Molecular mechanism of heterochromatin formation

Massachusetts General Hospital, Boston MA

Investigators

Abstract

Project Summary Eukaryotic heterochromatin is demarcated by histone H3 lysine 9 methylation (H3K9me) and is required for maintaining genomic stability. Loss of heterochromatin results aneuploidies and translocations and is associated with human diseases including cancers. The centromeres of the fission yeast, Schizosaccharomyces pombe, have served as a powerful model for uncovering the conserved chromatin- and RNA-based mechanisms governing heterochromatin formation. Historically, in this system, the recruitment of the highly conserved Suv39/Clr4, the sole histone H3 lysine 9 (H3K9) methyltransferase, to centromeres was thought to be guided by Argonaute-associated small RNAs (sRNAs) generated by processing of heterochromatic transcripts by the RNAi machinery. Additionally, the two highly conserved heterochromatic histone deacetylases, Sir2 and Clr3, deacetylate histones and prepare histones for Clr4-mediated methylation. Once targeted, Clr4 can not only methylate H3K9 but also bind to this mark, thus creating a positive feed forward loop, stabilizing its interaction and spreading along heterochromatin. Even though how centromeric heterochromatin is nucleated has been topic of many studies, all models converge on sRNAs as the only mechanism for nucleating Clr4 and triggering heterochromatin formation. My team on the other hand, inspired by the 20-year-old observation that in ago1D cells H3K9me is reduced but not eliminated at pericentromeres, asked whether an sRNA-independent mechanism could nucleate Clr4. In recent Cell paper, we define an sRNA-independent alternative axis to heterochromatin nucleation. This pathway uses the nuclear RNA quality control complex, MTREC which interacts with Clr4 and specifically recognizes a heterochromatic long noncoding RNA (lncRNA) called SPNCRNA.230 bivalently, via its Mmi1 (YTH-domain protein) and Mtl1 (RNA-helicase) components. More importantly, loss of MTREC together with RNAi leads to complete loss of Clr4 recruitment at centromeres, revealing that these two pathways operate in parallel to nucleate Clr4. Additionally, we showed that Sir2 and Clr3 work upstream of Clr4 to deacetylate H3K9 for Clr4 methylation and are recruited to pericentromeres, including SPNCRNA.230, by sRNA-independent mechanisms. Overall, these data reveal that three independent mechanisms amass Sir2, Clr3 and Clr4 at SPCNRNA.230 to nucleate and trigger heterochromatin formation. Here, we will (Aim 1) Decipher the mechanism(s) of Sir2 recruitment to heterochromatin; (Aim 2) Delineate how SPNCRNA.230 nucleates Clr4 and Clr3 independently of sRNAs and H3K9me; and (Aim 3) Reveal the mechanisms of lncRNA- mediated heterochromatin assembly using an inducible system. Overall, the proposed studies will reveal how distinct, parallel recruitment mechanisms nucleate heterochromatin machinery at lncRNAs to trigger heterochromatin formation. We predict that these studies will reshape our understanding of heterochromatin mechanisms and expose novel targets for manipulating heterochromatic state in eukaryotes.