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.
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ItemMechanistic and structure-function characterisation of the epigenetic regulator Smchd1( 2015)Epigenetic regulation of gene expression is fundamental in controlling biological processes in multicellular organisms. Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic repressor that plays critical roles in X chromosome inactivation, genomic imprinting and monoallelic expression of clustered protocadherin genes. In addition, SMCHD1 is crucial for suppressing the D4Z4 repeat implicated in pathogenesis of facioscapulohumeral muscular dystrophy (FSHD). However, the exact molecular mechanism by which Smchd1 regulates gene expression is unclear. Likewise, little is known about the structure and function of Smchd1 protein apart from that it contains two predicted domains, an N-terminal GHKL-ATPase domain and a C-terminal Smc hinge domain. To understand how Smchd1 mediates epigenetic regulation at the molecular level, I determined genome-wide Smchd1 binding sites in male murine neural stem cells by performing chromatin immunoprecipitation coupled with next-generation sequencing. Together with profiling gene expression and epigenetic marks in wild type and Smchd1-deficient cells, I found that Smchd1 binding at several of its known target genes is correlated with differential gene expression, concomitant with changes in epigenetic modifications. Unexpectedly, a significant proportion of Smchd1 occupancy overlaps with that of CCCTC-binding factor (Ctcf) at distal cis-regulatory elements, indicative of a functional relationship between Smchd1 and Ctcf. Indeed, I demonstrated that Smchd1 and Ctcf could evoke opposing effects on the expression of many protocadherin genes. As Ctcf is implicated in mediating chromatin interactions, these results indicate that Smchd1 may impart epigenetic regulation via physical association with chromatin and maintaining a repressive chromatin state that antagonises Ctcf facilitated chromatin interactions. In order to gain further mechanistic insights into how Smchd1 functions, I conducted structural-functional characterisation of the Smchd1 protein. I determined the domain boundaries and generated recombinant Smc hinge domain with its flanking coiled-coil and recombinant protein corresponding to the N-terminal region of Smchd1 encompassing the putative GHKL-ATPase domain. By performing small-angle X-ray scattering (SAXS) analysis, structural characteristics of those two domains were revealed for the first time. By utilizing a suite of biochemical and biophysical assays, I was able to show that the hinge domain could directly bind to nucleic acids in vitro. Furthermore, I found that a single-amino acid substitution within the hinge domain, equivalent to a modification implicated in FSHD pathogenesis, displayed significantly compromised binding activities. These results support the notion that the Smchd1-chromatin interaction via the hinge domain is critical for its role in epigenetic regulation. In addition, I demonstrated that a putative gain-of-function mutation of Smchd1, could potentially induce a conformational change and acquisition of ATP-binding activity of the N-terminal region of Smchd1. Preliminary results also suggest that this mutation could enhance Smchd1 function in the cellular context, potentially resulting from the altered structural-functional properties. Together, work from this thesis provides unprecedented molecular explanations for Smchd1’s action in epigenetic regulation, which are supported by structural-functional characteristics of the Smchd1 protein uncovered by this study. These results not only offer valuable insights into Smchd1 function, but also may inform future development of much needed therapy for FSHD disease where SMCHD1 is critically involved.