Histone Modifications in the Regulation of Expression at Imprinted Genes     Paige De Rosa

 

              Genomic imprinting occurs at certain genes in mammals. Imprinting leads to the expression of a gene from only one parental allele. Differentiating imprinting marks allow the parental alleles to be distinguished and properly expressed. In order to appropriately maintain imprinted expression, these marks are maintained in the somatic cells but reset in germ cells so that the individual can pass on the appropriate sex-specific mark to the next generation at fertilization. These differentiating imprinting marks can be epigenetic factors including DNA methylation and histone modification. DNA methylation is the attachment of a methyl group to certain nucleotides in the DNA. Histone modification refers to the attachment or removal of different chemical groups to the histone proteins which influences the way they compact the genomic DNA.

              Rasgrf1 is an example of an imprinted gene in mouse, the model organism studied in the Davis Lab. Rasgrf1 is a paternally expressed gene that is imprinted in a tissue-specific manner. In some tissues, Rasgrf1 is expressed from both parental alleles (biallelic expression) but is only expressed from the paternal allele in other tissues (monoallelic expression). At Rasgrf1, the epigenetic factor DNA methylation acts to label the paternal allele at an area called the imprinting control region. This methylation serves to block the action of a nearby enhancer which enables expression of the Rasgrf1 gene on the paternal allele. On the maternal allele, the imprinting control region is unmethylated which allows an enhancer-blocking protein to bind, preventing expression of the maternal allele. Previous research in the Davis Lab found that the paternal allele of the Rasgrf1 gene is methylated at the imprinting control region in both tissues with monoallelic expression and tissues with biallelic expression. This finding suggests that though methylation distinguishes the paternal allele of Rasgrf1, other elements influence the tissue-specific expression of this gene.

              It is possible that histone modifications are involved in the imprinted expression of the Rasgrf1 gene. Certain histone modifications are associated with higher expression and some modifications are associated with lower expression of genes. We have developed an assay to study histone modifications in Rasgrf1. The assay consists of a procedure called chromatin immunoprecipitation (ChIP) followed by allele-specific amplification of the DNA using quantitative PCR. We are able to differentiate between the parental alleles due to strain-specific DNA sequence differences between the strains of mice we use to produce the hybrid mice that we will study. This assay will let us characterize the histone modifications on the parental alleles in Rasgrf1 and better understand the role of histone modifications in the regulation of imprinted gene expression. Preliminary results from our lab suggests that the maternal allele of the Rasgrf1 gene has both activating and silencing types of histone modifications, which could explain the lack of expression from the maternal allele in monoallelic tissues while allowing for expression from the maternal allele in biallelic tissues.

We plan to validate the assay we have developed to study histone modifications by comparing our results at Rasgrf1 with those previously obtained at a well-characterized gene, H19. H19 is an example of a maternally expressed imprinted gene in mouse which produces a non-coding RNA. At H19, expressed alleles have been found to have activating modifications and non-expressed alleles typically have repressive modifications. Histone modifications at H19 have been examined in an allele-specific manner in many types of cells which will enable us to compare our Rasgrf1data from multiple tissues.