ORCID
0009-0005-5814-604X
Date of Award
2025
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Biochemistry
First Advisor
Karen Glass
Second Advisor
Bryan Ballif
Abstract
Histone post-translational modifications (PTMs) serve as an epigenetic signaling mechanism that regulates gene expression. Two of the most prevalent PTMs are acetylation, which can activate gene expression, and methylation, which can activate or repress gene expression depending on location. Histone acetylation is recognized by bromodomains (BRDs), while histone methylation is recognized by plant homeodomains (PHDs). The up-regulation and down-regulation of these histone modifications contribute to the pathogenesis of various cancers and inflammatory diseases by recruiting proteins that contribute to the underlying etiology of the disease, including bromodomain-containing proteins and PHD-finger-containing proteins. The Speckled Protein (SP) 110C (SP110C) contains multiple functional domains such as the caspase activation and recruitment domain (CARD) that mediates the formation of larger protein complexes, along with three chromatin reader domains Fsa). The bromodomain adjacent to zinc finger domain protein 1A (BAZ1A) functions as a non-catalytic regulatory subunit of select ISWI (imitation switch) chromatin remodeling complexes and contains multiple reader domains (PHD and BRD), along with WAC (named after proteins WSTF, Acf1, and CBP146) and DDT (DNA binding homeobox and Different Transcription factors) motifs. The reader domains found in these proteins bind modifications on DNA or histones to regulate cellular processes such as transcription, DNA repair and replication, and chromatin remodeling. The aim of these studies is to characterize how bromodomain-containing proteins up-reregulated in tamoxifen-resistant breast cancer are recruited to chromatin. There is a gap in knowledge surrounding the histone post-translational modifications that are recognized by the SP110C SAND-PHD-BRD triple domain and the BAZ1A-BRD, and how atypical bromodomains adjacent to other chromatin reader domains coordinate recognition of acetyllysine.To further characterize the structure and function of the chromatin reader domains of BAZ1A and SP110C, we expressed and purified the BAZ1A-BRD, the SP110C-SAND, and the SP110C SAND-PHD-BRD triple domain module. We performed circular dichroism (CD) spectroscopy with the BAZ1A-BRD and found that it is folded in a way that is canonically accurate. We also examined the binding activity of BAZ1A-BRD and found that it does not recognize acetylated histone ligands, likely due to a negatively charged gate-keeper residue found within the BRD binding pocket. Analysis of the SP110C SAND-PHD-BRD protein revealed that it is monomeric in solution. SP110C is predicted to recognize histone H3 that is acetylated on lysine 18 (H3K18ac). However, we were unable to detect binding between SP110C SAND-PHD-BRD and acetylated histone H3 and H4 ligands due to solubility issues with the protein sample. Using a combination of biochemical and computational techniques including analytical size exclusion chromatography, mass spectrometry, pyMOL, and AlphaFold, we found there are a number of solvent exposed cysteine residues that contribute to the insolubility of SP110C SAND-PHD-BRD. Optimization of the buffer conditions, expression system, and mutation of the solvent-exposed cysteine residues was carried out to improve protein expression and solubility for additional binding experiments using isothermal titration calorimetry. In summary, the results from this work offer insights into the characterization of bromodomain-containing proteins in the context of epigenetics and disease.
Language
en
Number of Pages
114 p.
Recommended Citation
Cook, Elizabeth, "Characterization of BAZ1A and SP110C: Insights into the Role of Epigenetic Regulation by Bromodomain-Containing Proteins in Cancer" (2025). Graduate College Dissertations and Theses. 2104.
https://scholarworks.uvm.edu/graddis/2104