Formation Of Silent Chromatin Research Articles

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.

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.

Molecular dynamics of de novo telomere heterochromatin formation in budding yeast

In the budding yeast Saccharomyces cerevisiae, heterochromatin structure is found at three chromosome regions, which are homothallic mating-type loci, rDNA regions and telomeres. To address how telomere heterochromatin is assembled under physiological conditions, we employed a de novo telomere addition system, and analyzed the dynamic chromatin changes of the TRP1 reporter gene during telomere elongation. We found that integrating a 255-bp, but not an 81-bp telomeric sequence near the TRP1 promoter could trigger Sir2 recruitment, active chromatin mark(s)' removal, chromatin compaction and TRP1 gene silencing, indicating that the length of the telomeric sequence inserted in the internal region of a chromosome is critical for determining the chromatin state at the proximal region. Interestingly, Rif1 but not Rif2 or yKu is indispensable for the formation of intra-chromosomal silent chromatin initiated by telomeric sequence. When an internal short telomeric sequence (e.g., 81 bp) gets exposed to become a de novo telomere, the herterochromatin features, such as Sir recruitment, active chromatin mark(s)' removal and chromatin compaction, are detected within a few hours before the de novo telomere reaches a stable length. Our results recapitulate the molecular dynamics and reveal a coherent picture of telomere heterochromatin formation.

Meeting report: The biology of genomes and proteomes

A mini-symposium of the Genetics Society of Korea was held at Dankook University, Korea on April 24, 2015. The theme of the mini-symposium was ‘The biology of genomes and proteomes.’ This was the first mini-symposium of 2015. There were two sessions that were chaired by Dr. Kyu-Dong Han (Dankook University, Korea) and Dr. Heui-Soo Kim (Pusan National University, Korea). In the first session, three speakers gave talks on genomics and epigenomics. The first speaker, Dr. Keunsoo Kang (Department of Microbiology, Dankook University, Korea), introduced ‘‘An automated analysis pipeline for a large set of ChIP-seq data: AutoChIP (Kim et al. 2014a, b).’’ Recent advances in genomic DNA sequencing have revolutionized the ways to examine molecular events inside cells in several aspects. Next-generation sequencing approaches, which enable the rapid and accurate sequencing of short DNA fragments, have changed the ways researchers approach biological problems. For example, researchers can identify the genome-wide binding sites of proteins by using chromatin immune precipitation coupled with parallel sequencing (ChIP-seq) without any prior knowledge (Johnson et al. 2007; Robertson et al. 2007). Although there are many applications available for the analysis of ChIP-seq data (Henry et al. 2014), users need to know some details about the installation, alignment, and peak-calling procedures in most cases. Dr. Kang summarized his recent advances with the AutoChip assay, which is an easy-to-use application for ChIP-seq analysis. Using the AutoChip, the user automatically downloads and installs programs on his or her computer before launching the actual data analysis. In addition, all procedures for ChIP-seq analyses, such as the alignment of unmapped reads to a reference genome and the identification of genome-wide binding sites for a given protein, can be done by two simple steps coupled with AutoChIP. An evaluation of the cocktail algorithm implemented in AutoChIP showed that the algorithm outperformed a single ChIP-seq tool in terms of the ratio of motif occurrences and the average height of the normalized read density over the identified peaks. In addition, the annotation of the identified peaks with information about known genes and repeat elements provides a comprehensive picture of the genome-wide binding sites of selected proteins. Overall, AutoChIP provides a comprehensive platform to analyze a large set of ChIPseq data in one step. The second speaker, Dr. Kyunghwan Kim (Department of Biology, Chungbuk National University, Korea), gave a presentation entitled ‘Epigenetics and cancer development.’ The regulation of gene expression and the maintenance of chromosome stability in eukaryotes are critically associated with the formation of silent chromatin. Inactive chromatin structures are frequently related to distinctive histone modifications (Suganuma and Workman 2011). Although recent studies have implicated histone modification as a key regulator of chromatin fluidity, the molecular mechanisms underlying such effects remain & Nam-Soo Kim kimnamsu@kangwon.ac.kr

A Nucleotide-Driven Switch Regulates Flanking DNA Length Sensing by a Dimeric Chromatin Remodeler

The ATP-dependent chromatin assembly factor (ACF) spaces nucleosomes to promote formation of silent chromatin. Two copies of its ATPase subunit SNF2h bind opposite sides of a nucleosome, but how these protomers avoid competition is unknown. SNF2h senses the length of DNA flanking a nucleosome via its HAND-SANT-SLIDE (HSS) domain, yet it is unclear how this interaction enhances remodeling. Using covalently connected SNF2h dimers we show that dimerization accelerates remodeling and that the HSS contributes to communication between protomers. We further identify a nucleotide-dependent conformational change in SNF2h. In one conformation the HSS binds flanking DNA, and in another conformation the HSS engages the nucleosome core. Based on these results, we propose a model in which DNA length sensing and translocation are performed by two distinct conformational states of SNF2h. Such separation of function suggests that these activities could be independently regulated to affect remodeling outcomes.

VprBP Has Intrinsic Kinase Activity Targeting Histone H2A and Represses Gene Transcription

Histone modifications play important roles in the regulation of gene expression and chromatin organization. VprBP has been implicated in transcriptionally silent chromatin formation and cell-cycle regulation, but the molecular basis underlying such effects remains unclear. Here we report that VprBP possesses an intrinsic protein kinase activity and is capable of phosphorylating histone H2A on threonine 120 (H2AT120p) in a nucleosomal context. VprBP is localized to a large set of tumor suppressor genes and blocks their transcription, in a manner that is dependent on its kinase activity toward H2AT120. The functional significance of VprBP-mediated H2AT120p is further underscored by the fact that RNAi knockdown and small-molecule inhibition of VprBP reactivate growth regulatory genes and impede tumor growth. Our findings establish VprBP as a major kinase responsible for H2AT120p in cancer cells and suggest that VprBP inhibition could be a new strategy for the development of anticancer therapeutics.

Recruitment of Rpd3 to the Telomere Depends on the Protein Arginine Methyltransferase Hmt1

In the yeast Saccharomyces cerevisiae, the establishment and maintenance of silent chromatin at the telomere requires a delicate balance between opposing activities of histone modifying enzymes. Previously, we demonstrated that the protein arginine methyltransferase Hmt1 plays a role in the formation of yeast silent chromatin. To better understand the nature of the Hmt1 interactions that contribute to this phenomenon, we carried out a systematic reverse genetic screen using a null allele of HMT1 and the synthetic genetic array (SGA) methodology. This screen revealed interactions between HMT1 and genes encoding components of the histone deacetylase complex Rpd3L (large). A double mutant carrying both RPD3 and HMT1 deletions display increased telomeric silencing and Sir2 occupancy at the telomeric boundary regions, when comparing to a single mutant carrying Hmt1-deletion only. However, the dual rpd3/hmt1-null mutant behaves like the rpd3-null single mutant with respect to silencing behavior, indicating that RPD3 is epistatic to HMT1. Mutants lacking either Hmt1 or its catalytic activity display an increase in the recruitment of histone deacetylase Rpd3 to the telomeric boundary regions. Moreover, in such loss-of-function mutants the levels of acetylated H4K5, which is a substrate of Rpd3, are altered at the telomeric boundary regions. In contrast, the level of acetylated H4K16, a target of the histone deacetylase Sir2, was increased in these regions. Interestingly, mutants lacking either Rpd3 or Sir2 display various levels of reduction in dimethylated H4R3 at these telomeric boundary regions. Together, these data provide insight into the mechanism whereby Hmt1 promotes the proper establishment and maintenance of silent chromatin at the telomeres.

Inheritance of Silent rDNA Chromatin Is Mediated by PARP1 via Noncoding RNA

Faithful propagation of specific chromatin states requires re-establishment of epigenetic marks after every cell division. How the original epigenetic signature is inherited after disruption during DNA replication is still poorly understood. Here, we show that the poly(ADP-ribose)-polymerase-1 (PARP1/ARTD1) is implicated in the maintenance of silent rDNA chromatin during cell division. We demonstrate that PARP1 associates with TIP5, a subunit of the NoRC complex, via the noncoding pRNA and binds to silent rRNA genes after their replication in mid-late S phase. PARP1 represses rRNA transcription and is implicated in the formation of silent rDNA chromatin. Silent rDNA chromatin is a specific substrate for ADP-ribosylation and the enzymatic activity of PARP1 is necessary to establish rDNA silencing. The data unravel a function of PARP1 and ADP-ribosylation that serves to allow for the inheritance of silent chromatin structures, shedding light on how epigenetic marks are transmitted during each cell cycle.

Small RNA-Mediated Epigenetic Myostatin Silencing

Myostatin (Mstn) is a secreted growth factor that negatively regulates muscle mass and is therefore a potential pharmacological target for the treatment of muscle wasting disorders such as Duchenne muscular dystrophy. Here we describe a novel Mstn blockade approach in which small interfering RNAs (siRNAs) complementary to a promoter-associated transcript induce transcriptional gene silencing (TGS) in two differentiated mouse muscle cell lines. Silencing is sensitive to treatment with the histone deacetylase inhibitor trichostatin A, and the silent state chromatin mark H3K9me2 is enriched at the Mstn promoter following siRNA transfection, suggesting epigenetic remodeling underlies the silencing effect. These observations suggest that long-term epigenetic silencing may be feasible for Mstn and that TGS is a promising novel therapeutic strategy for the treatment of muscle wasting disorders.

Silent information regulator 3: the Goldilocks of the silencing complex

A recent explosion of work surrounds the interactions between Sir3p (Silent Information Regulator 3) and chromatin. We review here the Sir3p functions related to its role in silencing in Saccharomyces cerevisiae. This unusual protein, which is absolutely required for silencing, is distantly related to the highly conserved replication initiator Orc1p, but is itself phylogenetically limited to "post-genome-duplicated" budding yeasts. Several recent studies revise earlier models for Sir3p action. Specifically, the N-terminal bromo-adjacent homology (BAH) domain plays a now well-defined role in silencing, and a picture is emerging in which both termini of Sir3p bind two locations on the nucleosome: (1) the loss of ribosomal DNA silencing (LRS) surface in the nucleosome core, and (2) the N-terminal histone tails for effective silencing at telomeres. We relate Sir3p structure and function, and summarize recent molecular studies of Sir3p/chromatin binding, Sir3p/Dot1p competition, and the possible role of O-Acetyl ADP ribose (O-AADPR) in Sir3p/chromatin binding. We emphasize recent genetic data that provide important new insights and settle controversies created by in vitro work. Finally, we synthesize these ideas to revise the model for how Sir3p mediates silent chromatin formation in yeast, in part through its affinity for the LRS region of the nucleosome, which must be "just right."

Histone H3 N-terminus regulates higher order structure of yeast heterochromatin

In budding yeast, telomeres and the mating type (HM) loci are found in a heterochromatin-like silent structure initiated by Rap1 and extended by the interaction of Silencing Information Regulator (Sir) proteins with histones. Binding data demonstrate that both the H3 and H4 N-terminal domains required for silencing in vivo interact directly with Sir3 and Sir4 in vitro. The role of H4 lysine 16 deacetylation is well established in Sir3 protein recruitment; however, that of the H3 N-terminal tail has remained unclear. To characterize the role of H3 in silent chromatin formation and compare it to H4 we have generated comprehensive high resolution genome-wide binding maps of heterochromatin proteins. We found that H4 lysine 16 deacetylation is required for the recruitment and spreading of heterochromatin proteins at all telomeres and HM loci. In contrast, the H3 N terminus is required for neither recruitment nor spreading of Sir proteins. Instead, deletion of the H3 tail leads to increased accessibility within heterochromatin of an ectopic bacterial dam methylase and the decreased mobility of an HML heterochromatic fragment in sucrose gradients. These findings indicate an altered chromatin structure. We propose that Sir proteins recruited by the H4 tail then interact with the H3 tail to form a higher order silent chromatin structure.

Silent chromatin at the middle and ends: lessons from yeasts

Eukaryotic centromeres and telomeres are specialized chromosomal regions that share one common characteristic: their underlying DNA sequences are assembled into heritably repressed chromatin. Silent chromatin in budding and fission yeast is composed of fundamentally divergent proteins tat assemble very different chromatin structures. However, the ultimate behaviour of silent chromatin and the pathways that assemble it seem strikingly similar among Saccharomyces cerevisiae (S. cerevisiae), Schizosaccharomyces pombe (S. pombe) and other eukaryotes. Thus, studies in both yeasts have been instrumental in dissecting the mechanisms that establish and maintain silent chromatin in eukaryotes, contributing substantially to our understanding of epigenetic processes. In this review, we discuss current models for the generation of heterochromatic domains at centromeres and telomeres in the two yeast species.

The establishment of gene silencing at single-cell resolution

The establishment of silencing in Saccharomyces cerevisiae is similar to heterochromatin formation in multi-cellular eukaryotes. Previous batch culture studies determined that the de novo establishment of silencing initiates during S phase and continues for up to 5 cell divisions for completion. To track silencing phenotypically, we developed an assay that introduces Sir3 protein into individual sir3Δ mutant cells synchronously and then detects the onset of silencing with single-cell resolution. Silencing was completed within the first one to two cell divisions in most cells queried. Moreover, we uncovered unexpected complexity in the contributions of a histone acetyltransferase (Sas2), two histone methytransferases (Dot1 and Set1), and one histone demethylase (Jhd2) to the dynamics of silencing. Our findings revealed that removal of methyl modifications at H3 K4 and H3 K79 were important steps in silent chromatin formation, and that Jhd2 and Set1 played competing roles in the process.

Genomewide Screen for Negative Regulators of Sirtuin Activity inSaccharomyces cerevisiaeReveals 40 Loci and Links to Metabolism

Sirtuins are conserved proteins implicated in myriad key processes including gene control, aging, cell survival, metabolism, and DNA repair. In Saccharomyces cerevisiae, the sirtuin Silent information regulator 2 (Sir2) promotes silent chromatin formation, suppresses recombination between repeats, and inhibits senescence. We performed a genomewide screen for factors that negatively regulate Sir activity at a reporter gene placed immediately outside a silenced region. After linkage analysis, assessment of Sir dependency, and knockout tag verification, 40 loci were identified, including 20 that have not been previously described to regulate Sir. In addition to chromatin-associated factors known to prevent ectopic silencing (Bdf1, SAS-I complex, Rpd3L complex, Ku), we identified the Rtt109 DNA repair-associated histone H3 lysine 56 acetyltransferase as an anti-silencing factor. Our findings indicate that Rtt109 functions independently of its proposed effectors, the Rtt101 cullin, Mms1, and Mms22, and demonstrate unexpected interplay between H3K56 and H4K16 acetylation. The screen also identified subunits of mediator (Soh1, Srb2, and Srb5) and mRNA metabolism factors (Kem1, Ssd1), thus raising the possibility that weak silencing affects some aspect of mRNA structure. Finally, several factors connected to metabolism were identified. These include the PAS-domain metabolic sensor kinase Psk2, the mitochondrial homocysteine detoxification enzyme Lap3, and the Fe-S cluster protein maturase Isa2. We speculate that PAS kinase may integrate metabolic signals to control sirtuin activity.

Insights into the Impact of Histone Acetylation and Methylation on Sir Protein Recruitment, Spreading, and Silencing in Saccharomyces cerevisiae

Silent chromatin formation in Saccharomyces cerevisiae begins with the recruitment of silent information regulator (Sir) proteins to silencers at the silent mating-type loci and to telomere ends. Next, Sir2/3/4 proteins propagate across these loci as histones are deacetylated by the NAD + -dependent histone deacetylase Sir2p, ultimately resulting in the cessation of transcription and in the loss of SET1- and DOT1-dependent methylation of histone H3 within silent chromatin. We analyzed the effects of modifiable lysine residues on histones H3 and H4 on experimentally defined steps in silencing: recruitment of Sir proteins to silencers, Sir protein spreading, and transcriptional repression. Loss of acetylation, but not methylation, facilitated both Sir recruitment and spreading, and Sir spreading across hypoacetylated chromatin could disrupt SET1- and DOT1-dependent histone methylation without silencing underlying genes. Our data indicate that loss of methylation of K4 and K79 on histone H3 reflects intermediate events during the formation of silent chromatin, and that retention of a positive charge at a single residue on histone H4 (K16) was both necessary and sufficient to permit Sir spreading beyond sites of their recruitment.

RSF Governs Silent Chromatin Formation via Histone H2Av Replacement

Human remodeling and spacing factor (RSF) consists of a heterodimer of Rsf-1 and hSNF2H, a counterpart of Drosophila ISWI. RSF possesses not only chromatin remodeling activity but also chromatin assembly activity in vitro. While no other single factor can execute the same activities as RSF, the biological significance of RSF remained unknown. To investigate the in vivo function of RSF, we generated a mutant allele of Drosophila Rsf-1 (dRsf-1). The dRsf-1 mutant behaved as a dominant suppressor of position effect variegation. In dRsf-1 mutant, the levels of histone H3K9 dimethylation and histone H2A variant H2Av were significantly reduced in an euchromatic region juxtaposed with heterochromatin. Furthermore, using both genetic and biochemical approaches, we demonstrate that dRsf-1 interacts with H2Av and the H2Av-exchanging machinery Tip60 complex. These results suggest that RSF contributes to histone H2Av replacement in the pathway of silent chromatin formation.

The Chromatin-Remodeling Factor FACT Contributes to Centromeric Heterochromatin Independently of RNAi

Centromeres exert vital cellular functions in mitosis and meiosis. A specialized histone and other chromatin-bound factors nucleate a dynamic protein assembly that is required for the proper segregation of sister chromatids. In several organisms, including the fission yeast, Schizosaccharomyces pombe, the RNAi pathway contributes to the formation of silent chromatin in pericentromeric regions. Little is known about how chromatin-remodeling factors contribute to heterochromatic integrity and centromere function. Here we show that the histone chaperone and remodeling complex FACT is required for centromeric-heterochromatin integrity and accurate chromosome segregation. We show that Spt16 and Pob3 are two subunits of the S. pombe FACT complex. Surprisingly, yeast strains deleted for pob3+ are viable and alleviate gene silencing at centromeric repeats and at the silent mating-type locus. Importantly, like heterochromatin and RNAi pathway mutants, Pob3 null strains exhibit lagging chromosomes on anaphase spindles. Whereas the processing of centromeric RNA transcripts into siRNAs is maintained in Pob3 mutants, Swi6-association with the centromere is reduced. Our studies provide the first experimental evidence for a role of the RNA polymerase II cofactor FACT in heterochromatin integrity and in centromere function.

The role of protein arginine methylation in the formation of silent chromatin

Establishment and maintenance of silent chromatin in the Saccharomyces cerevisiae involves a step-wise assembly of the SIR complex. Here we demonstrate a role for the protein arginine methyltransferase Hmt1 in this process. In the absence of catalytically active Hmt1, yeast cells display increased transcription from silent chromatin regions and increased mitotic recombination within tandem repeats of rDNA. At the molecular level, loss of Hmt1's catalytic activity results in decreased Sir2 and dimethylated Arg-3 histone H4 occupancy across silent chromatin regions. These data suggest a model whereby protein arginine methylation affects the establishment and maintenance of silent chromatin.

Asymmetric Positioning of Nucleosomes and Directional Establishment of Transcriptionally Silent Chromatin by Saccharomyces cerevisiae Silencers

In Saccharomyces cerevisiae, silencers flanking the HML and HMR loci consist of various combinations of binding sites for Abf1p, Rap1p, and the origin recognition complex (ORC) that serve to recruit the Sir silencing complex, thereby initiating the establishment of transcriptionally silent chromatin. There have been seemingly conflicting reports concerning whether silencers function in an orientation-dependent or -independent manner, and what determines the directionality of a silencer has not been explored. We demonstrate that chromatin plays a key role in determining the potency and directionality of silencers. We show that nucleosomes are asymmetrically distributed around the HML-I or HMR-E silencer so that a nucleosome is positioned close to the Abf1p side but not the ORC side of the silencer. This coincides with preferential association of Sir proteins and transcriptional silencing on the Abf1p side of the silencer. Elimination of the asymmetry in nucleosome positioning at a silencer leads to comparable silencing on both sides. Asymmetric nucleosome positioning in the immediate vicinity of a silencer is independent of its orientation and genomic context, indicating that it is the inherent property of the silencer. Moreover, it is also independent of the Sir complex and thus precedes the formation of silent chromatin. Finally, we demonstrate that asymmetric positioning of nucleosomes and directional silencing by a silencer depend on ORC and Abf1p. We conclude that the HML-I and HMR-E silencers promote asymmetric positioning of nucleosomes, leading to unequal potentials of transcriptional silencing on their sides and, hence, directional silencing.

Structure of the Sir3 protein bromo adjacent homology (BAH) domain from S. cerevisiae at 1.95 Å resolution

Sir3p is a silent-information-regulator (SIR) protein required for the assembly of a transcriptionally "silent" chromatin structure at telomeres and the cryptic HM mating-type loci in Saccharomyces cerevisiae. Sir3p contains a putative "bromo adjacent homology" (BAH) domain at its N terminus that shares strong sequence similarity with the BAH domain of a subunit of the origin recognition complex (ORC), Orc1p. The Orc1p-BAH domain forms a well-defined complex with the ORC interaction region (OIR) of another Sir protein, Sir1p, which targets formation of silent chromatin to the HM-loci. Interestingly, despite sequence similarity of the Sir3p and Orc1p BAH domains and Sir3p's established importance in silencing, Sir3p does not bind the Sir1p-OIR. Here we report the 1.95 A resolution crystal structure of the Sir3p-BAH domain. The structure reveals two key features that can account for Sir3p-BAH domain's inability to interact with Sir1p. First, several Orc1p-BAH domain residues known to directly contact Sir1p are altered in the Sir3p-BAH domain. Second, a critical OIR-binding pocket present on the surface of the Orc1p-BAH domain is "filled" in the Sir3p-BAH domain structure, potentially making it inaccessible to Sir1p. These findings imply that the Sir3p-BAH domain structure has evolved for functions distinct from those of the Orc1p-BAH domain.