Investigation of a novel Ercc1 transcript predominant in skin

Abstract

The integrity of an organism's genome is constantly threatened due to the damaging effects of external and internal environmental factors. Additionally, the DNA molecule also has an inherent chemical instability. All these factors contribute to the formation of DNA lesions which, if not repaired, could develop into permanent mutations and malignancy. Fortunately, many DNA repair systems exist to prevent the accumulation of lesions. Of these, the nucleotide excision repair system (NER) is capable of repairing the greatest range of lesions, including those caused by UV irradiation, cross -linking agents and free radicals. Defects in the NER pathway disrupt DNA repair and one human disorder characterised by defective NER is xeroderma pigmentosum (XP). Exposure to UV is extremely dangerous for individuals with XP since their disorder predisposes them to a greatly increased risk of developing skin cancer. The gene ERCC1, which has an essential role in the NER pathway, has not been linked to a human disorder but mice in which the Erccl gene has been inactivated are DNA repair deficient. These mice die, prior to weaning, of liver failure.Earlier work in the Melton laboratory had demonstrated that there was tissue-specific Erccl expression in the mouse and an additional novel, Erccl transcript was identified in the skin. The skin has a higher biological demand for repair of UV damaged DNA than other body tissues. As Erccl is required for repair of UV damaged DNA the novel transcript may be involved in meeting the increased demand for repair in this tissue. The difference between the skin-specific and normal transcripts was localised to the 5' end and is caused by differential initiation of transcription, with the skin-specific transcript initiating 5' to the normal transcript's promoter. This indicated the likely presence of a separate skin-specific promoter. To facilitate more detailed analysis of the upstream promoter region and the identification of sequences responsible for regulation of the skin -specific Erccl expression pattern a number of Erccl minigenes were constructed.In this project the skin-specific pattern of expression was studied by transfecting Erccl null cells with the minigenes. The Erccl null cells used were murine embryonic fibroblasts, PF24 + #2D -4; Chinese hamster ovary cells, CH043.3B; and murine keratinocytes, Ker. ( -/ -). UV survival studies were performed on transfected cells and RT -PCR was used to study the pattern of Erccl expression. Transfection with the Erccl minigenes corrected the UV sensitivity of the null cell lines. The Erccl expression pattern was usually, but not always, appropriate for the cell type transfected.Comparisons between the Erccl transcription of in vitro cultures and in vivo tissues taken from various stages of murine development were made by means of northern blotting. Irradiation of transfected cells and primary cultures with UV -B, UV -C and visible light was performed in an attempt to of transcript induction. Changes in Erccl transcription following irradiation of primary cultures were observed, but these changes did not conclusively prove that UV could induce Erccl transcription. Serum starvation experiments were performed upon primary cultures to further study differences in Erccl transcription in vitro. Release from serum starvation resulted in increased skin -specific transcript production and total Erccl expression. The size of the skin -specific Erccl transcript in keratinocytes was found to be larger than that of fibroblasts. Sequencing demonstrated that the size difference was in the CT repeat region of the gene. This region was deleted from one of the Erccl minigenes mentioned and results indicated that it may be required for correct expression of the novel transcript.