Role of CpG Binding Protein in the Regulation of Cytosine Methylation and Chromatin Structure

Indiana University, Bloomington IN

Investigators

Abstract

Cytosine methylation of CpG motifs is an important epigenetic modification of mammalian genomes. The molecular mechanisms that regulate this process remain unclear. The investigator's laboratory cloned a novel factor, the CpG binding protein (CGBP), that binds DNA containing unmethylated CpG dinucleotides. Disruption of the CGBP gene leads to embryonic death in mice, and CGBP-null embryonic stem (ES) cells exhibit elevated apoptosis, an inability to differentiate in vitro, decreased levels of global and locus-specific cytosine methylation (including deficient cytosine methylation at repetitive elements and imprinted loci), decreased total DNA methyltransferases (DNMT) activity, and altered patterns of histone modifications consistent with reduced levels of heterochromatin. However, expression of DNMT proteins (DNMT1, DNMT3a, DNMT3) is normal, and CGBP-null cells exhibit de novo DNMT activity. The phenotypes exhibited by CGBP-null ES cells are largely corrected upon introduction of a CGBP-expression vector. The finding in CGBP-null ES cells of decreased DNMT activity and cytosine methylation in the presence of normal levels of DNMT proteins is unique, and further analysis of this phenotype should provide important insight into the mechanisms that control cytosine methylation. The central hypothesis of this project is that CGBP is a regulator of cytosine methylation and chromatin structure. The specific aims are to (1) determine the functional significance of reduced DNMT activity in CGBP-null cells, and (2) identify proteins that interact with CGBP. In the first aim the significance of decreased DNMT activity to the phenotypes observed in CGBP-null cells (see above) will be assessed by introducing a DNMT1 expression vector into the CGBP-null cells to permit over-expression of this maintenance DNA methyltransferase. Clones that exhibit a normal level of total DNMT activity will be examined further. Rescue of a CGBP-null phenotype following re-establishment of normal DNMT activity will demonstrate a causal relationship. In the second aim a biochemical "pull-down" approach utilizing mass spectrometry will be used to identify proteins that interact with CGBP. These proteins should provide molecular insights into the mechanisms of CGBP function. DNA in living cells is associated with proteins to form a structure known as chromatin. Modulation of the exact make-up of chromatin structure is an important mechanism to regulate patterns of gene expression. The goal of this project is to gain insight into the molecular mechanisms that control chromatin structure. The Skalnik laboratory involves both undergraduate and graduate students, including those from underrepresented groups, in its research.