the treatment of patients with advanced prostate cancer and expect to file an IND in the second half of 2021. Citation Format: Anneleen Daemen, Jessica D. Sun, Aleksandr Pankov, Frank L. Duong, Natalie Yuen, Shravani Barkund, Shelly Kaushik, Jae H. Chang, David M. Briere, Niranjan Sudhakar, Andrew Calinisan, Aaron Burns, John M. Ketcham, Matthew A. Marx, Peter Olson, James G. Christensen, Melissa R. Junttila, Lori S. Friedman. ORIC-944, a potent and selective allosteric PRC2 inhibitor, demonstrates robust in vivo activity in prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1131.

Identifying a Serum Exosomal-Associated lncRNA/circRNA-miRNA-mRNA Network in Coronary Heart Disease

Background Accumulating evidence supports the importance of noncoding RNAs and exosomes in coronary heart disease (CHD). However, exosomal-associated competing endogenous RNA- (ceRNA-) mediated regulatory mechanisms in CHD are largely unexplored. The present study aimed to explore exosomal-associated ceRNA networks in CHD. Methods Data from 6 CHD patients and 32 normal controls were downloaded from the ExoRBase database. CHD and normal controls were compared by screening differentially expressed mRNAs (DEMs), lncRNAs (DELs), and circRNAs (DECs) in serum exosomes. MicroRNAs (miRNAs) targeting DEMs were predicted using the Targetscan and miRanda databases, and miRNAs targeted by DELs and DECs were predicted using the miRcode and starBase databases, respectively. The biological functions and related signaling pathways of DEMs were analyzed using the David and KOBAS databases. Subsequently, a protein-protein interaction (PPI) network was established to screen out on which hub genes enrichment analyses should be performed, and a ceRNA network (lncRNA/circRNA-miRNA-mRNA) was constructed to elucidate ceRNA axes in CHD. Results A total of 312 DEMs, 43 DELs, and 85 DECs were identified between CHD patients and normal controls. Functional enrichment analysis showed that DEMs were significantly enriched in “chromatin silencing at rDNA,” “telomere organization,” and “negative regulation of gene expression, epigenetic.” PPI network analysis showed that 25 hub DEMs were closely related to CHD, of which ubiquitin C (UBC) was the most important. Hub genes were mainly enriched in “cellular protein metabolic process” functions. The exosomal-associated ceRNA regulatory network incorporated 48 DEMs, 73 predicted miRNAs, 10 DELs, and 15 DECs. The LncRNA/circRNA-miRNA-mRNA interaction axes (RPL7AP11/hsa-miR-17-5p/UBC and RPL7AP11/hsa-miR-20b-5p/UBC) were obtained from the network. Conclusions Our findings provide a novel perspective on the potential role of exosomal-associated ceRNA network regulation of the pathogenesis of CHD.

An enhancer screen identifies new suppressors of small-RNA-mediated epigenetic gene silencing

Small non-protein coding RNAs are involved in pathways that control the genome at the level of chromatin. In Schizosaccharomyces pombe, small interfering RNAs (siRNAs) are required for the faithful propagation of heterochromatin that is found at peri-centromeric repeats. In contrast to repetitive DNA, protein-coding genes are refractory to siRNA-mediated heterochromatin formation, unless siRNAs are expressed in mutant cells. Here we report the identification of 20 novel mutant alleles that enable de novo formation of heterochromatin at a euchromatic protein-coding gene by using trans-acting siRNAs as triggers. For example, a single amino acid substitution in the pre-mRNA cleavage factor Yth1 enables siRNAs to trigger silent chromatin formation with unparalleled efficiency. Our results are consistent with a kinetic nascent transcript processing model for the inhibition of small-RNA-directed de novo formation of heterochromatin and lay a foundation for further mechanistic dissection of cellular activities that counteract epigenetic gene silencing.

Timing cues epigenomic reprogramming

Signal Transduction Different temporal dynamics of activation of the transcription factor nuclear factor κB (NF-κB) can influence the inflammatory response of activated macrophages. Cheng et al. report a mechanism by which oscillatory and sustained NF-κB signaling may produce distinct transcriptional responses (see the Perspective by Nandagopal et al. ). Oscillatory activation of NF-κB activated poised enhancers to transcribe inflammatory genes in mouse macrophages. However, sustained activation of NF-κB produced in cells activated by other stimuli acted on the epigenome. These stimuli relieved chromatin silencing at enhancers and enabled regulation of additional genes. Science , abc0269, this issue p. [1349][1]; see also abj2040, p. [1263][2] [1]: /lookup/doi/10.1126/science.abc0269 [2]: /lookup/doi/10.1126/science.abj2040

Post-diapause transcriptomic restarts: insight from a high-latitude copepod

BackgroundDiapause is a seasonal dormancy that allows organisms to survive unfavorable conditions and optimizes the timing of reproduction and growth. Emergence from diapause reverses the state of arrested development and metabolic suppression returning the organism to an active state. The physiological mechanisms that regulate the transition from diapause to post-diapause are still unknown. In this study, this transition has been characterized for the sub-arctic calanoid copepod Neocalanus flemingeri, a key crustacean zooplankter that supports the highly productive North Pacific fisheries. Transcriptional profiling of females, determined over a two-week time series starting with diapausing females collected from > 400 m depth, characterized the molecular mechanisms that regulate the post-diapause trajectory.ResultsA complex set of transitions in relative gene expression defined the transcriptomic changes from diapause to post-diapause. Despite low temperatures (5–6 °C), the switch from a “diapause” to a “post-diapause” transcriptional profile occurred within 12 h of the termination stimulus. Transcriptional changes signaling the end of diapause were activated within one-hour post collection and included the up-regulation of genes involved in the 20E cascade pathway, the TCA cycle and RNA metabolism in combination with the down-regulation of genes associated with chromatin silencing. By 12 h, females exhibited a post-diapause phenotype characterized by the up-regulation of genes involved in cell division, cell differentiation and multiple developmental processes. By seven days post collection, the reproductive program was fully activated as indicated by up-regulation of genes involved in oogenesis and energy metabolism, processes that were enriched among the differentially expressed genes.ConclusionsThe analysis revealed a finely structured, precisely orchestrated sequence of transcriptional changes that led to rapid changes in the activation of biological processes paving the way to the successful completion of the reproductive program. Our findings lead to new hypotheses related to potentially universal mechanisms that terminate diapause before an organism can resume its developmental program.

Polycomb repressive complex 2 mutations predict survival benefit in advanced cancer patients treated with immune checkpoint inhibitors.

BackgroundNumerous biomarkers are being tested to enhance the ability of clinicians to predict responses and prognosis after treatment with immune checkpoint inhibitors (ICIs). Polycomb repressive complex 2 (PRC2) is a histone methyltransferase family that plays a major role in chromatin silencing. Preclinical evidence implicates PRC2 components such as enhancer of zeste homolog 2 (EZH2) in immune resistance. This study aimed to assess the clinical relevance of PRC2 mutations in the clinical outcome of ICI-treated patients.Materials and methodsNext-generation sequencing (NGS) data from tumor samples of patients treated with ICIs (anti-PD-1/PD-L1, anti-CTLA-4 or both) were interrogated for alterations in PRC2-related genes. The Kaplan–Meier method was used to assess the association between altered and unaltered PRC2-related genes with overall survival.ResultsSomatic NGS data from 1662 advanced-stage, ICI-treated patients with various primaries (lung, melanoma, bladder, kidney, head neck, esophagogastric, glioma, colorectal, breast, unknown primary) were examined. Seventy patients (4%) harbored truncating or missense mutations or fusions in EZH2 (2.4%), EZH1 (1.2%), SUZ12 (0.9%) or EED (0.7%) genes. Patients carrying alterations in PRC2 genes had significantly longer median overall survival (44 months) compared with those with unaltered tumors (18 months, log-rank P=0.0174). These findings were validated in two additional cohorts of patients (n=313) with various primaries (melanoma, lung, bladder, head neck, anal, sarcoma) who were treated with ICIs.ConclusionsInactivating mutations in the PRC2 chromatin silencing machinery, although rare, may predict favorable outcomes in ICI-treated patients with metastatic cancers. This warrants prospective confirmation, and suggests that epigenetic regulators could serve as surrogate markers to guide ICI treatment decisions.

Inhibition of histone lysine demethylases with TACH101, a first-in-class pan-inhibitor of KDM4.

3105 Background: The KDM4 family of histone lysine demethylases consists of four main isoforms (KDM4A, B, C, D), all of which have been identified as key oncogenic drivers. They function as epigenetic regulators and control transitions between transcriptionally silent and active chromatin states via removal of methyl marks on histone H3K9 and histone H3K36. KDM4 isoforms play an important role in the epigenetic dysregulation in various cancers and is linked to more aggressive disease and poorer clinical outcomes. Functional redundancy and cross-activity have been observed across KDM4 family members, thus, selective inhibition of one isoform appears to not be effective. TACH101 is a novel, first-in-class pan inhibitor of KDM4 that simultaneously targets multiple isoforms of KDM4. Here we present data that show TACH101 has promising pre-clinical and pharmacologic properties as a cancer therapeutic. Methods: TACH101 was evaluated in in vitro and in vivo studies including cell-proliferation assays in multiple cancer cell lines, apoptotic and cell cycle analyses, and efficacy studies in various xenograft tumor models and patient-derived organoid models. Results: In vitro, TACH101 was broadly effective in killing 67% (200 out of 300) of cancer cell lines screened. TACH101 demonstrated potent increase of H3K36me3 levels (EC50 &lt; 0.001 mM, HTRF) in KYSE-150 cell line engineered to overexpress KDM4C and potent anti-proliferative activity in multiple cell lines in OncoPanel. TACH101 treatment increased cancer cell population in S-phase in multiple cancer cell lines indicating cell-cycle arrest. TACH101 induced apoptosis in human colorectal (HT-29), esophageal (KYSE-150), and triple negative breast cancer (MDA-MB-231) cell lines with EC50s ranging from 0.033-0.092 µM. In vivo, TACH101 triggered effective tumor control in xenograft models including colorectal, esophageal, gastric, breast, and lymphoma with tumor growth inhibition of up to 100%. Further evaluation using a panel of patient-derived colorectal models and patient-derived organoids showed a strong correlation of TACH101 sensitivity with MSI-H status (IC50 ranges 1-150 nM). TACH101 also reduced tumorigenic potential by 4.4-fold as determined by FACS analysis using sorted CD44High EpCAM+ population in Limiting Dilution Assays in vivo, suggesting that reduction of cancer stem cells by TACH101 may be effective in therapy-resistant settings. Pharmacologic studies showed TACH101 demonstrated favorable cell permeability, good oral bioavailability, and high metabolic stability. Conclusions: Extensive preclinical work on TACH101 KDM4 inhibitor shows compelling data and broad applicability as a potential anti-cancer agent. Further evaluation is ongoing to advance the molecule into clinical trials.

HDAC1 SUMOylation promotes Argonaute-directed transcriptional silencing in C. elegans.

Eukaryotic cells use guided search to coordinately control dispersed genetic elements. Argonaute proteins and their small RNA cofactors engage nascent RNAs and chromatin-associated proteins to direct transcriptional silencing. The small ubiquitin-like modifier (SUMO) has been shown to promote the formation and maintenance of silent chromatin (called heterochromatin) in yeast, plants, and animals. Here, we show that Argonaute-directed transcriptional silencing in Caenorhabditis elegans requires SUMOylation of the type 1 histone deacetylase HDA-1. Our findings suggest how SUMOylation promotes the association of HDAC1 with chromatin remodeling factors and with a nuclear Argonaute to initiate de novo heterochromatin silencing.

Reproductive Isolation Between Taxonomically Controversial Forms of the Gray Voles (Microtus, Rodentia; Arvicolinae): Cytological Mechanisms and Taxonomical Implications.

The formation of hybrid sterility is an important stage of speciation. The voles of the genus Microtus, which is the most speciose genus of rodents, provide a good model for studying the cytological mechanisms of hybrid sterility. The voles of the “mystacinus” group of the subgenus Microtus (2n = 54) comprising several recently diverged forms with unclear taxonomic status are especially interesting. To resolve the taxonomic status of Microtus mystacinus and Microtus kermanensis, we crossed both with Microtus rossiaemeridionalis, and M. kermanensis alone with Microtus arvalis “obscurus” and M. transcaspicus and examined the reproductive performance of their F1 hybrids. All interspecies male hybrids were sterile. Female M. kermanensis × M. arvalis and M. kermanensis × M. transcaspicus hybrids were sterile as well. Therefore, M. mystacinus, M. kermanensis, and M. rossiaemeridionalis could be considered valid species. To gain an insight into the cytological mechanisms of male hybrid sterility, we carried out a histological analysis of spermatogenesis and a cytological analysis of chromosome synapsis, recombination, and epigenetic chromatin modifications in the germ cells of the hybrids using immunolocalization of key meiotic proteins. The hybrids showed wide variation in the onset of spermatogenesis arrest stage, from mature (although abnormal) spermatozoa to spermatogonia only. Chromosome asynapsis was apparently the main cause of meiotic arrest. The degree of asynapsis varied widely across cells, individuals, and the crosses—from partial asynapsis of several small bivalents to complete asynapsis of all chromosomes. The asynapsis was accompanied by a delayed repair of DNA double-strand breaks marked by RAD51 antibodies and silencing of unpaired chromatin marked by γH2A.X antibodies. Overall, the severity of disturbances in spermatogenesis in general and in chromosome synapsis in particular increased in the hybrids with an increase in the phylogenetic distance between their parental species.

R-loop resolution promotes co-transcriptional chromatin silencing

RNA-mediated chromatin silencing is central to genome regulation in many organisms. However, how nascent non-coding transcripts regulate chromatin is poorly understood. Here, through analysis of Arabidopsis FLC, we show that resolution of a nascent-transcript-induced R-loop promotes chromatin silencing. Stabilization of an antisense-induced R-loop at the 3′ end of FLC enables an RNA binding protein FCA, with its direct partner FY/WDR33 and other 3′-end processing factors, to polyadenylate the nascent antisense transcript. This clears the R-loop and recruits the chromatin modifiers demethylating H3K4me1. FCA immunoprecipitates with components of the m6A writer complex, and m6A modification affects dynamics of FCA nuclear condensates, and promotes FLC chromatin silencing. This mechanism also targets other loci in the Arabidopsis genome, and consistent with this fca and fy are hypersensitive to a DNA damage-inducing drug. These results show how modulation of R-loop stability by co-transcriptional RNA processing can trigger chromatin silencing.

JASMONATE-ZIM DOMAIN proteins engage Polycomb chromatin modifiers to modulate Jasmonate signaling in Arabidopsis

Jasmonate (JA) regulates various aspects of plant growth and development and stress responses, with prominent roles in male reproductive development and defenses against herbivores and necrotrophic pathogens. JASMONATE-ZIM DOMAIN (JAZ) proteins are key regulators in the JA signaling pathway and function to repress the expression of JA-responsive genes. Here, we show that JAZ proteins directly interact with several chromatin-associated Polycomb proteins to mediate repressive chromatin modifications at JA-responsive genes and, thus, their transcriptional repression in Arabidopsis. Genetic analyses revealed that the developmental defects, including anther and pollen abnormalities, resulting from loss or block of JA signaling were partially rescued by loss of Polycomb protein-mediated chromatin silencing (Polycomb repression). We further found that JAZ-mediated transcriptional repression during anther and pollen development requires Polycomb proteins at four key regulatory loci. Analysis of genome-wide occupancy of a Polycomb factor and transcriptome reprogramming in response to JA revealed that Polycomb repression is involved in the repression of various JA-responsive genes. Taken together, our study reveals an important chromatin-based mechanism for JAZ-mediated transcriptional repression and JA signaling in plants.

Large-scale reconstruction of chromatin structures of maize temperate and tropical inbred lines.

Maize is a model plant species often used for genetics and genomics research because of its genetic diversity. There are prominent morphological, genetic, and epigenetic variations between tropical and temperate maize lines. However, the genome-wide chromatin conformations of these two maize types remain unexplored. We applied a Hi-C approach to compare the genome-wide chromatin interactions between temperate inbred line D132 and tropical line CML288. A reconstructed maize three-dimensional genome model revealed the spatial segregation of the global A and B compartments. The A compartments contain enriched genes and active epigenome marks, whereas the B compartments are gene-poor, transcriptionally silent chromatin regions. Whole-genome analyses indicated that the global A compartment content of CML288 was 3.12% lower than that of D132. Additionally, global and A/B sub-compartments were associated with differential gene expression and epigenetic changes between two inbred lines. About 25.3% of topologically associating domains (TADs) were determined to be associated with complex domain-level modifications that induced transcriptional changes, indicative of a large-scale reorganization of chromatin structures between the inbred maize lines. Furthermore, differences in chromatin interactions between the two lines correlated with epigenetic changes. These findings provide a solid foundation for the wider plant community to further investigate the genome-wide chromatin structures in other plant species.

21P Polycomb repressive complex 2 mutations predict survival benefit in advanced cancer patients treated with immune checkpoint inhibitors

ICI have become key in the treatment armamentarium for pts with advanced cancer. Besides PD-L1, numerous biomarkers are being tested to enhance clinicians' ability to predict responses and prognosis. The polycomb repressive complex 2 (PKC2) is a histone methtyltransferase family that plays a major role in chromatin silencing. Preclinical evidence implicates PKC2 components such as EZH2 in immune resistance. This study aimed to assess the clinical relevance of PKC2 mutations (mts) in outcomes of ICI-treated pts.

Epstein-Barr virus inactivates the transcriptome and disrupts the chromatin architecture of its host cell in the first phase of lytic reactivation.

Epstein-Barr virus (EBV), a herpes virus also termed HHV 4 and the first identified human tumor virus, establishes a stable, long-term latent infection in human B cells, its preferred host. Upon induction of EBV’s lytic phase, the latently infected cells turn into a virus factory, a process that is governed by EBV. In the lytic, productive phase, all herpes viruses ensure the efficient induction of all lytic viral genes to produce progeny, but certain of these genes also repress the ensuing antiviral responses of the virally infected host cells, regulate their apoptotic death or control the cellular transcriptome. We now find that EBV causes previously unknown massive and global alterations in the chromatin of its host cell upon induction of the viral lytic phase and prior to the onset of viral DNA replication. The viral initiator protein of the lytic cycle, BZLF1, binds to >105 binding sites with different sequence motifs in cellular chromatin in a concentration dependent manner implementing a binary molar switch probably to prevent noise-induced erroneous induction of EBV’s lytic phase. Concomitant with DNA binding of BZLF1, silent chromatin opens locally as shown by ATAC-seq experiments, while previously wide-open cellular chromatin becomes inaccessible on a global scale within hours. While viral transcripts increase drastically, the induction of the lytic phase results in a massive reduction of cellular transcripts and a loss of chromatin-chromatin interactions of cellular promoters with their distal regulatory elements as shown in Capture-C experiments. Our data document that EBV’s lytic cycle induces discrete early processes that disrupt the architecture of host cellular chromatin and repress the cellular epigenome and transcriptome likely supporting the efficient de novo synthesis of this herpes virus.

Ablation of SMUG1 Reduces Cell Viability and Increases UVC-Mediated Apoptosis in Hepatocarcinoma HepG2 Cells.

Uracil is an unavoidable aberrant base in DNA sequences, the repair of which takes place by a highly efficient base excision repair mechanism. The removal of uracil from the genome requires multiple biochemical steps with conformational changes of DNA that inhibit DNA replication and interfere with transcription. However, the relevance of uracil in DNA for cellular physiology and transcriptional regulation is not fully understood. We investigated the functional roles of SMUG1 using knock-down (KD) and knock-out (KO) models. The proliferation ratio of SMUG1 KD and KO cells was decreased compared to WT control cells, and the cell cycle was arrested in the G2/M phases before the transition to mitosis. The apoptotic cell death was increased in KD and KO cell lines through the increase of BAX and active caspase 3 expression. Phospho-gamma-H2AX expression, which reflected accumulated DNA damage, was also increased in KO cells. Moreover, the apoptotic cells by DNA damage accumulation were markedly increased in SMUG1 KD and KO cells after ultraviolet C irradiation. Transcriptomic analysis using RNA-seq revealed that SMUG1 was involved in gene sets expression including cell cycle transition and chromatin silencing. Together, the results implicate SMUG1 as a critical factor in cell cycle and transcriptional regulation.