Defense Date

2020

Document Type

Thesis

Degree Name

Master of Science

Department

Human and Molecular Genetics

First Advisor

Dr. Joyce Lloyd

Abstract

β-hemoglobinopathies affect millions of people around the world. Research into treatments for these conditions has focused on methods to increase γ-globin expression, because increased levels of γ-globin ameliorate or reduce the severity of symptoms. As more and more studies have been done, a few proteins have emerged as having crucial roles in γ-globin repression and have been established as key genes to study. These are Krüppel-like factor 1 (KLF1), B cell CLL/lymphoma 11A (BCL11A), and methyl binding domain 2 (MBD2). The roles of these proteins in the switch from fetal to adult hemoglobin and in repressing γ-globin expression have been investigated extensively. However, there are still questions that remain unanswered and knowledge gaps that need to be filled. This thesis focuses on the gaps for each of these three genes and presents approaches to answer these questions. Some of the questions discussed are how variants of KLF1 affect its function, if BCL11A regulates the γ-globin gene by binding at a distance or at its promoter, and how MBD2 binds at the β-globin locus. One of the questions that is universal to the three genes is how they each interact with the β-globin locus and if they each interact with each other in order to repress γ-globin expression. We propose knocking down different combinations of two of the three genes simultaneously to determine if the combinations have an additive or synergistic effect on increasing γ-globin expression, which could indicate whether the proteins are working in the same or different pathways. We provide evidence that dual knockdown of BCL11A and MBD2 in HUDEP-2 cells results in a significantly larger increase in γ-globin mRNA than single knockdown of either gene. Our evidence supports our rationale that further experiments on combinations of the KLF1, BCL11A, and MBD2 gene knockdowns in HUDEP-2 cells and then further verification in CD34+ cells could lead to future effective treatments for β-hemoglobinopathies.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

8-4-2020

Available for download on Wednesday, August 04, 2021

Included in

Genetics Commons

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