Defense Date

2006

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Microbiology & Immunology

First Advisor

Dr. Gordon D. Ginder

Abstract

The genes of the human β-globin locus are located on chromosome 11 in the order of their expression during development: 5' ε, γ, β 3'. During development, silencing of the 5' gene occurs with activation of the immediate 3' gene. This process occurs twice and is termed hemoglobin switching. The exact mechanism(s) of this process have not been fully described. Herein, we describe a role for DNA methylation and methyl binding domain protein 2 in the transcriptional regulation of the human embryonic and fetal beta type globin genes. Adult mice containing the entire human β-globin locus as a yeast artificial chromosome (βYAC) express very low levels of the fetal γ-globin gene. However, treatment of adult βYAC transgenic mice with the DNA methyltransferase inhibitor, 5-azacytidine, induces a >10-fold increase γ-globin mRNA levels. In addition, βYAC transgenic mice null for methyl binding domain protein 2 (MBD2) express a similar level of γ-globin mRNA. DNA methylation and MBD2 appear to induce γ-globin expression via the same pathway(s), as treatment of MBD2 null βYAC transgenic mice do not show an additive boost in γ-globin expression. MBD2 does not bind to the γ-globin promoter region in vivo indicating MBD2 mediated transcriptional silencing does not occur by recruitment of transcriptional repression complexes to the γ-globin gene promoter. Additionally, these transgenic mice contain only the 5' portion of the β-globin locus through the ε-globin, and do not express the ε-globin genes as adults. However, treatment with 5-azacytidine or loss of MBD2 induces expression of the ε-globin gene in adult transgenic mice. A similar induction of ε-globin is seen in βYAC transgenic mice under the same conditions. The level of expression of the ε-globin gene is much lower than the γ-globin gene, indicating the powerful effect of the cis elements mediating transcriptional repression of the ε-globin gene. These studies indicate DNA methylation and MBD2 contribute to the transcriptional repression of the human embryonic and fetal β-type globin genes. Additionally, MBD2 has been identified as a potential target for the therapeutic induction of fetal hemoglobin for the treatment of hemoglobinopathies.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

June 2008

Share

COinS