RAD6-Mediated Transcription-Coupled H2B Ubiquitylation Directly Stimulates H3K4 Methylation in Human Cells

The Rockefeller University, New York, NY 10065, USA.
Cell (Impact Factor: 32.24). 06/2009; 137(3):459-71. DOI: 10.1016/j.cell.2009.02.027
Source: PubMed


H2B ubiquitylation has been implicated in active transcription but is not well understood in mammalian cells. Beyond earlier identification of hBRE1 as the E3 ligase for H2B ubiquitylation in human cells, we now show (1) that hRAD6 serves as the cognate E2-conjugating enzyme; (2) that hRAD6, through direct interaction with hPAF-bound hBRE1, is recruited to transcribed genes and ubiquitylates chromatinized H2B at lysine 120; (3) that hPAF-mediated transcription is required for efficient H2B ubiquitylation as a result of hPAF-dependent recruitment of hBRE1-hRAD6 to the Pol II transcription machinery; (4) that H2B ubiquitylation per se does not affect the level of hPAF-, SII-, and p300-dependent transcription and likely functions downstream; and (5) that H2B ubiquitylation directly stimulates hSET1-dependent H3K4 di- and trimethylation. These studies establish the natural H2B ubiquitylation factors in human cells and also detail the mechanistic basis for H2B ubiquitylation and function during transcription.

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Available from: Jung-Shin Lee, Jan 03, 2015
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    • "Recombinant chromatin was assembled as described (Tang et al, 2013). In vitro H2B ubiquitylation was established as described with minor modifications (Kim et al, 2009). Briefly, complete reaction containing 100 ng E1, 100 ng E2, 200 ng RNF20/ 40 complex, 2.5 lg ubiquitin, and 1.0 lg histone octamer or 100 ng recombinant chromatin in 20 ll reaction buffer (50 mM Tris–Cl pH 7.9, 5 mM MgCl 2 , 2 mM NaF, 0.4 mM DTT, and 4 mM ATP) was incubated at 37°C for 1 h and then subjected to SDS–PAGE and Western blot using the indicated antibodies. "
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    ABSTRACT: The Mediator complex orchestrates multiple transcription factors with the Pol II apparatus for precise transcriptional control. However, its interplay with the surrounding chromatin remains poorly understood. Here, we analyze differential histone modifications between WT and MED23(-/-) (KO) cells and identify H2B mono-ubiquitination at lysine 120 (H2Bub) as a MED23-dependent histone modification. Using tandem affinity purification and mass spectrometry, we find that MED23 associates with the RNF20/40 complex, the enzyme for H2Bub, and show that this association is critical for the recruitment of RNF20/40 to chromatin. In a cell-free system, Mediator directly and substantially increases H2Bub on recombinant chromatin through its cooperation with RNF20/40 and the PAF complex. Integrative genome-wide analyses show that MED23 depletion specifically reduces H2Bub on a subset of MED23-controlled genes. Importantly, MED23-coupled H2Bub levels are oppositely regulated during myogenesis and lung carcinogenesis. In sum, these results establish a mechanistic link between the Mediator complex and a critical chromatin modification in coordinating transcription with cell growth and differentiation. © 2015 The Authors.
    Full-text · Article · Sep 2015 · The EMBO Journal
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    • "It is possible that the proteins downstream of BRE1/HUB bring about the diversity in histone methylation. H2B ubiquitylation has been shown to directly stimulate Dot1/ hDot1L-mediated H3K79 methylation (Dover et al., 2002; McGinty et al., 2008) and SET1/hSET1 complex-mediated H3K4 di-and trimethylation (Shilatifard, 2006; Kim et al., 2009) in yeast and human cells. But nothing is known about the proteins mediating the crosslink between H2Bub1 and H3 Lys methylation in plant. "
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    ABSTRACT: Histone H2B monoubiquitination (H2Bub1) is an important regulatory mechanism in eukaryotic gene transcription and is essential for normal plant development. However, the function of H2Bub1 in reproductive development remains elusive. Here, we report OsHUB1 (Oryza sativa HISTONE MONO-UBIQUITINATION1) and OsHUB2, the homologues of Arabidopsis HUB1 and HUB2 proteins which function as E3 ligases in H2Bub1, are involved in late anther development in rice. oshub mutants exhibit abnormal tapetum development and aborted pollen in postmeiotic anthers. Knock-out of OsHUB1 or OsHUB2 results in the loss of H2Bub1, and a reduction in the levels of dimethylated lysine 4 on histone 3 (H3K4me2). Anther transcriptome analysis revealed that several key tapetum degradation-related genes including OsC4, OsCP1 and UDT1 were down-regulated in the mutants. Further, chromatin immunoprecipitation assays demonstrate that H2Bub1 directly targets OsC4, OsCP1 and UDT1 genes and enrichment of H2Bub1 and H3K4me2 in the targets is consistent to some degree. Our studies suggest that histone H2B monoubiquitination, mediated by OsHUB1 and OsHUB2, is an important epigenetic modification that in concert with H3K4me2 modulates transcriptional regulation of anther development in rice. Copyright © 2015, American Society of Plant Biologists.
    Full-text · Article · Jul 2015 · Plant physiology
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    • "K11 Ub chains were observed in vitro but clear data about the implication of UBE2B for building these chains in vivo are still lacking (Hibbert et al., 2011). PolyUb chain formation is not the only signal UBE2B can promote, as its activity is restricted to monoubiquitination of PCNA and histone H2B when combined with the E3 ligases Rad18 and Bre1 respectively (Kim et al., 2009; Hibbert et al., 2011). "
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