The long-term goal of this work is to understand how the RNA interference (RNAi) pathway regulates heterochromatic gene silencing in fission yeast. RNAi is a widespread silencing mechanism that acts at both the posttranscriptional and transcriptional levels and is triggered by double stranded RNA molecules that are processed to small interfering RNAs (called siRNAs). siRNAs guide the inactivation of complementary target nucleic acids by effector complexes. Our laboratory has purified the RITS (RNA-Induced Transcriptional Gene Silencing) complex, containing the Ago1, Chp1, and Tas3 proteins, which physically links the RNAi pathway to heterochromatin. In addition, we have purified the RNAi complexes that synthesize dsRNA, process it into siRNAs, and help load siRNAs onto the RITS complex. We have provided evidence that these RNAi complexes localize to specific chromosome regions via base pairing interactions between siRNAs and nascent non-coding transcripts. This localization leads to recruitment of a histone H3 methyltransferase complex, called the CRC (Clr4-Rik1-Cul4) complex, which methylates histone H3 on lysine-9 and promotes heterochromatin formation. Our recent results suggest that cis-synthesis of dsRNA, at sites of heterochromatin assembly, is required for H3 lysine-9 methylation. The goals of this proposal are to understand how RITS and dsRNA synthesis recruit the CRC histone methyltransferase complex and how the nascent RNA transcript controls the spreading of heterochromatin. In addition, we will perform a biochemical dissection of steps that are involved in the formation of heterochromatic siRNAs.

Public Health Relevance

This project addresses the role of RNA interference (RNAi), a conserved RNA silencing pathway, in gene silencing in the nucleus. Components of this pathway are involved in regulation of cell differentiation and cancer in humans. A basic understanding of the role of RNAi in gene silencing will not only provide a frame work for understanding how the process can fail, but also provides the substrate and knowledge to design therapeutic strategies based on intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM072805-08
Application #
8225222
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
2005-02-01
Project End
2013-03-31
Budget Start
2012-02-01
Budget End
2013-03-31
Support Year
8
Fiscal Year
2012
Total Cost
$347,397
Indirect Cost
$142,443
Name
Harvard University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Iglesias, Nahid; Currie, Mark A; Jih, Gloria et al. (2018) Automethylation-induced conformational switch in Clr4 (Suv39h) maintains epigenetic stability. Nature 560:504-508
Yu, Ruby; Wang, Xiaoyi; Moazed, Danesh (2018) Epigenetic inheritance mediated by coupling of RNAi and histone H3K9 methylation. Nature 558:615-619
Wang, Xiaoyi; Moazed, Danesh (2017) DNA sequence-dependent epigenetic inheritance of gene silencing and histone H3K9 methylation. Science 356:88-91
Jih, Gloria; Iglesias, Nahid; Currie, Mark A et al. (2017) Unique roles for histone H3K9me states in RNAi and heritable silencing of transcription. Nature 547:463-467
Jain, Ruchi; Iglesias, Nahid; Moazed, Danesh (2016) Distinct Functions of Argonaute Slicer in siRNA Maturation and Heterochromatin Formation. Mol Cell 63:191-205
Banday, Shahid; Farooq, Zeenat; Rashid, Romana et al. (2016) Role of Inner Nuclear Membrane Protein Complex Lem2-Nur1 in Heterochromatic Gene Silencing. J Biol Chem 291:20021-9
Behrouzi, Reza; Lu, Chenning; Currie, Mark A et al. (2016) Heterochromatin assembly by interrupted Sir3 bridges across neighboring nucleosomes. Elife 5:
Ragunathan, Kaushik; Jih, Gloria; Moazed, Danesh (2015) Epigenetics. Epigenetic inheritance uncoupled from sequence-specific recruitment. Science 348:1258699
Holoch, Daniel; Moazed, Danesh (2015) RNA-mediated epigenetic regulation of gene expression. Nat Rev Genet 16:71-84
Martienssen, Robert; Moazed, Danesh (2015) RNAi and heterochromatin assembly. Cold Spring Harb Perspect Biol 7:a019323

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