Background
Structural maintenance of chromosomes (SMC) flexible hingedomain-containing1(SMCHD1) is a non-canonical SMC family protein, which mainly contains an N-terminal ATPase domain, C-terminal Hinge domain and an uncharacterized central domain. SMCHD1 is an epigenetic repressor and plays critical role of gene repression on inactive X and many autosomal loci e.g., D4Z4 repeats and Hox gene cluster. Perturbation of functional SMCHD1 could disrupt repression of its targets, but the underlying mechanisms that how SMCHD1 plays its role remains unknown.
SMCHD1 has also been implicated in human developmental disease muscular dystrophy Facioscapulohumeral muscular dystrophy (FSHD). Missense and nonsense mutations on SMCHD1 were found in FSHD patients and relevant with the onset of disease. These mutations spread the whole protein, and some impair SMCHD1’s function e.g., ATPase activity or nucleic acid binding ability. However, the exact mechanism of how SMCHD1 exert its function and the role of its ATPase activity and nucleic acid binding ability is still mysterious.
Research methodologies and Major finding
To unravel the role of SMCHD1’s nucleic binding ability and its underlying mechanism, I plan to combine genomic and live cell imaging methods to get insights of both genomic and protein dynamic aspects. First, I developed inducible mice strains which conditional expresses either wildtype SMCHD1-GFP or SMCHD1-GFP contains a patient mutation R1867G, which has been proved impair the nucleic binding ability of SMCHD1.
After validation of the inducible mice system, I used immunofluorescence to see whether this R1867G mutation dramatically influences SMCHD1’s distribution in cell. The IF result shows no change of SMCHD1 mutant’s distribution in the nucleus, but a significant decrease of binding on inactive X. This indicates that SMCHD1R1867G-GFP might has a lower loading rate to Xi, or higher disassociation rate from Xi. This promotes us to use the following live-cell imaging methods e.g., FRAP, FLIM-FRET and FFS to further answer these questions. Besides, I will use genomic assays including ChIP-seq, RNA-seq and Hi-C to decipher the influence of SMCHD1 mutant in genome level.
Conclusion
By creating and using inducible mice strain harbors a patient mutation which impairs SMCHD1’s nucleic acid binding ability, we preliminarily found that this mutant has a decrease binding on inactive X. This encourages us to further investigate the role of SMCHD1’s nucleic binding ability of SMCHD1’s biological functions and protein dynamics by using genomics and live-cell imaging methods.