Date of Award
2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Cellular, Molecular and Biomedical Sciences
First Advisor
Robert J. Kelm
Second Advisor
Sylvie Doublié
Abstract
Purβ and Purα are the two most abundant purine-rich element binding (PUR) proteins in humans and are known to play a role in various diseases such as cardiovascular disease (CVD), repeat expansion diseases, and cancer. Moreover, mutations in the gene encoding Purα cause a severe neurodevelopmental disorder called PURA syndrome characterized by hypotonia and neurodevelopmental delay. The mechanisms by which PUR proteins contribute to these diseases are largely unknown, however, some studies have pointed to their gene regulatory activities and unique ability to bind single-stranded DNA as driving factors. This dissertation refines the structural basis for the DNA binding and gene regulatory properties of mammalian PUR proteins, elaborates the role of Purβ in CVD, and defines the genomic targets of Purα and Purβ relevant to cancer. To investigate the molecular basis of PUR protein transcription factor (TF) activity, we used a model system of mouse Purβ-mediated repression of the smooth muscle alpha actin gene (Acta2). A recent crystal structure of an arthropod PUR protein indicated that aromatic residues - stacked with nucleobases enabling DNA binding. We hypothesized that aromatic residues in mammalian PUR proteins may serve a similar function based on homology modeling. We mutated selected aromatic residues to alanine in mouse Purβ and assessed the impact on protein stability, TF activity and DNA binding. Residues Y59 and F155 were necessary for efficient repression of the Acta2 promoter and high affinity DNA binding, consistent with a key role in the molecular function of mammalian PUR proteins. Acta2 is among one of several genes encoding contractile proteins that are regulated by PUR proteins in cell types comprising the heart. Indeed, studies have implicated Purα and Purβ in CVD through modulation of cell phenotype. However, no one has yet examined the impact of Purβ loss of function (LOF) in an animal model. Therefore, we employed a mouse model of myocardial infarction (MI) to test the effect of global Purb knockout on cardiac function after MI. However, no significant differences were detected in mice devoid of Purb expression compared to wild-type mice. This finding suggests that Purβ LOF has no impact on the response of the heart to ischemic injury in mice. In addition to CVD, PUR proteins have also been implicated in cancer. However, cancer-specific regulatory targets of PUR proteins are largely unknown. We sought to determine targets of PUR proteins that contribute to cancer pathogenesis by screening the cancer cell genome for Purα and Purβ binding sites using CUT&RUN. We identified ~43,000 high confidence genomic binding sites for Purα and ~29,000 for Purβ in MCF7 human breast cancer cells. Purα and Purβ bound primarily to promoter regions supporting the TF function of these proteins. Bioinformatics analysis revealed biological processes such as cell cycle transition and DNA repair as putative targets of Purα and/or Purβ. The findings presented in this dissertation offer new insight into the structural and functional properties of mammalian Purβ and Purα. Specifically, the mutation analysis of Purβ provides direction for development of PUR protein inhibitors. The mouse studies point to the necessity of exploring the effect of Purβ LOF in other CVD models. The genomic targets and pathways identified in the CUT&RUN analysis support the investigation of Purα and/or Purβ as modulators of the response of the cancer cell to DNA damaging therapies. Collectively, this dissertation delineates a plausible roadmap for future study of the regulatory functions of Purα and Purβ in CVD and cancer.
Language
en
Number of Pages
311 p.
Recommended Citation
Foote, Andrea, "Investigating the DNA Binding and Gene Regulatory Properties of PUR Proteins and Their Contribution to Cardiovascular Disease and Cancer" (2024). Graduate College Dissertations and Theses. 1887.
https://scholarworks.uvm.edu/graddis/1887