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...

During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the homeodomain factor Nkx2-5. We now show that feedback repression of Bmp2/Smad1 signaling by Nkx2-5 critically regulates SHF proliferation and outflow tract (OFT) morphology. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification (including Bmp2) and maintenance of the progenitor state were upregulated, leading initially to progenitor overspecification, but subsequently to failed SHF proliferation and OFT truncation. In Smad1 mutants, SHF proliferation and deployment to the OFT were increased, while Smad1 deletion in Nkx2-5 mutants rescued SHF proliferation and OFT development. In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies were also rescued by Smad1 deletion. Our findings demonstrate that Nkx2-5 orchestrates the transition between periods of cardiac induction, progenitor proliferation, and OFT morphogenesis via a Smad1-dependent negative feedback loop, which may be a frequent molecular target in CHD.

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.

Set1 is the catalytic subunit and the central component of the evolutionarily conserved Set1 complex (Set1C) that methylates histone 3 lysine 4 (H3K4). Here we have determined protein/protein interactions within the complex and related the substructure to function. The loss of individual Set1C subunits differentially affects Set1 stability, complex integrity, global H3K4 methylation, and distribution of H3K4 methylation along active genes. The complex requires Set1, Swd1, and Swd3 for integrity, and Set1 amount is greatly reduced in the absence of the Swd1-Swd3 heterodimer. Bre2 and Sdc1 also form a heteromeric subunit, which requires the SET domain for interaction with the complex, and Sdc1 strongly interacts with itself. Inactivation of either Bre2 or Sdc1 has very similar effects. Neither is required for complex integrity, and their removal results in an increase of H3K4 mono-and dimethylation and a severe decrease of trimethylation at the 5 end of active coding regions but a decrease of H3K4 dimethylation at the 3 end of coding regions. Cells lacking Spp1 have a reduced amount of Set1 and retain a fraction of trimethylated H3K4, whereas cells lacking Shg1 show slightly elevated levels of both di-and trimethylation. Set1C associates with both serine 5-and serine 2-phosphorylated forms of polymerase II, indicating that the association persists to the 3 end of transcribed genes. Taken together, our results suggest that Set1C subunits stimulate Set1 catalytic activity all along active genes.

Background: Understanding the design logic of living systems requires the understanding and comparison of proteomes. Proteomes define the commonalities between organisms more precisely than genomic sequences. Because uncertainties remain regarding the accuracy of proteomic data, several issues need to be resolved before comparative proteomics can be fruitful.

Existing evidence indicates that SET2, the histone 3 lysine 36 methyltransferase of Saccharomyces cerevisiae, is a transcriptional repressor. Here we show by five main lines of evidence that SET2 is involved in transcriptional elongation. First, most, if not all, subunits of the RNAP II holoenzyme co-purify with SET2. Second, all of the co-purifying RNAP II subunit, RPO21, was phosphorylated at serines 5 and 2 of the C-terminal domain (CTD) tail, indicating that the SET2 association is specific to either the elongating or SSN3 repressed forms (or both) of RNAP II. Third, the association of SET2 with CTD phosphorylated RPO21 remained in the absence of ssn3. Fourth, in the absence of ssn3, mRNA production from gal1 required SET2. Fifth, SET2 was detected on gal1 by in vivo crosslinking after, but not before, the induction of transcription. Similarly, SET2 physically associated with the transcribed region of pdr5 but was not detected on gal1 or pdr5 promoter regions. Since SET2 is also a histone methyltransferase, these results suggest a role for histone 3 lysine 36 methylation in transcriptional elongation.

Histone 3 lysine 4 (H3 Lys 4 ) methylation in Saccharomyces cerevisiae is mediated by the Set1 complex (Set1C) and is dependent upon ubiquitinylation of H2B by Rad6. Mutually exclusive methylation of H3 at Lys 4 or Lys 9 is central to chromatin regulation; however, S. cerevisiae lacks Lys 9 methylation. Furthermore, a different H3 Lys 4 methylase, Set 7/9, has been identified in mammals, thereby questioning the relevance of the S. cerevisiae findings for eukaryotes in general. We report that the majority of Lys 4 methylation in Schizosaccharomyces pombe, like in S. cerevisiae, is mediated by Set1C and is Rad6-dependent. S. pombe Set1C mediates H3 Lys 4 methylation in vitro and contains the same eight subunits found in S. cerevisiae, including the homologue of the Drosophila trithorax Group protein, Ash2. Three additional features of S. pombe Set1C each involve PHD fingers. Notably, the Spp1 subunit is dispensable for H3 Lys 4 methylation in budding yeast but required in fission yeast, and Sp_Set1C has a novel proteomic hyperlink to a new complex that includes the homologue of another trithorax Group protein, Lid (little imaginal discs). Thus, we infer that Set1C is highly conserved in eukaryotes but observe that its links to the proteome are not.

We employed a combination of tandem affinity purification and mass spectrometry for deciphering protein complexes and the protein interaction network in budding yeast. 53 genes were epitope-tagged, and their interaction partners were isolated by two-step immunoaffinity chromatography from whole cell lysates. 38 baits pulled down a total of 220 interaction partners, which are members of 19 functionally distinct protein complexes. We identified four proteins shared between complexes of different functionality thus charting segments of a protein interaction network. Concordance with the results of genome-wide two-hybrid screening was poor (14% of identified interactors overlapped) suggesting that the two ap-