Trichothiodystrophy causative TFIIEβ mutation affects transcription in highly differentiated tissue

Abstract

The rare recessive developmental disorder Trichothiodystrophy (TTD) is characterized by brittle hair and nails. Patients also present a variable set of poorly explained additional clinical features, including ichthyosis, impaired intelligence, developmental delay and anemia. About half of TTD patients are photosensitive due to inherited defects in the DNA repair and transcription factor II H (TFIIH). The pathophysiological contributions of unrepaired DNA lesions and impaired transcription have not been dissected yet. Here, we functionally characterize the consequence of a homozygous missense mutation in the general transcription factor II E, subunit 2 (GTF2E2/TFIIEβ) of two unrelated non-photosensitive TTD (NPS-TTD) families. We demonstrate that mutant TFIIEβ strongly reduces the total amount of the entire TFIIE complex, with a remarkable temperature-sensitive transcription defect, which strikingly correlates with the phenotypic aggravation of key clinical symptoms after episodes of high fever. We performed induced pluripotent stem (iPS) cell reprogramming of patient fibroblasts followed by in vitro erythroid differentiation to translate the intriguing molecular defect to phenotypic expression in relevant tissue, to disclose the molecular basis for some specific TTD features. We observed a clear hematopoietic defect during late-stage differentiation associated with hemoglobin subunit imbalance. These new findings of a DNA repair-independent transcription defect and tissue-specific malfunctioning provide novel mechanistic insight into the etiology of TTD.

Figure 1.

TFIIE protein levels and stability is reduced in TFIIEβ-mutated TTD cells. (A) Sanger sequencing profiles of TFIIEβ cDNA. TTD218UT shows homozygosity for the c.G559T missense mutation. (B,C) TFIIEβ mutations segregating in the patients’ families. (D–F) Immuno-fluorescence analysis of TTD218UT and TTD251HE fibroblasts compared with wild-type control (C5RO) cells, stained for (D) TFIIEβ, (E) TFIIEα or (F) XPB (TFIIH) and DNA was stained with DAPI (blue). Quantification of the mean intensities (n = 50 nuclei), expressed as percentage of the mean intensity in normal cells, is shown beneath the representative images. Error bars indicate SEM. (G) Immuno-blot analysis to determine protein stability of TFIIEβ after cycloheximide (100 µM) treatment (for indicated times in hours) of Sv40-immoratlized patient cells (TTD218UT_sv) compared with two wild-type controls (MRC5_sv, C5RO-sv). (H) Quantification of TFIIEβ band intensities normalized to tubulin were expressed as percentages of t=0 values.

Figure 2.

Transcription and cell survival is reduced at 40 °C in TFIIEβ-mutated TTD cells. (A) Transcription levels after incubation for 72 h at 37 °C or 40 °C, measured by pulse-labeling with ethynyl-uridine (EU) and subsequent fluorescent staining of incorporated EU, of Sv40-immortalized patient cells (TTD218UT_sv), compared with wild-type controls (MRC5_sv, C5RO-sv). DNA was stained with DAPI (blue). (B) Quantification of the mean intensities (n = 50), expressed as percentages of intensity in MRC5_sv. Error bars indicate SEM. (C,D) Coomassie staining and quantification of colonies (D) of TTD218UT_sv, MRC5_sv and C5RO-sv cells formed after 12 days culturing at 37 °C (blue bars) or 40 °C (red bars) (n = 2).

Figure 3.

Affected Hematopoiesis in iPS-derived erythroid cells of TTD patients. In vitro erythroblast differentiation performed on iPS cells derived from TTD218UT (TTD) fibroblasts, control fibroblasts (control F) and peripheral blood mononuclear cell derived erythroblasts (control E).(A) Cytospins and Giemsa-Grünwald staining of in vitro differentiated erythroblasts. Arrows indicate multinucleated cells. (B) Quantification of multinucleated cells, expressed as a percentage of total cells [>500 cells, counted by two independent researchers (N = 2)]. (C) Dot plots of flow-cytometric analysis of differentiated erythroid control and TTD cells. Lower bar graph depicts the quantification of the gated dot-plots [Q8=FSC^low/SSC^low (normal size and intracellular structures); Q5=FSC^high/SSC^low (bigger cells); Q6=FSC^high/SSC^high (bigger cells, more intracellular structures); Q7=FSC^low/SSC^high (normal size, more intracellular structure); N = 5 for each. Error bars represent standard deviation] (D) HPLC analysis of different hemoglobin variants. Bar graphs depict the percentage of hemoglobin variants: “HbF total” = sum of HbF1 and HbF2, Hb-HbH = sum of all non-aberrant hemoglobin variants, HbA2, HbE, HbH represent the peaks identified as HbH, probably representing HbBarts (beta tetramers or gamma tetramers respectively) and “Hb Total” = sum of all the peaks. Experiments represent an N = 3 for TTD and N = 6 for control iPS-derived erythroid cells. Error bars represent standard deviation and student t-test was performed to calculate significance, * <0.05 and ***< 0.01.