SUMMARY 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 (i) that hRAD6 serves as the cognate E2 conjugating enzyme, (ii) that hRAD6, through direct interaction with hPAF-bound hBRE1, is recruited to transcribed genes and ubiquitylates chromatinized H2B at lysine 120, (iii) 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, (iv) that H2B ubiquitylation per se does not affect the level of hPAF-, SII- and p300-dependent transcription and likely functions downstream and (v) that H2B ubiquitylation directly stimulates hSET1-dependent H3K4 di- and tri-methylation. These studies establish the natural H2B ubiquitylation factors in human cells and also detail the mechanistic basis for H2B ubiquitylation and function during transcription.

Numerous post-translational modifications of histones have been described in organisms ranging from yeast to humans. Growing evidence for dynamic regulation of these modifications, position- and modification-specific protein interactions, and biochemical crosstalk between modifications has strengthened the 'histone code' hypothesis, in which histone modifications are integral to choreographing the expression of the genome. One such modification, ubiquitylation of histone H2B (uH2B) on lysine 120 (K120) in humans, and lysine 123 in yeast, has been correlated with enhanced methylation of lysine 79 (K79) of histone H3 (refs 5-8), by K79-specific methyltransferase Dot1 (KMT4). However, the specific function of uH2B in this crosstalk pathway is not understood. Here we demonstrate, using chemically ubiquitylated H2B, a direct stimulation of hDot1L-mediated intranucleosomal methylation of H3 K79. Two traceless orthogonal expressed protein ligation (EPL) reactions were used to ubiquitylate H2B site-specifically. This strategy, using a photolytic ligation auxiliary and a desulphurization reaction, should be generally applicable to the chemical ubiquitylation of other proteins. Reconstitution of our uH2B into chemically defined nucleosomes, followed by biochemical analysis, revealed that uH2B directly activates methylation of H3 K79 by hDot1L. This effect is mediated through the catalytic domain of hDot1L, most likely through allosteric mechanisms. Furthermore, asymmetric incorporation of uH2B into dinucleosomes showed that the enhancement of methylation was limited to nucleosomes bearing uH2B. This work demonstrates a direct biochemical crosstalk between two modifications on separate histone proteins within a nucleosome.

Transcriptional coactivators that modify histones represent an increasingly important group of regulatory factors, although their ability to modify other factors as well precludes common assumptions that they necessarily act by histone modification. In an extension of previous studies showing a role for acetyltransferase p300/CBP in p53 function, we have used systems reconstituted with recombinant chromatin templates and (co)activators to demonstrate (1) the additional involvement of protein arginine methyltransferases PRMT1 and CARM1 in p53 function; (2) both independent and ordered cooperative functions of p300, PRMT1, and CARM1; and (3) mechanisms that involve direct interactions with p53 and, most importantly, obligatory modifications of corresponding histone substrates. ChIP analyses have confirmed the ordered accumulation of these (and other) coactivators and cognate histone modifications on the GADD45 gene following ectopic p53 expression and/or UV irradiation. These studies thus define diverse cofactor functions, as well as underlying mechanisms involving distinct histone modifications, in p53-dependent gene activation.

SUMMARY Genetic and cell-based studies have implicated the PAF1 complex (PAF1C) in transcription-associated events, but there has been no evidence showing a direct role in facilitating transcription of a natural chromatin template. Here, we demonstrate an intrinsic ability of human PAF1C (hPAF1C) to facilitate activator (p53)- and histone acetyltransferase (p300)-dependent transcription elongation from a recombinant chromatin template in a biochemically defined RNA polymerase II transcription system. This represents a PAF1C function distinct from its established role in histone ubiquitylation and methylation. Importantly, we further demonstrate a strong synergy between hPAF1C and elongation factor SII/TFIIS and an underlying mechanism involving direct hPAF1C-SII interactions and cooperative binding to RNA polymerase II. Apart from a distinct PAF1C function, the present observations provide a molecular mechanism for the cooperative function of distinct transcription elongation factors in chromatin transcription.

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. Here, we report the identification of a functional human homolog, hBRE1, of the yeast BRE1 E3 ubiquitin ligase. hBRE1 specifically increases the global level of H2B ubiquitylation at lysine 120 and enhances activator-dependent transcription. Moreover, reduction of hBRE1 by RNAi decreases endogenous H2B ubiquitylation, activator-dependent transcription, and interestingly, H3-K4 and -K79 methylation. Of special significance, we show that hBRE1 directly interacts with p53 and that it is recruited to the mdm2 promoter in a p53-dependent manner. These studies suggest that hBRE1 is an H2B-specific E3 ubiquitin ligase and that it functions, through direct activator interactions, as a transcriptional coactivator. Importantly, they thus provide a paradigm for BRE1 recruitment and function in both yeast and higher eukaryotes.