The Transcriptional Regulation And Function Of The Checkpoint Protein Nbs1

Nbs1, whose gene mutated in Nijmegen Breakage Syndrome patients, is an important factor in cells in response to DNA damages especially DNA double strand breakage (DSB). Nbs1 plays important roles in DSB detection, DNA damage responses, cell cycle checkpoints, DNA damage repair and DNA replication. It therefore makes crucial contributions to the maintenance of genome stability and integrity, as well as cell survival. Although it has been reported that c-Myc regulates Nbs1 gene expression, the molecular mechanisms governing Nbs1 transcription is largely unknown. c-Myc is a ubiquitous transcription factor that regulates a wide variety of genes involved in the control of cell proliferation, growth, apoptosis, energy metabolism and differentiation. Abnormal c-Myc expression is also critical to carcinogenesis and cancer treatment. However, the molecular mechanisms by which c-Myc affects the sensitivity of cancer cells to anti-cancer drugs are also unclear. In this thesis, we uncovered the transcriptional regulation of Nbs1 gene expression and the significance of the mechanism, and investigated the relationship of c-Myc to the Nbs1 related DNA repair and the drug sensitivity of cancer cells. Our results confirmed that Nbs1 is a target gene of c-Myc. The two E-box elements located in the Nbs1 promoter are necessary for the binding by c-Myc. Our results also demonstrated that histone acetyltransferase p300 is involved in regulating Nbs1 gene expression. Overexpression of p300 increased Nbs1 transcription. Results obtained from chromatin immunoprecipitation (ChIP) assay revealed that c-Myc can recruit p300 complex to the promoter region of Nbs1 gene, resulting in an increased level of acetylated histone H4 in the region. Through point mutation and chromatin immunoprecipitation assay, we confirmed that the two E-box elements located in the Nbs1 promoter are also important for c-Myc to recruit p300. Through colony formation assay, we found that overexpression of c-Myc or Nbs1 attenuated the ability of doxorubicin in reduction of colony formation in U2OS cells. By using the small RNA interference and compensation experiments, we found that the attenuating effect of c-Myc is associated with the fact that c-Myc regulates Nbs1. And we propose that the increased ability of DNA damage repair may be a main cause of the attenuating effect of c-Myc on the efficacy of doxorubicin. We also found that the Nbs1 RNAi is more potent than c-Myc RNAi in increasing doxorubicin sensitivity in the cells overexpressing c-Myc. Our study, for the first time, elucidated the mechanisms by which c-Myc regulates Nbs1 gene expression, and the mechanisms by which c-Myc affects the drug resistance of U2OS cells, from a point of view of DNA damage repair. Our findings will be helpful for cancer treatment.