Proteomic analysis demonstrates activator- and chromatin-specific recruitment to promoters

Abstract

In-depth characterization of RNA polymerase II preinitiation complexes remains an important and challenging goal. We used quantitative mass spectrometry to explore context-dependent Saccharomyces cerevisiae preinitiation complex formation at the HIS4 promoter reconstituted on naked and chromatinized DNA templates. The transcription activators Gal4-VP16 and Gal4-Gcn4 recruited a limited set of chromatin-related coactivator complexes, namely the chromatin remodeler Swi/Snf and histone acetyltransferases SAGA and NuA4, suggesting that transcription stimulation is mediated through these factors. Moreover, the two activators differentially recruited the coactivator complexes, consistent with specific activator-coactivator interactions. Chromatinized templates suppressed recruitment of basal transcription factors, thereby amplifying the effect of activators, compared with naked DNA templates. This system is sensitive, highly reproducible, and easily applicable to mapping the repertoire of proteins found at any promoter.

FIGURE 1.

Proteomic analysis of PICs. A, workflow for cysteine peptide capture in conjunction with iTRAQ labeling and LC-MS/MS. Protein samples are reduced with DTT, digested with trypsin, and desalted by reversed phase chromatography. Cysteine-containing peptides are captured on thiol-activated Sepharose beads, washed, and labeled with iTRAQ stable isotope reagents. Samples from different conditions are processed in parallel, and labeled peptides are eluted with DTT, combined, and analyzed by three-dimensional high-pH reversed phase/strong anion exchange/low-pH reversed phase (RP-SAX-RP) tandem mass spectrometry. B, schematic representation of the immobilized promoter experiment shown in Fig. 2. C, iTRAQ reporter ion region from a representative MS/MS spectra of a RPA135 peptide, detected at equal quantities in each sample, and a Gal4 peptide, detected only in the activated transcription sample.

FIGURE 2.

Sequence- and activator-dependent recruitment of factors to the HIS4 promoter. A, scatter plot of each protein ratio for HIS4 promoter/promoterless (log₂(116/114)). Log₂ ratios were derived from iTRAQ reporter ions across replicate experiments; threshold log₂ ratios for proteins enriched or excluded with probability ≥0.6 are +1.0 and −1.0, respectively (supplemental Fig. S2C, left panel). The peach circle represents Gal4. Other proteins are color-coded based on their membership in basal or coactivator complexes. Gray circles represent proteins that are not classified as basal factors or coactivators. B, transcription-related proteins from A organized by basal or coactivator complex. RPA, replication protein A. C, scatter plot of log₂ ratios for proteins detected in association with the HIS4 promoter with or without Gal4-VP16 (116/115). Threshold log₂ ratios for proteins enriched or excluded with probability ≥0.6 are +0.82 and −0.96, respectively (supplemental Fig. S2C, right panel). D, data from C showing transcription-related proteins organized by complex. E, data from C showing basal initiation factors. F, immunoblots for the largest subunit of RNAPII, TBP, Gal4, and Ada1 (a subunit of SAGA), after immobilized template pulldown assays with the promoterless or HIS4 templates, in the presence or absence of Gal4-VP16.

FIGURE 3.

Proteomic analysis of different activated transcription complexes. A, schematic diagram of Gal4-VP16 and Gal4-Gcn4 activator proteins used in this study. Both have a common DNA-binding domain from Gal4, and each has a unique activation domain, as described previously (11). B, in vitro transcription reactions were performed in nuclear extract using the immobilized HIS4 promoter templates, which included ∼150 nucleotides downstream of the transcription start site. Reactions were performed in the absence of activator or in the presence of Gal4-Gcn4 or Gal4-VP16. C, schematic representation of the immobilized promoter reactions analyzed by iTRAQ-based quantitative mass spectrometry. RP-SAX-RP, high-pH reversed phase/strong anion exchange/low-pH reversed phase. D, scatter plot of the average protein ratio for reactions with and without Gal4-Gcn4 (log₂(117/115)). Threshold log₂ ratios for proteins enriched or excluded with probability ≥0.6 are +0.40 and −0.88, respectively (supplemental Fig. S3C, left panel). The peach circle represents Gal4. Other proteins are color-coded based on their membership in coactivator complexes. Gray circles represent proteins that are not members of coactivator complexes. E, NuA4, SAGA, and Swi/Snf subunits from D organized by complex. Two other Nua4 subunits (Epl1 and Yaf9) were identified only in a single replicate, but likewise were recruited more strongly by Gal4-VP16 (supplemental Table S2). F, scatter plot of the average protein ratio for Gal4-Gcn4/Gal4-VP16 (log₂(117/116)). Proteins are color-coded as described for D. Threshold log₂ ratios for proteins enriched or excluded with probability ≥0.6 are +0.65 and +0.067, respectively (supplemental Fig. S3C, right panel). G, NuA4, SAGA, and Swi/Snf subunits from F organized by complex.

FIGURE 4.

Proteomic analysis of chromatinized DNA templates. A, workflow for proteomic analysis of PIC assembly on a chromatinized HIS4 template. B, proteins that were specifically enriched on chromatinized templates are listed, along with their description and log₂ ratio. C, Scatter plot of protein ratios on chromatinized versus naked templates. Log₂ ratios were derived from iTRAQ reporter ions across replicate experiments; threshold log₂ ratios for proteins enriched or excluded with probability ≥0.6 are +0.66 and −1.2, respectively (supplemental Fig. S4C). Complexes are color-coded as described in the legend to Fig. 2. D, the values from C for known transcription factors are grouped by complex.

FIGURE 5.

Analyses of activated transcription on chromatinized DNA templates. A, scatter plot of ratios for proteins binding to chromatinized HIS4 templates in the presence or absence of Gal4-VP16. Log₂ ratios were derived from iTRAQ reporter ions across replicate experiments; threshold log₂ ratios for proteins enriched or excluded with probability ≥0.6 are +0.65 and −0.78, respectively (supplemental Fig. S5E). B, the values from A for basal transcription factors, coactivators, and chromatin remodelers are organized by complex. C, immunoblot (IB) analysis to validate that chromatin suppresses PIC assembly. The assembly reaction was performed with naked and chromatinized templates in the presence or absence of Gal4-VP16, and eluted proteins were probed with specific antibodies as indicated. D, in vitro transcription with naked versus chromatinized DNA template. Transcription templates were incubated with or without Gal4-VP16, followed by in vitro transcription in yeast nuclear extracts. Transcripts were analyzed by PAGE phosphorimaging (left panel). Relative transcript intensities were evaluated by densitometry (right panel). Gal4-VP16 gave a 3.25-fold activation on naked templates and 7.24-fold on chromatinized templates.

FIGURE 6.

Comparison of two different activators on chromatinized DNA templates. A, workflow for proteomic analysis of PIC assembly with different activators on chromatinized HIS4 templates. B, in vitro transcription reaction using chromatinized templates showing response to the indicated activators. C, scatter plot of protein ratios on chromatinized templates with and without Gal4-Gcn4 (115/114). Log₂ ratios were derived from iTRAQ reporter ions across replicate experiments (supplemental Fig. S6, A and B). Threshold log₂ ratios for proteins enriched or excluded with probability ≥0.6 are +0.4 and −0.7, respectively (supplemental Fig. S6C). Complexes are color-coded as described in the legend to Fig. 2. D, the values from C for known transcription factors are grouped by complex. E, scatter plot of protein ratios on chromatinized templates comparing Gal4-Gcn4 with Gal4-VP16 (115/116). Log₂ ratios were derived from iTRAQ reporter ions across replicate experiments (supplemental Figs. S5D and S6, A and B). Threshold log₂ ratios for proteins enriched or excluded with probability ≥0.6 are +0.3 and −0.5, respectively (supplemental Fig. S6D). F, the values from D for known transcription factors are grouped by complex.