Hugh P. Cam

Comprehensive analysis of heterochromatin- and RNAi-mediated epigenetic control of the fission yeast genome

The organization of eukaryotic genomes into distinct structural and functional domains is important for the regulation and transduction of genetic information. Here, we investigated heterochromatin and euchromatin profiles of the entire fission yeast genome and explored the role of RNA interference (RNAi) in genome organization. Histone H3 methylated at Lys4, which defines euchromatin, was not only distributed across most of the chromosomal landscape but was also present at the centromere core, the site of kinetochore assembly. In contrast, histone H3 methylated at Lys9 and its interacting protein Swi6/HP1, which define heterochromatin, coated extended domains associated with a variety of repeat elements and small islands corresponding to meiotic genes. Notably, RNAi components were distributed throughout all these heterochromatin domains, and their localization depended on Clr4/Suv39h histone methyltransferase. Sequencing of small interfering RNAs (siRNAs) associated with the RITS RNAi effector complex identified hot spots of siRNAs, which mapped to a diverse array of elements in these RNAi-heterochromatin domains. We found that Clr4/Suv39h predominantly silenced repeat elements whose derived transcripts, transcribed mainly by RNA polymerase II, serve as a source for siRNAs. Our analyses also uncover an important role for the RNAi machinery in maintaining genomic integrity.

RITS acts in cis to promote RNA interference–mediated transcriptional and post-transcriptional silencing

RNA interference is a conserved mechanism by which double-stranded RNA is processed into short interfering RNAs (siRNAs) that can trigger both post-transcriptional and transcriptional gene silencing. In fission yeast, the RNA-induced initiation of transcriptional gene silencing (RITS) complex contains Dicer-generated siRNAs and is required for heterochromatic silencing. Here we show that RITS components, including Argonaute protein, bind to all known heterochromatic loci. At the mating-type region, RITS is recruited to the centromere-homologous repeat cenH in a Dicer-dependent manner, whereas the spreading of RITS across the entire 20-kb silenced domain, as well as its subsequent maintenance, requires heterochromatin machinery including Swi6 and occurs even in the absence of Dicer. Furthermore, our analyses suggest that RNA interference machinery operates in cis as a stable component of heterochromatic domains with RITS tethered to silenced loci by methylation of histone H3 at Lys9. This tethering promotes the processing of transcripts and generation of additional siRNAs for heterochromatin maintenance.

Cell cycle control of centromeric repeat transcription and heterochromatin assembly.

Heterochromatin in eukaryotic genomes regulates diverse chromosomal processes including transcriptional silencing. However, in Schizosaccharomyces pombe RNA polymerase II (RNAPII) transcription of centromeric repeats is essential for RNA-interference-mediated heterochromatin assembly. Here we study heterochromatin dynamics during the cell cycle and its effect on RNAPII transcription. We describe a brief period during the S phase of the cell cycle in which RNAPII preferentially transcribes centromeric repeats. This period is enforced by heterochromatin, which restricts RNAPII accessibility at centromeric repeats for most of the cell cycle. RNAPII transcription during S phase is linked to loading of RNA interference and heterochromatin factors such as the Ago1 subunit of the RITS complex and the Clr4 methyltransferase complex subunit Rik1 (ref. 7). Moreover, Set2, an RNAPII-associated methyltransferase that methylates histone H3 lysine 36 at repeat loci during S phase, acts in a pathway parallel to Clr4 to promote heterochromatin assembly. We also show that phosphorylation of histone H3 serine 10 alters heterochromatin during mitosis, correlating with recruitment of condensin that affects silencing of centromeric repeats. Our analyses suggest at least two distinct modes of heterochromatin targeting to centromeric repeats, whereby RNAPII transcription of repeats and chromodomain proteins bound to methylated histone H3 lysine 9 mediate recruitment of silencing factors. Together, these processes probably facilitate heterochromatin maintenance through successive cell divisions.

A Role for TFIIIC Transcription Factor Complex in Genome Organization

Eukaryotic genome complexity necessitates boundary and insulator elements to partition genomic content into distinct domains. We show that inverted repeat (IR) boundary elements flanking the fission yeast mating-type heterochromatin domain contain B-box sequences, which prevent heterochromatin from spreading into neighboring euchromatic regions by recruiting transcription factor TFIIIC complex without RNA polymerase III (Pol III). Genome-wide analysis reveals TFIIIC with Pol III at all tRNA genes, many of which cluster at pericentromeric heterochromatin domain boundaries. However, a single tRNA(phe) gene with modest TFIIIC enrichment is insufficient to serve as boundary and requires RNAi-associated element to restrain heterochromatin spreading. Remarkably, we found TFIIIC localization without Pol III at many sites located between divergent promoters. These sites appear to act as chromosome-organizing clamps by tethering distant loci to the nuclear periphery, at which TFIIIC is concentrated into several distinct bodies. Our analyses uncover a general genome organization mechanism involving conserved TFIIIC complex.

Distinct roles of HDAC complexes in promoter silencing, antisense suppression and DNA damage protection

Histone acetylation is important in regulating DNA accessibility. Multifunctional Sin3 proteins bind histone deacetylases (HDACs) to assemble silencing complexes that selectively target chromatin. We show that, in fission yeast, an essential HDAC, Clr6, exists in two distinct Sin3 core complexes. Complex I contains an essential Sin3 homolog, Pst1, and other factors, and predominantly targets gene promoters. Complex II contains a nonessential Sin3 homolog, Pst2, and several conserved proteins. It preferentially targets transcribed chromosomal regions and centromere cores. Defects in complex II abrogate global protective functions of chromatin, causing increased accessibility of DNA to genotoxic agents and widespread antisense transcripts that are processed by the exosome. Notably, the two Clr6 complexes differentially repress forward and reverse centromeric repeat transcripts, suggesting that these complexes regulate transcription in heterochromatin and euchromatin in similar manners, including suppression of spurious transcripts from cryptic start sites.

Host genome surveillance for retrotransposons by transposon-derived proteins

Transposable elements and their remnants constitute a substantial fraction of eukaryotic genomes. Host genomes have evolved defence mechanisms, including chromatin modifications and RNA interference, to regulate transposable elements. Here we describe a genome surveillance mechanism for retrotransposons by transposase-derived centromeric protein CENP-B homologues of the fission yeast Schizosaccharomyces pombe. CENP-B homologues of S. pombe localize at and recruit histone deacetylases to silence Tf2 retrotransposons. CENP-Bs also repress solo long terminal repeats (LTRs) and LTR-associated genes. Tf2 elements are clustered into ‘Tf’ bodies, the organization of which depends on CENP-Bs that display discrete nuclear structures. Furthermore, CENP-Bs prevent an ‘extinct’ Tf1 retrotransposon from re-entering the host genome by blocking its recombination with extant Tf2, and silence and immobilize a Tf1 integrant that becomes sequestered into Tf bodies. Our results reveal a probable ancient retrotransposon surveillance pathway important for host genome integrity, and highlight potential conflicts between DNA transposons and retrotransposons, major transposable elements believed to have greatly moulded the evolution of genomes.

Transcriptional Scaffolds for Heterochromatin Assembly

Heterochromatin is dynamically regulated during the cell cycle and in response to developmental signals. Recent findings from diverse systems suggest an extensive role for transcription in the assembly of heterochromatin, highlighting the emerging theme that transcription and noncoding RNAs can provide the initial scaffold for the formation of heterochromatin, which serves as a versatile recruiting platform for diverse factors involved in many cellular processes.

Fission Yeast Homologs of Human Histone H3 Lysine 4 Demethylase Regulate a Common Set of Genes with Diverse Functions

Schizosaccharomyces pombe contains two proteins, SWIRM1 and SWIRM2, with close homology to human histone H3 lysine 4 demethylase. Both proteins contain the amino oxidase catalytic domain and a recently described DNA interaction SWIRM domain. Here we describe the biochemical isolation and the functional characterization of SWIRM1 and SWIRM2. Our results indicate that while SWIRM2 is an essential gene, cells lacking SWIRM1 are viable. We found that SWIRM1 and SWIRM2 are stably associated in a multiprotein complex, but intriguingly, unlike their human counterpart, S. pombe SWIRM complex contains neither a histone deacetylase nor any detectable demethylase activity. Genome-wide chromatin immunoprecipitation unexpectedly showed the absence of both SWIRM proteins from heterochromatic domains. Instead, consistent with biochemical analyses, SWIRM1 and SWIRM2 co-localize to a common set of target gene promoters whose functions are implicated in diverse processes including mitochondrial metabolism and transcriptional regulation. Importantly, we show that SWIRM1 is not only required for optimum transcription of its target genes but also display a global role in regulation of antisense transcription.