DOI
https://doi.org/10.25772/VTBQ-RA49
Defense Date
2020
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Clinical and Translational Sciences
First Advisor
Gordon D. Ginder
Abstract
Humans undergo two developmental switches in the predominantly expressed β-like globin chain during embryogenesis and fetal development. The first switch from embryonic (ε) to fetal (γ) occurs around week 5 of embryonic development, while the second switch from fetal to adult (β) globin occurs shortly after birth. By adulthood, fetal hemoglobin represents only 1-2% of total hemoglobin in the blood. As sufficiently elevated levels of fetal hemoglobin are beneficial for improving clinical outcomes in sickle cell disease and β-thalassemia, the mechanisms that enforce silencing of fetal hemoglobin expression postnatally are of great clinical significance. The methyl-CpG binding domain protein MBD2 and members of its associated co-repressor, the Nucleosome Remodeling and Deacetylase (NuRD) complex, have previously been shown to be important silencers of embryonic and fetal β-like globin genes in multiple erythroid systems. Herein we demonstrate that CRISPR/Cas9 mediated knockout of MBD2, but not the related protein MBD3, results in high level fetal hemoglobin expression in the adult phenotype Human Umbilical cord Derived Erythroid Progenitor 2 (HUDEP-2) cell model. MBD2 Knockout (MBD2KO) HUDEP-2 cells make approximately 50% γ/γ + β mRNA levels and 40% HbF by HPLC, while knockout of MBD3 had no appreciable effect on γ-globin gene expression. Knockdown of MBD2 in primary human adult CD34 + erythroid progenitor cells results in up to 40% γ/γ+β expression and a large increase in γ-globin protein. Importantly, depletion of MBD2 has no deleterious effect on markers of erythroid differentiation as determined by RNA-sequencing and flow cytometric analyses. MBD2 interacts with NuRD members GATAD2A/B and CHD4 through a C-terminal coiled-coil domain, while interaction with the HDAC sub-complex is mediated via an intrinsically disordered region (IDR). Expression of wild-type MBD2 in MBD2KO HUDEP-2 cells partially represses γ-globin expression, while expression of MBD2 with mutations in the coiled-coil or IDR domains fails to rescue the effect of MBD2 depletion on γ-globin expression, functionally validating the importance of these regions in MBD2-NuRD mediated gene silencing and pointing to the protein-protein interfaces of MBD2-NuRD as potential therapeutic targets for the β-hemoglobinopathies. In the last section of this work, we examine the mechanisms of MBD2-NuRD mediated fetal hemoglobin silencing and explore interactions between MBD2-NuRD and other established regulators of fetal hemoglobin switching.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
5-21-2020
Included in
Biology Commons, Congenital, Hereditary, and Neonatal Diseases and Abnormalities Commons, Genomics Commons, Molecular Genetics Commons