Cluster analysis of mass spectrometry data reveals a novel component of SAGA

Abstract

The SAGA histone acetyltransferase and TFIID complexes play key roles in eukaryotic transcription. Using hierarchical cluster analysis of mass spectrometry data to identify proteins that copurify with components of the budding yeast TFIID transcription complex, we discovered that an uncharacterized protein corresponding to the YPL047W open reading frame significantly associated with shared components of the TFIID and SAGA complexes. Using mass spectrometry and biochemical assays, we show that YPL047W (SGF11, 11-kDa SAGA-associated factor) is an integral subunit of SAGA. However, SGF11 does not appear to play a role in SAGA-mediated histone acetylation. DNA microarray analysis showed that SGF11 mediates transcription of a subset of SAGA-dependent genes, as well as SAGA-independent genes. SAGA purified from a sgf11 Delta deletion strain has reduced amounts of Ubp8p, and a ubp8 Delta deletion strain shows changes in transcription similar to those seen with the sgf11 Delta deletion strain. Together, these data show that Sgf11p is a novel component of the yeast SAGA complex and that SGF11 regulates transcription of a subset of SAGA-regulated genes. Our data suggest that the role of SGF11 in transcription is independent of SAGA's histone acetyltransferase activity but may involve Ubp8p recruitment to or stabilization in SAGA.

FIG. 1.

Cluster analysis of proteomic data identifies YPL047W as a candidate subunit of SAGA. The cluster diagram on the left shows the unsupervised hierarchical clustering of proteomic data from replicate immunoaffinity purifications of each component of TFIID and control purifications (41). The enlargement on the right expands the cluster display where a pattern of proteins copurifying with a distinct set of TAFs is observed. This cluster of proteins includes all of the previously characterized components of SAGA and a protein corresponding to the uncharacterized ORF (uORF) YPL047W. The SAGA proteins are clustering together with the five TAFs known to be shared by TFIID and SAGA. These shared TAFs are labeled with an asterisk. The estimated relative abundance of each identified protein, PAF (see Materials and Methods), is designated by a shade of red. PAFs of proteins identified in control experiments are indicated by shades of green. A square is black if the protein was not detected from the computational analysis of the experiment's mass spectrometry data. The cluster diagram on the left suggest additional groups of protein associated with specific TFIID subunits that are not addressed in this report (McAfee et al., unpublished).

FIG. 2.

SAGA components copurify with TAP-Sgf11p from yeast extracts. (A) Proteins recovered after purification from a TAP-SGF11 yeast strain were separated by SDS-PAGE and silver stained. (B) The proteins purified with TAP-Sgf11p were identified by DALPC mass spectrometry. None of the listed proteins were identified from control purifications. The number of nonredundant spectra identifying each protein and a PAF (the spectra were normalized to molecular weight [MW] of the cognate protein [10⁴]) are shown. The complete set of proteins identified by mass spectrometry is available at http://linklab.mc.vanderbilt.edu.

FIG. 3.

Sgf11p copurifies with the SAGA subunits Gcn5p and Spt7p. Immunoaffinity copurification assays were performed with yeast strains containing either a plasmid expressing V5-tagged-SGF11 (pYes-SGF11) or the empty vector (pYes-DEST52). Proteins were analyzed by immunoblotting (IB) as follows. The numbers on the left of each panel represent molecular weight standards. (A) Cell lysates were probed with an anti-V5 antibody. The V5 fusion protein is seen at the expected molecular mass of 13 kDa. (B and C) Proteins immunoaffinity purified (IP) with anti-Gcn5p and anti-Spt7p antibodies, respectively, were probed with an anti-V5 antibody. V5-Sgf11p was recovered with both Gcn5p and Spt7p. (D) Proteins purified with protein A-Sepharose alone were probed with an anti-V5 antibody. No V5-Sgf11p was recovered. (E) Proteins immunoaffinity purified (IP) with an anti-V5 antibody were probed with an anti-Spt7p antibody. Spt7p was recovered with V5-Sgf11p.

FIG. 4.

Association of Ubp8p with SAGA is reduced in a sgf11 deletion strain. (A) Proteins immunoaffinity purified (IP) with an anti-Gcn5p antibody from wild-type and sgf11Δ deletion strains were probed with an anti-Ubp8p antibody. The recovery of Ubp8p was reduced in the purification from the sgf11 deletion strain. The input of Ubp8p in cell lysates is also shown. (B) Proteins immunoaffinity purified (IP) with an anti-Gcn5p antibody from wild-type and sgf11 deletion strains were also probed with antibodies for Taf12p, Gcn5p, Tra1p, and Taf10p.

FIG. 5.

Sgf11p is not required for SAGA's HAT activity. SAGA complexes were immunoaffinity purified from the indicated strains with an anti-Gcn5p antibody and used in HAT assays containing nucleosomal histones and ³H-acetyl-CoA. (A) Aliquots from each HAT assay were subjected to SDS-PAGE, followed by autoradiography to reveal labeled histones. (B) Aliquots of the same reactions were subjected to immunoblotting (IB) with the anti-Gcn5p antibody to show recovery of Gcn5p.

FIG. 6.

SGF11 and UBP8 regulate transcription. RNA isolated from wild-type and mutant strains with sgf11 or ubp8 deletions was used as the template in RT-PCRs. The products from cycle 15 were separated on a polyacrylamide gel and stained with ethidium bromide. The numbers of the left indicate base pair standards. Changes in the expression of MATα1 and CDC8 were detected in the mutant strains. Amplified products of TDH3 were used as a loading control.