Porter Elizabeth 2019 4-19-19.pdf (3.58 MB)
ELUCIDATING THE ROLE OF POLYBROMO-1 IN TARGETING THE PBAF COMPLEX UNDER STRESS
thesisposted on 2019-05-15, 19:23 authored by Elizabeth G PorterElizabeth G Porter
DNA organization is an intricate and dynamic process. The approximately two meters of DNA in a single cell is wrapped around small proteins called histones. Histones can be compacted into dense coils or loosely distributed along DNA, allowing for cells to control gene expression. This combination of DNA and histones forms chromatin. This work has focused on understanding the role of Polybromo1 (PBRM1), which is a member of a chromatin remodeling complex. PBRM1 is mutated in 3% of all human cancers and is mutated in 40% of renal clear cell carcinomas (ccRCC), the most common type of kidney cancer. Through my work characterizing PBRM1 as a tumor suppressor, we have found PBRM1 acts as a stress sensor. PBRM1 is a member of the Polybromo1 BRG1 associating factors (PBAF) complex which is a subtype of the larger BAF family of chromatin remodelers. Although BAF is essential for cell viability, knockdown of PBRM1 shows minor phenotypic changes in many cell types under standard cell culturing conditions. However, when cells without PBRM1 experience external stress, the reactive oxygen species levels in the cells are elevated and remain high compared to cells with wild type PBRM1. Depending on the cellular environment of the cell, increase in ROS can be growth promoting or growth inhibiting. PBRM1 is a structurally unique protein, containing two bromo-adjacent homologs, a high mobility group and six tandem bromodomains. Due to the multiple reader domains, it is likely PBRM1 acts to target the complex. Taking advantage of a RCCC cell line not expressing PBRM1, we re-expressed full length PBRM1 containing an asparagine to alanine mutation in each bromodomain, disrupting the acetyl-lysine binding. We have found that the bromodomains are cooperative and are facilitating binding of PBAF to chromatin. We found defects in PBRM1’s ability to suppress growth, bind to chromatin, and regulate gene expression when any of the bromodomains were mutated besides the third bromodomain. These results correlated with patient data. Using acetylated histone peptides, we have identified potential combinations of marks that PBRM1 prefers over single marks. Further work needs to be done to characterize how these histone modifications are altered under stress and they contribute to the role of PBRM1 in stress response.
- Doctor of Philosophy
- Medicinal Chemistry and Molecular Pharmacology
- West Lafayette