Selective Regulation of Vitamin D Receptor-Responsive Genes by TFIIH Pascal Drané, Emmanuel Compe, Philippe Catez, Pierre Chymkowitch, Jean-Marc Egly  Molecular Cell  Volume 16, Issue 2, Pages 187-197 (October 2004) DOI: 10.1016/j.molcel.2004.10.007

Figure 1 VitD Response Is Altered by XPD/R683W Mutation VitD-mediated response of CYP24 (A) and osteopontin (B), two VDR-target genes, in XPD-deficient cells. RT-PCR analysis was performed 8 hr and 16 hr after vitD addition (10−7 M). The results are the mean of three independent experiments and represent the relative expression level of the two genes versus 18S. (C) UV survival of XPD-complemented cells. XPJCLO (XPD/R683W) (closed diamonds), XPJCLO+LXPDN (open squares), and GM3348D (NHF) (open circles) cells were (104 cells/well) cultured overnight and UV irradiated (254nm) at the indicated dose (J.m−2). Cells were stained two-weeks later using crystal violet dye. Results of three independent experiments are expressed as percentage of cell survival relative to nonirradiated cells. (D) VitD-mediated response of CYP24 in XPD complemented cells. The kinetic of vitD-mediated CYP24 expression was measured in cells depicted in (C). Molecular Cell 2004 16, 187-197DOI: (10.1016/j.molcel.2004.10.007)

Figure 2 XPD/R683W Mutation Affects VDR Recruitment on CYP24 Promoter (A) ChIP analysis on the CYP24 (left) and osteopontin (right) promoters in HeLa and HD2. Soluble chromatin was prepared from cells treated with vitD (10−7 M) for the indicated time and immunoprecipitated with an antibody against VDR. The genomic DNAs were analyzed by quantitative PCR. (B) Similar ChIP assays were performed to investigateTFIIH (XPB), RNA pol II, TRAP220, and TIF2 recruitment on CYP24 . Molecular Cell 2004 16, 187-197DOI: (10.1016/j.molcel.2004.10.007)

Figure 3 DNA Binding Activity of VDR Is Not Affected by XPD/R683W Mutation (A) Western blot analyses of VDR, RXR, TFIIH (p62), RNA pol II, TIF2, and TRAP220 from nuclear extracts (NE) of HeLa and HD2 cells treated with vitD (10−7 M). Pol-IIo and Pol-IIA are the hyper- and hypophosphorylated form of RNA polII, respectively. (B) VDR from HD2 cells is able to bind to a CYP24-responsive element. Left: NE prepared from HeLa cells treated for 16 hr with vitD (10−7 M) was incubated with a 32P-labeled VDR-CYP24 responsive element (lane 3) and with an antibody against VDR or RXR (lanes 1 and 2, respectively), or with a 50× molar excess of either specific (S, lane 4) or nonspecific (NS, lane 5) cold oligonucleotides to be analyzed by EMSA. Right: NE from HeLa (lanes 7–10) and HD2 cells (lanes 11–14) treated with vitD (10−7 M) were analyzed by EMSA, as described in (A). NC1 is a heterodimeric complex containing VDR and RXR, and the asterisk indicates nonspecific complexes. Molecular Cell 2004 16, 187-197DOI: (10.1016/j.molcel.2004.10.007)

Figure 4 XPD/R683W Mutation Affects Activity of an Ets Binding Site (A) Transfections in HeLa and HD2 of reporter vectors containing vitD-responsive element 1 and 2 (VDRE1 and 2), and either the wild-type Ets binding site (EBS) or the mutated one (mEBS) as indicated at the left of the panel. Activity of these constructs as well as of those containing the luciferase gene under the control of an SV40 promoter (SVE) and either VDRE1 (pGL3-VDRE1) or a retinoic acid receptor responsive element RARE (pGL3-RARE) was measured in cells treated with either 10−7 M vitD or 10−7 M all-trans retinoic acid, respectively. The results are expressed as fold activation relative to nontreated cells. (B) EBS participates in VDR recruitment. The VDR recruitment on the indicated plasmid transfected in HeLa and HD2 cells was monitored by ChIP, 8 hr after vitD addition. (C) The Ets1 activity is impaired in HD2 cells. The pMdm2 construct was transfected in HeLa and HD2 cells with an expression vector encoding Ets1. Molecular Cell 2004 16, 187-197DOI: (10.1016/j.molcel.2004.10.007)

Figure 5 Physical and Functional Interaction between TFIIH and Ets1 (A) In vivo phosphorylation of endogenous Ets1 (lanes 1 and 2), overexpressed Ets1 wt (lanes 3 and 4) or Ets1 T38A (lanes 5 and 6) in HeLa and HD2, after immunoprecipitation. Western blot (WB), and autoradiography (Auto.). Note that the exposure of the autoradiograph of Ets1 T38A (lanes 3 and 4) was six times longer than the one of Ets1. (B) The Sf9 cells were coinfected with baculoviruses encoding Ets1 alone (panel I) or in combination with TFIIHwt (II and V) or with TFIIH containing either a XPD/R683W (III) or a XPD/R722W (IV) mutated subunit. Extracts from infected cells were pretreated (V) with calf intestinal phosphatase. Proteins were fractionated by 2D-PAGE, and Ets1 was detected by immunobloting. The position of the Ets1 isoforms is indicated by arrows (a–d). (C) 1 μg of purified Ets1 (lanes 3–5), Ets1/T38A (lanes 6–8), RARα (lanes 9–11), or VDR (lanes 12–14) was incubated HeLa TFIIH or recombinant CAK. Lanes 1 and 2 show CAK and TFIIH, respectively, incubated alone in the reaction buffer. Blue staining of the gels (stain.) is shown to compare level of each protein. (D) The Sf9 cells were coinfected with baculoviruses encoding all the TFIIH subunits and Ets1. Immunoprecipitation was done by using an antibody directed against TFIIH/p44 subunit (p44) or a control antibody (C). The bound proteins were analyzed by Western blot by using antibodies against three subunits of TFIIH (cdk7, p62 and XPD) or Ets1. The position of the antibody heavy chain is shown (H). The input lanes (1–4) represent 5% of the total volume of extract used in each immunoprecipitation. (E) Interaction between TFIIH and VDR was investigated by using a baculovirus encoding VDR as described in (D). (F) Interaction of Ets1 with the core TFIIH. A GST-Ets1 recombinant protein was incubated with extracts from cells infected with baculovirus encoding the core subunits. Washing were performed with buffer containing 300 mM KCl. The input lanes represent 5% of the total volume of the extract used for the pulldowns. Molecular Cell 2004 16, 187-197DOI: (10.1016/j.molcel.2004.10.007)

Figure 6 Role of Ets1 T38 Phosphorylation in CYP24 Promoter Regulation (A) The Ets1 phosphorylation is important for CYP24 promoter activity. Hela and HD2 cells were cotransfected with the indicated promoter construct in combination with an empty vector or a vector encoding Ets1 or Ets1/T38A. The cells were then treated with vitD (10−7 M). Promoter activity was checked as described in Figure 4. (B) The Ets1 phosphorylation is crucial for VDR recruitment. The cells were transfected with the pCYP24wt construct and then treated for 8 hr with vitD, and VDR recruitment is checked by ChIP assays. Molecular Cell 2004 16, 187-197DOI: (10.1016/j.molcel.2004.10.007)