Identification of Imprinting Regulators at the Meg3 Differentially Methylated Region

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

Identification of Imprinting Regulators at the Meg3 Differentially Methylated Region

Genomic imprinting at the Delta-like 1 (Dlk1)-Maternally expressed gene 3 (Meg3) locus is regulated by the Meg3 differentially methylated region (DMR), but the mechanism by which this DMR acts is unknown. The goal of this study was to analyze the Meg3 DMR during imprinting establishment and maintenance for the presence of histone modifications and trans-acting DNA binding proteins using chromatin immunoprecipitation. In embryonic stem (ES) cells, where Meg3 is biallelically expressed, the DMR showed 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 Meg3 is maternally expressed, the paternal Meg3 DMR was methylated, and activating histone modifications were specific to the maternal DMR. DNA-binding proteins that represent potential regulatory factors were identified in both ES cells and embryos

Localizing Transcriptional Regulatory Elements at the Mouse Dlk1 Locus

Much effort has focused recently on determining the mechanisms that control the allele-specific expression of genes subject to genomic imprinting, yet imprinting regulation is only one aspect of configuring appropriate expression of these genes. Imprinting control mechanisms must interact with those regulating the tissue-specific expression pattern of each imprinted gene in a cluster. Proper expression of the imprinted Delta-like 1 (Dlk1) - Maternally expressed gene 3 (Meg3) gene pair is required for normal fetal development in mammals, yet the mechanisms that control tissue-specific expression of these genes are unknown. We have used a combination of in vivo and in vitro expression assays to localize cis-regulatory elements that may regulate Dlk1 expression in the mouse embryo. A bacterial artificial chromosome transgene encompassing the Dlk1 gene and 77 kb of flanking sequence conferred expression in most endogenous Dlk1-expressing tissues. In combination with previous transgenic data, these experiments localize the majority of Dlk1 cis-regulatory elements to a 41 kb region upstream of the gene. Cross-species sequence conservation was used to further define potential regulatory elements, several of which functioned as enhancers in a luciferase expression assay. Two of these elements were able to drive expression of a lacZ reporter transgene in Dlk1-expressing tissues in the mouse embryo. The sequence proximal to Dlk1 therefore contains at least two discrete regions that may regulate tissue-specificity of Dlk1 expression