Reduced levels of prostaglandin I2 synthase: a distinctive feature of the cancer-free trichothiodystrophy

Abstract

The cancer-free photosensitive trichothiodystrophy (PS-TTD) and the cancer-prone xeroderma pigmentosum (XP) are rare monogenic disorders that can arise from mutations in the same genes, namely ERCC2/XPD or ERCC3/XPB Both XPD and XPB proteins belong to the 10-subunit complex transcription factor IIH (TFIIH) that plays a key role in transcription and nucleotide excision repair, the DNA repair pathway devoted to the removal of ultraviolet-induced DNA lesions. Compelling evidence suggests that mutations affecting the DNA repair activity of TFIIH are responsible for the pathological features of XP, whereas those also impairing transcription give rise to TTD. By adopting a relatives-based whole transcriptome sequencing approach followed by specific gene expression profiling in primary fibroblasts from a large cohort of TTD or XP cases with mutations in ERCC2/XPD gene, we identify the expression alterations specific for TTD primary dermal fibroblasts. While most of these transcription deregulations do not impact on the protein level, very low amounts of prostaglandin I₂ synthase (PTGIS) are found in TTD cells. PTGIS catalyzes the last step of prostaglandin I₂ synthesis, a potent vasodilator and inhibitor of platelet aggregation. Its reduction characterizes all TTD cases so far investigated, both the PS-TTD with mutations in TFIIH coding genes as well as the nonphotosensitive (NPS)-TTD. A severe impairment of TFIIH and RNA polymerase II recruitment on the PTGIS promoter is found in TTD but not in XP cells. Thus, PTGIS represents a biomarker that combines all PS- and NPS-TTD cases and distinguishes them from XP.

Keywords: NER-defective disorders; PTGIS; TFIIH transcription.

Fig. 1.

Schematic representation of the genes differentially expressed in TTD7PV skin fibroblasts compared to those from the healthy TTD7PVmother. (A and B) In total, 718 and 730 genes are differentially expressed in TTD7PV fibroblasts cultured under basal condition (A) or 2 h after 10 J/m² UV irradiation (B), respectively. Red and blue bars indicate the up- and down-regulated genes, respectively. (C) Venn diagram and schematic representation of the transcript coding genes whose expression is modified following UV irradiation in TTD7PVmother (green, 332 genes) or TTD7PV (yellow, 502 genes) fibroblasts. The expression of 250 genes is commonly modified in TTD and control cells in response to UV light, whereas the transcriptional alteration of 82 and 252 genes occurs specifically in the control and patient fibroblasts, respectively. Red and blue bars indicate the up- and down-regulated genes, respectively. (D) Scatter plots in logarithmic scale representing the comparison of the 174 messenger RNA levels [log₂(−ΔCt)] found in the TTD patient RNA pool versus healthy parent RNA pool from fibroblasts cultured in basal condition (Left) and upon UV irradiation (Right). Only values with a log₂ fold change higher than ±|2| are indicated (gene names and fold change values, SI Appendix, Table S12). The previously identified deregulated gene (MMP1) in TTD cells and the most down-regulated gene found in this study (WISP2) have been pinpointed. The black line indicates fold changes of 1 (=no deregulation), whereas the pink lines correspond to log₂ fold changes ±|2|.

Fig. 2.

Gene expression deregulation in XP-D cells. (A and B) Transcription profile of the UV-responsive genes EGR1, ID3, c-Jun, IER3, ANGPTL4, GADD45B, and GADD45A (A) and the UV-unresponsive genes IL20RB, PTGIS, JunD, CLU, WISP2, WNT4, and ID1 (B). Graphs indicate the mean values of the transcript levels analyzed by real-time RT-PCR in single-strain fibroblasts from healthy control (CTR; namely, TTD12PV-15PV parents, TTD11PV parents, and TTD7PV parents; black bars), TTD/XP-D (namely, TTD12PV, TTD15PV, TTD11PV, TTD7PV, TTD23PV, TTD20PV, and TTD8PV; red bars), and XP/XP-D (XP15PV, XP-16PV, XP17PV, XP49BR, and XP1BR; blue bars) individuals. Fibroblasts were cultured under basal condition (not treated [NT]; solid bars) or after UV irradiation (UV; empty bars). The single-strain values normalized to the expression of the GAPDH housekeeping gene are indicated in SI Appendix, Figs. S2 and S3. The reported values represent the means of at least two independent experiments, whose samples have been analyzed in triplicate (*P < 0.05, **P < 0.01, and ***P < 0.001; Student’s t test; only the significant differences are shown). Bars indicate the SEs.

Fig. 3.

PTGIS protein amount in XP-D human and mouse cell/tissues. (A–D) Immunoblot analysis of PTGIS in total protein extracts from (A) healthy human control (C3PV and TTD12-15PV parents; black empty bars) and TTD/XP-D (TTD12PV and TTD15PV; black solid bars) primary skin fibroblasts cultured under basal condition (NT) and 2 h after UV irradiation; (B) healthy control (C3PV; black empty bar), TTD/XP-D (TTD20PV and TTD23PV; black bars) primary skin fibroblasts cultured under basal condition (NT) and 2 h after UV irradiation; (C) healthy control (C3PV; black empty bar) and TTD/XP-D (TTD11PV, TTD24PV, and TTD35PV; black solid bars) primary skin fibroblasts; and (D) healthy controls (C3PV, mother and father of XP15-16PV siblings; black empty bars) and XP/XP-D (XP15PV and XP16PV; gray bars) fibroblasts cultured in basal condition (NT) and 2 h after UV irradiation. The signals were quantified and normalized to the γ-tubulin amount. (E and F) Immunoblot analysis of PTGIS in total protein extracts from (E) wild-type (WT; white bars) and PS-TTD/XP-D (black bars) MEF; (F) WT (black empty bars) and TTD/XP-D (black solid bars) mouse skin. γ-tubulin was used as the loading control. The diagram below each blot represents the mean values of at least three independent experiments. Bars indicate SEs. Arbitrary unit, au.

Fig. 4.

PTGIS deregulation in PS-TTD and NPS-TTD. (A) Graphs indicate the mean values of the transcript levels analyzed by real-time RT-PCR in single-strain fibroblasts from healthy controls (CTR; white bars), TTD (PS-TTD/XP-D, PS-TTD/XP-B, PS-TTD/TTD-A, NPS-TTD/MPLKIP, NPS-TTD/GTF2E2, and NPS-TTD/TARS1; black bars), and XP-XP-D (gray bar). The single-strain values were normalized to the expression of the GAPDH housekeeping gene. The reported values represent the means of at least two independent experiments, whose samples have been analyzed in triplicate. Bars indicate the SEs. (B) Immunoblot analysis of PTGIS in total protein extracts from healthy control (TTD12-15PV father and C3PV; white bar), PS-TTD (TTD6VI/XP-B, TTD12PV/XP-D, TTD14PV/TTD-A, TTD1BR/TTD-A, and TTD28PV/TTD-A; black bars), and NPS-TTD (TTD16PV/MPLKIP, TTD31PV/MPLKIP, TTD379BE/GTF2E2, TTD18PV/TARS1, and RD251/CARS1; black bars) skin fibroblasts. The signals were quantified and normalized to the γ-tubulin amount. The diagrams below represent the mean values of at least three independent experiments. Bars indicate SEs. (C) Schematic organization of the genomic fragment included between nucleotides −3,000 + 5,571 of PTGIS gene (GenBank accession number NG_007940.1). The spotted and tessellated boxes are the CT-rich and GC-rich regions of PTGIS promoter, respectively. Introns are shrunk to a minimal length. Exons are indicated with solid black boxes. The light gray box indicates the 3′ untranscribed region. Horizontal broken lines delimitate the position of the fragment amplified by quantitative ChIP assays (amplicon location: I +205/+324). (D) RNAP IIA and TFIIH (XPB) occupancy on the proximal intron of PTGIS promoter in healthy (white bars), PS- and NPS-TTD (black bars), and XP/XP-D (gray bar) primary dermal fibroblasts. The values are the mean of at least two independent experiments (**P < 0.005, ***P < 0.001; Student’s t test).