Trimethylation of histone H3 at lysine 4 (H3K4me3) is regarded as a hallmark of active human promoters, but it remains unclear how this posttranslational modification links to transcriptional activation. Using a stable isotope labeling by amino acids in cell culture (SILAC)-based proteomic screening we show that the basal transcription factor TFIID directly binds to the H3K4me3 mark via the plant homeodomain (PHD) finger of TAF3. Selective loss of H3K4me3 reduces transcription from and TFIID binding to a subset of promoters in vivo. Equilibrium binding assays and competition experiments show that the TAF3 PHD finger is highly selective for H3K4me3. In transient assays, TAF3 can act as a transcriptional coactivator in a PHD finger-dependent manner. Interestingly, asymmetric dimethylation of H3R2 selectively inhibits TFIID binding to H3K4me3, whereas acetylation of H3K9 and H3K14 potentiates TFIID interaction. Our experiments reveal crosstalk between histone modifications and the transcription factor TFIID. This has important implications for regulation of RNA polymerase II-mediated transcription in higher eukaryotes.

The SET domain proteins, SUV39 and G9a have recently been shown to be histone methyltransferases speci®c for lysines 9 and 27 (G9a only) of histone 3 (H3). The SET domains of the Saccharomyces cerevisiae Set1 and Drosophila trithorax proteins are closely related to each other but distinct from SUV39 and G9a. We characterized the complex associated with Set1 and Set1C and found that it is comprised of eight members, one of which, Bre2, is homologous to the trithorax-group (trxG) protein, Ash2. Set1C requires Set1 for complex integrity and mutation of Set1 and Set1C components shortens telomeres. One Set1C member, Swd2/Cpf10 is also present in cleavage polyadenylation factor (CPF). Set1C methylates lysine 4 of H3, thus adding a new speci®city and a new subclass of SET domain proteins known to methyltransferases. Since methylation of H3 lysine 4 is widespread in eukaryotes, we screened the databases and found other Set1 homologues. We propose that eukaryotic Set1Cs are H3 lysine 4 methyltransferases and are related to trxG action through association with Ash2 homologues. Keywords: chromatin/epigenetic/histone methyltransferase/telomere/trithorax-group IntroductionMembers of the trithorax group (trxG) have been identi®ed by genetic screens of Drosophila for mutations that suppress phenotypes caused by disregulation of polycomb-group (PcG) action or mimic loss-of-function homeotic mutant phenotypes. As expected from these complex genetic screens, the trxG appears to encompass several subclasses of gene regulatory factors (Kennison, 1995). One subclass involves chromatin remodelling activity. The realization that the trxG member Brahma (BRM) is a homologue of Saccharomyces cerevisiae Swi2/Snf2 (Peterson and Herskowitz, 1992;Carlson and Laurent, 1994;Elfring et al., 1994) led to the de®nition of the SWI/SNF complex as a chromatin remodelling machine (Cote et al., 1994;Logie and Peterson, 1997) and the identi®cation of another trxG member, moira, as a further component of the Drosophila SWI/SNF complex (Papoulas et al., 1998). Another trxG subclass encompasses the DNA binding proteins, zeste and GAGA factor. Although these proteins act independently, both appear to play similar roles in the stabilization of higher-order chromatin looping (Chen and Pirrotta, 1993;Katsani et al., 1999). A third subclass within the trxG (called here trxG3) that remains poorly understood includes trithorax itself, ash1 and ash2 (Shearn, 1989).Insight into the potential molecular actions of trxG3 members came from the identi®cation of several domains within their protein sequences (Mazo et al., 1990;Stassen et al., 1995;Adamson and Shearn, 1996;Tripoulas et al., 1996). Both trithorax (Trx) and Ash1 include a SET domain, which was identi®ed through its occurrence in the chromatin factors, Su(var)3-9, enhancer of zeste [E(Z)] and Trx (Jones and Gelbart, 1993;Tschiersch et al., 1994). All three trxG3 members also include one or more PHD ®ngers (Aasland et al., 1995) and Ash2 includes a SPRY domain (Ponting et al., 1997). Of these domains, the S...

Set3 is one of two proteins in the yeast Saccharomyces cerevisiae that, like Drosophila Trithorax, contains both SET and PHD domains. We found that Set3 forms a single complex, Set3C, with Snt1, YIL112w, Sif2, Cpr1, and two putative histone deacetylases, Hos2 and NAD-dependent Hst1. Set3C includes NAD-dependent and independent deacetylase activities when assayed in vitro. Homology searches suggest that Set3C is the yeast analog of the mammalian HDAC3/SMRT complex. Set3C represses genes in early/middle of the yeast sporulation program, including the key meiotic regulators ime2 and ndt80. Whereas Hos2 is only found in Set3C, Hst1 is also present in a complex with Sum1, supporting previous characterizations of Hst1 and Sum1 as repressors of middle sporulation genes during vegetative growth. However, Hst1 is not required for meiotic repression by Set3C, thus implying that Set3C (−Hst1) and not Hst1-Sum1, is the meiotic-specific repressor of early/middle sporulation genes.

The Ccr4-Not complex is evolutionarily conserved and important for regulation of mRNA synthesis and decay. The composition of the yeast complex has been well described. Orthologues of the yeast Ccr4-Not components have been identified in human cells including multiple subunits with mRNA deadenylase activity. In the present study, we examine the composition of the human Ccr4-Not complex in an in-depth proteomic approach using stable cell lines expressing tagged CNOT proteins. We find at least four different variants of the human complex, consisting of seven stable core proteins and mutually exclusive associated mRNA deadenylase subunits. Interestingly, human CNOT4 is in a separate approximately 200 kDa complex. Furthermore, analyses of associated proteins indicate involvement of Ccr4-Not complexes in splicing, transport and localization of RNA molecules. Taken together, human Ccr4-Not complexes are heterogeneous in composition owing to differences in their deadenylase subunits, which may reflect the multi-functionality of these complexes in cellular processes.

The yeast SAGA (Spt-Ada-Gcn5-acetyltransferase) coactivator complex exerts functions in gene expression, including activator interaction, histone acetylation, histone deubiquitination, mRNA export, chromatin recognition, and regulation of the basal transcription machinery. These diverse functions involve distinct modules within this multiprotein complex. It has now become clear that yeast SAGA has diverged during metazoan evolution into two related complexes, SAGA and ATAC, which exist in two flavors in vertebrates. The compositions of metazoan ATAC and SAGA complexes have been characterized, and functional analyses indicate that these complexes have important but distinct roles in transcription, histone modification, signaling pathways, and cell cycle regulation.