Members of the trithorax group (trxG) have been identified 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 definition of the SWI/SNF complex as a chromatin remodelling machine (Cote et al., 1994; Logie and Peterson, 1997) and the identification 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 identification 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 identified 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 fingers (Aasland et al., 1995) and Ash2 includes a SPRY domain (Ponting et al., 1997). Of these domains, the SET domain in Su(var)3-9 homologues, in particular mammalian SUV39H1 and Schizosaccharomyces pombe Clr4, was recently identified as the first histone lysine methyltrans ferase (Rea et al., 2000), thus suggesting that other proteins containing SET domains could also be histone lysine methyltransferases. Subsequently, two other SET domain proteins, human G9a (Tachibana et al., 2001) and Drosophila Ash1 (C.Beisel, A.Imhof and F.Sauer, in preparation) have been shown to possess histone lysine methyltransferase activities. Notably each of these proteins have an additional cysteine-rich motif immediately N-terminal to their SET domains, previously referred to as a Cys-rich cluster or a preSET region. The presence of the preSET region in SUV39H1 is required for methyltransferase activity (Rea et al., 2000). Trx homologs contain a different type of preSET region, termed ATA2 (Prasad et al., 1997). Whether the absence of a SUV39-type preSET domain is the reason why Trx and E(Z) so far have tested negative in methyltransferase assays (Rea et al., 2000; C.Beisel, A.Imhof and F.Sauer, in preparation) is not yet clear.
By sequence alignments, the genome of S.cerevisiae encodes six genes with significant matches to the SET domain (now termed Set1–6; Pijnappel et al., 2001). Of these, the SET domain of Set1 is more similar to Trx SET domains than any other. Set1 does not appear to include a preSET Cys-rich region. In fact, S.cerevisiae does not appear to have a clear Su(var)3-9 homologue. set1 is not essential to yeast, however thorough analyses of its mutant phenotype revealed a variety of defects, including roles in silencing at mating-type loci and telomeres, metabolism, maintenance of telomere length (Nislow et al., 1997) and DNA repair (Corda et al., 1999; Schramke et al., 2001). Notably, expression of mammalian E(Z) homologues, either human EZH2 or murine Ezh1, in set1 strains restored the loss of gene repression at telomeres (Laible et al., 1997). To explore further Set1 function in S.cerevisiae, we characterized the Set1 complex (Set1C) and its proteomic environment using a sequential tagging and mass spectrometry approach (Rigaut et al., 1999; Shevchenko et al., 1999). We define Set1C as a complex of eight members which includes two proteins that display a SPRY domain and a PHD finger respectively. Thus it appears that Set1C incorporates an Ash2 analogue by physically associating two proteins that each carry a part of Ash2. Here we show that Set1C has histone methyltransferase activity specific for lysine 4 of histone 3 (H3) and is thus both the first histone lysine methyltransferase described in S.cerevisiae and the first from the Set1/Trx branch of SET domains. Together, these results imply that the Cys-rich preSET region is not essential for histone lysine methyltransferase activity in certain contexts and that unexpected aspects of trxG3 action also exist in S.cerevisiae.