Identification of Imprinting Regulators as the Meg3 Differentially Methylated Region
Erin Nichole McMurray, PhD
Department of Biological Sciences
University of Illinois at Chicago
Chicago, Illinois (2012)
Dissertation Chairperson: Dr. Teresa Orenic, PhD
Genomic imprinting is the differential expression of two alleles of a gene based on the parent of origin. The imprinted Dlk1-Meg3 locus is located on distal mouse chromosome 12, and contains the paternally expressed Dlk1 and the maternally expressed Meg3 genes. Three differentially methylated regions (DMRs) are present at the Dlk1-Meg3 locus, and they are the Dlk1 DMR, the intergenic DMR, and the Meg3 DMR. Several studies have indicated that the Meg3 DMR plays a role in the expression and imprinting of genes at the Dlk1-Meg3 locus.
The goal of this work was to determine the factors present at the Meg3 DMR that may prove necessary for proper imprinting of the Dlk1-Meg3 locus. Chromatin immunoprecipitation (ChIP) was used to analyze histone modifications and trans-acting DNA binding proteins at the Meg3 DMR during imprinting establishment and maintenance. In embryonic stem (ES) cells, where imprints are being established, Meg3 is biallelically expressed, and the DMR shows variable DNA methylation, with biallelic methylation at one region but paternal allele-specific methylation at another. All histone modifications detected at the Meg3 DMR of ES cells were biallelic. In embryonic day 12.5 (e12.5) embryos, where imprints are already established and are maintained in an allele-specific manner, Meg3 is maternally expressed, the paternal Meg3 DMR is methylated, and activating histone modifications are specific to the maternal DMR. Also in this study, DNA binding proteins that represent potential regulatory factors were identified in both ES cells and e12.5 embryos.
In addition, a combination of methods including electrophoretic mobility shift assays, DNA affinity chromatography, and mass spectrometry were used to determine the presence of novel trans-acting DNA binding proteins at the Meg3 DMR that may prove to be necessary for imprinting regulation of the Dlk1-Meg3 locus. Using these methods, the protein PARP1 was detected at the Meg3 DMR of e12.5 embryos. PARP1 binding of the Meg3 DMR was confirmed in vitro using supershift analysis and in vivo using ChIP. The histone modifications and trans-acting DNA binding proteins detected at the Meg3 DMR in these studies provide a more complete picture of how imprinting is regulated at the Dlk1-Meg3 locus
