Polymeric nature of tandemly repeated genes enhances assembly of constitutive heterochromatin in fission yeast.

Motivated by our recent experiments that demonstrate that the tandemly repeated genes become heterochromatin, here we show a theory of heterochromatin assembly by taking into account the connectivity of these genes along the chromatin in the kinetic equations of small RNA production and histone methylation, which are the key biochemical reactions involved in the heterochromatin assembly. Our theory predicts that the polymeric nature of the tandemly repeated genes ensures the steady production of small RNAs because of the stable binding of nascent RNAs produced from the genes to RDRC/Dicers at the surface of nuclear membrane. This theory also predicts that the compaction of the tandemly repeated genes suppresses the production of small RNAs, consistent with our recent experiments. This theory can be extended to the small RNA-dependent gene silencing in higher organisms.

Tandemly repeated genes promote RNAi-mediated heterochromatin formation via an antisilencing factor, Epe1, in fission yeast.

In most eukaryotes, constitutive heterochromatin, defined by histone H3 lysine 9 methylation (H3K9me), is enriched on repetitive DNA, such as pericentromeric repeats and transposons. Furthermore, repetitive transgenes also induce heterochromatin formation in diverse model organisms. However, the mechanisms that promote heterochromatin formation at repetitive DNA elements are still not clear. Here, using fission yeast, we show that tandemly repeated mRNA genes promote RNA interference (RNAi)-mediated heterochromatin formation in cooperation with an antisilencing factor, Epe1. Although the presence of tandemly repeated genes itself does not cause heterochromatin formation, once complementary small RNAs are artificially supplied in trans, the RNAi machinery assembled on the repeated genes starts producing cognate small RNAs in cis to autonomously maintain heterochromatin at these sites. This "repeat-induced RNAi" depends on the copy number of repeated genes and Epe1, which is known to remove H3K9me and derepress the transcription of genes underlying heterochromatin. Analogous to repeated genes, the DNA sequence underlying constitutive heterochromatin encodes widespread transcription start sites (TSSs), from which Epe1 activates ncRNA transcription to promote RNAi-mediated heterochromatin formation. Our results suggest that when repetitive transcription units underlie heterochromatin, Epe1 generates sufficient transcripts for the activation of RNAi without disruption of heterochromatin.

Two secured FACT recruitment mechanisms are essential for heterochromatin maintenance.

FACT (facilitate chromatin transcription) is involved in heterochromatic silencing, but its mechanisms and function remain unclear. We reveal that the Spt16 recruitment mechanism operates in two distinct ways in heterochromatin. First, Pob3 mediates Spt16 recruitment onto the heterochromatin through its Spt16 dimerization and tandem PH domains. Without Pob3, Spt16 recruitment is partially reduced, exhibiting a silencing defect and impaired H2A/H2B organization. Second, heterochromatin protein 1 (HP1)/Swi6 mediates Spt16 recruitment onto the heterochromatin by physical interaction of the Swi6 chromo-shadow domain (CSD) and Spt16 peptidase-like domains. Several CSD mutants are tested for Spt16 binding activity, and the charged loop connecting ß1 and ß2 is critical for Spt16 binding and heterochromatic silencing. Loss of these pathways causes a severe defect in H3K9 methylation and HP1/Swi6 localization in the pericentromeric region, exhibiting transcriptional silencing defects and disordered heterochromatin. Our findings suggest that FACT and HP1/Swi6 work intimately to regulate heterochromatin organization.

Construction and characterization of a zinc-inducible gene expression vector in fission yeast.

Gene expression vectors are useful and important tools that are commonly used in a variety of experiments, including expression of foreign genes, functional analysis of genes of interest and complementation experiments. In this study, a hybrid promoter, combining the adh1+ upstream activating sequence (UAS) of fission yeast and the GAL10 core promoter of budding yeast, was constructed to enable high level expression depending on the presence of zinc in culture medium for fission yeast. When the hybrid promoter was cloned on the multicopy plasmid, it was fully induced and repressed within 10 h in the presence and absence of zinc, respectively. The kinetics of induction and reduction were similar to those of the endogenous adh1+ mRNA. In contrast, native adh1+ promoter lost its tight repression in zinc-depleted condition when it was cloned on the plasmid. Because adh1+ UAS-specific transcription factors have not yet been identified, we identified UAS elements involved in zinc sensing by characterizing this hybrid promoter. We also found that the expression level increased by the TATA box mutation, GATAA, in the presence of zinc.

Sfh1, an essential component of the RSC chromatin remodeling complex, maintains genome integrity in fission yeast.

Abp1 is a fission yeast CENP-B homologue that contributes to centromere function, silencing at pericentromeric heterochromatin and silencing of retrotransposons. We identified the sfh1 gene, encoding a core subunit of the fission yeast chromatin remodeling complex RSC as an Abp1-interacting protein. Because sfh1 is essential for growth, we isolated temperature-sensitive sfh1 mutants. These mutants showed defects in centromere functions, reflected by sensitivity to an inhibitor of spindle formation and minichromosome instability. Sfh1 localized at both kinetochore and pericentromeric heterochromatin regions. Although sfh1 mutations had minor effect on silencing at these regions, they decreased the levels of cohesin on centromeric heterochromatin. Sfh1 also localized at a retrotransposon, Tf2, in a partly Abp1-dependent manner, and assisted in silencing of Tf2 by Abp1 probably in the same pathway as a histone chaperon, HIRA, which is also known to involve in Tf2 repression. Furthermore, sfh1 mutants were sensitive to several DNA-damaging treatments (HU, MMS, UV and X-ray). Increase in spontaneous foci of Rad22, a recombination Mediator protein Rad52 homologue, in sfh1 mutant suggests that RSC functions in homologous recombination repair of double-stranded break downstream of the Rad22 recruitment. These results indicate that RSC plays multiple roles in the maintenance of genome integrity.