Medical Biology - Theses
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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.