Cdyl, a New Partner of the Inactive X Chromosome and Potential Reader of H3K27me3 and H3K9me2

Abstract

X chromosome inactivation is a remarkable example of chromosome-wide gene silencing and facultative heterochromatin formation. Numerous histone posttranslational modifications, including H3K9me2 and H3K27me3, accompany this process, although our understanding of the enzymes that lay down these marks and the factors that bind to them is still incomplete. Here we identify Cdyl, a chromodomain-containing transcriptional corepressor, as a new chromatin-associated protein partner of the inactive X chromosome (Xi). Using mouse embryonic stem cell lines with mutated histone methyltransferase activities, we show that Cdyl relies on H3K9me2 for its general association with chromatin in vivo. For its association with Xi, Cdyl requires the process of differentiation and the presence of H3K9me2 and H3K27me3, which both become chromosomally enriched following Xist RNA coating. We further show that the removal of the PRC2 component Eed and subsequent loss of H3K27me3 lead to a reduction of both Cdyl and H3K9me2 enrichment on inactive Xi. Finally, we show that Cdyl associates with the H3K9 histone methyltransferase G9a and the MGA protein, both of which are also found on Xi. We propose that the combination of H3K9me2 and H3K27me3 recruits Cdyl to Xi, and this, in turn, may facilitate propagation of the H3K9me2 mark by anchoring G9a.

ACKNOWLEDGMENTS

We thank members of the Heard lab, in particular, E. Nora and R. Duffié, for helpful advice and discussions, C. Picard for early experiments with Cdyl, and A.-V. Gendrel for critical input on Western blot analysis. We thank A. Wutz for Xist-inducible ESCs and S. Khochbin for Cdyl constructs and critical feedback.

M.E.-D.-A. was supported by an FRM fellowship. This work was supported by Ligue contre le Cancer, the ANR, the EpiGeneSys FP7 257082 Network of Excellence, ERC Advanced Investigator award 250367, and EU FP7 SYBOSS grant 242129 (E.H.). The Vermeulen lab is supported by a grant from the Netherlands Organization for Scientific Research (NWO-VIDI; project no. 864.09.003).

Contributions were as follows: biochemical and microscopy experiments, M.E.-D.-A. and S.T.D.R.; cell culture, M.E.-D.-A.; generation of plasmids and cell lines, M.E.-D.-A.; mass spectrometry, C.G.S., A.H.S., and M.V.; FRAP, O.R. and M.E.-D.-A.; experimental design, interpretation, and writing, E.H., M.E.-D.-A., M.V., and M.R.

We have no conflict of interest to declare.