The dynamic regulation of higher-order chromosome structure governs diverse cellular processes ranging from stable inheritance of gene expression patterns to other aspects of global chromosome structure essential for preserving genomic integrity. Our earlier studies revealed that RNA interference (RNAi), whereby double-stranded RNAs silence cognate genes, plays a critical role in targeting of heterochromatin, a specialized form of chromatin that can inhibit transcription and recombination across large chromosomal domains, to specific locations in the fission yeast Schizosaccharomyces pombe genome. Subsequent genetic and biochemical investigations identified RNAi-induced transcriptional gene silencing (RITS) complex that provided direct link between small RNAs and heterochromatin formation. These studies also uncovered surprising interdependency between heterochromatin and RNAi mechanism and led to the discovery of an elegant self-reinforcing RNAi loop mechanism that ensures both transcriptional and post-transcriptional silencing in cis. In this loop mechanism, RNAi machinery operates as a stable component of the heterochromatic domains via tethering of RNAi complexes (such as RITS) to heterochromatin marks (including histone H3 methylated at lysine 9) to destroy repeat transcripts that escape heterochromatin-mediated transcriptional silencing. The processing of transcripts by RNAi machinery generate small interfering RNAs (siRNAs) that are utilized for further targeting of heterochromatin complexes, so the mechanism continues. We have extended these analyses to gain insights into full spectrum of target sequences across the entire S. pombe genome affected by the RNAi and heterochromatin machineries. In a comprehensive study, we developed a high-resolution map of the heterochromatin distribution across the entire S. pombe genome. These analyses together with mapping of RNAi components and large scale sequencing of siRNAs associated with an RNAi effector RITS complex, involved in heterochromatic silencing, have yielded novel insights into the epigenetic profile of this model eukaryotic genome. In an interesting new development, our recent work suggests that heterochromatic structures are dynamically regulated during the cell cycle. In particular, heterochromatic repeat elements are transcribed during a brief window during the S-phase. Importantly, we have discovered that the transcription of repeats by RNA polymerase II is coupled to the recruitment of heterochromatin complexes, supporting a prominent role for transcriptional machinery in determining the epigenetic makeup of the genome. In another surprising finding, we have discovered that low levels of heterochromatin factors localize broadly across euchromatic regions containing genes and cooperate with RNAi machinery to regulate expression of RNA polymerase II transcripts across large portions of the genome. In particular, we have found that heterochromatin/RNAi factors prevent accumulation of potentially deleterious antisense RNAs. Heterochromatin and RNAi factors are partially redundant in this regard with a histone H2A variant H2A.Z. Loss of Clr4/Suv39h-containing heterochromatin silencing complex or an Argonaute protein alone has little effect on antisense transcript levels, but cells lacking either of these factors and H2A.Z show markedly increased levels of antisense RNAs that are normally degraded by the exosome. These analyses suggest that in addition to performing other functions, heterochromatin and RNAi factors cooperate with H2A.Z to suppress antisense transcripts, which has important implications for diverse chromosomal processes. In another important finding, we have discovered a novel heterochromatin assembly pathway that relies on transcription and RNAs but does not require RNAi machinery. We have discovered that facultative heterochromatin is established at genes required for gametogenesis (which are repressed in vegetative cells), and that its formation is dependent on conserved RNA degradation factors, including a protein complex involved in polyadenylation of transcripts and the exosome that degrade gene transcripts. Importantly, heterochromatin formation by this pathway is modulated in response to signals that induce gametogenesis. This groundbreaking study has paved the way for understanding the more complex regulatory networks at work in higher eukaryotes including that involve polycomb silencing, and has provided a foundation for understanding the large scale reprogramming of the genome in response to developmental and environmental cues that occur through modifications of heterochromatin.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010523-11
Application #
8763127
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
11
Fiscal Year
2013
Total Cost
$1,488,146
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Sugiyama, Tomoyasu; Thillainadesan, Gobi; Chalamcharla, Venkata R et al. (2016) Enhancer of Rudimentary Cooperates with Conserved RNA-Processing Factors to Promote Meiotic mRNA Decay and Facultative Heterochromatin Assembly. Mol Cell 61:747-59
Zofall, Martin; Smith, Deborah R; Mizuguchi, Takeshi et al. (2016) Taz1-Shelterin Promotes Facultative Heterochromatin Assembly at Chromosome-Internal Sites Containing Late Replication Origins. Mol Cell 62:862-74
Chalamcharla, Venkata R; Folco, H Diego; Dhakshnamoorthy, Jothy et al. (2015) Conserved factor Dhp1/Rat1/Xrn2 triggers premature transcription termination and nucleates heterochromatin to promote gene silencing. Proc Natl Acad Sci U S A 112:15548-55
Mizuguchi, Takeshi; Barrowman, Jemima; Grewal, Shiv I S (2015) Chromosome domain architecture and dynamic organization of the fission yeast genome. FEBS Lett 589:2975-86
Mizuguchi, Takeshi; Fudenberg, Geoffrey; Mehta, Sameet et al. (2014) Cohesin-dependent globules and heterochromatin shape 3D genome architecture in S. pombe. Nature 516:432-5
Aygun, Ozan; Mehta, Sameet; Grewal, Shiv I S (2013) HDAC-mediated suppression of histone turnover promotes epigenetic stability of heterochromatin. Nat Struct Mol Biol :
Zofall, Martin; Yamanaka, Soichiro; Reyes-Turcu, Francisca E et al. (2012) RNA elimination machinery targeting meiotic mRNAs promotes facultative heterochromatin formation. Science 335:96-100
Reyes-Turcu, Francisca E; Grewal, Shiv Is (2012) Different means, same end-heterochromatin formation by RNAi and RNAi-independent RNA processing factors in fission yeast. Curr Opin Genet Dev 22:156-63
Zhang, Ke; Fischer, Tamas; Porter, Rebecca L et al. (2011) Clr4/Suv39 and RNA quality control factors cooperate to trigger RNAi and suppress antisense RNA. Science 331:1624-7
Ni, Ting; Tu, Kang; Wang, Zhong et al. (2010) The prevalence and regulation of antisense transcripts in Schizosaccharomyces pombe. PLoS One 5:e15271

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