Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
Diverse histone modifications play important roles in transcriptional regulation throughout eukaryotes, and recent studies have implicated histone H2B ubiquitylation in active transcription. The necessity of at least three enzymes (E1-E3), as well as ongoing transcription events, for efficient H2B ubiquitylation complicates mechanistic studies of H2B ubiquitylation relative to other histone modifications. Here we describe experimental protocols for preparation of human H2B ubiquitylation factors, ubiquitylation substrates and transcription factors, as well as the use of these factors to establish H2B ubiquitylation mechanisms during transcription. The methods include reliable protein interaction and E3 ubiquitylation assays that can be widely applied to confirm cognate E2-E3 pairs in other protein ubiquitylation systems, optimized in vitro ubiquitylation assays for various histone substrates, and a transcription-coupled H2B ubiquitylation assay in a highly purified transcription system. These comprehensive analyses have revealed (i) that RAD6 serves as the cognate E2 for the BRE1 complex in human cells, as previously established in yeast, (ii) that RAD6, through direct interaction with the BRE1 complex, ubiquitylates chromatinized H2B at lysine 120 and (iii) that PAF1 complex-mediated transcription is required for efficient H2B ubiquitylation. This experimental system permits detailed mechanistic analyses of H2B ubiquitylation during transcription by providing information concerning both precise enzyme functions and physical interactions between the transcription and histone modification machineries.
An,
Ordered cooperative functions of PRMT1, p300, and CARM1 in transcriptional activation by p53.
2004, Pubmed
An,
Ordered cooperative functions of PRMT1, p300, and CARM1 in transcriptional activation by p53.
2004,
Pubmed Bitter,
Purification of DNA-dependent RNA polymerase II from Saccharomyces cerevisiae.
1983,
Pubmed Dignam,
Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
1983,
Pubmed Dover,
Methylation of histone H3 by COMPASS requires ubiquitination of histone H2B by Rad6.
2002,
Pubmed Ge,
Activator-dependent transcription by mammalian RNA polymerase II: in vitro reconstitution with general transcription factors and cofactors.
1996,
Pubmed Goebl,
The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme.
1988,
Pubmed Guermah,
Synergistic functions of SII and p300 in productive activator-dependent transcription of chromatin templates.
2006,
Pubmed Guermah,
Transcription of in vitro assembled chromatin templates in a highly purified RNA polymerase II system.
2009,
Pubmed Henry,
Transcriptional activation via sequential histone H2B ubiquitylation and deubiquitylation, mediated by SAGA-associated Ubp8.
2003,
Pubmed Hershko,
The ubiquitin system.
1998,
Pubmed Hwang,
A conserved RING finger protein required for histone H2B monoubiquitination and cell size control.
2003,
Pubmed Ito,
ACF consists of two subunits, Acf1 and ISWI, that function cooperatively in the ATP-dependent catalysis of chromatin assembly.
1999,
Pubmed Ito,
ACF, an ISWI-containing and ATP-utilizing chromatin assembly and remodeling factor.
1997,
Pubmed Jentsch,
The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme.
,
Pubmed Kao,
Rad6 plays a role in transcriptional activation through ubiquitylation of histone H2B.
2004,
Pubmed Kim,
Direct Bre1-Paf1 complex interactions and RING finger-independent Bre1-Rad6 interactions mediate histone H2B ubiquitylation in yeast.
2009,
Pubmed Kim,
RAD6-Mediated transcription-coupled H2B ubiquitylation directly stimulates H3K4 methylation in human cells.
2009,
Pubmed Kim,
The human PAF1 complex acts in chromatin transcription elongation both independently and cooperatively with SII/TFIIS.
2010,
Pubmed Kim,
The human homolog of yeast BRE1 functions as a transcriptional coactivator through direct activator interactions.
2005,
Pubmed Kundu,
Activator-dependent transcription from chromatin in vitro involving targeted histone acetylation by p300.
2000,
Pubmed Li,
The role of chromatin during transcription.
2007,
Pubmed Lorick,
RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination.
1999,
Pubmed Luger,
Characterization of nucleosome core particles containing histone proteins made in bacteria.
1997,
Pubmed
,
Xenbase Malik,
Isolation and functional characterization of the TRAP/mediator complex.
2003,
Pubmed McGinty,
Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation.
2008,
Pubmed
,
Xenbase Minsky,
Monoubiquitinated H2B is associated with the transcribed region of highly expressed genes in human cells.
2008,
Pubmed Ng,
The Rtf1 component of the Paf1 transcriptional elongation complex is required for ubiquitination of histone H2B.
2003,
Pubmed Owen-Hughes,
Persistent site-specific remodeling of a nucleosome array by transient action of the SWI/SNF complex.
1996,
Pubmed Robzyk,
Rad6-dependent ubiquitination of histone H2B in yeast.
2000,
Pubmed Shi,
Paf1p, an RNA polymerase II-associated factor in Saccharomyces cerevisiae, may have both positive and negative roles in transcription.
1996,
Pubmed Sun,
Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast.
2002,
Pubmed Sung,
The RAD6 protein of Saccharomyces cerevisiae polyubiquitinates histones, and its acidic domain mediates this activity.
1988,
Pubmed Tan,
Recruitment of a ROC1-CUL1 ubiquitin ligase by Skp1 and HOS to catalyze the ubiquitination of I kappa B alpha.
1999,
Pubmed Wood,
The Paf1 complex is essential for histone monoubiquitination by the Rad6-Bre1 complex, which signals for histone methylation by COMPASS and Dot1p.
2003,
Pubmed Wood,
Bre1, an E3 ubiquitin ligase required for recruitment and substrate selection of Rad6 at a promoter.
2003,
Pubmed Workman,
Alteration of nucleosome structure as a mechanism of transcriptional regulation.
1998,
Pubmed
