Yeast homolog of a cancer-testis antigen defines a new transcription complex

Abstract

We have isolated a new yeast gene (PCC1) that codes for a factor homologous to human cancer-testis antigens. We provide evidence that Pcc1p is a new transcription factor and that its mutation affects expression of several genes, some of which are involved in cell cycle progression and polarized growth. Mutation of Pcc1p also affects the expression of GAL genes by impairing the recruitment of the SAGA and Mediator co-activators. We characterize a new complex that contains Pcc1p, a kinase, Bud32p, a putative endopeptidase, Kae1p and two additional proteins encoded by ORFs YJL184w and YMLO36w. Genetic and physical interactions among these proteins strongly suggest that this complex is a functional unit. Chromatin immunoprecipitation experiments and multiple genetic interactions of pcc1 mutants with mutants of the transcription apparatus and chromatin modifying enzymes underscore the direct role of the complex in transcription. Functional complementation experiments indicate that the transcriptional function of this set of genes is conserved throughout evolution.

Figure 1

Real-time RT–PCR analysis of mRNA levels produced by α-factor-inducible and galactose-inducible genes in wild type and pcc1-4 mutant cells. Wild type and pcc1-4 mutant cells were preincubated for 3 h at 37°C before the addition of α-factor for analysis of FUS1, FAR1 and STE2 transcripts, or in raffinose-containing medium before the addition of galactose for analysis of GAL1 transcripts. Samples were taken at the indicated time points. Signals (arbitrary units) were normalized to U4 snRNA, the abundance of which was not affected by mutation of Pcc1p.

Figure 2

(A) RNAPII recruitment to transcriptionally active genes is affected in the pcc1-4 mutant. Wild type and pcc1-4 mutant cells were preincubated for 3 h at 37°C before addition of galactose or α-factor. Chromatin was immunoprecipitated with anti-Rbp3p antibodies. (B) Recruitment to the GAL1 locus of the Gal4p activator, SAGA (Gcn5p), Mediator (Rgr1p), and TBP (Spt15p) in pcc1-4 mutant cells. Antibodies against Gal4p, myc and HA epitopes (all from Santa Cruz Biotech) were used, respectively, to probe Gal4p, Gcn5p-myc and Spt15-HA. IgG Sepharose was used to immunoprecipitate Rgr1-TAP. In (A, B), immunoprecipitated DNAs were analyzed by real-time PCR with primers for GAL1 and FUS1 genes, respectively (the position of primers is schematically shown) and expressed relative to input DNA (arbitrary units). Averages and standard deviations were obtained from three independent experiments.

Figure 3

Pcc1p associates with the chromatin of transcriptionally active genes. (A) PCC1-TAP and isogenic untagged cells were grown at 30°C to early exponential phase before addition of α-factor. Cells were harvested for ChIP preparations after 15 min of incubation with α-factor. Immunoprecipitated and input (total) DNA were analyzed with primers located at the 5′ coding regions of the FUS1, FUS3, STE2 and FAR1 genes. Averages and standard deviations were obtained from three independent experiments. (B) PCC1-TAP and isogenic untagged cells were grown at 30°C in raffinose to early exponential phase before addition of galactose. After 30′ of galactose induction, growth was shifted to glucose-containing media to shutoff transcription of GAL genes. Cells were harvested for ChIP analysis at the indicated time points in the three different growth conditions. Immunoprecipitated DNAs were analyzed by real-time PCR with primers spanning the entire GAL1–GAL10 region and expressed relative to input DNA (arbitrary units). Immunoprecipitations were performed with IgG-Sepharose. The high level of Pcc1p occupancy at the UAS region in raffinose growth conditions was not consistently observed. Values are averages from duplicate points.

Figure 4

Isolation and characterization of the EKC complex. (A) TAP purification of the Pcc2p-associated complex. Co-purifying proteins were identified by mass spectrometry. (B) Gel filtration of whole-cell extracts from the indicated strains. Western blots of the indicated fractions were probed with anti-myc, anti-HA and PAP (peroxydase-anti peroxidase) complex. Note that the first two Western blot stripes derive from a doubly tagged Bud32-HA/Kae1-myc strain, which most likely accounts for the slightly different hydrodynamic properties of the complex compared to the single TAP-tagged Kae1p, Pcc1p and Pcc2p/Gon7p strains. I: Input. (C) Gel filtration of the affinity-purified EKC complex. The Pcc2-TAP complex was bound to IgG-Sepharose and eluted by cleavage with the TEV protease. The eluate was subjected to Superdex 200 gel filtration and the proteins in individual fractions were visualized by Coomassie blue staining after 5–20% SDS–PAGE. The identity of the bands was confirmed by mass spectrometry. The band marked with ^* in fractions 13–14 was identified as Pcc2p/Gon7p-CBP by combined MS and MS/MS analysis and does not appear to contain Bud32p. These two proteins comigrate in some gels (see fraction 12 and data not shown). The open arrows indicate two proteins that have been identified as the Hsp70 chaperones Ssa2p and Ssb1p. The protein found in fractions 17–18 is an unidentified contaminant. Apparent molecular weights are indicated by filled arrows.

Figure 5

Kae1p contains a highly conserved and functionally critical Zn-binding endoprotease motif. (A) Overall identity of Kae1p homologs from several species to the S. cerevisiae sequence. The sequence of the putative Zn-binding domain is shown in the right column. (B) The human OSGEP gene partially complements the kae1Δ mutant. Two independent clones bearing the OSGEP gene expressed from a Tet promoter-based centromeric plasmid were introduced into a kae1∷HIS, pCM188(URA)-KAE1 shuffle strain and assayed for growth on 5-FOA plates at 30 and 37°C as indicated. Human OSGEP partially complements the growth defect of the kae1∷HIS3 mutant at 30°C, but not 37°C. (C) Mutation of the two histidines in the putative Zn-binding is incompatible with Kae1p function. Three different histidine mutants (kae1-hh1, kae1-hh2 and kae1-hh3) were introduced into the kae1∷HIS, pCM188(URA)-KAE1 shuffle strain and assayed for growth on 5-FOA plates at 30°C. Clones kae1-hh1, kae1-hh2 and kae1-hh3 bear respectively the H176L/H180G, H176V/H180V and H176V/H180T mutations.