Compact heterochromatin is essential for genome stability and hence cell survival. Studies in many organisms including humans underline the importance of pericentromeric heterochromatin in centromere function. Fission yeast centromeres share a common structural organisation with those of their metazoan counterparts. The fission yeast model has been pivotal in understanding many key events in the pathway leading to the assembly of pericentromeric heterochromatin. In particular, studies in this system have revealed that the RNA interference (RNAi) pathway connects with the chromatin modification machinery to impart proper heterochromatin formation. Transcription of the pericentromeres by RNA polymerase II (Pol II) produces double stranded RNA (ds RNA) which is processed by Dicer(Dcr1) into small interfering RNAs (siRNAs). These siRNAs are loaded onto the Argonaute protein Ago1, and target the Ago1- containing RITS (RNA-Induced Transcriptional Silencing) complex to the pericentromeres via complementary base-pairing of the siRNA to the nascent centromeric transcript. RITS then recruits the sole Histone H3-K9-methyl transferase, Clr4, as part of the Clr4-complex, CLRC. The resulting H3K9-methyl marks further result in the recruitment of downstream chromatin binding proteins including the HP1- homolgue Swi6 which plays a key role in cohesin retention. Additionally, the H3K9- methyl marks are required for stabilising the association of CLRC and RITS, thereby promoting a reinforcing loop within the RNAi-mediated heterochromatin pathway. Thus crosstalk between RITS and CLRC is important in establishing and maintaining silent chromatin at the pericentromeres. Stc1 has been proposed to act as a critical link that connects the RITS and CLRC complexes. Stc1 is required for heterochromatin establishment and maintenance at the pericentromere and association of RITS with CLRC is lost in the absence of Stc1. Moreover, Stc1 directly interacts with Ago1 and is essential for siRNA production. These and other previous observations (Bayne et al. 2010) highlight the key role played by Stc1 in the RNAi-mediated heterochromatin pathway. To understand how Stc1 mediates the specific cross-talk between RNAi and chromatin modification, I have investigated the nature of Stc1 interactions with the RNAi and chromatin modification machineries. Using in-vitro binding assays, I found that Stc1 directly interacts with the CLRC subunits Dos2 and Clr4. I also identified the RITS subunit Tas3 as a potential interactor of Stc1, in addition to Ago1. A collaborating research group elucidated the structure of Stc1 using NMR (He et al. 2013) and my study provides evidence for interactions via the distinct domains of Stc1. Stc1 utilises its disordered C-terminus to bind to Dos2 while the N-terminus, which contains a tandem zinc finger domain, acts as a multi-protein interaction interface binding the CLRC subunit Clr4 and RITS subunits Ago1 and Tas3, opening up possibilities for Stc1-containing distinct-complexes. My work provides new insights into the role of Stc1 and opens up future avenues of research key to understanding how heterochromatin domains are defined and maintained.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:738714 |
| Date | January 2017 |
| Creators | Sreedharan Pillai, Sreerekha |
| Contributors | Bayne, Elizabeth ; Stancheva, Irina |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/28733 |
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