Faculty Bibliography

Methylation marks on the lysine residues of histone proteins are thought to contribute to epigenetic phenomena in part because of their apparent irreversibility. Will this view change with the recent discovery of histone lysine demethylases that reversibly remove methyl marks?

The Paf1 complex in yeast has been reported to influence a multitude of steps in gene expression through interactions with RNA polymerase II (Pol II) and chromatin-modifying complexes; however, it is unclear which of these many activities are primary functions of Paf1 and are conserved in metazoans. We have identified and characterized the Drosophila homologs of three subunits of the yeast Paf1 complex and found striking differences between the yeast and Drosophila Paf1 complexes. We demonstrate that although Drosophila Paf1, Rtf1, and Cdc73 colocalize broadly with actively transcribing, phosphorylated Pol II, and all are recruited to activated heat shock genes with similar kinetics; Rtf1 does not appear to be a stable part of the Drosophila Paf1 complex. RNA interference (RNAi)-mediated depletion of Paf1 or Rtf1 leads to defects in induction of Hsp70 RNA, but tandem RNAi-chromatin immunoprecipitation assays show that loss of neither Paf1 nor Rtf1 alters the density or distribution of phosphorylated Pol II on the active Hsp70 gene. However, depletion of Paf1 reduces trimethylation of histone H3 at lysine 4 in the Hsp70 promoter region and significantly decreases the recruitment of chromatin-associated factors Spt6 and FACT, suggesting that Paf1 may manifest its effects on transcription through modulating chromatin structure

Defining the protein factors that directly recognize post-translational, covalent histone modifications is essential toward understanding the impact of these chromatin 'marks' on gene regulation. In the current study, we identify human CHD1, an ATP-dependent chromatin remodeling protein, as a factor that directly and selectively recognizes histone H3 methylated on lysine 4. In vitro binding studies identified that CHD1 recognizes di- and trimethyl H3K4 with a dissociation constant (Kd) of approximately 5 microm, whereas monomethyl H3K4 binds CHD1 with a 3-fold lower affinity. Surprisingly, human CHD1 binds to methylated H3K4 in a manner that requires both of its tandem chromodomains. In vitro analyses demonstrate that unlike human CHD1, yeast Chd1 does not bind methylated H3K4. Our findings indicate that yeast and human CHD1 have diverged in their ability to discriminate covalently modified histones and link histone modification-recognition and non-covalent chromatin remodeling activities within a single human protein

In yeast, histone H2B monoubiquitination is a cotranscriptional event regulating histone H3 methylation at lysines 4 and 79. However, mammalian H2B monoubiquitination remains poorly understood. We report that in humans, the 600 kDa RNF20/40 complex is the E3 ligase and UbcH6 is the ubiquitin E2-conjugating enzyme for H2B-Lys120 monoubiquitination. RNF20 and RNF40 are both homologs of Bre1, the E3 ligase in the yeast case. UbcH6 physically interacts with RNF20/40 and with the hPAF complex. Formation of a trimeric complex with hPAF stimulates H2B monoubiquitination activity in vitro. Accordingly, UbcH6, RNF20/40, and the hPAF complex are recruited to transcriptionally active genes in vivo. RNF20 overexpression leads to elevated H2B monoubiquitination, subsequently higher levels of methylation at H3 lysines 4 and 79, and stimulation of HOX gene expression. In contrast, RNAi against the RNF20/40 complex or hPAF complex reduces H2B monoubiquitination, lowers methylation levels at H3 lysines 4 and 79, and represses HOX gene expression

Downstream elements are a newly appreciated class of core promoter elements of RNA polymerase II-transcribed genes. The downstream core element (DCE) was discovered in the human beta-globin promoter, and its sequence composition is distinct from that of the downstream promoter element (DPE). We show here that the DCE is a bona fide core promoter element present in a large number of promoters and with high incidence in promoters containing a TATA motif. Database analysis indicates that the DCE is found in diverse promoters, supporting its functional relevance in a variety of promoter contexts. The DCE consists of three subelements, and DCE function is recapitulated in a TFIID-dependent manner. Subelement 3 can function independently of the other two and shows a TFIID requirement as well. UV photo-cross-linking results demonstrate that TAF1/TAF(II)250 interacts with the DCE subelement DNA in a sequence-dependent manner. These data show that downstream elements consist of at least two types, those of the DPE class and those of the DCE class; they function via different DNA sequences and interact with different transcription activation factors. Finally, these data argue that TFIID is, in fact, a core promoter recognition complex

The yeast PAF (yPAF) complex interacts with RNA polymerase II and coordinates the setting of histone marks associated with active transcription. We report the isolation and functional characterization of the human PAF (hPAF) complex. hPAF shares four subunits with yPAF (hCtr9, hPaf1, hLeo1, and hCdc73), but contains a novel higher eukaryotic-specific subunit, hSki8. RNAi against hSki8 or hCtr9 reduces the cellular levels of other hPAF subunits and of mono- and trimethylated H3-Lys 4 and dimethylated H3-Lys 79. The hSki8 subunit is also a component of the human SKI (hSKI) complex. Yeast SKI complex is cytoplasmic and together with Exosome mediates 3'-5' mRNA degradation. However, hSKI complex localizes to both nucleus and cytoplasm. Immunoprecipitation experiments revealed that hPAF and hSKI complexes interact, and ChIP experiments demonstrated that hSKI associates with transcriptionally active genes dependent on the presence of hPAF. Thus, in addition to coordinating events during transcription (initiation, promoter clearance, and elongation), hPAF also coordinates events in RNA quality control

Methylation of lysine residues of histones is an important epigenetic mark that correlates with functionally distinct regions of chromatin. We present here the crystal structure of a ternary complex of the enzyme Pr-Set7 (also known as Set8) that methylates Lys 20 of histone H4 (H4-K20). We show that the enzyme is exclusively a mono-methylase and is therefore responsible for a signaling role quite distinct from that established by other enzymes that target this histone residue. We provide evidence from NMR for the C-flanking domains of SET proteins becoming ordered upon addition of AdoMet cofactor and develop a model for the catalytic cycle of these enzymes. The crystal structure reveals the basis of the specificity of the enzyme for H4-K20 because a histidine residue within the substrate, close to the target lysine, is required for completion of the active site. We also show how a highly variable component of the SET domain is responsible for many of the enzymes' interactions with its target histone peptide and probably also how this part of the structure ensures that Pr-Set7 is nucleosome specific

Downstream core promoter elements are an expanding class of regulatory sequences that add considerable diversity to the promoter architecture of RNA polymerase II-transcribed genes. We set out to determine the factors necessary for downstream promoter element (DPE)-dependent transcription and find that, against expectations, TFIID and the GTFs are not sufficient. Instead, the protein kinase CK2 and the coactivator PC4 establish DPE-specific transcription in an in vitro transcription system containing TFIID, Mediator, and the GTFs. Chromatin immunoprecipitation analyses using the DPE-dependent IRF-1 and TAF7 promoters demonstrated that CK2, and PC4 are present on these promoters in vivo. In contrast, neither PC4 nor CK2 were detected on the TAF1-dependent cyclin D promoter, which contains a DCE type of downstream element. Our findings also demonstrate that CK2 activity alters TFIID-dependent recognition of DCE sequences. These data establish that CK2 acts as a switch, converting the transcriptional machinery from functioning on one type of downstream element to another

Polycomb group protein Ezh2, one of the key regulators of development in organisms from flies to mice, exerts its epigenetic function through regulation of histone methylation. Here, we report the existence of the cytosolic Ezh2-containing methyltransferase complex and tie the function of this complex to regulation of actin polymerization in various cell types. Genetic evidence supports the essential role of cytosolic Ezh2 in actin polymerization-dependent processes such as antigen receptor signaling in T cells and PDGF-induced dorsal circular ruffle formation in fibroblasts. Revealed function of Ezh2 points to a broader usage of lysine methylation in regulation of both nuclear and extra-nuclear signaling processes