Graduation Year

2020

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Jiandong Chen, Ph.D.

Committee Member

Jianfeng Cai, Ph.D.

Committee Member

Lixin Wan, Ph.D.

Committee Member

Eric Lau, Ph.D.

Committee Member

Gary W. Daughdrill, Ph.D.

Keywords

IDR, MDMX, NT, p53, T55

Abstract

The p53 tumor suppressor is a sequence-specific DNA binding protein that activates gene transcription to regulate cell survival and proliferation. The activation process involves post-translational modifications that suppress p53 degradation by MDM2 and increase p53 DNA binding affinity. p53 is mutated in ~50% of human tumors, with higher frequency in specific tumor types and after relapse. Mutated p53 loses transcriptional activity and gains new functions that drive tumor progression. Both N-terminus (NT) and C-terminus (CT) of p53 contain intrinsically disordered regions. The p53 CT has well-documented effects in regulating DNA binding. CT truncated p53 mutants showed defective DNA binding and transcriptional activities. However, the effects of NT on p53 DNA binding and its underlying mechanisms are elusive. Hence, this study is to elucidate the role of the p53 NT as an intrinsically disordered region in p53 regulation.

The goal of the first part of this study was to investigate the effects of NT on p53 DNA binding and its molecular mechanisms. We showed that transactivation domain 2 (TAD2) and proline-rich region (PRR) inhibit DNA binding by directly interacting with the sequence-specific DNA binding domain (DBD). NMR spectroscopy revealed that TAD2 and PRR interact with the DBD at or near the DNA binding surface, possibly acting as a nucleic acid mimetic to competitively block DNA binding. In vitro and in vivo DNA binding analyses showed that the NT reduced p53 DNA binding affinity but improved the ability of p53 to distinguish between specific and nonspecific sequences.

The second part of this study was to explore the regulation and significance of the NT-DBD interaction in both wild-type and mutant p53. MDMX inhibits p53 binding to specific target promoters but stimulates binding to nonspecific chromatin sites. The phosphomimetic mutations of T55 alter the intramolecular interactions in p53. Furthermore, T55 phosphorylation is induced by DNA damaging compounds and T55A mutant p53 manifests defective growth suppression activities. The results suggest that p53 NT regulates the affinity and specificity of DNA binding by the DBD. p53 NT-interacting proteins and post-translational modifications may regulate DNA binding partly by modulating the NT-DBD interaction in p53. Also, we demonstrated stronger NT-DBD interaction in mutant p53, especially the conformational mutations.

Overall, our results established a novel form of p53 regulation via the NT-DBD interaction in p53, highlighting the importance of N terminus in p53 DNA binding and function.

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