Date of Award
Bachelor of Science
Cells use multiple distinct pathways to respond to cellular stress depending on context and the particular stress. The integrated stress response pathway (ISR) controls the cellular response to numerous types of extrinsic and intrinsic stressors, such as exposure to environmental toxins and viral infection, through modulation of gene expression. Transcription factors of the ISR pathway promote the restoration of intracellular homeostasis or programmed cell death if homeostasis cannot be restored. These transcription factors act spatially and temporally to control the activity of regulatory regions such as enhancers and promoters. We aimed to study how promoters of genes activated by the ISR, such as the common stress response gene ATF3, are regulated in response to stress. To this end, we sought to define the most plausible promoter sequence of the ATF3 gene and determine the transcription factors that regulate this sequence. The boundaries and important sequences within a promoter are often uncertain, therefore we used several techniques such as mutagenesis, molecular cloning, and reporter gene assays, to measure the transcriptional activity of various candidate promoter sequences. We determined a 1340 bp region (hereafter called “exon”) to represent the entirety of the ATF3 promoter as it had the most robust transcriptional response. Both shorter and longer regions lacked robust responses, including a previously published putative promoter region (Kilberg). Utilizing these two constructs, we additionally aimed to recognize the functions of several transcription factors such as ATF4 and TFAP2A/2B in response to cellular stress and their role within the ISR pathway. ATF4 and TFAP2A/2B’s contribution to transcriptional activity of the ATF3 promoter was measured and analyzed utilizing wildtype and mutant promoter variants in human colorectal cancer wildtype cells (HCT116WT). Results suggest that one ATF4 binding site, the critical amino acid response element (CARE), functions as an inducer of promoter activity when under stress conditions whereas ATF/CRE, as a common target site among the transcriptional ATF family, is responsible for basal levels of activation. Mutations to the TFAP2A/2B motif resulted in no significant change compared to the wild-type, suggesting that in this context, these transcription factors are unlikely to contribute to promoter activity. These results continue to highlight the importance of context, such as cell type and the particular cell stress, in the regulation of promoter and enhancer elements.
Merchant, Lauren, "Determining Regulatory Sequences of the ATF3 Promoter within the Integrated Stress Response Pathway" (2021). Biological Sciences. 71.