Catalysis-dependent stabilization of Bre1 fine-tunes histone H2B ubiquitylation to regulate gene transcription

Wozniak, Glenn G.; Strahl, Brian D.

doi:10.1101/gad.243121.114

Cited by 19 publications

(18 citation statements)

References 39 publications

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“…Subsequent genetic and biochemical studies in yeast have shown that Rtf1 functions as a component of the polymerase-associated factor 1 (Paf1) complex (PAF1C) containing Paf1, Ctr9, Leo1, and Cdc73 (2)(3)(4)(5). The PAF1C is a multifunctional protein complex whose primary role is to facilitate histone modifications, such as H2B monoubiquitination at K123 and H3 methylation at K4 and K79 (6)(7)(8)(9)(10)(11)(12). The PAF1C also plays important roles in transcription elongation through chromatin, as well as on naked DNA (13)(14)(15)(16).…”

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confidence: 99%

Characterization of the Human Transcription Elongation Factor Rtf1: Evidence for Nonoverlapping Functions of Rtf1 and the Paf1 Complex

Cao

Yamamoto

Isobe

et al. 2015

Molecular and Cellular Biology

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bRestores TBP function 1 (Rtf1) is generally considered to be a subunit of the Paf1 complex (PAF1C), a multifunctional protein complex involved in histone modification and transcriptional or posttranscriptional regulation. Rtf1, however, is not stably associated with the PAF1C in most species except Saccharomyces cerevisiae, and its biochemical functions are not well understood. Here, we show that human Rtf1 is a transcription elongation factor that may function independently of the PAF1C. Rtf1 requires "Rtf1 coactivator" activity, which is most likely unrelated to the PAF1C or DSIF, for transcriptional activation in vitro. A mutational study revealed that the Plus3 domain of human Rtf1 is critical for its coactivator-dependent function. Transcriptome sequencing (RNA-seq) and chromatin immunoprecipitation studies in HeLa cells showed that Rtf1 and the PAF1C play distinct roles in regulating the expression of a subset of genes. Moreover, contrary to the finding in S. cerevisiae, the PAF1C was apparently recruited to the genes examined in an Rtf1-independent manner. The present study establishes a role for human Rtf1 as a transcription elongation factor and highlights the similarities and differences between the S. cerevisiae and human Rtf1 proteins. Restores TBP (TATA box-binding protein) function 1 (Rtf1) was identified as a suppressor of a TBP mutant in Saccharomyces cerevisiae (1). Subsequent genetic and biochemical studies in yeast have shown that Rtf1 functions as a component of the polymerase-associated factor 1 (Paf1) complex (PAF1C) containing Paf1, Ctr9, Leo1, and Cdc73 (2-5). The PAF1C is a multifunctional protein complex whose primary role is to facilitate histone modifications, such as H2B monoubiquitination at K123 and H3 methylation at K4 and K79 (6-12). The PAF1C also plays important roles in transcription elongation through chromatin, as well as on naked DNA (13-16). Furthermore, the PAF1C is involved in transcription termination and 3= processing of polyadenylated and nonpolyadenylated .Paf1, Ctr9, Leo1, Cdc73, and Rtf1 are highly conserved in eukaryotes; however, the subunit compositions of the PAF1C vary among species. Purification of the PAF1C from human HeLa cells yielded a five-subunit complex lacking Rtf1 but containing Ski8 as an additional subunit (22,26,27). Similarly, coimmunoprecipitation studies in zebrafish, Drosophila, and Schizosaccharomyces pombe showed that PAF1C homologs in these species lack a stably associated Rtf1 subunit (28-30). At the functional level, however, Rtf1 and other PAF1C subunits have a number of similarities. For example, knockout or knockdown of Rtf1 and other PAF1C subunits results in similar defects in histone modifications in all the species examined (27,28,31). At the organismal level, inhibition of Rtf1 and other PAF1C subunits causes similar defects in epidermal morphogenesis in Caenorhabditis elegans and in somitogenesis and cardiomyocyte development in zebrafish (29,32,33). Rtf1 and Paf1 colocalize with each other and with RNA polymerase II (RNAPII) in...

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confidence: 99%

Characterization of the Human Transcription Elongation Factor Rtf1: Evidence for Nonoverlapping Functions of Rtf1 and the Paf1 Complex

Cao

Yamamoto

Isobe

et al. 2015

Molecular and Cellular Biology

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“…We hypothesize that the HMD serves as a cofactor in the H2B ubiquitylation process by enhancing the ability of Bre1 to direct Rad6 to the appropriate lysine on H2B and/or by stimulating the catalytic ability of Rad6 in the presence of Bre1. This hypothesis does not exclude additional roles for Rtf1 or other Paf1C subunits in facilitating recruitment of Rad6-Bre1, stimulating H2Bub by an effect on elongation, or stabilizing Bre1 at the protein level (Wozniak and Strahl, 2014). Our results provide mechanistic insight into a crucial histone modification pathway.…”

Section: Discussionmentioning

confidence: 99%

“…An antibody against human H2B K120ub (Cell Signaling #5546; 1:1000 dilution) was used to detect H2B K123ub in yeast (Wozniak and Strahl, 2014). Lack of antibody reactivity in H2B K123R and bre1Δ mutant strains confirms specificity of the antibody for the appropriate lysine.…”

Section: Methodsmentioning

confidence: 99%

The Histone Modification Domain of Paf1 Complex Subunit Rtf1 Directly Stimulates H2B Ubiquitylation through an Interaction with Rad6

et al. 2016

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Summary The five-subunit yeast Paf1 Complex (Paf1C) regulates all stages of transcription and is critical for the monoubiquitylation of histone H2B (H2Bub), a modification that broadly influences chromatin structure and eukaryotic transcription. Here we show that the histone modification domain (HMD) of Paf1C subunit Rtf1 directly interacts with the ubiquitin conjugase Rad6 and stimulates H2Bub independently of transcription. We present the crystal structure of the Rtf1 HMD and use site-specific, in vivo crosslinking to identify a conserved Rad6 interaction surface. Utilizing ChIP-exo analysis, we define the localization patterns of the H2Bub machinery at high resolution and demonstrate the importance of Paf1C in targeting the Rtf1 HMD, and thereby H2Bub, to its appropriate genomic locations. Finally, we observe HMD-dependent stimulation of H2Bub in a transcription-free, reconstituted in vitro system. Taken together, our results argue for an active role for Paf1C in promoting H2Bub and ensuring its proper localization in vivo.

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“…RNF20/RNF40 exists as a tetramer containing two copies of each protein (25). They are reported to be required for the transcription of some genes (26–28), and play vital roles in some fundamental biological processes such as stem cell differentiation (29–31) and tumorigenesis (26,32). Whether H2Bub or its related E3 ligase involved in meiotic recombination or spermatogenesis in mammals is unknown.…”

Section: Introductionmentioning

confidence: 99%

H2B ubiquitination regulates meiotic recombination by promoting chromatin relaxation

Song

et al. 2016

Nucleic Acids Res

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Meiotic recombination is essential for fertility in most sexually reproducing species, but the molecular mechanisms underlying this process remain poorly understood in mammals. Here, we show that RNF20-mediated H2B ubiquitination is required for meiotic recombination. A germ cell-specific knockout of the H2B ubiquitination E3 ligase RNF20 results in complete male infertility. The Stra8-Rnf20−/− spermatocytes arrest at the pachytene stage because of impaired programmed double-strand break (DSB) repair. Further investigations reveal that the depletion of RNF20 in the germ cells affects chromatin relaxation, thus preventing programmed DSB repair factors from being recruited to proper positions on the chromatin. The gametogenetic defects of the H2B ubiquitination deficient cells could be partially rescued by forced chromatin relaxation. Taken together, our results demonstrate that RNF20/Bre1p-mediated H2B ubiquitination regulates meiotic recombination by promoting chromatin relaxation, and suggest an old drug may provide a new way to treat some oligo- or azoospermia patients with chromatin relaxation disorders.

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Catalysis-dependent stabilization of Bre1 fine-tunes histone H2B ubiquitylation to regulate gene transcription

Cited by 19 publications

References 39 publications

Characterization of the Human Transcription Elongation Factor Rtf1: Evidence for Nonoverlapping Functions of Rtf1 and the Paf1 Complex

Characterization of the Human Transcription Elongation Factor Rtf1: Evidence for Nonoverlapping Functions of Rtf1 and the Paf1 Complex

The Histone Modification Domain of Paf1 Complex Subunit Rtf1 Directly Stimulates H2B Ubiquitylation through an Interaction with Rad6

H2B ubiquitination regulates meiotic recombination by promoting chromatin relaxation

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