Date of Degree

9-2022

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Jill Bargonetti

Committee Members

Jayne Raper

Kevin Gardner

Wenwei Hu

Olorunseun Ogunwobi

Subject Categories

Biochemistry | Cancer Biology | Cell Biology | Molecular Biology

Keywords

p53, oligomerization, activation, transcription, tetramer, replication

Abstract

The tumor suppressor p53 (TP53) gene is often mutated in cancer, with missense mutations found in the central DNA binding domain, and less often in the oligomerization domain (OD) and C-terminal domain (CTD). The OD and CTD have been found to be critical for the tumor suppressor functionality of wild-type p53 (wtp53). Specific missense mutations in the DNA binding domain have been found to confer new gain-of-function (GOF) activities. Mutations that destabilize tetramer formation, or deletion of key lysine residues within the CTD, downregulate the ability of wtp53 to transactivate (increase the rate of transcription of) its target genes. We previously found that mutant p53 (mtp53) R273H, which has lost tumor suppression function and has GOF, associates with replicating DNA and promotes the chromatin association of replication-associated proteins mini-chromosome maintenance 2 (MCM2), and poly ADP-ribose polymerase 1 (PARP1). Herein, we created dual mutants to test if the oligomerization state and/or deletion of the CTD of mtp53 R273H played a role in chromatin binding oncogenic gain-of-function (GOF) activities. We used site-directed mutagenesis to introduce point mutations in the OD of wtp53 (R337C, A347D, L344P), and mtp53 R273H (R273H-R337C, R273H-A347D, R273H-L344P), expressing plasmids. We also used the CRISPR/Cas9 gene editing system to generate two endogenous mutants in MDA-MB-468 cells which encode mtp53 R273H. One with deletion within the mtp53 CTD (R273HΔ381-388) and the other an OD-CTD deletion mutant (R273HΔ347-393). The glutaraldehyde crosslinking assay revealed that both exogenously expressed wtp53 and mtp53 R273H formed predominantly tetramers, while the single OD mutant A347D, and the dual mtp53 R273H-A347D, formed predominantly dimers. The R337C, L344P, mtp53 R273H-R337C, and mtp53 R273H-L344P proteins formed predominantly monomers. The exogenously expressed mtp53 R273H and the dual OD mutants, R273H-A347D, R273H-R337C, and R273H-L344P were able to interact with chromatin. They were also found to be in close nuclear proximity to PARP1 and MCM2 via the proximity ligation assay (PLA). The MDA-MB-468 endogenously expressed mtp53 R273HΔ381-388 and R273HΔ347-393 exhibited decreased chromatin interaction and less interaction with PARP1 and MCM2 as compared to the full-length R273H. In vivo mouse xenograft model experiments with MDA-MB-468 cells orthotopically implanted into NOD scid gamma (NSG) mice demonstrated that R273HΔ347-393 mtp53, and a mtp53 knockdown cell line R273Hfs387, formed smaller tumors and less circulating tumor cells compared to the parental cells. This suggested that the absence of the CTD, or depletion of mtp53 R273H, decreases GOF R273H mediated tumor formation with a significant decrease in the levels of circulating tumor cells assessed via flow cytometry. Our findings suggest that while mtp53 R273H can form tetramers, tetramer formation is not required for the GOF-associated chromatin interactions, rather residues within the CTD are critical for the observed GOF mtp53 oncogenic abilities.

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