Independent Mechanisms Target SMCHD1 to Trimethylated Histone H3 Lysine 9-Modified Chromatin and the Inactive X Chromosome

Abstract

The chromosomal protein SMCHD1 plays an important role in epigenetic silencing at diverse loci, including the inactive X chromosome, imprinted genes, and the facioscapulohumeral muscular dystrophy locus. Although homology with canonical SMC family proteins suggests a role in chromosome organization, the mechanisms underlying SMCHD1 function and target site selection remain poorly understood. Here we show that SMCHD1 forms an active GHKL-ATPase homodimer, contrasting with canonical SMC complexes, which exist as tripartite ring structures. Electron microscopy analysis demonstrates that SMCHD1 homodimers structurally resemble prokaryotic condensins. We further show that the principal mechanism for chromatin loading of SMCHD1 involves an LRIF1-mediated interaction with HP1γ at trimethylated histone H3 lysine 9 (H3K9me3)-modified chromatin sites on the chromosome arms. A parallel pathway accounts for chromatin loading at a minority of sites, notably the inactive X chromosome. Together, our results provide key insights into SMCHD1 function and target site selection.

ACKNOWLEDGMENTS

We thank Mickael Ploquin and Rob Klose for help with protein expression and purification, Graham Kay for the gift of the Smchd1 cDNA, and members of the Brockdorff, Klose, and Nasmyth laboratories for discussion and advice.

This work was funded by Wellcome Trust grants 081385 and 103768. The OPIC electron microscopy facility was founded by a Wellcome Trust JIF award (060208/Z/00/Z) and is supported by a Wellcome Trust equipment grant (093305/Z/10/Z).

N.J.B., A.-V.G., and T.B.N. generated plasmids and cell lines; N.J.B., H.C., and A.-V.G. performed gel filtration and sucrose gradient sedimentation; A.-V.G., K.B., J.D., and R.A.P. performed mass spectrometry; C.A.S performed electron microscopy; N.J.B. performed protein expression, purification, and biochemical assays; N.J.B. and H.C. performed immunofluorescence microscopy; N.J.B., H.C., A.-V.G., and N.B. designed the experiments and interpreted the data; and N.J.B. and N.B. wrote the manuscript.

We declare that we have no conflicts of interest.