UV-induced inhibition of transcription involves repression of transcription initiation and phosphorylation of RNA polymerase II

Rockx, Davy; Mason, Rebecca; Hoffen, Anneke van; Barton, Michelle C.; Citterio, Elisabetta; Bregman, David B.; Zeeland, A.A. van; Vrieling, Harry; Mullenders, Leon H.F.

doi:10.1073/pnas.180169797

Cited by 183 publications

(180 citation statements)

References 29 publications

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“…The dephosphorylation was substantially inhibited in UV S S-A cells 4-6 h after UV-irradiation (Fig. 4c, N20-antibody, lanes 1-4), as previously reported for CS cells 24 , and was restored following expression of the UVSSA cDNA (Fig. 4c, N20-antibody, lanes 5-8).…”

Section: Vhs-domain-proteins Have Been Recently Implicated In Ubiquitsupporting

confidence: 86%

Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair

et al. 2012

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We initially performed exome-sequencing 11 of the two UV S S-A patients, Kps3 and XP24KO (details described in Methods, Supplementary Note, Table 2c). The patients were homozygous for c.367A>T mutation in UVSSA, which led to a premature termination, p.Lys123* (Fig. 1a, b). We identified the same homozygous mutation in Kps2 (sib. of Kps3), and a homozygous c.87delG, causing a frameshift p.Ile31Phefs*9, in an Israeli patient UV S S24TA (Fig. 1b, c, Supplementary Note, Supplementary Fig. 1). The identified mutations are summarized in Fig. 1d. We did not detect the 80kDa UVSSA protein in any of the UV S S-A patients (Fig. 1e). We additionally examined several mild xeroderma pigmentosum (XP) cases; in one such case, XP70TO 12 (Supplementary Table 1), we identified a homozygous p.Cys32Arg, in the UVSSA (Fig. 1c, d), implying that XP70TO is also in the UV S S-A group. The mutant protein was stably expressed in XP70TO cells (Fig. 1f, Supplementary Fig. 2a-d). 4Allele frequencies of the identified mutations in a control population were examined (Supplementary Note, Supplementary Fig. 3a). Haploinsufficiency for UVSSA is negligible as the parents of Kps2/Kps3 showed no symptoms 4 . In parallel with exome-sequencing, we performed whole-genome SNP-genotyping to identify runs-of-homozygosity (ROH) shared among the patients. We identified three overlapping-ROHs (> 1Mbps) on autosomes, one of which encompasses the UVSSA locus (Fig. 1g, Supplementary Table 3a, b, Supplementary Fig. 3b, c). No chromosome copy number variation was detected (Supplementary Fig. 3d).The above findings strongly suggest that the mutations in UVSSA in the UV S S-A patients are causal for the disease; we therefore, next examined the NER-activities in the UV S S-A cells (Fig. 2). Unscheduled-DNA-synthesis (UDS 13 , defective in XP) was nearly normal; however, RNA-synthesis-recovery (RRS 14 , defective in UV S S and in CS) was reduced in all cell-strains mutated in UVSSA ( Fig. 2a, b; UDS and RRS were measured using a recently-developed rapid non-radioactive system 15,16 ). Similarly, siRNA-based depletion of the UVSSA gene (Fig. 2c) caused a drastic reduction of RRS (Fig. 2d, Supplementary Fig. 4), whereas UDS was unaffected (Fig. 2e). Ectopic-expression of the wild-type UVSSA cDNA in UV S S-A cells restored normal RRS ( Fig. 2f; V5-tagged-UVSSA immunofluorescent-staining shown in Fig. 2g), while it did not affect RRS-level in normal, CS-A, or CS-B cells; neither ERCC8 nor ERCC6 cDNA expression in UV S S-A cells restored the RRS-level.We conclude that KIAA1530/UVSSA is the causal gene for UV S S-A.ERCC8 and ERCC6 genes are responsible for both CS and UV S S 7,8 . To evaluate whether UVSSA mutations may also result in CS-phenotypes, we sequenced 5 the UVSSA gene of 61 CS-patients whose genetic defects had not yet been determined (Supplementary Table 4). We found no obvious mutations except for four novel heterozygous changes. These changes as well as the SNPs, also found in control and UV S S-A individuals, do not affect the RRS-activity (Suppleme...

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Section: Vhs-domain-proteins Have Been Recently Implicated In Ubiquitsupporting

confidence: 86%

Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair

et al. 2012

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“…One possibility is that transcriptional arrest may only be a passive consequence of lesions located in the transcribed strand, blocking RNA elongation. However, the reduction in RNA synthesis may also be caused by the repression of transcription initiation 19 and RNA polymerase II degradation. 14 base pairs around CPD.…”

Section: Discussionmentioning

confidence: 99%

Photorepair of RNA polymerase arrest and apoptosis after ultraviolet irradiation in normal and XPB deficient rodent cells

et al. 2002

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Cyclobutane pyrimidine dimers (CPDs) are directly involved in signaling for UV-induced apoptosis in mammalian cells. Failure to remove these lesions, specially those located at actively expressing genes, is critical, as cells defective in transcription coupled repair have increased apoptotic levels. Thus, the blockage of RNA synthesis by lesions is an important candidate event triggering off active cell death. In this work, wild-type and XPB mutated Chinese hamster ovary (CHO) cells expressing a marsupial photolyase, that removes specifically CPDs from the damaged DNA, were generated, in order to investigate the importance of this lesion in both RNA transcription blockage and apoptotic induction. Photorepair strongly recovers RNA synthesis in wild-type CHO cell line, although the resumption of transcription is decreased in XPB deficient cells. This recovery is accompanied by the prevention of cells entering into apoptosis. These results demonstrate that marsupial photolyase has access to CPDs blocking RNA synthesis in vivo, and this may be affected by the presence of a mutated XPB protein.

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“…One possibility is that the primary defect in CS cells might be related to a defect in transcription initiation rather than a defect in TC-NER. In vitro transcription assays revealed that transcription initiation is persistently inhibited in extracts of UV-irradiated CS-B cells due to depletion of the transcription initiating RNA polymerase IIa (RNAPIIa) [59]. Proietti-De-Sanctis et al showed a defective reinitiation of transcription of housekeeping genes in CS-B cells due to impairment of transcription initiation complex formation [37].…”

Section: Tc-ner and Transcription Response: A Complex Relationshipmentioning

confidence: 99%

“…The interaction between CSB and the transcription machinery was stabilized by DNA damage [73]. Moreover, recruitment of CSB to chromatin was obvious from fractionation of nuclear extracts prepared from UV-irradiated cells [59,41]. ChIP analysis of chromatin-bound RNAPIIo isolated from in vivo crosslinked cells [41], confirmed the previously reported observations and provided direct evidence for the association of CSB with RNAPIIo in the absence of DNA damage; in contrast, CSA was not found to interact with RNAPIIo consistent with in vitro [74] and in vivo data [41].…”

Section: Cs Proteins Associate With Uv-stalled Rnapiiomentioning

confidence: 99%

Transcription-coupled nucleotide excision repair in mammalian cells: molecular mechanisms and biological effects

2008

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The encounter of elongating RNA polymerase II (RNAPIIo) with DNA lesions has severe consequences for the cell as this event provides a strong signal for P53-dependent apoptosis and cell cycle arrest. To counteract prolonged blockage of transcription, the cell removes the RNAPIIo-blocking DNA lesions by transcription-coupled repair (TC-NER), a specialized subpathway of nucleotide excision repair (NER). Exposure of mice to UVB light or chemicals has elucidated that TC-NER is a critical survival pathway protecting against acute toxic and long-term effects (cancer) of genotoxic exposure. Deficiency in TC-NER is associated with mutations in the CSA and CSB genes giving rise to the rare human disorder Cockayne syndrome (CS). Recent data suggest that CSA and CSB play differential roles in mammalian TC-NER: CSB as a repair coupling factor to attract NER proteins, chromatin remodellers and the CSA-E3-ubiquitin ligase complex to the stalled RNAPIIo. CSA is dispensable for attraction of NER proteins, yet in cooperation with CSB is required to recruit XAB2, the nucleosomal binding protein HMGN1 and TFIIS. The emerging picture of TC-NER is complex: repair of transcription-blocking lesions occurs without displacement of the DNA damage-stalled RNAPIIo, and requires at least two essential assembly factors (CSA and CSB), the core NER factors (except for XPC-RAD23B), and TC-NER specific factors. These and yet unidentified proteins will accomplish not only efficient repair of transcription-blocking lesions, but are also likely to contribute to DNA damage signalling events.

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UV-induced inhibition of transcription involves repression of transcription initiation and phosphorylation of RNA polymerase II

Cited by 183 publications

References 29 publications

Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair

Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair

Photorepair of RNA polymerase arrest and apoptosis after ultraviolet irradiation in normal and XPB deficient rodent cells

Transcription-coupled nucleotide excision repair in mammalian cells: molecular mechanisms and biological effects

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