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Hyperactivation of HUSH complex function by Charcot–Marie–Tooth disease mutation in MORC2

Abstract

Dominant mutations in the MORC2 gene have recently been shown to cause axonal Charcot–Marie–Tooth (CMT) disease, but the cellular function of MORC2 is poorly understood. Here, through a genome-wide CRISPR–Cas9-mediated forward genetic screen, we identified MORC2 as an essential gene required for epigenetic silencing by the HUSH complex. HUSH recruits MORC2 to target sites in heterochromatin. We exploited a new method, differential viral accessibility (DIVA), to show that loss of MORC2 results in chromatin decompaction at these target loci, which is concomitant with a loss of H3K9me3 deposition and transcriptional derepression. The ATPase activity of MORC2 is critical for HUSH-mediated silencing, and the most common alteration affecting the ATPase domain in CMT patients (p.Arg252Trp) hyperactivates HUSH-mediated repression in neuronal cells. These data define a critical role for MORC2 in epigenetic silencing by the HUSH complex and provide a mechanistic basis underpinning the role of MORC2 mutations in CMT disease.

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Figure 1: A genome-wide CRISPR–Cas9-mediated forward genetic screen identifies an essential role for MORC2 in transgene silencing by the HUSH complex.
Figure 2: The ATPase, CW and coiled-coil domains of MORC2 are required for HUSH complex function.
Figure 3: The HUSH complex recruits MORC2 to heterochromatic target sites.
Figure 4: Loss of MORC2 results in chromatin decompaction at HUSH-target sites.
Figure 5: Chromatin decompaction in MORC2-knockout cells is accompanied by a loss of H3K9me3 and transcriptional depression.
Figure 6: The p.Arg252Trp alteration in MORC2, which is associated with CMT disease, hyperactivates HUSH-mediated epigenetic repression.

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Acknowledgements

We are indebted to CIMR core facilities: R. Schulte and his team for FACS, and M. Gratian and M. Bowen for microscopy. We thank S. Andrews for assistance with data analysis with SeqMonk, and S. Kundu and M. Tolstorukov for helpful discussions. We thank B. Cochran (Tufts University), F. Randow (MRC-LMB), D. Rubinsztein (CIMR) and M. Brenner (Harvard Medical School) for providing materials. This work was supported by the Wellcome Trust, through a Principal Research Fellowship to P.J.L. (101835/Z/13/Z), a Senior Research Fellowship to Y.M. (101908/Z/13/Z), a Sir Henry Wellcome Postdoctoral Fellowship to R.T.T. (201387/Z/16/Z) and a PhD studentship to I.A.T., and by the BBSRC, through a Future Leader Fellowship to C.H.D. I.A.T. is supported as a Damon Runyon Fellow by the Damon Runyon Cancer Research Foundation (DRG-2277-16). The CIMR is in receipt of a Wellcome Trust strategic award.

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Authors

Contributions

I.A.T., R.T.T. and P.J.L. conceived the study. Except for the in vitro expression and purification experiments, which were carried out by C.H.D., I.A.T. and R.T.T. performed all of the experiments and, together with Y.M. and P.J.L., analyzed the data and wrote the manuscript. G.D., R.C.R. and R.E.K. contributed essential reagents.

Corresponding author

Correspondence to Paul J Lehner.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10. (PDF 2738 kb)

Supplementary Table 1

Composition of MORC2 mutants. (XLSX 10 kb)

Supplementary Table 2

Full DIVA data quantifying viral accessibility across all genomic loci in wild-type versus MORC2 knockout HeLa cells. (XLSX 24640 kb)

Supplementary Table 3

Oligonucleotide sequences. (XLSX 11 kb)

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Tchasovnikarova, I., Timms, R., Douse, C. et al. Hyperactivation of HUSH complex function by Charcot–Marie–Tooth disease mutation in MORC2. Nat Genet 49, 1035–1044 (2017). https://doi.org/10.1038/ng.3878

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