STRUCTURE AND FUNCTION STUDIES OF METHYL-CPG BINDING DOMAIN PROTEINS AND THEIR COMPLEXES

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  • March 20, 2019
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  • Sperlazza, Mary
    • Affiliation: College of Arts and Sciences, Department of Chemistry
Abstract
  • DNA methylation is an epigenetic mechanism of transcriptional silencing of increasing interest for treating human disease. Methyl-CpG binding domain (MBD) proteins recognize 5 methylcytosines (mC) primarily in symmetrical CpG (mCG) dinucleotides. Seven proteins comprise the MBD family, MBD1-6 and MeCP2. MeCP2 is primarily expressed in the brain and plays a critical role in neuron maturation, as mutations disrupting its function account for up to 80% of Rett Syndrome cases. MBD2/3 associates with the nucleosome remodeling and deacetylase complex (NuRD) and modulates gene expression through alteration of the chromatin architecture surrounding the mC mark. In these studies, we examine the behavior of the MBDs of MeCP2 and MBD2, in addition to further characterizing the protein-protein interactions between subunits of NuRD. Recent work suggests the primary effects of MeCP2 on gene expression in the developing mammalian brain are mediated by binding asymmetrically methylated and hydroxymethylated CpA (h/mCA) dinucleotides. This work establishes that the MeCP2 MBD binds mCA with high affinity in a strand specific and orientation dependent manner. This preference is specific to MeCP2, as the MBD2 MBD does not show high affinity or methyl-specific binding to mCA. Introduction of the Rett Syndrome-associated mutations T158M, R106W and P101S destabilized the MeCP2 MBD and lessened recognition of mCG and mCA equally. Finally, hydroxymethylation of a high affinity mCA site did not dramatically change binding properties, however hemi-hydroxylation of the same cytosine in mCG significantly decreased affinity. We suggest a model for MeCP2 recognition of mCA and for hydroxymethylation as an epigenetic switch to redistribute MeCP2 among mCG and mCA loci. Blocking recruitment of NuRD by MBD2 restores expression of developmentally silenced fetal hemoglobin and aberrantly silenced tumor suppressor genes. Additionally, knockdown of the NuRD helicase, CHD4, results in cancer cells growth arrest and increased sensitivity to DNA damage. Therefore, targeting MBD2-NuRD presents a promising avenue for treating β-hemoglobinopathies and cancer. Towards understanding the recruitment of the NuRD components to the complex, this study characterizes the GATA-like zinc-finger domains of the NuRD components GATAD2A and MTA2. We propose a model of NuRD in which MTA2 binds DNA and GATAD2A serves to bridge MBD2 and CHD4.
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  • In Copyright
Advisor
  • Kuhlman, Brian
  • Brustad, Eric
  • Erie, Dorothy
  • Waters, Marcey
  • Williams, David
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2017
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