The cAMP signaling pathway regulates Epe1 protein levels and heterochromatin assembly.

The epigenetic landscape of a cell frequently changes in response to fluctuations in nutrient levels, but the mechanistic link is not well understood. In fission yeast, the JmjC domain protein Epe1 is critical for maintaining the heterochromatin landscape. While loss of Epe1 results in heterochromatin expansion, overexpression of Epe1 leads to defective heterochromatin. Through a genetic screen, we found that mutations in genes of the cAMP signaling pathway suppress the heterochromatin defects associated with Epe1 overexpression. We further demonstrated that the activation of Pka1, the downstream effector of cAMP signaling, is required for the efficient translation of epe1+ mRNA to maintain Epe1 overexpression. Moreover, inactivation of the cAMP-signaling pathway, either through genetic mutations or glucose deprivation, leads to the reduction of endogenous Epe1 and corresponding heterochromatin changes. These results reveal the mechanism by which the cAMP signaling pathway regulates heterochromatin landscape in fission yeast.

The cooperative assembly of shelterin bridge provides a kinetic gateway that controls telomere length homeostasis.

Shelterin is a six-protein complex that coats chromosome ends to ensure their proper protection and maintenance. Similar to the human shelterin, fission yeast shelterin is composed of telomeric double- and single-stranded DNA-binding proteins, Taz1 and Pot1, respectively, bridged by Rap1, Poz1 and Tpz1. The assembly of the proteinaceous Tpz1-Poz1-Rap1 complex occurs cooperatively and disruption of this shelterin bridge leads to unregulated telomere elongation. However, how this biophysical property of bridge assembly is integrated into shelterin function is not known. Here, utilizing synthetic bridges with a range of binding properties, we find that synthetic shelterin bridge lacking cooperativity requires a linker pair that matches the native bridge in complex lifespan but has dramatically higher affinity. We find that cooperative assembly confers kinetic properties on the shelterin bridge allowing disassembly to function as a molecular timer, regulating the duration of the telomere open state, and consequently telomere lengthening to achieve a defined species-specific length range.

The histone H3K9M mutation synergizes with H3K14 ubiquitylation to selectively sequester histone H3K9 methyltransferase Clr4 at heterochromatin.

Oncogenic histone lysine-to-methionine mutations block the methylation of their corresponding lysine residues on wild-type histones. One attractive model is that these mutations sequester histone methyltransferases, but genome-wide studies show that mutant histones and histone methyltransferases often do not colocalize. Using chromatin immunoprecipitation sequencing (ChIP-seq), here, we show that, in fission yeast, even though H3K9M-containing nucleosomes are broadly distributed across the genome, the histone H3K9 methyltransferase Clr4 is mainly sequestered at pericentric repeats. This selective sequestration of Clr4 depends not only on H3K9M but also on H3K14 ubiquitylation (H3K14ub), a modification deposited by a Clr4-associated E3 ubiquitin ligase complex. In vitro, H3K14ub synergizes with H3K9M to interact with Clr4 and potentiates the inhibitory effects of H3K9M on Clr4 enzymatic activity. Moreover, binding kinetics show that H3K14ub overcomes the Clr4 aversion to H3K9M and reduces its dissociation. The selective sequestration model reconciles previous discrepancies and demonstrates the importance of protein-interaction kinetics in regulating biological processes.

The INO80 Complex Regulates Epigenetic Inheritance of Heterochromatin.

One key aspect of epigenetic inheritance is that chromatin structures can be stably inherited through generations after the removal of the signals that establish such structures. In fission yeast, the RNA interference (RNAi) pathway is critical for the targeting of histone methyltransferase Clr4 to pericentric repeats to establish heterochromatin. However, pericentric heterochromatin cannot be properly inherited in the absence of RNAi, suggesting the existence of mechanisms that counteract chromatin structure inheritance. Here, we show that mutations of components of the INO80 chromatin-remodeling complex allow pericentric heterochromatin inheritance in RNAi mutants. The ability of INO80 to counter heterochromatin inheritance is attributed to one subunit, Iec5, which promotes histone turnover at heterochromatin but has little effects on nucleosome positioning at heterochromatin, gene expression, or the DNA damage response. These analyses demonstrate the importance of the INO80 chromatin-remodeling complex in controlling heterochromatin inheritance and maintaining the proper heterochromatin landscape of the genome.

Effects of the microtubule nucleator Mto1 on chromosomal movement, DNA repair, and sister chromatid cohesion in fission yeast.

Although the function of microtubules (MTs) in chromosomal segregation during mitosis is well characterized, much less is known about the role of MTs in chromosomal functions during interphase. In the fission yeast Schizosaccharomyces pombe, dynamic cytoplasmic MT bundles move chromosomes in an oscillatory manner during interphase via linkages through the nuclear envelope (NE) at the spindle pole body (SPB) and other sites. Mto1 is a cytoplasmic factor that mediates the nucleation and attachment of cytoplasmic MTs to the nucleus. Here, we test the function of these cytoplasmic MTs and Mto1 on DNA repair and recombination during interphase. We find that mto1Δ cells exhibit defects in DNA repair and homologous recombination (HR) and abnormal DNA repair factory dynamics. In these cells, sister chromatids are not properly paired, and binding of Rad21 cohesin subunit along chromosomal arms is reduced. Our findings suggest a model in which cytoplasmic MTs and Mto1 facilitate efficient DNA repair and HR by promoting dynamic chromosomal organization and cohesion in the nucleus.

Noncatalytic Function of a JmjC Domain Protein Disrupts Heterochromatin.

Chromatin-modifying enzymes are frequently overexpressed in cancer cells, and their enzymatic activities play important roles in changing the epigenetic landscape responsible for tumorigenesis. However, many of these proteins also execute noncatalytic functions, which are poorly understood. In fission yeast, overexpression of Epe1, a histone demethylase homolog, causes heterochromatin defects. Interestingly, in our recent work, we discovered that overexpressed Epe1 recruits SAGA, a histone acetyltransferase complex important for transcriptional regulation, to disrupt heterochromatin, independent of its demethylase activity. Our findings suggest that overexpressed chromatin-modifying enzymes can alter the epigenetic landscape through changing their proteomic environments, an area that needs to be further explored in dissecting disease etiology associated with overexpression of chromatin regulators.

Anti-silencing factor Epe1 associates with SAGA to regulate transcription within heterochromatin.

Heterochromatin is a highly condensed form of chromatin that silences gene transcription. Although high levels of transcriptional activities disrupt heterochromatin, transcription of repetitive DNA elements and subsequent processing of the transcripts by the RNAi machinery are required for heterochromatin assembly. In fission yeast, a JmjC domain protein, Epe1, promotes transcription of DNA repeats to facilitate heterochromatin formation, but overexpression of Epe1 leads to heterochromatin defects. However, the molecular function of Epe1 is not well understood. By screening the fission yeast deletion library, we found that heterochromatin defects associated with Epe1 overexpression are alleviated by mutations of the SAGA histone acetyltransferase complex. Overexpressed Epe1 associates with SAGA and recruits SAGA to heterochromatin regions, which leads to increased histone acetylation, transcription of repeats, and the disruption of heterochromatin. At its normal expression levels, Epe1 also associates with SAGA, albeit weakly. Such interaction regulates histone acetylation levels at heterochromatin and promotes transcription of repeats for heterochromatin assembly. Our results also suggest that increases of certain chromatin protein levels, which frequently occur in cancer cells, might strengthen relatively weak interactions to affect the epigenetic landscape.

Molecular basis for the role of oncogenic histone mutations in modulating H3K36 methylation.

Histone H3 lysine 36 methylation (H3K36me) is critical for epigenetic regulation and mutations at or near H3K36 are associated with distinct types of cancers. H3K36M dominantly inhibits H3K36me on wild-type histones, whereas H3G34R/V selectively affects H3K36me on the same histone tail. Here we report the crystal structures of SETD2 SET domain in complex with an H3K36M peptide and SAM or SAH. There are large conformational changes in the substrate binding regions of the SET domain, and the K36M residue interacts with the catalytic pocket of SETD2. H3G34 is surrounded by a very narrow tunnel, which excludes larger amino acid side chains. H3P38 is in the trans configuration, and the cis configuration is incompatible with SETD2 binding. Finally, mutations of H3G34 or H3P38 alleviate the inhibitory effects of H3K36M on H3K36me, demonstrating that the stable interaction of H3K36M with SETD2 is critical for its inhibitory effects.