Two ubiquitin-conjugating enzymes,Rhp6 and UbcX,regulate heterochromatin silencing in Schizosaccharomyces pombe

Methylation of histone H3 has been linked to the assembly of higher-order chromatin structures. Very recently, several examples, including the Schizosaccharomyces pombe mating-type region, chicken beta-globin locus, and inactive X-chromosome, revealed that H3-Lys9-methyl (Me) is associated with silent chromatin while H3-Lys4-Me is prominent in active chromatin. Surprisingly, it was shown that homologs of Drosophila Su(var)3-9 specifically methylate the Lys9 residue of histone H3. Here, to identify putative enzymes responsible for destabilization of heterochromatin, we screened genes whose overexpressions disrupt silencing at the silent mat3 locus in fission yeast. Interestingly, we identified two genes, rhp6(+) and ubcX(+) (ubiquitin-conjugating enzyme participating in silencing), both of which encode ubiquitin-conjugating enzymes. Their overexpression disrupted silencing at centromeres and telomeres as well as at mat3. Additionally, the overexpression interfered with centromeric function, as confirmed by elevated minichromosome loss and antimicrotubule drug sensitivity. On the contrary, deletion of rhp6(+) or ubcX(+) enhanced silencing at all heterochromatic regions tested, indicating that they are negative regulators of silencing. More importantly, chromatin immunoprecipitation showed that their overexpression alleviated the level of H3-Lys9-Me while enhancing the level of H3-Lys4-Me at the silent regions. On the contrary, their deletions enhanced the level of H3-Lys9-Me while alleviating that of H3-Lys4-Me. Taken together, the data suggest that two ubiquitin-conjugating enzymes, Rhp6 and UbcX, affect methylation of histone H3 at silent chromatin, which then reconfigures silencing.     

To SIR with Polycomb: linking silencing mechanisms

Yeast SIR2, the most evolutionarily conserved deacetylase, plays an essential role in epigenetic silencing at the silent mating type loci and telomeres. SIR2 has been implicated in chromatin silencing and lifespan determination in several organisms. Discovery that Drosophila SIR2 is also involved in epigenetic silencing mediated by the Polycomb group proteins and is physically associated with a complex containing the E(Z) histone methyltransferase has wide implications. These findings suggest possible link of Polycomb system to diverse cellular processes including senescence.     

Schizosaccharomyces pombe Hat1 (Kat1) Is Associated with Mis16 and Is Required for Telomeric Silencing

The Hat1 histone acetyltransferase has been implicated in the acetylation of histone H4 during chromatin assembly. In this study, we have characterized the Hat1 complex from the fission yeast Schizosaccharomyces pombe and have examined its role in telomeric silencing. Hat1 is found associated with the RbAp46 homologue Mis16, an essential protein. The Hat1 complex acetylates lysines 5 and 12 of histone H4, the sites that are acetylated in newly synthesized H4 in a wide range of eukaryotes. Deletion of hat1 in S. pombe is itself sufficient to cause the loss of silencing at telomeres. This is in contrast to results obtained with an S. cerevisiae hat1Δ strain, which must also carry mutations of specific acetylatable lysines in the H3 tail domain for loss of telomeric silencing to occur. Notably, deletion of hat1 from S. pombe resulted in an increase of acetylation of histone H4 in subtelomeric chromatin, concomitant with derepression of this region. A similar loss of telomeric silencing was also observed after growing cells in the presence of the deacetylase inhibitor trichostatin A. However, deleting hat1 did not cause loss of silencing at centromeres or the silent mating type locus. These results point to a direct link between Hat1, H4 acetylation, and the establishment of repressed telomeric chromatin in fission yeast.     

Epigenetic Regulation of Chromatin States in Schizosaccharomyces pombe

This article discusses the advances made in epigenetic research using the model organism fission yeast Schizosaccharomyces pombe. S. pombe has been used for epigenetic research since the discovery of position effect variegation (PEV). This is a phenomenon in which a transgene inserted within heterochromatin is variably expressed, but can be stably inherited in subsequent cell generations. PEV occurs at centromeres, telomeres, ribosomal DNA (rDNA) loci, and mating-type regions of S. pombe chromosomes. Heterochromatin assembly in these regions requires enzymes that modify histones and the RNA interference (RNAi) machinery. One of the key histone-modifying enzymes is the lysine methyltransferase Clr4, which methylates histone H3 on lysine 9 (H3K9), a classic hallmark of heterochromatin. The kinetochore is assembled on specialized chromatin in which histone H3 is replaced by the variant CENP-A. Studies in fission yeast have contributed to our understanding of the establishment and maintenance of CENP-A chromatin and the epigenetic activation and inactivation of centromeres.     

The CHD remodeling factor Hrp1 stimulates CENP-A loading to centromeres

Centromeres of fission yeast are arranged with a central core DNA sequence flanked by repeated sequences. The centromere-associated histone H3 variant Cnp1 (SpCENP-A) binds exclusively to central core DNA, while the heterochromatin proteins and cohesins bind the surrounding outer repeats. CHD (chromo-helicase/ATPase DNA binding) chromatin remodeling factors were recently shown to affect chromatin assembly in vitro. Here, we report that the CHD protein Hrp1 plays a key role at fission yeast centromeres. The hrp1Δ mutant disrupts silencing of the outer repeats and central core regions of the centromere and displays chromosome segregation defects characteristic for dysfunction of both regions. Importantly, Hrp1 is required to maintain high levels of Cnp1 and low levels of histone H3 and H4 acetylation at the central core region. Hrp1 interacts directly with the centromere in early S-phase when centromeres are replicated, suggesting that Hrp1 plays a direct role in chromatin assembly during DNA replication.     

Bypassing the catalytic activity of SIR2 for SIR protein spreading in Saccharomyces cerevisiae

Sir protein spreading along chromosomes and silencing in Saccharomyces cerevisiae requires the NAD+-dependent histone deacetylase activity of Sir2p. We tested whether this requirement could be bypassed at the HM loci and telomeres in cells containing a stably expressed, but catalytically inactive mutant of Sir2p, sir2-345p, plus histone mutants that mimic the hypoacetylated state normally created by Sir2p. Sir protein spreading was rescued in sir2-345 mutants expressing histones in which key lysine residues in their N-termini had been mutated to arginine. Mating in these mutants was also partially restored upon overexpression of Sir3p. Together, these results indicate that histone hypoacetylation is sufficient for Sir protein spreading in the absence of production of 2'-O-acetyl-ADP ribose by sir2p and Sir2p's enzymatic function for silencing can be bypassed in a subset of cells in a given population. These results also provide genetic evidence for the existence of additional critical substrates of Sir2p for silencing in vivo.