Heterochromatin is a quantitative trait associated with spontaneous epiallele formation

Epialleles are meiotically heritable variations in expression states that are independent from changes in DNA sequence. Although they are common in plant genomes, their molecular origins are unknown. Here we show, using mutant and experimental populations, that epialleles in Arabidopsis thaliana that result from ectopic hypermethylation are due to feedback regulation of pathways that primarily function to maintain DNA methylation at heterochromatin. Perturbations to maintenance of heterochromatin methylation leads to feedback regulation of DNA methylation in genes. Using single base resolution methylomes from epigenetic recombinant inbred lines (epiRIL), we show that epiallelic variation is abundant in euchromatin, yet, associates with QTL primarily in heterochromatin regions. Mapping three-dimensional chromatin contacts shows that genes that are hotspots for ectopic hypermethylation have increases in contact frequencies with regions possessing H3K9me2. Altogether, these data show that feedback regulation of pathways that have evolved to maintain heterochromatin silencing leads to the origins of spontaneous hypermethylated epialleles.

SUV39 SET domains mediate crosstalk of heterochromatic histone marks

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.

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

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.

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.

RNA-silencing induces target gene relocalization toward a specialized nuage domain

Argonaute small RNA pathways maintain genome integrity and fertility by enforcing the transgenerational silencing of transposons as well as many developmentally regulated germline genes 1. To propagate silencing, Argonaute pathways coordinate heterochromatin silencing with cycles of small RNA amplification 2. In animal germlines, mRNA surveillance is thought to occur within cytoplasmic perinuclear domains called nuage 3. In C. elegans 20-50 nuage droplets called P granules surround each pachytene germline nucleus. P granules are known to host many of the Argonaute small RNA systems that carry out transcriptome surveillance, but what if any specific roles P granules might play in Argonaute silencing have remained mysterious. Here we show that RNAi triggers the expansion of a unique P granule, which accumulates large amounts of the target RNA. As transcriptional silencing ensues, both alleles of the target gene relocate near the inner nuclear membrane (INM) directly adjacent this enlarged P granule. Similarly, during piRNA-mediated silencing, both alleles of a target gene reside adjacent to a P granule containing target RNA sequences. In an Argonaute mutant defective in piRNA silencing, the target RNA is released from nuage, and the target alleles dissociate from each other and from the INM. Our findings suggest that transcriptome-surveillance tasks are sub-divided between nuage domains that become specialized to coordinate small RNA silencing signals to their heterochromatin targets.

Ectopic expression of pericentric HSATII RNA results in nuclear RNA accumulation, MeCP2 recruitment, and cell division defects

Within the pericentric regions of human chromosomes reside large arrays of tandemly repeated satellite sequences. Expression of the human pericentric satellite HSATII is prevented by extensive heterochromatin silencing in normal cells, yet in many cancer cells, HSATII RNA is aberrantly expressed and accumulates in large nuclear foci in cis. Expression and aggregation of HSATII RNA in cancer cells is concomitant with recruitment of key chromatin regulatory proteins including methyl-CpG binding protein 2 (MeCP2). While HSATII expression has been observed in a wide variety of cancer cell lines and tissues, the effect of its expression is unknown. We tested the effect of stable expression of HSATII RNA within cells that do not normally express HSATII. Ectopic HSATII expression in HeLa and primary fibroblast cells leads to focal accumulation of HSATII RNA in cis and triggers the accumulation of MeCP2 onto nuclear HSATII RNA bodies. Further, long-term expression of HSATII RNA leads to cell division defects including lagging chromosomes, chromatin bridges, and other chromatin defects. Thus, expression of HSATII RNA in normal cells phenocopies its nuclear accumulation in cancer cells and allows for the characterization of the cellular events triggered by aberrant expression of pericentric satellite RNA.

Rbm10 facilitates heterochromatin assembly via the Clr6 HDAC complex

Splicing factors have recently been shown to be involved in heterochromatin formation, but their role in controlling heterochromatin structure and function remains poorly understood. In this study, we identified a fission yeast homologue of human splicing factor RBM10, which has been linked to TARP syndrome. Overexpression of Rbm10 in fission yeast leads to strong global intron retention. Rbm10 also interacts with splicing factors in a pattern resembling that of human RBM10, suggesting that the function of Rbm10 as a splicing regulator is conserved. Surprisingly, our deep-sequencing data showed that deletion of Rbm10 caused only minor effect on genome-wide gene expression and splicing. However, the mutant displays severe heterochromatin defects. Further analyses indicated that the heterochromatin defects in the mutant did not result from mis-splicing of heterochromatin factors. Our proteomic data revealed that Rbm10 associates with the histone deacetylase Clr6 complex and chromatin remodelers known to be important for heterochromatin silencing. Deletion of Rbm10 results in significant reduction of Clr6 in heterochromatin. Our work together with previous findings further suggests that different splicing subunits may play distinct roles in heterochromatin regulation.

Chromatin Regulation and Genome Maintenance by Mammalian SIRT7

Members of the Sirtuin family of enzymes are important regulators of genomic stability, stress responses, and metabolic programs that impact on human physiology, aging, and age-related disease processes. We previously showed that the mammalian Sirtuins SIRT6 and SIRT7 have high-selectivity histone deacetylase activities at chromatin, and inactivation of SIRT6 or SIRT7 results in dysregulated histone acetylation states and gene expression programs, with pathological consequences at the cellular and whole organism levels. Recently, we have been exploring novel functions of SIRT6 and SIRT7 in silencing of heterochromatic regions of the genome, the deregulation of which has been linked to aging and cancer biology. We found that pericentric heterochromatin silencing by SIRT6 prevents acute cellular senescence that is triggered by pathologic pericentric transcripts. We also uncovered a second novel trigger of human cellular senescence, ribosomal DNA instability in nucleoli, and we showed that SIRT7 guards against senescence induced by this instability. In our studies, a long-term focus has been identifying substrates of SIRT6 and SIRT7 and their roles in aging and disease pathways. In new work, are studying a novel physiologic substrate of SIRT7 at chromatin, H3K36Ac, and we are characterizing the genomic landscape of this SIRT7-dependent deacetylation target, and its downstream chromatin and nuclear signaling mechanisms. We will also discuss mechanistic insights into the functions of SIRT6 and SIRT7 from new proteomic, cellular, and mouse model studies.

DNA polymerase epsilon is required for heterochromatin maintenance in Arabidopsis

Background Chromatin organizes DNA and regulates its transcriptional activity through epigenetic modifications. Heterochromatic regions of the genome are generally transcriptionally silent, while euchromatin is more prone to transcription. During DNA replication, both genetic information and chromatin modifications must be faithfully passed on to daughter strands. There is evidence that DNA polymerases play a role in transcriptional silencing, but the extent of their contribution and how it relates to heterochromatin maintenance is unclear. Results We isolate a strong hypomorphic Arabidopsis thaliana mutant of the POL2A catalytic subunit of DNA polymerase epsilon and show that POL2A is required to stabilize heterochromatin silencing genome-wide, likely by preventing replicative stress. We reveal that POL2A inhibits DNA methylation and histone H3 lysine 9 methylation. Hence, the release of heterochromatin silencing in POL2A-deficient mutants paradoxically occurs in a chromatin context of increased levels of these two repressive epigenetic marks. At the nuclear level, the POL2A defect is associated with fragmentation of heterochromatin. Conclusion These results indicate that POL2A is critical to heterochromatin structure and function, and that unhindered replisome progression is required for the faithful propagation of DNA methylation throughout the cell cycle.

Swi6/HP1 binding to RNA-DNA hybrids initiates heterochromatin assembly at the centromeric dg-dh repeats in Fission Yeast

ABSTRACTCanonically, 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 chromodomain. Subsequent self-association of Swi6/HP1 on adjacent nucleosomes leads to folded heterochromatin structure. An alternate model suggests a cooperative interaction between Clr4 and Swi6/HP1 in heterochromatin assembly. HP1 binding to RNA has also been invoked for heterochromatin silencing in metazoans. Recruitment of Swi6/HP1 to centromere has been shown to be dependent on the RNAi pathway in fission yeast. Here we show that Swi6/HP1 exhibits a hierarchy of binding affinity to RNAs, ranging from promiscuous, low-affinity binding to mRNAs, to moderate-affinity binding to the RNAi-generated siRNAs corresponding to the dg-dh repeats present in pericentromeric heterochromatin regions, to high affinity binding to the RNA-DNA hybrids to the cognate dg-dh repeats. Together with sensitivity of Swi6 localization and silencing to RNaseH, our results suggest a dynamic control of localization of Swi6/HP1 towards the dg-dh repeats versus euchromatic regions. This is mediated by its binding to RNA-DNA hybrid at the dg-dh repeats, as an RNAi-dependent and Me2/me3-K9-H3-independent mechanism of recruitment, leading to heterochromatin formation and silencing.