Medical Biology - Theses
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ItemMechanistic insights into how the epigenetic regulator Smchd1 interacts with and alters the chromatin( 2018)Structural Maintenance of Chromosomes, Hinge Domain containing 1 (Smchd1) is critical for the maintenance of X Chromosome Inactivation (XCI), and transcriptional repression at a subset of autosomal loci (Blewitt et al., 2008; Mould et al., 2013; Gendrel et al., 2013). Gain and loss of function mutations in SMCHD1 have been found to underlie Bosma arhinia micropthalmia syndrome (BAMS) and Facioscapulohumoral muscular dystrophy 2 (FSHD2), respectively - two distinct developmental disorders (Lemmers et al., 2012; Gordon et al., 2017; Shaw et al., 2017). Currently little is known about molecular mechanisms underlying the involvement of Smchd1 in transcriptional repression or disease. This project aimed to better understand how Smchd1 associates with and influences the chromatin. There has been growing evidence in the literature to suggest that Smchd1 and the non-coding RNA Xist might interact directly (Nozawa et al., 2013; Kelsey et al., 2015; Minajigi et al., 2015). We have previously shown that the hinge domain of Smchd1 binds synthetic DNA and RNA oligonucleotides in vitro (Chen et al., 2015). I was therefore interested in whether Smchd1 directly associates with endogenous nucleic acids, and whether such interactions could be important for Smchd1's localisation to the chromatin. To this end, I performed PAR-CLIP to determine whether Smchd1 binds endogenous RNAs genome-wide. I find Smchd1-RNA interactions to be non-specific, and are therefore unlikely to act as a targeting mechanism. I also find that while Smchd1 is dependent on Xist for its localisation to the Xi, this is not due to a direct protein-RNA interaction, but rather due to a dependency on the downstream HnrnpK-polycomb pathway. Evidence from our lab has suggested that Smchd1 may be involved in regulating higher order chromatin organisation (Chen et al., 2015). To investigate changes to the chromatin architecture in the absence of Smchd1, I have performed in-situ Hi-C and ATAC-seq in Smchd1 wild-type and deleted neural stem cells. For the first time my data have demonstrated a role for Smchd1 in chromatin organisation of the Hox cluster, but also the inactive X chromosome. Furthermore, I have identified that in the absence of Smchd1, Hox genes are dysregulated, implicating Smchd1 in Hox gene silencing via a role in chromatin conformation. Taken together the results from the body of work I present here allow me to put forward a model, in which Smchd1 is recruited to target loci by the recognition of a PRC1-mediated chromatin structure. At these sites, I propose that Smchd1 is involved in the maintenance of long-range repressive chromatin structures, which limit promoter-enhancer interactions that are permissive for transcription, potentially by preventing binding of Ctcf, and therefore Ctcf-mediated looping events.