Date of Award

January 2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Archana Dhasarathy

Second Advisor

Sergei Nechaev

Abstract

Transcription factors (TFs) are a class of proteins that recognize and bind to specific DNA sequences to regulate gene transcription. This process often involves the recruitment of other proteins, including chromatin remodelers and RNA polymerase. Because of their crucial role in gene expression, transcription factor activity is generally tightly controlled. Aberrant transcription factor activity has been implicated in the pathogenesis of most diseases. Techniques such as chromatin immunoprecipitation followed by sequencing have helped researchers determine which genomic sites are bound by TFs.Experimentally determined TF binding sites are much fewer than the total number of predicted DNA binding sites present in the genome. This phenomenon led us to ask what other factors may be involved in a) targeting TFs to their binding sites and b) controlling TF regulation of gene expression. Because the function of a protein is largely dependent on its structure, TFs can be sub-classified based on the structural domains they contain. Zinc finger domain-containing proteins comprise the largest subgroup, and of these, the most common type of zinc finger domain is the Cys-Cys-His-His or C2H2 zinc finger domain, which is stabilized by one or more zinc ions. Interestingly, C2H2 zinc finger domains have also been implicated in RNA binding, and a few C2H2 zinc finger proteins have been shown to bind both DNA and RNA. The biological significance of this dual binding capability has not been well-studied, but researchers speculate it may a) cause competitive binding of RNAs, which act as “decoys” and thus reverse a TF’s influence on gene expression, b) allow TFs to be guided by RNA coactivators or RNA corepressors to specific genomic sites, and c) allow for regulation of non-coding RNAs, which influence gene expression on a separate level. However, despite structural similarities, only a few of the plethora of C2H2 zinc finger proteins have been studied with regards to their dual-binding function. Further, due to the dearth of experimental evidence, the biological function of this dual-binding ability remains largely speculative rather than experimentally validated. I will characterize RNA targets of the C2H2 zinc finger transcription factor SNAIL, which is known to regulate the Epithelial to Mesenchymal Transition (EMT), a process necessary for development, wound healing, and early metastasis. I have focused my research efforts on three main goals that address the topic of the dissertation proposal: First, I will perform a literature review of C2H2 zinc finger proteins, specifically comparing their DNA and RNA binding abilities. I will discuss the proven biological relevance of the dual-binding ability of these proteins and will hypothesize what other functions this dual-binding ability may have in terms of control of gene expression. Additionally, I will use phylogeny-based analysis to study the evolution of dual-binding zinc finger domains. Secondly, experimental evidence regarding the dual-binding role of these C2H2 zinc finger TFs is greatly lacking. Much of this is due to the dearth of experimental techniques to study simultaneous dual DNA and RNA binding, but part of this can also be attributed to the unique biochemistry of each protein, which makes it difficult to establish a central standardized protocol for RNA immunoprecipitation to study each protein’s RNA targets. I will optimize a method that will define which RNA targets SNAIL binds to in vitro. Finally, I will determine which RNA targets are bound by SNAIL in both endogenous and exogenously expressed conditions. I will parse through the targets to determine which types of RNAs SNAIL binds to, and how they compare to known DNA targets of SNAIL. Characterization of these RNA targets will also aid in hypothesizing why SNAIL binds RNA in addition to DNA, and how this may influence its role on gene expression, particularly during the process of EMT. Further, I will investigate the mechanism of SNAIL RNA binding. Combined, these experiments will help us illuminate SNAIL’s RNA binding ability and aid in determining what the function of this RNA binding may be, particularly with regards to SNAIL’s well-known role in regulating the process of EMT.

Available for download on Saturday, August 26, 2023

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