June 10, 2013
Histone modification studies at the imprinted Rasgrf1 and H19 genes
Epigenetics is the study of changes in gene expression facilitated by molecular modifications of DNA. In genomic imprinting, DNA modifications regulate gene expression monoallelically in a parent of origin-specific manner. Thus, imprinted genes are either expressed exclusively from the maternal allele, or exclusively from the paternal allele.
Genomic imprinting is largely controlled by parent-specific cytosine methylation at clusters of CpG dinucleotides. Imprinting control regions (ICRs) develop differential methylation during gametogenesis, and differentially methylated regions (DMRs) develop DNA methylation arises post-fertilization. ICRs and DMRs often work cooperatively to promote expression of one parental allele while simultaneously repressing expression of the other parental allele.
Histone modifications also contribute to the expression of some imprinted genes. Under unmodified conditions, negatively charged DNA coils around positively charged histones. Histone acetylation is associated with gene expression, and histone methylation can either repress or activate expression.
The imprinted murine Rasgrf1 gene is paternally methylated in all tissues, but different tissues have been shown to exhibit either monoallelic or biallelic expression. Consequently, Rasgrf1 expression appears to have other molecular controls. We designed a chromatin immunoprecipitation (ChIP) assay to analyze the potential role of histone modifications in Rasgrf1expression.
Chromatin immunoprecipitation can be used to analyze gene-protein interactions. The procedure cross-links chromatin and then shears it into smaller strands. Primary antibodies then attaches to modified histones and their associated DNA. A secondary antibody then attaches to the primary antibodies, which selectively precipitates the modified histones and DNA. The precipitated DNA is then analyzed via quantitative PCR to determine the relative concentrations of maternal versus paternal DNA in the sample. This approach allows us to determine whether a modified histone is preferentially located on one parental allele or equally located on both parental alleles.
Our initial ChIP explorations, performed in the 2012-2013 academic year, yielded variable data that was sometimes inconsistent with published literature, which suggests that our assay may not function properly. To analyze the quality of our data and the efficacy of our assay, we will perform identical experiments on the imprinted murine H19 gene. H19 contains well-documented histone modification. If our assay shows histone modification at H19 that correlate with data from prior studies, then the accuracy of both our data and our assay will be confirmed.