My lab focuses on understanding the mechanism of genomic imprinting. Genomic imprinting is a mammalian-specific phenomenon whereby the expression of a subset of genes depends on their parental origin. In other words, although we inherit one copy of every gene from our mothers and one copy from our fathers, there are a small number of genes for which only the maternally inherited copy is expressed and a small number for which only the paternally inherited copy is expressesd. There are two major consequences of this unusual form of gene regulation. First, mutations in imprinted genes act in a dominant, parent of origin-specific fashion since there is not a second copy whose wild-type expression can compensate for the mutation. Second, every mammal needs to have a genetic contribution from both a male and a female parent – otherwise, genes critical for normal development will not be expressed. Failure to achieve genomic imprinting can result in developmental disorders such as Beckwith-Wiedemann, Prader-Willi and Angelman syndromes.
One main question in the field of genomic imprinting is: how does the cellular machinery distinguish the maternally inherited allele from the paternally inherited allele so that it knows which copy should be expressed and which copy should remain silent? The simple answer is that the maternal and paternal alleles must be marked so that they appear to be different from each other. To date, the best candidate for the imprinting mark is DNA methylation. In mammals, DNA methylation is a modification of cytosines that are present in CG dinucleotides, such that the cytosines have a methyl group covalently attached at the 5′ position. This type of modification is called epigenetic because it is a modification of the DNA structure but does not alter the DNA sequence. The reason DNA methylation stands out as a candidate for the imprinting mark is that most imprinted genes are associated with a region of differential methylation – for example, the silent paternal allele of an imprinted gene may be methylated while the expressed maternal allele is unmethylated.
As mentioned above, all imprinted genes are associated with a primary region of differential DNA methylation which serves as an imprinting control region. However, the precise regulation of imprinted genes requires additional epigenetic modifications, including secondary differentially methylated regions (DMRs) and differential distribution of modified histones on the parental alleles, or copies, of imprinted genes. Secondary DMRs are regions at which differential methylation is not inherited via the gamete; rather, allele-specific methylation at secondary DMRs is acquired post-fertilization. One aspect of my research is focused on understanding when methylation is acquired during embryogenesis, and how these secondary DMRs differ from primary imprinting control regions. To do this, my lab conducts analysis of DNA methylation patterns at imprinted genes during various stages of development in the mouse. Thus far, we have analyzed the acquisition of DNA methylation at secondary DMRs associated with the imprinted genes Cdkn1c, Gtl2 and Dlk1. We have learned that Cdkn1c acquires DNA methylation on its paternal allele at a different developmental stage than Gtl2 and Dlk1, and that the DNA methylation pattern at Dlk1 continues to change during development. These results indicate that there is not a single developmental stage during which allele-specific methylation is established. More recently, we observed that the DNA methylation has an unexpected high level of asymmetry on the complementary strands of the Dlk1 gene. We are currently conducting experiments to determine if this asymmetry is unique to Dlk1 or whether it is a common feature of secondary DMRs, and to determine the biochemical basis for this asymmetry.
While it is clear that DNA methylation plays a role in regulating the expression of imprinted genes, it is also clear that differential DNA methylation cannot be the only factor distinguishing the maternal and paternal alleles from each other. Rasgrf1 is an imprinted gene at which the paternal allele is marked with DNA methylation. However, the DNA methylation pattern at Rasgrf1 does not directly correlate with its expression pattern. Rasgrf1 is an example of a tissue-specific imprinted gene: it is expressed solely from the paternally inherited copy in some tissues, such as brain, but is expressed from both the paternal and the maternal copy in other tissues, such as lung. Therefore, there must be other factors responsible for regulating the tissue-specific imprinting of this gene. Histone modification is one candidate that may be playing a role in the complex regulation of Rasgrf1. Histones are proteins that DNA wraps around in order to achieve the first level of chromosome compaction. The addition of different chemical groups, such as methyl and acetyl groups, to histone proteins affects the structure of the chromatin and the degree of DNA compaction. My lab is currently investigating the distribution of modified histones on the parental alleles of Rasgrf1in both imprinted and non-imprinted tissues in order to determine if they play a role in achieving imprinting at this gene.
There are opportunities for student research in my lab during the course of the academic year as well as in the summer.