关键词: 连接介导PCR, DNaseⅠ高敏感位点, egfr基因, 启动子

Abstract: Objective To predict the binding sites of transcription factors in DNaseⅠ hypersensitive site(DHS) of epidermal growth factor receptor(EGFR) gene promoter and to elucidate the regulation mechanism of egfr gene transcription, we used a new molecular technique to locate the precise DNaseⅠ cutting sites. Methods Cervical cancer cell line HeLa and breast cancer cell line MDA-MB-231, which are both positive for EGFR were tested; while leukemia cell line K562, which is EGFR negative, was used for the negative control. The HeLa, MDA-MB-231 and K562 cell nuclei were all treated with DNaseⅠ at the concentrations of 5 kU/L and 10 kU/L separately. Then ligation-mediated PCR(LM-PCR) technique was performed as follows. DNA extracted from DNase Ⅰ-treated or non-treated cell nuclei were filled-in to form blunt ends, and were ligated to the adaptors. The target DNA was then amplified by nest PCR, using the adaptors-specific primers and egfr promoter-specific primers. DHSs were predicted by the length of nest PCR products. Ten clones from each PCR products were randomly picked up and sequenced to confirm that the products were from egfr gene promoter. After analysis of the sequencing results, the precise DNaseⅠ cutting sites in the promoter of egfr gene were obtained. To further predict the binding sites of the transcription factors in DHS, we used bioinformatics software called transcription element search software(TESS) to analyze potential transcription binding sites on DHS. Results After the analysis of sequencing results, we have detected eight different precise DNaseⅠ cutting sites in the egfr gene promoter of HeLa and MDA-MB-231, but no specific DNaseⅠ cutting sites was found in the egfr gene promoter of K562. In the egfr gene promoter of HeLa and MDA-MB-231, the DHS distribution was similar, mainly located from 300 bp to 500 bp (-300 bp~-500 bp ) upstream of the transcription start site, which may be the key regulation region of the egfr transcription. Results showed that 16 transcription factors possibly bind to DHS in the promoter of egfr gene in the HeLa and MDA-MB- 231 cell lines. Among these transcription factors predicted by TESS, three of them, namely Sp1, TCF and YY1 were already reported by others. In our experiment, we found eight DNaseⅠ cutting sites in the egfr gene promoter in the HeLa and MDA-MB-231, but no specific site was found from the K562 cells. The results showed that in the DHS of EGFR⁺ cells, the chromatin conformation were looser and more accessible to the transcription factors, but which were not in the EGFR^- cells. By analyzing the sequence of DHS, we predicted more potential transcription factors possibly binding to DHS in the promoter of egfr gene. Conclusion We have established a simple method to detect the DHS in gene promoter region. Meanwhile, we can obtain more precise DHS location using this method. This method will be useful for the research of the regulation mechanism of specific gene transcription.

Key words: ligation-mediated PCR(LM-PCR), DNaseⅠhypersensitive site, egfr gene, promoter