Date of Graduation

Spring 5-15-2025

Document Type

Thesis

Degree Name

Master of Science in Biology

College/School

College of Arts and Sciences

Department/Program

Biology

First Advisor

Christina Tzagarakis-Foster, Ph.D.

Second Advisor

Cary Lai, Ph.D.

Third Advisor

Sangman Kim, Ph.D.

Abstract

The DAX-1 gene (Dosage-Sensitive Sex Reversal, Adrenal Hypoplasia Congenita, Critical Region on the X chromosome, gene 1) encodes for an orphan nuclear hormone receptor and its mutation is implicated in multiple diseases including congenital adrenal hypoplasia, adrenal cancer, and breast cancer. Previous research has linked DAX-1 downregulation to tumor initiation in breast tissue, suggesting the gene acts as a tumor suppressor with respect to breast cancer. Additional studies completed by the Tzagarakis-Foster laboratory have shown that methylation of the DAX-1 promoter region is heavily influential in breast cancer development, with release of epigenetic repression resulting in slowing of cellular proliferation and tumor progression. Subsequently, epigenetic mechanisms controlling DAX-1 gene expression in breast cell lines were investigated.

Methylation patterns in both normal human breast and breast cancer cell lines were initially determined via a bisulfite sequencing workflow including techniques such as bisulfite conversion, PCR, and plasmid vector ligation. While bisulfite sequencing the DAX-1 promoter returned results below a certain confidence threshold, nanopore sequencing was attempted and is under further review. The binding affinity of DNA methylating proteins along the DAX-1 promoter were measured via chromatin immunoprecipitation and quantitative PCR. Preliminary results show differential levels of expression in the various methyltransferases throughout the promoter region; moreover, expression of methyl binding domains suggested that there are additional, untested DNA methylating proteins that may play significant roles in the methylation of the DAX-1 promoter. Introductory studies attempting to identify chemical inhibitors of methylating proteins were performed, as DNA methyltransferase inhibitors have shown to restore expression of tumor suppressor genes and induce expression of endogenous retroviruses that lead to a viral mimicry state, potentiating host immune response. A proof-of-concept study was performed using 5-aza-2′-deoxycytidine, a universal demethylating compound, and Trichostatin A, a histone deacetylase inhibitor. Additional chemical inhibitors were also identified as potential therapeutic compounds for inhibiting DAX-1 activity based on their affinity for the identified methylating proteins.

The goal of these experiments was to map out epigenetic modifications and begin preliminary trials to identify chemical inhibitors that would lead to restoration of the normal phenotype in human breast cancer cells. Preliminary studies with the nanopore sequencing technology have been completed and results are currently undergoing analysis. With comparative analyses of normal and cancerous epitypes, differential expression patterns of associated DNA methyltransferases, and direction for therapeutic compound testing, such results provide a systematic basis for further investigation into the role epigenetics plays in DAX-1-mediated breast cancer initiation.

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