KNOCKDOWN OF USF LEADS TO LONG-TERM CHANGES IN THE GENE EXPRESSION RESPONSE TO DNA DAMAGE
Kimberly Bellingham-Johnstun, North Carolina State University
Kimberly Bellingham-Johnstun1, John King1, Kevin Gerrish2, Pierre Bushel3, and Michael
1. Department of Biological Sciences, North Carolina State University, Raleigh, NC
2. Molecular Genomics Branch, National Institute of Environmental Health Sciences, Durham, NC
3. Computational Biology Branch, National Institute of Environmental Health Sciences, Durham, NC.
After receiving genotoxic insult, cells rapidly activate P53-dependent programs that pause the cell cycle and direct either DNA repair or apoptosis. Beyond P53, the long-term transcriptional programs that direct either cell recovery or senescence, depending on the success of DNA repair, are unclear. In this study, we used RNAi to investigate the potential of USF1 and USF2 stressresponse transcription factors as directors of long-term DNA damage responses in the P53-deficient mouse B lymphocyte cell line, M12. Microarray analysis revealed 765 differentially expressed genes (≥1.50-fold change, n=3) in USF-depleted cells when compared with cells expressing a scrambled shRNA. In contrast, 7 days after cells were exposed to ionizing radiation, 2866 genes now showed altered expression in the USF-depleted cells. Microarray findings were recapitulated in separate biological replicates, and further confirmed for a panel of constitutively altered and IR-induced genes by RT-qPCR. In particular, USF-depletion led to the up-regulation of a number of genes critical for immune function and strongly linked to cancer development, including genes for the DNA mutator AID, the CD300a lipid receptor, the Ig kinases BLK and BLNK, and multiple members of the NFκB transcription factor family REL-A, REL-B, c-REL, IκBα and IκBδ. Together, our results provide the first evidence of a novel role for USF in regulating long-term response to DNA damage, while loss of USF results in overexpression of genes associated with B cell activation and tumorigenesis.