SET domain proteins in S. cerevisiae. (A) Cartoons of the six yeast SET domain proteins identified. Each sequence was retrieved from SGD (http://genome-www.stanford.edu/Saccharomyces/) by the systematic yeast protein names given. The length of each protein and the position in each protein of the PHD fingers and SET domains are also indicated. Set5 and Set6 have large insertions (173 and 192 amino acids, respectively) indicated as light green boxes in the SET icon. E-values obtained when using the Set1 SET domain as a probe in a PSI-BLAST search (four iterations; Altschul et al. 1997) against the yeast proteome, are indicated. (B) A multiple alignment of the six SET domains in yeast. The alignment was generated with CLUSTAL_X (Thompson et al. 1997) and subsequently manually refined. The alignment is color coded to highlight conserved features using the color scheme implemented in CLUSTAL_X. The two positions in Set5 and Set6 with large insertions are indicated. Below the alignment is shown a consensus featuring positions conserved in at least five of the six sequences. The six positions in the alignment that correspond to the motif common to SET domains and methyltransferases (Rea et al. 2000) are indicated with red dots. The alignment includes the C-terminal SET domain-associated cysteine cluster, postSET (position 203–217 in the alignment).

Set3 and Hos2 are central components of the Set3C but Hst1 is not. Coomassie-stained polyacrylamide SDS gels showing TAP purifications of Sif2–TAP in a Δset3 background (left), YIL112–TAP in a Δhos2 background (middle), and Sif2–TAP in Δhst1 background (right). The tagged proteins with remaining TAP fusion are indicated in bold.

Set3C is a repressor of meiosis. Wild type, Δset3, or Δhos2 diploid strains were sporulated and assayed for meiotic landmarks at the times indicated (in hours). (A) Premeiotic DNA synthesis as measured by FACS analysis. X-axis, fluorescence intensity; Y-axis, number of cells. (Left peaks) 2n DNA; (right peaks) 4n DNA (as confirmed by microscopic analysis of sorted cells). (Blue graphs) wild type; (orange graphs) Δset3; (green graphs) Δhos2. (B) Cells completing meiosis I, meiosis II, and ascus formation (Asci) were counted using a fluorescence microscope. At least 300 cells per time point per strain were counted. (C) Northern blot analysis of meiotic genes during sporulation of wild-type (wt) or Δset3 cells. Data were obtained from a separate experiment as in A and B, and the absolute time of sporulation onset is therefore slightly different. Relative differences between wild type and Δset3 are similar to those in B. Genes examined are indicated next to the expression data, as are corresponding gene clusters (Primig et al. 2000) and gene functions. The mRNA coding for the ribosomal protein Tcm1 was used as control for RNA. Decreased expression of this gene very early during sporulation, followed by stable expression levels, is as reported (Chu and Herskowitz 1998). Expression of asc1 and dal7 confirmed the quality of the RNA at these early time points. (D) As in C, except RNA was harvested from wild-type, Δhst1, and Δhst1,Δhos2 strains.

The Set3 protein complex. (A) Set3–TAP purification. Coomassie-stained 7%–25% polyacrylamide SDS gel showing the purified complex (Set3–TAP). Cartoons of MS-identified proteins are shown with full-length protein sizes in amino acids (aa). Protein domains are also indicated in amino acids. The tagged protein, Set3, is indicated in bold together with its C-terminal remnant of the TAP tag. Protein size markers (broad range, NEB) are indicated in kilodaltons. Ssa1 and Ssb1 are heat-shock proteins also found in mock purifications, and therefore represent unspecific contaminants. (B) Verification of the complex. As in A, showing the complexes obtained by TAP purification of Snt1, YIL112w, Sif2, or Hos2, as indicated. The tagged proteins with remaining TAP fusions are indicated in bold. TRiC complex in Hos2–TAP: 8, Cct6; 9, Cct8; 10, Cct2; 11, Cct3; 12, Cct4; 13, Cct7. (C) Defining a proteomic border of Set3C. As in A and B, showing TAP purification of Hst1 and Sum1, as indicated. The presence of many bands, marked by asterisks, was later found to be due to the batches of calmodulin beads used. All the bands marked by an asterisk were identified by MS, and are nonspecific contaminants, also visible in Figure 3. (They are, from top to bottom, in the Hst1–TAP panel: J04692; Ssa2: Ssb1: HSP60; ef1-α; enolase2; phosphoglycerate kinase; 60s ribosomal protein [rp] l4-B; fructose-biphosphate aldolase; glyceraldehyde 3-phosphate dehydrogenase; 60s rp l2; mixture of 40s rp s1-B, 60s rp l8-A, 40s rp s4; mixture of 60s rp l1, triosephosphate isomerase; mixture of 60 ns rp l7-A, 60 s rp l10; mixture of 40s rp s7-A, 60s rp l13-A; 60 s rp l20; and in the SUM1–TAP panel: mixture of sum1, ef2; hsp82; jo4692; sum1; ssa2; ssb2; pyruvate kinase 1; mixture of sum1, pyruvate decarboxylase isozyme; ef1α; mixture of enolase 2, 60s rp l3; 60s rp l4-A; glyceraldehyde3-phosphate dehydrogenase; 60s rpl2; mixture of 40s rp s1-B, 40s rp s4, 60s rp l8-B; mixture of 60s rp l7-B, 60s rp l10, 60s rp l1; mixture of 60s l13-B, 40s rp s7-A; 60s rp l20; mixture of 40s rp s24, 40s rp s17-A, 60s rpl25, 40s rp s19-B).

The Set3 and Hst1 complexes contain both NAD-independent and NAD-dependent histone deacetylase activities. IgG Sepharose eluates from the strains indicated were assayed for histone deacetylase activity as described in Materials and Methods. The presence or absence of 0.5 mM NAD or NADH in the reaction is also indicated. Data represent ³H release as a percentage of input, and are means ± SEM.

The proteomic environment of Set3. The proteomic environment of Set3, associated factors, and their regulation of part of the sporulation program is presented. The proteins involved are shown as colored objects below the regulatory interactions. Question marks indicate uncertainties in the regulation circuitry. Note that Set3C repression applies in meiosis and Hst1–Sum1 repression in vegetative growth. (MSE) Middle sporulation element. The Set3C includes two molecules of each Snt1 and Sif2 in a tetramer. Protein interactions in Set3C are shown as black boxes, probable interactions as gray boxes. The +/− beside Hst1 and YOR279 indicates the likelihood that these proteins are not always present in their respective complexes. Those proteins that are also found free are indicated, as is the relationship between Hst1 and NAD.