Background
SMCHD1 is an epigenetic repressor and plays critical role of gene repression on inactive X and autosomal locus e.g., D4Z4repeats and Hox gene cluster. It 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.
Research methodologies and Major finding
Here we developed inducible mice strains which conditional expresses either wildtype SMCHD1-GFP or SMCHD1-GFP contains a patient mutation R1867G, which has been proved impairs the nucleic binding ability of SMCHD1. Then we 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. Besides, we performed FRAP experiments on both Xi and nucleus regions. We found SMCHD1-GFP mutant carries R1867G mutation shows higher protein turnover rate than wildtype on the Xi, but similar free diffusion rate. We also performed bulk RNA-seq and found the R1867G mutant shows loss of function among the expression of SMCHD1 sensitive genes.
We are doing ChIP-seq and more live cell imaging methods e.g., FCS and FLIM-FRET to further investigate SMCHD1 protein dynamics and binding profile on the chromatin and answer how SMCHD1 works.
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 but more frequent protein dynamic on inactive X. And we are doing more to further understand the mechanism of SMCHD1.