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. 2012 Aug;32(16):3358-69.
doi: 10.1128/MCB.00416-12. Epub 2012 Jun 18.

Phosphorylation-dependent regulation of cyclin D1 and cyclin A gene transcription by TFIID subunits TAF1 and TAF7

Susan L Kloet  1 , Jennifer L Whiting, Phil Gafken, Jeff Ranish, Edith H Wang
Affiliations

Phosphorylation-dependent regulation of cyclin D1 and cyclin A gene transcription by TFIID subunits TAF1 and TAF7

Susan L Kloet et al. Mol Cell Biol. 2012 Aug.

Abstract

The largest transcription factor IID (TFIID) subunit, TBP-associated factor 1 (TAF1), possesses protein kinase and histone acetyltransferase (HAT) activities. Both enzymatic activities are essential for transcription from a subset of genes and G(1) progression in mammalian cells. TAF7, another TFIID subunit, binds TAF1 and inhibits TAF1 HAT activity. Here we present data demonstrating that disruption of the TAF1/TAF7 interaction within TFIID by protein phosphorylation leads to activation of TAF1 HAT activity and stimulation of cyclin D1 and cyclin A gene transcription. Overexpression and small interfering RNA knockdown experiments confirmed that TAF7 functions as a transcriptional repressor at these promoters. Release of TAF7 from TFIID by TAF1 phosphorylation of TAF7 increased TAF1 HAT activity and elevated histone H3 acetylation levels at the cyclin D1 and cyclin A promoters. Serine-264 of TAF7 was identified as a substrate for TAF1 kinase activity. Using TAF7 S264A and S264D phosphomutants, we determined that the phosphorylation state of TAF7 at S264 influences the levels of cyclin D1 and cyclin A gene transcription and promoter histone H3 acetylation. Our studies have uncovered a novel function for the TFIID subunit TAF7 as a phosphorylation-dependent regulator of TAF1-catalyzed histone H3 acetylation at the cyclin D1 and cyclin A promoters.

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Figures

Fig 1
Fig 1
TAF7 functions as a transcriptional repressor at a subset of promoters. (A) HeLa cells were treated with 50 nM control (white bars) or TAF7 (black bars) siRNA for 72 h. Total RNA was collected, and transcript levels for TAF7, cyclin D1, cyclin A, cyclin E, GAPDH, and c-fos were determined by qRT-PCR. Results are averaged from 3 independent experiments, each done in triplicate. *, P < 0.05; **, P < 0.005; ***, P < 0.001. (B) HeLa cells were treated with siRNA as described for panel A. TFIID complexes were immunoprecipitated using an anti-TBP antibody. Precipitated proteins were separated on SDS-polyacrylamide, and the indicated TFIID subunits were detected by immunoblotting. (C) HeLa cells were transfected with pGLUE empty vector (white bars) or TAP-TAF7 expression plasmid (black bars). Forty-eight hours posttransfection, total RNA was collected and analyzed by qRT-PCR as described for panel A. Results are averaged from 3 independent experiments, each done in triplicate. ***, P < 0.001. (D) HeLa cells were transfected with TAP-TAF7 expression plasmid. After 48 h, immunoprecipitations were carried out using an anti-TBP antibody. Precipitated proteins were analyzed by SDS-PAGE and immunoblotted for the indicated TFIID subunits.
Fig 2
Fig 2
TAF7 is a regulator of early S phase progression. HeLa cells were transiently transfected with YFP-TAF7 expression plasmid. After 72 h, cells were fixed and stained with propidium iodide. YFP fluorescence and DNA content were determined by flow cytometry. The cell cycle profiles of YFP-negative (A) and YFP-positive (B) cells from one representative experiment are provided. Percentages of cells in each cell cycle phase, as defined by the indicated gates, are provided. (C) Cell cycle distribution of YFP-negative and YFP-positive cells from 11 independent transfections is shown. ***, P < 0.001; n.s., not significant.
Fig 3
Fig 3
Inverse correlation between TAF1 and TAF7 binding at the cyclin D1 and cyclin A promoters. (A) HeLa cells were synchronized by thymidine/nocodazole block and collected at the indicated time points after drug removal. Percentage of G1 cells was determined by propidium iodide staining and flow cytometry. (B) Chromatin immunoprecipitation experiments were performed using anti-TAF1 or anti-TBP antibody, and samples were analyzed by qPCR using primers spanning the cyclin D1 and cyclin A promoters. The y axis shows the average percent input detected from one representative experiment carried out in triplicate. Time period when cells are predominantly in G1 is indicated by the shaded gray box. (C) ChIP experiments using anti-TAF7 antibody were carried out and analyzed as described for panel B.
Fig 4
Fig 4
TAF7 phosphorylation by TAF1 kinase hinders their interaction. (A) HeLa cells were synchronized using a thymidine/nocodazole block and collected at the indicated time points after drug removal. For TAF1 kinase activity, HeLa nuclear extracts were prepared and immunoprecipitated with anti-TBP Sepharose beads. Bound proteins were added to in vitro kinase assays using recombinant His-TAF7 as the substrate. Phosphorylated TAF7 was detected by autoradiography (p-TAF7). Total TAF1 and His-TAF7 protein levels were monitored by Western blotting. (B) Phosphorylated TAF7 in kinase assays was quantified using ImageJ software. TAF1 kinase activity is expressed as intensity of TAF7 phosphorylation corrected for total TAF1 protein. Kinase activity was compared to TAF7 ChIP data presented in Fig. 3C. (C) Purified baculovirus-expressed TAF1 fragments, GST-CTK and GST-C.RAP, and bacterially expressed His-TAF7 were visualized by Coomassie blue staining. (D) Purified His-TAF7 preincubated with buffer (mock) or TAF1 CTK (pre-phos) was isolated using Ni-agarose beads (input) and incubated with C-terminal TAF1 fragment (GST-C.RAP). Proteins coprecipitating with GST-C.RAP (IP: Glut-Seph) were detected by immunoblotting with the indicated antibody. (E) TAF1 binding was quantified using ImageJ software. *, P < 0.05; n = 5. (F) Schematic diagram of full-length TAF1 and TAF1 fragments used in panels D and E are shown. Domains shown are as follows: NTK, N-terminal kinase; HAT, histone acetyltransferase domain; RAPiD, TAF7 interaction domain; and CTK, C-terminal kinase.
Fig 5
Fig 5
Phosphorylation disrupts TAF7 binding and stimulates TAF1 HAT activity. (A) TFIID was immunoprecipitated from fractionated HeLa nuclear extracts (enriched for TFIID) with an anti-TBP antibody and visualized by silver staining (SS). Proteins precipitated in the absence of antibody also are shown. In vitro kinase assays were performed with the precipitated proteins. Reaction products were separated on SDS-polyacrylamide, transferred to nitrocellulose, visualized by autoradiography (kinase), and subjected to immunoblotting (IB). The positions of molecular weight standards are shown. (B) TFIID was immunoprecipitated as described for panel A. In vitro kinase assays using cold ATP were carried out with TFIID immobilized on anti-TBP Sepharose beads. The supernatant (S) and bound (B) fractions were collected, and the amount of TAF1, TBP, and TAF7 present was determined by immunoblotting. (C) Signal intensities were quantified using ImageJ software, and the percent total for each protein in the bound and supernatant fractions was calculated. *, P < 0.05; n = 3. (D) Immunoprecipitated TFIID was incubated in the absence (TFIID) or presence (pre-phos TFIID) of cold ATP prior to assaying for HAT activity. [3H]acetyl-CoA incorporation into a human histone H3 peptide (aa 1 to 20) was measured by liquid scintillation. Results from one representative experiment are shown. **, P < 0.005; n = 3. (E) HeLa cells were treated with 50 nM control (white bars) or TAF7 (black bars) siRNA for 72 h. ChIPs were performed using anti-histone H3 K9K14Ac antibody and analyzed by qPCR at the indicated promoters. The y axis shows fold change in H3 K9K14Ac relative to control-treated cells. *, P < 0.05; ***, P < 0.001; n = 3.
Fig 6
Fig 6
TAF1 phosphorylation of TAF7 at serine-264 disrupts their protein interaction. (A) His-tagged WT and TAF7 mutant proteins were expressed in bacteria, affinity purified, and used as the substrates for TAF1 CTK. Phosphorylated TAF7 was detected by autoradiography (pTAF7). Equal loading of TAF7 substrates was confirmed by silver staining (TAF7). (B) The indicated His-tagged WT and TAF7 mutant proteins (input) were incubated with GST-TAF1 C.RAP. Pulldowns with glutathione-Sepharose were performed, and precipitated proteins were subjected to SDS-PAGE and immunoblotting. (C) TAF1 binding was quantified as described for Fig. 4E. ***, P < 0.001; n = 4. (D) The YFP-tagged TAF7 WT or S264D mutant was transiently expressed in HeLa cells for 48 h. Nuclear extracts were prepared, and total levels of the YFP-tagged TAF7 WT and S264D mutant were determined by immunoblotting (input) using anti-YFP antibody. TFIID was immunoprecipitated using an anti-TBP antibody, and the indicated subunits were detected by immunoblotting.
Fig 7
Fig 7
TAF7 S264 phosphorylation stimulates cyclin D1 and cyclin A gene transcription and promoter H3 acetylation. (A) HeLa cells were transfected with TAP-tagged control (empty), TAP-TAF7 (WT), TAP-S264A, or TAP-S264D plasmid. After 48 h, whole-cell extracts were prepared and expression levels of TAF7 proteins were determined by immunoblotting using an anti-HA antibody. GAPDH protein levels were measured to ensure equal protein loading. (B to D) For RNA expression (left panels), total RNA was collected and transcript levels for cyclin D1 (B), cyclin A (C), and c-fos (D) were measured by qRT-PCR. The y axis shows mRNA levels relative to cells transfected with the empty control plasmid (n = 3). (A to D, right panels) In parallel transfections, H3 K9 acetylation levels at the indicated promoters were determined by ChIP using anti-histone H3 K9Ac antibody and qPCR. The y axis shows H3 acetylation levels relative to cells transfected with the empty control plasmid for each promoter (n = 3). *, P < 0.05; **, P < 0.005; ***, P < 0.001.
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