The School of Molecular and Cellular Biology at the University of Illinois at Urbana-Champaign

My laboratory is interested in the process of genomic imprinting. Genomic imprinting is a phenomenon that leads to the repression of one of the two copies of a gene depending on which parent it is inherited from. In other words, genes that are subject to genomic imprinting are transcribed from only the maternal or paternal chromosome. These functionally haploid genes are an important and puzzling exception, as most genes are transcribed equally from both parental alleles.There are about 40 genes that have been identified as being imprinted in mammals. Alteration in the imprinting status of some of these genes leads to genetic diseases in humans such as Beckwith-Wiedemann, Prader-Willi and Angelmann syndromes. My lab focuses on the insulin-like growth factor II (Igf2) and H19 gene locus. Igf2 is an essential fetal growth factor and its misregulation plays a role in Beckwith-Wiedemann syndrome. H19 is an enigmatic untranslated RNA whose function is still unknown.

The central aspects of genomic imprinting concern how a cell can distinguish two almost identical copies of a gene and how it represses only one of them. At present, differences in DNA methylation of CpG dinucleotides appear to be a key element for distinguishing the two parental chromosomes. The differences in DNA methylation between the two alleles are established during the maturation of oocytes and sperm. At the Igf2/H19 locus, for example, a sequence between H19 and Igf2 known as the imprinting control region (ICR) is completely methylated in sperm and completely unmethylated in oocytes. This indicates that during spermatogenesis, a male’s unmethylated maternal ICR must become methylated, whereas during oogenesis in females, pre-existing paternal methylation must be removed. This methylation difference is then maintained after fertilization and throughout development. How these methylation differences are established and maintained, however, is still unknown. By creating mice with mutations in the ICR, we can determine which sequences are necessary for both methylation and demethylation. The next step will be to identify the proteins and the DNA methyltransferases that regulate the methylation process.

For Igf2/H19, the differentially methylated ICR is necessary for imprinted transcription of both genes. When methylated it acts as a repressor of paternal H19, and when unmethylated it prevents expression of maternal Igf2. One focus of the lab is to determine how the ICR can have two completely different activities that are determined by its methylation state. We know that methylation is essential for repression of paternal H19 and would like to determine the exact sequences and proteins that convert this signal into changes in transcription.

While we know little about the methylated ICR, the unmethylated ICR is a chromatin boundary. Chromatin boundaries or insulators are DNA elements that are thought to divide the genome into separately regulated units. They can prevent the spread of heterochromatin and can contain the activity of enhancers to regions of DNA between two boundary elements. Our research into the chromatin boundary activity of the ICR focuses primarily on CTCF. CTCF is a zinc finger transcription factor that binds to the mouse ICR in four places and is essential for blocking activation of Igf2 100kb away. It is not known how CTCF opposes enhancer activity, but it is likely to achieve this by modifying chromatin structure. Using a mutant ICR that does not bind CTCF, we can compare the physical and biochemical structure of chromatin from mutant and wild type ICR’s. In addition, differences in chemical modifications (methylation, acetylation) of the histones can be examined. Future plans also include identifying the domains of CTCF necessary for enhancer blocking and the proteins that interact with them.