Defense Date


Document Type


Degree Name

Doctor of Philosophy


Clinical and Translational Sciences

First Advisor

Gordon D. Ginder


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.


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Available for download on Tuesday, May 20, 2025