Graduation Date

Fall 12-16-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Genetics, Cell Biology & Anatomy

First Advisor

Dr. Vimla Band

Abstract

The ADA3 (Alteration/Deficiency in Activation 3) protein is a transcriptional adaptor protein that was initially discovered as a component of several HAT (Histone Acetyltransferase) complexes, the enzyme complex responsible for histone acetylation, which is a prerequisite for transcription. Earlier the studies from Dr. Band’s laboratory and that of others’ have deciphered a crucial role of ADA3 in cell cycle regulation (both through G1/S and G2/M phase transitions) and in maintaining the genomic stability.

While our laboratory investigated the mechanism behind the role of ADA3 in G1/S transition, the same remained unknown for G2/M phase transition. Based on this prior knowledge about ADA3, I started out my Ph.D. thesis work in Dr. Band’s laboratory directed towards examining the role of ADA3 in mitosis. During my doctoral research, I demonstrated that ADA3 governs the recruitment of a key centromeric protein CENP-B on to the centromeres and regulates the chromosome segregation during mitosis.

ADA3 protein has the potential to undergo posttranslational modification, including acetylation, and in the course of my Ph.D. research, I became interested in how these modifications might regulate the function of ADA3. I showed that ADA3 acetylation is regulated by coordinated actions of its associated HATs, GCN5, PCAF and p300, and a new partner I discovered, the deacetylase SIRT1. We used mass-spectrometry and site-directed mutagenesis to identify major sites of ADA3 acetylated by GCN5 and p300 and found that acetylation defective mutants were capable of interacting with HATs and other components of HAT complexes but deficient in their ability to restore ADA3-dependent global or locus-specific histone acetylation marks and cell proliferation in Ada3 deleted MEFs.

A parallel focus of my studies was to define the role of ADA3 in HER2+ breast cancers, which basically emanates from a clinical study from our laboratory that revealed that ADA3 is overexpressed/mislocalized in these types of aggressive tumors. By using cell culture models I have established a link between ADA3 and HER2 signaling pathways. In these cell lines, I found that ADA3 is a downstream target of HER2 and discovered a novel phospho-AKT-phospho-p300-Ac-ADA3 signaling pathway. Importantly, ADA3 knockdown in these cells recapitulates the cell cycle inhibitory effects of a tyrosine kinase inhibitor lapatinib such as accumulation of CDK inhibitor p27 and reduced mitotic index. Taken together these results highlight the importance of ADA3 as a marker for treatment efficacy and a promising therapeutic target. Given the key importance of ADA3-containing HAT complexes in the regulation of various biological processes, including cell cycle, my thesis work provides an insight for the regulation of the function of these complexes through dynamic ADA3 acetylation.

Download

Share

COinS