Author ORCID Identifier

0000-0002-3328-915X

Date of Graduation

5-2022

Document Type

Dissertation (PhD)

Program Affiliation

Biomedical Sciences

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Margarida I. Albuquerque Almeida Santos, Ph.D.

Committee Member

Mark T. Bedford, Ph.D.

Committee Member

Jeffrey N. Myers, M.D., Ph.D., F.A.C.S

Committee Member

Joya Chandra, Ph.D.

Committee Member

Sean M. Post, Ph.D.

Abstract

Plant Homeodomain Finger Protein 20 (PHF20) and its homolog PHF20 Like 1 (PHF20L1) are known subunits of the Non-Specific Lethal (NSL) complex, which acetylates lysine residues on histone H4 and regulates gene expression. The current model assumes that PHF20 and PHF20L1 are present together in the NSL complex, although it has never been tested. Performing extensive biochemical analysis, we observed that PHF20 and PHF20L1 were exclusively and independently associated with the NSL complex. Our protein domain analysis showed that the C-termini of PHF20 and PHF20L1 are crucial for their interactions with the respective complexes. Furthermore, enrichment sites of PHF20 and PHF20L1 on chromatin were also identified by chromatin-precipitation (ChIP) followed by next-generation sequencing (ChIP-seq) and mapped to the promoters of genes that were highly expressed in cells. Results from ChIP followed by quantitative RT-PCR (ChIP-qPCR) for PHF20 or PHF20L1 carrying mutated protein domains indicated that the Tudor 2 domains are required for their chromatin binding. However, when PHF20 and/or PHF20L1 were lost, their target genes' transcription, the NSL complex's recruitment, and the histone H4 acetylation at target genes’ promoters remained unchanged. In short, we defined two distinct NSL complexes: one with PHF20 and one with PHF20L1. This finding challenges the current model that the NSL complex contains both homologs and lays a foundation for further studies on the differential roles of PHF20 and PHF20L1 in regulating the NSL complex in both physiological and diseased states.

Keywords

transcriptional regulation, epigenetics, histone acetylation, lysine methylation, housekeeping genes

Available for download on Friday, January 27, 2023

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