1. Volume 6, Issue 1, Pages 127-137 (July 2000)
Activation of Estrogen Receptor α by S118 Phosphorylation Involves a Ligand- Dependent Interaction with TFIIH and Participation of CDK7 
Dongsheng Chen, Thilo Riedl, Elinor Washbrook, Paul E. Pace, R.Charles Coombes, Jean-Marc Egly, Simak Ali 
Molecular Cell 
Volume 6, Issue 1, Pages (July 2000)
DOI: /S (05)

2. Figure 1
Transactivation by ERα Is Increased by Overexpression of Cdk7 in Concert with MAT1
(A) The amino acid sequence of human ERα (HEG0) is schematically represented and shows positions of the functional domains, activation function 1 (AF1), the ligand binding domain (LBD)/activation function-2 (AF2), and the DNA binding domain (DBD). The position of helix 12 is displayed. Also shown are the positions of mapped phosphorylation sites within AF1. Mutant ERα proteins lacking the LBD (HE15) or AF1 (HEG19), which were used in this study, are also represented. The amino acid sequence around identified AF1 phosphorylation sites is also shown. The sequence of the peptide used to raise α-P-S118 is underlined.
(B) COS-1 cells were transfected with the ERE-containing CAT reporter gene, 17M-ERE-TATA-CAT (4 μg), and wild-type ERα (500 ng of HEG0), without (lanes 1–4) or with 4 μg of Cdk7 (lanes 5–8, 17–24, and 29–32), MAT1 (lanes 9–12, 17–20, and 25–32), and cyclin H (Cyc H; lanes 13–16, and 21–32). 17β-estradiol (E2, 10−8 M), 4-hydroxytamoxifen (OHT, 10−7 M), or ICI 182, 780 (ICI, 10−7 M) was added as indicated. Since the ligands were prepared in ethanol, an equal volume of ethanol was added to the no ligand controls. The results of three independent experiments are displayed in the form of a bar chart. Transcription activation by HEG0 in the presence of E2 (lane 2) was taken as 100%. All other activities are shown relative to this.
(C) COS-1 cells were transfected with 4 μg of 17M-ERE-TATA-CAT or 17M-TATA-CAT in the presence of HEG0 (500 ng), either without (−) or with 4 μg of Cdk7 and MAT1 (Cdk7+MAT1). Different concentrations of E2, ranging from 10−14 M to 10−6 M were added, as shown. Ethanol was added to the no ligand (0) controls. The results of three independent experiments are shown. Transcription activation by HEG0 with 17M-ERE-TATA-CAT in the presence of 10−8 M E2 was taken as 100%.
(D) COS-1 cells were cotransfected with 4 μg of 17M-ERE-TATA-CAT together with 500 ng of pSG5 (lane 1), HEG0 (lanes 2 and 3), HEG0106A (lanes 4 and 5), HEG0102N, 104P, 106A (lanes 6 and 7), HEG0102N, 104P, 106A, 118A (lanes 8 and 9), HEG0118A (lanes 10 and 11), HEG0118E (lanes 12 and 13), or HEG0167A (lanes 14 and 15), and Cdk7 and MAT1 were also cotransfected, as indicated. E2 (10−8 M) was added in all cases. The results of three independent experiments are shown. Transcription activation by HEG0 (lane 2) was taken as 100%.
(E) COS-1 cells were transfected with 17M-ERE-TATA-CAT and HEG0, as above. Cdk7, Cdk7M and MAT1 were cotransfected, as indicated. E2 (10−8 M) was added, as shown. The results of four independent experiments are shown. Transcription activation by HEG0 (lane 2) was taken as 100%.
Molecular Cell 2000 6, DOI: ( /S (05) )

3. Figure 2 Phosphorylation of ERα by Cdk7 In Vivo
(A) COS-1 cells transfected with 5 μg of the parental vector (pSG5) or HEG0, together with Cdk7 and/or MAT1, were labeled with [32P]-phosphoric acid in the absence of ligand or in the presence of 10−8 M E2, 10−7 M OHT, or 10−7 M ICI. Whole-cell extracts were immunoprecipitated with monoclonal antibody B10. The immunoprecipitates were resolved by SDS-PAGE, and autoradiography (upper panel) and immunoblotting using B10 (lower panel) were performed.
(B) COS-1 cells transfected with pSG5 or HEG0 without or with Cdk7 (lanes 6–13) and MAT1 were treated with ethanol, E2, OHT, ICI, and/or phorbol myristate acetate (PMA) (100 ng/ml) for 30 min prior to harvesting. The extracts were resolved by SDS-PAGE and immunoblotted using antisera specific to ERα phosphorylated at S118 (P-S118) or B10 for detection of total ERα (HEG0). Western blotting with antibodies against Cdk7 and MAT1 was performed as controls.
Molecular Cell 2000 6, DOI: ( /S (05) )

4. Figure 3 In Vitro Phosphorylation of ERα at S118 by TFIIH or CAK
(A) Purified ERα (0.01 pmoles) was phosphorylated by incubation with 1, 2.5, or 5 μl of purified CAK or with 1 μl of TFIIH in the absence of ligand, or in the presence of 10−7 M E2, 10−7 M OHT, or 10−7 M ICI. Lane 1 shows CAK (5 μl) incubated in the reaction buffer in the absence of ERα. Lane 2 shows ERα, incubated in the absence of CAK or TFIIH. Autoradiography (top panel) and immunoblotting using B10 (α-ERα; middle panel) and α-Cdk7 (bottom panel) were performed to compare levels of each protein.
(B) Whole-cell extracts of COS-1 cells transfected with pSG5, HEG0, HE15, or HEG19, immunoprecipitated using monoclonal antibodies B10 (lanes 1–12) or F3 (lanes 13–16) were phosphorylated with highly purified TFIIH. Increasing amounts of TFIIH (0.5 μl, lanes 2, 6, 10, and 14; 1 μl, lanes 3, 7, 11, and 15; and 2 μl, lanes 4, 8, 12, and 16) were added. Phosphorylated proteins were visualized by autoradiography (upper panel) and immunoblotting with anti-ERα B10 (lanes 1–12) or F3 (for HEG19; lanes 13–16), as appropriate (lower panel). Lanes 1, 5, 9, and 13 were incubated in the absence of TFIIH. E2 (10−7 M) was present in all samples throughout the procedure.
(C) Mutants of HEG0 in which serine residues corresponding to known phosphorylation sites within AF1 were replaced by non-phosphorylatable residues were phosphorylated using 1 μl of TFIIH. Autoradiography (upper panel) and immunoblotting results using B10 (lower panel) are shown. E2 (10−7 M) was present in all samples throughout the procedure.
Molecular Cell 2000 6, DOI: ( /S (05) )

5. Figure 4 Association of In Vitro Synthesized ERα with TFIIH
(A) In vitro synthesized HEG0 was incubated with purified TFIIH, followed by immunoprecipitation using antibodies against p62 and XPB subunits of TFIIH. The proteins were resolved by SDS-PAGE and visualized by autoradiography. Controls involved immunoprecipitation in the absence of TFIIH. E2 was present at a concentration of 10−7 M. The input lane represents 10% of the total volume of the lysate used in each immunoprecipitation.
(B) [35S]-labeled HEG0, HE15, and HEG19, synthesized by in vitro translation, were incubated with TFIIH in the presence of 10−7 M E2 and immunoprecipitated using antibodies against p62, Cdk7, and the FLAG epitope (M2). The input lane represents 10% of the total volume of the lysate used in each immunoprecipitation.
(C) [35S]-labeled HEG0 was immunoprecipitated using anti-p62 or B10 in the presence (lanes 2, 4, 6, and 8) or absence of TFIIH. E2, OHT, or ICI were present at a concentration of 10−7 M. The input lane represents 10% of the total volume of the lysate used in each immunoprecipitation.
(D) Purified ERα was phosphorylated by incubation with purified TFIIH or purified CAK and immunoprecipitation using anti-Cdk7 (lanes 2–5, and 7–9) or anti-FLAG (M2, lanes 6 and 10). The immunoprecipitates were divided into four equal portions, fractionated on SDS-PAGE, and immunoblotted to detect P-S118, total ERα, or CDK7. The fourth gel was dried down and autoradiographed. The input lane represents 10% of the total amount of the sample used in each immunoprecipitation.
(E) Purified ERα was incubated with recombinant purified core TFIIH (rIIH5), in the presence of 10−7 M E2, followed by immunoprecipitation using antibodies against p62 and Cdk7. The input lane represents 10% of the amount of ERα used in each immunoprecipitation.
(F) Sf9 cells were coinfected with baculoviruses encoding the nine subunits of TFIIH (rIIH9) or the six subunits of core TFIIH containing XPD (rIIH6) in combination with or without a baculovirus encoding FLAG-tagged human ERα. Cell lysates were immunoprecipitated in the presence of 10−7 M E2 using anti-FLAG antibody. Bound proteins (B) were analyzed by SDS-PAGE and immunoblotting using antibodies against various subunits of core TFIIH, the CAK subcomplex, or ERα (anti-FLAG) as indicated on the left. The load lane (L) represents 8% of the total volume of lysate used in each immunoprecipitation.
Molecular Cell 2000 6, DOI: ( /S (05) )

6. Figure 5 ERα Association with Subunits of TFIIH
(A) Pulldowns of in vitro synthesized Cdk7, MAT1, cyclin H, p62, XPD, and XPB using GST or GST-AF2 were performed. 10−7 M E2 was present throughout the procedure. The input lanes represent 10% of the total volume of the lysate used for the pulldowns for each protein.
(B) GST pulldowns of [35S]-labeled p62 and XPD were performed using GST and GST-AF2 in the absence of ligand or in the presence of 10−7 M E2, OHT, or ICI. The input lanes represent 10% of the total volume of the lysate used for the pulldowns for each protein.
(C) GST pulldowns of [35S]-labeled p62 (upper panel) and XPD (lower panel) were performed using GST and GST-AF2 in the presence of 10−7 M E2. Pulldowns were also performed using AF2 containing mutations within the AF2 core/helix 12 region of the LBD. The input lane represents 10% of the total volume of lysate used for the pulldowns for each protein.
(D) Pulldowns were performed using GST, GST-AF2, or GST-AF2M in the presence of 10−7 M E2. GST-AF2M contains alanines at positions 543 and 544 of murine ERα in place of leucines. A peptide corresponding to amino acid residues 91–106 of human p62 (P1) was used to compete with p62 for binding to GST-AF2. The leucine residues corresponding to amino acids 100 and 101 were replaced with alanines in peptide P2. Peptides P1 and P2 were added at concentrations of 2 (lanes 5 and 9), 6 (lanes 6 and 10), 12 (lanes 7 and 11), and 24 μM (lanes 8 and 12). The input lane represents 10% of the total volume of the lysate used for the pulldowns.
(E) HEG0 synthesized in vitro in the presence of [35S]-labeled methionine was incubated with purified TFIIH in the presence of 10−7 M E2, followed by immunoprecipitation using antibodies against the FLAG epitope (M2) or p62. Increasing concentrations of P1 and P2 (as in D) were used to compete with TFIIH for binding to HEG0. The input lane represents 10% of the total volume of the HEG0 lysate used in each immunoprecipitation.
(F) Yeast two-hybrid assay was used to assess the ability of the small region of p62, which, containing the p62 LXXLL motif, fused to the GAL4 DNA binding domain (DBD-p62 89–105), to interact with murine ERα AF2, fused to the VP16 activation domain (AF2-AD), in the presence and absence of 10−6 M E2. The interaction was also tested with vector (VP16 only) or VP16-AF2 containing a substitution of leucine residues at positions 543 and 544 to alanines (AF2M-AD). p53-AD and the SV40 T antigen fused to the GAL4 DBD (DBD-T) were used as positive controls for interaction. Interactions were assayed by determining the activity of an integrated GAL4-regulated β-galactosidase gene. The results of four independent experiments are displayed in the form of a bar chart.
(G) Yeast two-hybrid assays were performed as in (F) using DBD-p62 89–105 containing amino acid susbtitutions as shown, together with AF2-AD in the presence of 10−6 M E2. The results of four independent experiments are displayed in the form of a bar chart.
(H) Pulldowns were performed using GST, GST-p62, or GST-p62AA in the presence or absence of 10−7 M E2. Leucine residues at positions 101 and 102 of p62 were replaced with alanines to give GST-p62AA. Immunoblotting was performed to reveal ERα. The input lane represents 10% of the amount of purified ERα used for the pulldowns.
Molecular Cell 2000 6, DOI: ( /S (05) )

7. Figure 6
Phosphorylation of Serine 118 Is Regulated by Association of TFIIH with AF2
(A) Purified ERα (0.01 pmoles) was phosphorylated using TFIIH (1 μl) or CAK (5 μl), as described for Figure 3A, except that peptides P1 (lanes 4 and 8) or P2 (lanes 5 and 9) were added to the phosphorylation reactions at a concentration of 24 μM. E2 (10−7 M) was added as shown.
(B) Extracts of COS-1 cells transfected with HEG0 or HEG0539A, 540A were immunoprecipitated using monoclonal antibody B10 and phosphorylated with purified CAK (5 μl) or TFIIH (1 μl). Phosphorylated proteins were visualized by autoradiography (upper panel) and immunoblotting with B10 (lower panel).
(C) COS-1 cells were transfected with 5 μg of pSG5, HEG0118A, HEG0, or HEG0539A, 540A, with CL100, p62, or p62101A, 102A (p62-AA), as appropriate. E2 (10−7 M) and PMA (100 ng/ml) was added 30 min prior to harvesting, where indicated. Cell lysates were resolved by SDS-PAGE, and immunoblotting was performed for total ERα (B10), ERα phosphorylated at S118 (P-S118), total ERK2, or phosphorylated ERK1/ERK2 (P-ERK1/P-ERK2).
Molecular Cell 2000 6, DOI: ( /S (05) )