Local UV-induced DNA damage in cell nuclei results in local transcription inhibition

Abstract

UV-induced DNA damage causes cells to repress RNA synthesis and to initiate nucleotide excision repair (NER). NER and transcription are intimately linked processes. Evidence has been presented that, in addition to damaged genes, undamaged loci are transcriptionally inhibited. We investigated whether RNA synthesis from undamaged genes is affected by the presence of UV damage elsewhere in the same nucleus, using a novel technique to UV irradiate only part of a nucleus. We show that the basal transcription/repair factor TFIIH is recruited to the damaged nuclear area, partially depleting the undamaged nuclear area. Remarkably, this sequestration has no effect on RNA synthesis. This result was obtained for cells that are able to carry out NER and for cells deficient in NER. We conclude that cross talk between NER and transcription occurs only over short distances in nuclei of living cells.

Fig. 1. (A) Detection of locally induced UV damage in cell nuclei. A UV-blocking polycarbonate filter containing pores of 5 µm in diameter was used to cover a monolayer of cells. The filter-covered cells were UV irradiated with 30 J/m² and CPDs were subsequently detected by immunofluorescent labelling. Dotted lines denote the contours of individual cell nuclei. Two nuclei show labelling of CPDs in discrete nuclear areas. (B) Effect of local nuclear UV damage on the distribution of TFIIH. Human primary fibroblasts were locally UV irradiated with 100 J/m² UV light, using a filter with 10 µm pores. Following irradiation, cells were grown for 1 h and immunolabelled against the p62 subunit of TFIIH. The top-left nucleus displays the characteristic labelling pattern of TFIIH in unirradiated cells, whereas the two remaining nuclei exhibit a TFIIH accumulation in UV-damaged nuclear areas, and a reduction in TFIIH signal outside these areas. A single confocal optical section is shown. (C) Colocalization of TFIIH (green) and CPDs (red). Human primary fibroblasts were locally irradiated with 30 J/m² UV light, using a filter with 8 µm pores. Following irradiation, cells were grown for 30 min and dual labelled against both CPDs and the p62 subunit of TFIIH. Bars represent 10 µm.

Fig. 2. Effect of local nuclear UV damage on transcription. Normal human primary fibroblasts (WT; NER-proficient) and immortalized primary fibroblasts from patients suffering from xeroderma pigmentosum group A (XP-A; no NER), group C (XP-C; no GGR) or Cockayne syndrome group B (CS-B; no TCR) were studied. Exponentially growing cells were locally UV irradiated with 50 J/m² using filters containing 10 µm pores. After irradiation, cells were cultured for either 1 or 12 h. Subsequently, cells were allowed to incorporate BrUTP into nascent RNA during run-on transcription labelling. Nascent RNA (green) and CPDs (red) were dual labelled by immunostaining. Single confocal optical sections are shown. Bars represent 5 µm.