Post-translational histone modifications have important regulatory roles in chromatin structure and function1,2,3. One example of such modifications is histone ubiquitination, which occurs predominately on histone H2A and H2B. Although the recent identification of the ubiquitin ligase for histone H2A has revealed important roles for H2A ubiquitination in Hox gene silencing4,5,6 as well as in X-chromosome inactivation7,8, the enzyme(s) involved in H2A deubiquitination and the function of H2A deubiquitination are not known. Here we report the identification and functional characterization of the major deubiquitinase for histone H2A, Ubp-M (also called USP16). Ubp-M prefers nucleosomal substrates in vitro, and specifically deubiquitinates histone H2A but not H2B in vitro and in vivo. Notably, knockdown of Ubp-M in HeLa cells results in slow cell growth rates owing to defects in the mitotic phase of the cell cycle. Further studies reveal that H2A deubiquitination by Ubp-M is a prerequisite for subsequent phosphorylation of Ser 10 of H3 and chromosome segregation when cells enter mitosis. Furthermore, we demonstrate that Ubp-M regulates Hox gene expression through H2A deubiquitination and that blocking the function of Ubp-M results in defective posterior development in Xenopus laevis. This study identifies the major deubiquitinase for histone H2A and demonstrates that H2A deubiquitination is critically involved in cell cycle progression and gene expression.
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We thank S. L. Berger and M. A. Osley for yeast strains; J. J. Hayes for XP-10 plasmid; and J. Wei for the initial cloning of Ubp-M. We also thank D. Crawford and W. S. Brooks for suggestions on cell cycle and apoptosis analysis; T. Townes and W. S. Brooks for critical reading of the manuscript; E. F. Keyser for assistance with FACS analysis; and A. Nazarian for help with mass spectrometric analysis. This work was supported by a start-up grant (to H.W.) and NCI Cancer Center Support Grant (to P.T.).
Author Contributions H.W. designed the experimental strategy, performed parts of the purification and wrote the paper; H.-Y.J. performed most of the purification, determined the substrate preference and investigated the role of Ubp-M in cell cycle progression and gene expression; L.Z. determined the substrate specificity of Ubp-M and performed the in vitro kinase reaction and nucleosome pull-down experiments; C.Y. cloned the Xenopus Hoxd10 gene and generated the RNAi-resistant Ubp-M constructs; S.N. and C.C. performed the Xenopus injection and in situ hybridization; H.E.-B. and P.T. performed mass spectrometric analysis.
This file contains Supplementary Figures S1-S15 with Legends.
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Archives of Pharmacal Research (2019)