Histone modifications have emerged as important regulators of transcription1,2. Histone H2B monoubiquitination has also been implicated in transcription3,4; however, better understanding of the biological significance of this modification in mammalian cells has been hindered by the lack of suitable reagents, particularly antibodies capable of specifically recognizing ubiquitinated H2B (ubH2B). Here, we report the generation of anti-ubH2B monoclonal antibodies using a branched peptide as immunogen. These antibodies provide a powerful tool for exploring the biochemical functions of H2B monoubiquitination at both a genome-wide and gene-specific level. Application of these antibodies in high resolution chromatin immunoprecipitation (ChIP)-chip experiments in human cells, using tiling arrays, revealed preferential association of ubiquitinated H2B with the transcribed regions of highly expressed genes. Unlike dimethylated H3K4, ubH2B was not associated with distal promoter regions. Furthermore, experimental modulation of the transcriptional activity of the tumour suppressor p53 was accompanied by rapid changes in the H2B ubiquitination status of its p21 target gene, attesting to the dynamic nature of this process. It has recently been demonstrated that the apparent extent of gene expression often reflects elongation rather than initiation rates5; thus, our findings suggest that H2B ubiquitination is intimately linked with global transcriptional elongation in mammalian cells.
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We are grateful to M. Fridkin for peptide design and synthesis, O. Leitner and A. Bren for immunizations and hybridoma fusion and growth, and S. Horn-Saban for DNA amplification and array hybridization. N.M. thanks R. Roeder for his support and encouragement. Supported in part by grant R37 CA40099 from the National Cancer Institute, a Prostate Cancer Foundation (Israel) Center of Excellence, the Dr. Miriam and Sheldon Adelson Medical Research Foundation, and the Yad Abraham Center for Cancer Diagnosis and Therapy.
The authors declare no competing financial interests.
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Minsky, N., Shema, E., Field, Y. et al. Monoubiquitinated H2B is associated with the transcribed region of highly expressed genes in human cells. Nat Cell Biol 10, 483–488 (2008). https://doi.org/10.1038/ncb1712
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