The Role of Histone H2B Ubiquitylation and its Related Factors in Transcriptional Regulation in Mammalian Cells

Diverse histone modifications such as acetylation, methylation, and phosphorylation play important roles in transcriptional regulation throughout eukaryotes, and recent studies in yeast also have implicated H2B ubiquitylation in the transcription of specific genes. However, a systematic study of H2B ubiquitylation in mammalian cells has been hindered by the lack of information about mammalian homologues of the yeast enzymes responsible for H2B ubiquitylation. I report identification of a functional human homologue, the hBRE1A/B complex, of the yeast BRE1 E3 ubiquitin ligase. hBRE1A, which forms a complex with hBRE1B, specifically increases the global level of H2B ubiquitylation at lysine 120 and enhances activator dependent transcription in vivo. An extensive screening of cognate E2 ubiquitin conjugating enzyme for the hBRE1A/B complex revealed that hRAD6A and hRAD6B specifically interact with the N-terminal region of hBRE1A, and ubiquitylate H2B at lysine 120 in the presence of hBRE1A/B in vitro. Moreover, reduction of hBRE1A, hBRE1B and hRAD6 proteins by RNAi decreases endogenous H2B ubiquitylation, activator-dependent transcription, and both H3-K4 and H3-K79 methylation. Of special significance, I show that hBRE1A/B directly interacts with p53 and that it is recruited to the mdm2 promoter in a p53-dependent manner. These studies suggest that hBRE1A/B is an H2B-specific E3 ubiquitin ligase and that it functions, at least in part, through direct activator interactions, as a transcriptional coactivator. In addition, hBRE1A/B directly interacts with the hPAF complex to bring hRAD6 to the transcription machinery. I also found that a direct interaction between the hPAF complex and the previously characterized transcription elongation factor SII enhances their mutual association with RNA polymerase II. In an in vitro transcription assay with highly purified transcription factors, the hPAF complex and SII showed significant synergistic effects on activator- and histone acetyltransferase-dependent transcription from a chromatin template. However, addition of the H2B ubiquitylation factors to the in vitro transcription reaction led to an unexpected reduction in transcription. These results suggest that the reconstituted system lacks additional histone modifying and/or chromatin remodeling activities that link H2B ubiquitylation and gene activation. Taken together, my new findings set a stage for studying the molecular mechanisms of H2B ubiquitylation in transcriptional control in mammalian cells