Fig. 1

Defective transcription in cells harboring TFIIE mutations. a Increasing amounts of whole-cell lysates isolated from fibroblasts of patients with TFIIEβ/A150P, /D187Y, and of unaffected parents were used for immunoblot analysis of TFIIE (IIEα and IIEβ), TFIIH (XPB, XPD, and CDK7), TFIIA (p35), TFIIB (p33), TFIID (TAF1, TAF4, and TBP), TFIIF (RAP74 and RAP30), and Pol II (the hypo IIA- phosphorylated and hyper IIO-phosphorylated forms of RPB1). β-actin was used as loading control. b, c Wild-type (gray boxes) and TTD fibroblasts (open boxes) with the mutation IIEβ/A150P (panel b) and IIEβ/D187Y (panel c) have been treated with t-RA (10 µM). Relative RARβ2 gene expression have been measured by RT-PCR after 0, 6, and 8 h of t-RA treatment (panels b and c). The mRNA levels were normalized to the 18S RNA amount. The results (n = 9, means ± s.d.) are presented as n-fold induction relative to non-treated cells. d–i: ChIP experiments have been done 0, 6, and 8 h post-t-RA treatment from wild-type (gray boxes) and TTD fibroblasts (open boxes) with the mutation IIEβ/A150P (panels d, f, and h) and IIEβ/D187Y (panels e, g, and i) to analyze the recruitment of Pol II (RPB1, d and e), TFIIH (CDK7, f and g) and TFIIE (TFIIEβ, h and i) at the RARB2 proximal promoter. The values (n = 8, means ± s.d.) are expressed as percentage of immunoprecipitated DNA relative to the input. j Relative RARβ2 gene expression after 0, 6, and 8 h of t-RA treatment in IIEβ/WT (gray boxes) and KI-IIEβ/A150P (open boxes) cells. The mRNA levels were normalized to the 18S RNA amount. The results (n = 9, means ± s.d.) are presented as n-fold induction relative to non-treated cells. k–x ChIP experiments have been done 0, 6, and 8 h post-t-RA treatment from IIEβ/WT (gray boxes) and KI-IIEβ/A150P (open boxes) cells. We analyzed at the RARB2 proximal promoter (panels k–q) and exon 4 (panels r–x) the recruitment of Pol II (RPB1, k, r), TFIIH (CDK7, l, s), TFIIB (p33, m, t), TFIIEβ (n, u), Ser5-P (o, v), Ser2-P (p, w), and DSIF (SPT5, q, x). The values (n = 8, means ± s.d.) are expressed as percentage of immunoprecipitated (IP) DNA relative to the input. Source data are provided as a Source Data file