Author ORCID Identifier

Date of Graduation


Document Type

Dissertation (PhD)

Program Affiliation

Epigenetics and Molecular Carcinogenesis

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Mark T. Bedford

Committee Member

Taiping Chen

Committee Member

David G. Johnson

Committee Member

Shawn Bratton

Committee Member

Bin Wang


Post-translational modifications (PTMs) drive signal transduction by interacting with "reader" proteins. Protein domain microarray is a high throughput platform to identify novel readers for PTMs. In this dissertation, I applied two protein domain microarrays identifying novel readers for histone H2Aub1 and H2Bub1, and H3TM K4me3. Ubiquitinations of histone H2A at K119 (H2Aub1) and histone H2B at K120 (H2Bub1) function in distinct transcription regulation and DNA damage repair pathways, likely mediated by specific "reader" proteins. There are only two H2Aub1-specific readers identified and no known H2Bub1-specific readers. Using a ubiquitin-binding domain microarray, I discovered the phospholipase A2-activating protein (PLAA) PFU domain as a novel H2Bub1-specific reader. PFU domain interacts with H2Bub1 in the context of histone acid extracts but not recombinant nucleosomes, suggesting that PLAA may require additional partners for chromatin binding or PLAA only interacts with free H2Bub1. PLAA knockout cells show decreased H2Aub1 and H2Bub1, and an accumulation of a 15 kDa ubiquitin-like protein in the cytoplasm. PLAA co-localizes with laser microirradiation-induced DNA damage sites, suggesting PLAA's function in DNA damage repair. PHD fingers recognize the histone H3 N-terminal tail harboring either H3K4me3 or H3K4me0. Structural studies have identified common features among different H3K4me3 effector PHDs: Cleaved initiator methionine: a groove that fits the R2 residue, and an aromatic cage that engages the K4me3. We hypothesize that some PHDs engage with non-histone ligands whose N-termini adhere to the three rules. A search of the human proteome revealed a striking enrichment of chromatin-binding proteins, and we termed these H3 N-terminal mimicry proteins (H3TMs). We selected seven H3TMs and synthesized the methylated forms of their N-termini. Using a methyl reader microarray, we found that they can bind known PHD and Tudor H3K4me3 effector proteins. We focused on the interaction between the kinase VRK1 and the PHF2 PHD domain. Several H3TMs peptides, in their unmethylated form, interact with NuRD complex components. These findings provide in vitro evidence that methylation of H3TMs can promote novel interactions with PHD finger- and Tudor domain-containing proteins and block interactions with the NuRD complex. We propose that these interactions can occur in vivo as well.


Epigenetics, histone ubiquitination, methylation, protein-protein interactions, protein domain microarray



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