Transcription factors constitutively bind to nucleosome-free regions of the gadd45 gene during basal and induced expression

Transcriptional upregulation of the gadd45 gene may help co-ordinate DNA repair with the cell cycle. To identify and monitor changes in DNA regions relevant to this transcriptional control, gadd45 gene expression was induced using ionizing radiation (IR) or ultraviolet radiation (UV). DNaseI was used to measure the accessibility of the gadd45 gene to proteins in the context of chromatin. Two regions hypersensitive to DNaseI (DHS) were identified in the gadd45 gene, one corresponding to the promoter, the other to an intron 3 region. Both regions were equally hypersensitive in non-irradiated and irradiated cells. These two DHS regions were analyzed using ligation-mediated PCR for DNA-protein interactions in vivo. DNA-protein interactions at two Oct-1 sites and a CCAAT box within the gadd45 promoter were identified as were DNA-protein interactions at an AP-1 site and a p53 binding site within intron 3. These interactions were present in non-irradiated and irradiated cells. Additionally, cycloheximide treatment of ML-1 cells prior to IR or UV exposure did not block increases in gadd45 mRNA levels, suggesting that protein synthesis was not required for gadd45 upregulation. These data support the idea that the rapid transcriptional upregulation of the gadd45 gene is due to DNA-damage induced activation of preformed transcription complexes.; Additionally, a putative CpG island was identified within the gadd45 gene. As my results suggested that the gadd45 gene assumes a chromatin structure that favors transcription factor binding in two regulatory regions, and that neither this chromatin structure nor transcription factor binding changes following upregulation, the chromatin structure of the gadd45 gene was investigated further. To define this chromatin structure and to determine the role of CpG island-associated chromatin structure in the regulation of gadd45 expression, the organization of nucleosomes within the gadd45 gene was investigated using MNase. These studies revealed regularly spaced nuclesome-like structures spanning the gadd45 CpG island. Taken together, these data suggested that gadd45 expression is rapidly induced not in association with gross changes in transcription factor binding or chromatin structure, but rather likely by modification of transcription factors whose binding is facilitated by the CpG island chromatin structure of the gene.