X chromosome inactivation (XCI) is a coordinated set of epigenetic mechanisms that lead to the inactivation of the majority of genes on one of the two X chromosomes in female mammals. Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic modifier that plays an important role in XCI, autosomal gene silencing, and is also implicated in several diseases in humans. Thus far little is known about the structure and function of native Smchd1 in the nucleus.
We made use of advanced microscopy to understand the macromolecular structure and dynamics of native Smchd1 in the nucleus. We performed fluorescence recovery after photobleaching (FRAP) to understand the dynamics of Smchd1 binding to the inactive X chromosome. FRAP data shows that Smchd1 has a very slow turnover on the inactive X. Comparison of the dynamics between wild type Smchd1 and our unique gain of function mutant showed faster dynamics which may explain the gain of function observed in the mutant and suggests the turnover rate is important for Smchd1 function. We also used FILM-FRET to understand Smchd1 binding ability in the inactive X chromosome vs the nuclear landscape during interphase. Using lattice light-sheet microscopy for the first time we show that Smchd1 unloads from the inactive X chromosome during mitosis and reloads once cell division is complete. Finally, to understand the structure of Smchd1, we tagged both ends of Smchd1 and performed 3D-direct stochastic optical reconstruction microscopy (3D-dSTORM). We found that Smchd1 is 40nm in length.
Together these approaches of studying both wild-type and mutant forms of native Smchd1 will further our understanding of the molecular mechanisms by which Smchd1 contributes to normal gene silencing and may shed a light on how things go wrong in disease.