Epigenetic regulation of the eukaryotic genome

PhD Students
Surabhi

The chromatin structure organization contributes towards establishment and maintenance of the expression profile of a differentiated cell and allows the same genetic information to be interpreted differentially in a cell-specific and stage -specific manner. The goal of this project is to identify the cis and trans acting factors involved in chromatin organization and epigenetic regulation and to understand the mechanism by which they influence the gene expression. The loci that exhibit monoallelic expression are particularly useful for such studies. The present focus is to understand the mechanisms by which cis acting insulator elements organize chromatin and regulate enhancer-promoter interaction. We are using the insulator at the Igf2/H19 locus as a model. This insulator is responsible for paternal allele specific expression of Igf2. Loss of monoallelic expression of Igf2 leads to developmental disorders and is also associated with several tumors and cancers. The objectives of the project are (i) analysing the mechanism by which the insulator prevents enhancer-promoter interaction at its endogenous Igf2/H19 locus and organizes transcriptional domains, (ii) evaluating the ability of the insulator to alter the chromatin structure of a heterologous locus and interfere with DNA-DNA interactions involved in VDJ recombination and (iii) identification of new cis/trans acting factors responsible for epigenetic regulation

At its endogenous location, the mechanism of H19/Igf2 insulator will be determined in context of its ability to act as a chromatin boundary and its ability to direct the Igf2 locus to transcriptionally inactive regions of the nucleus. Together, the complete analysis will address the interdependence of histone modifications, nuclear localization and expression of a gene as affected by a transcriptional insulator especially in the context of Igf2 regulation. Towards this goal, we are currently optimising the Chromatin immunoprecipitation assays to analyse specific histone tail modifications and thus determine the ability of the insulator to define a chromatin boundary. Also, the regions of H19 and Igf2 useful for synthesizing DNA and RNA probes for FISH analysis have been sub-cloned. The next step would be to standardize the FISH reactions to detect nascent RNA and DNA to delineate the ability of the insulator to direct the Igf2 allele to specific nuclear locations.

To investigate the alteration in chromatin structure and its effect on VDJ recombination, we propose to knock-in the H19/Igf2 insulator region at the TCR-b locus. As the first step towards this goal, using PCR based strategy, we designed probes to screen a BAC library that carries mouse gDNA. Several TCR-b clones were picked up during screening of the BAC library by colony hybridization. Using these BAC clones, we are now beginning to design the targeting constructs for creating mouse mutations that carry a knock-in of the insulator at the TCR-b locus. Also, earlier investigators have used miniloci that carry V,D,J,C and enhancer regions as substrates for recombination in transgenic or cell line based systems. Although, limited in scope for providing information about recombination process in vivo at the TCR-b locus, these studies have revealed important principles underlying VDJ recombination. We are also manipulating miniloci to complement our mutant based approach. In addition to giving insights about insulator function, our experiments will be useful to study the process of VDJ recombination at the molecular level in the chromatin context.

Publications

Original peer-reviewed articles

1.     ¶Srivastava M, Frolova E, Rottinghaus B, Boe S, Grinberg A, Lee E, Love P and Pfeifer K (2003) Imprint control element mediated secondary methylation imprints at the Igf2/H19 locus. J Biol Chem 278:5977-5983 (¶on deputation/work done elsewhere).