Geles et al. 10.1073/pnas.0510764103. |
Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Supporting Materials and Methods
Supporting Dataset 1
Supporting Dataset 2
Supporting Figure 6
Fig. 6. TAF4b association with TFIID subunits in stable SIGC lines. (A) Immunoprecipitation (IP) of total cell extracts from f-TAF4b stable cells using, as a control, anti-TBP and anti-Flag antibodies followed by immunoblotting with anti-TAF1 and anti-Flag antibodies. TAF1 and Flag-TAF4b were found to coimmunoprecipitate from total cell extracts by using antibodies against TBP and the FLAG epitope but not a control nonspecific IgG. (B) IP of f-TAF4b and flag total cell extracts using a control and anti-TAF4 antibodies, followed by immunoblotting with anti-TAF4 and anti-Flag antibodies. The presence of both Flag-TAF4b and TAF4 from f-TAF4b stable cell extracts was detected in coprecipitates from the anti-TAF4 IP but not from a control IP by Western blot analysis.
Fig. 7. Overexpression of TAF4b in NIH/3T3 cells. (A) Immunoblotting of Flag-TAF4b in SIGCs and NIH/3T3 cells using an anti-Flag antibody and anti-GAPDH antibodies to confirm equal protein loading. (B) IP of Flag-TAF4b in NIH/3T3 cells using an anti-TAF4b monoclonal antibody and a control antibody, followed by immunoblotting with an anti-TAF4b monoclonal antibody. The endogenous TAF4b and Flag-TAF4b were detectable in these cells only after immunoprecipitation (lanes 3 and 4). This low level of TAF4b in the control flag cells (lane 3) was not unexpected, because little Taf4b mRNA is detected in nongonadal tissues.
Fig. 8. Activation of gene expression in f-TAF4b stable cells. (A) Quantitative real-time RT-PCR was performed on a subset of genes identified by DNA microarray analysis (Table 1). Fold induction was calculated by comparing transcript levels in f-TAF4b (gray bars) versus control flag (white bars) cells that were normalized to Gapd expression. Error bars represent standard deviations from three independent experiments. The expression of matrix metalloproteinase (Mmp-3), vimentin (Vim), fibronectin (Fn1), latent transforming growth factor b-binding protein (Ltbp-1), fos-related antigen (Fra-1), and vascular endothelial growth factor (Vegf-B) were all up-regulated in the f-TAF4b stable cells compared with the control flag cells.
Fig. 9. A model depicting an alternative transcription pathway using TAF4b-containing TFIID complexes to stimulate clusters of c-Jun/AP-1 target genes. (A) TAF4b may coordinate the pituitary–ovarian reproductive axis. In wild-type animals, pituitary FSH stimulates the production of cAMP in granulosa cells, which ultimately induces gene expression and activates ovarian follicle growth. Potentially, in response to FSH signaling, TAF4b-containing TFIID complexes regulate granulosa cell (GC) transcription, in part, by inducing c-jun expression. Subsequently, a specific subset of target genes is then activated to stimulate folliculogenesis.
Materials and Methods
Plasmids, Antibodies, and Cell Lines. IMAGE clone 1498874 contains the full-length cDNA sequence of murine (m) TAF4b. mTAF4b was subcloned into the p3XFLAG-CMV-10 expression vector (Sigma), giving pFlag-TAF4b. The (–1,596 to –1) Ccnd2 and (–1,000 to –1) Fst promoters with the translation start sites being (+1) were PCR amplified from mouse genomic DNA and cloned into the Luciferase Reporter pGL3-Basic Vector (Promega). Luciferase reporter constructs for rat Inha (1), Inhba (2), and Inhbb-subunit (3) promoters were kindly provided by K. Mayo (Northwestern University, Evanston, IL). The control p21/waf1 (4) and Ube1L (W.-L.L., unpublished data) luciferase reporter constructs were previously described. DNA fragments for Inhba and c-jun RPA probes were cloned into the pCR4Blunt-Topo vector (Invitrogen). Primer sequences are available upon request. DNA sequencing was performed to verify each construct. pTRI-Actin-Mouse and pTRI-Gapdh-Rat control RPA plasmids were purchased from Ambion.
The rat spontaneously immortalized granulosa cell line (SIGC) was a gift from R. Burghardt (Texas A&M University, College Station, TX) and cultured as described in ref. 5. NIH/3T3 cells were obtained from the American Type Culture Collection. The p3XFLAG vector and pFlag-TAF4b were transfected into SIGCs and NIH/3T3 cells by using Effectene reagent (Qiagen, Valencia, CA). Stable transfected cell lines were selected in the presence of 0.6 mg/ml Geneticin (GIBCO). Clonal cell lines were isolated and maintained in 0.2–0.6 mg/ml Geneticin.
Mouse monoclonal and rabbit polyclonal antibodies were generated against amino acids 17–237 of rat TAF4b fused in frame with an N-terminal GST-tag (Amersham Pharmacia Biotech). Anti-TAF4b antibodies also cross-reacted with mouse TAF4b.
Chromatin Immunoprecipitation (ChIP) Assays.
After cross-linking, cells were collected in PBS containing 1 mM PMSF and processed as previously described without EGTA but containing 1× Complete protease inhibitors (Roche). Nuclei were sonicated in sonication buffer [50 mM Tris (pH7.9) 10 mM EDTA, 0.1 M NaCl, 1% Triton X-100, and 1× Complete). Centrifugation in cesium chloride was omitted. Precleared chromatin was incubated with anti-Flag M2, anti-TAF4, anti-Jun (BD Biosciences), and, as a control, anti-myc (Sigma) antibodies in sonication buffer for 2 h at 4°C, and then immune complexes were recovered. Immunoprecipitates were washed four times in buffer A [50 mM Hepes (pH7.6), 1 mM EDTA, 0.25 M LiCl, 1% Triton X-100, 0.5% Nonidet P-40, 0.1 mM PMSF, 1 mM Na-metabisulfite, and 1 mM benzamidine] and four times in buffer B [50 mM Hepes (pH7.6), 1 mM EDTA, 0.5 M LiCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and protease inhibitors]. Thirty-five cycles of PCR were performed in 50 ml with 10 ml of immunoprecipitated material and 10 pmol of each primer set. Primer sequences are available upon request.Immunoprecipitation.
Immunoprecipitation of whole-cell extracts from f-TAF4b cells with anti-TBP or anti-Flag M2 antibodies, followed by detection with anti-TAF1 and anti-Flag M2 antibodies was performed essentially as described in ref. 6. Control anti-DREF antibody was described in ref. 7. Whole-cell extracts from Flag control and f-TAF4b cells were prepared and immunoprecipitated with anti-TAF4 monoclonal antibody (BD Biosciences) and the control anti-Actin antibody (Santa Cruz Biotechnology). Western blot analysis was performed with anti-TAF4 and anti-Flag M2 antibodies.DNA Microarray
. The experiment and results presented in Table 1 were designed to identify genes up-regulated in stable f-TAF4b SIGC cells versus the flag control cell line. RNA was isolated from stable SIGC f-TAF4b and control Flag cell lines in log phase of growth by using TRIzol reagent according to manufacturer’s instructions (Invitrogen). Double-stranded cDNA was generated from 30 mg of total RNA by using standard protocols described in the GeneChip Expression Analysis Technical Manual (Affymetrix). Synthesis of biotin-labeled cRNA was performed with the BioArray HighYield RNA transcript labeling kit (Enzo Diagnostics). The control oligonucleotide B2 and eukaryotic hybridization controls (bioB, bioC, bioD, and cre) were added to each chip hybridization reaction according to manufacturer’s instructions (Affymetrix). RG-U34A oligonucleotide microarrays (P/N 900249) were hybridized, washed, and stained as suggested by the manufacturer (Affymetrix). Microarray analysis was performed in duplicate from independent RNA isolations. Each chip was scaled so that the average intensity was 500. For comparison analysis, baseline values for gene expression in f-TAF4b cells were set to the control flag cell line intensities by using the genechip software (Affymetrix). A four-way comparison of gene expression changes from the two individual hybridization experiments was performed with the data mining tool software to identify genes up-regulated by 2-fold or more (Affymetrix). Average fold inductions were calculated from a four-pair-wise comparison of two individual f-TAF4b versus two individual flag control hybrizations.1. Pei, L., Dodson, R., Schoderbek, W. E., Maurer, R. A. & Mayo, K. E. (1991) Mol. Endocrinol. 5, 521–534.
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