Covalent modification of histones is important in regulating chromatin dynamics and transcription. One example of such modification is ubiquitination, which mainly occurs on histones H2A and H2B. Although recent studies have uncovered the enzymes involved in histone H2B ubiquitination and a 'cross-talk' between H2B ubiquitination and histone methylation, the responsible enzymes and the functions of H2A ubiquitination are unknown. Here we report the purification and functional characterization of an E3 ubiquitin ligase complex that is specific for histone H2A. The complex, termed hPRC1L (human Polycomb repressive complex 1-like), is composed of several Polycomb-group proteins including Ring1, Ring2, Bmi1 and HPH2. hPRC1L monoubiquitinates nucleosomal histone H2A at lysine 119. Reducing the expression of Ring2 results in a dramatic decrease in the level of ubiquitinated H2A in HeLa cells. Chromatin immunoprecipitation analysis demonstrated colocalization of dRing with ubiquitinated H2A at the PRE and promoter regions of the Drosophila Ubx gene in wing imaginal discs. Removal of dRing in SL2 tissue culture cells by RNA interference resulted in loss of H2A ubiquitination concomitant with derepression of Ubx. Thus, our studies identify the H2A ubiquitin ligase, and link H2A ubiquitination to Polycomb silencing.
Bibliographical noteFunding Information:
Acknowledgements ThisworkwassupportedbyACI‘Biologiedudéveloppementetphysiologie intégrative’ from the Ministère de la Recherche. F.R. is supported by INSERM, and R.X. is supported by Fondation des Treilles and previously by CNRS and Fondation Electricité de France. We thank M. Boudinot for the continuously improving Faas software, L. Collet for artwork, A. Klarsfeld and A. Lamouroux for critical reading of the manuscript, and J. Champagnat for his support. We thank J. Hall, P. Taghert and T. Siegmund for providing pdf-Gal4, C929-Gal4 and Mai179-Gal4 lines, respectively, as well as R. Stanewsky for anti-PER serum. We are grateful to H. Otsuna and K. Ito for sharing unpublished observations with the Mz520-Gal4 line, and to M. Rosbash and colleagues for communicating their unpublished manuscript.
Acknowledgements We thank J. Kim for help with mass spectrometry. This work was supported by NIH grants to Y.Z., R.S.J. and P.T.