Epigenetic regulation of the eukaryotic genome

Project Associates/Assistants
Sarita Negi

Ph D Students
Surabhi Srivastava
Vikrant Singh

Chromatin structure plays a crucial role in defining the specificity of gene expression as it has the ability to regulate accessibility of the trans acting factors to enhancers and promoters. Consequently, temporally and spatially distinct genetic expression profiles are established and maintained during development which allow differential interpretation of identical genetic information present in all cells of an organism. In recent years, there is growing recognition of the functional role of chromatin organization in regulation of gene expression. We are interested in understanding gene regulation as regulated by chromatin structure. Our current focus is on understanding the nature of transcriptional insulators-cis DNA elements that have the ability to abrogate promoter-enhancer interactions. We would like to address the molecular mechanisms by which transcriptional insulators are able to organize chromatin and prevent enhancer-promoter interactions in a position dependent manner. We have chosen to use mammalian Igf2 insulator for our studies as this insulator regulates monoallelic expression of Igf2. Loss of monoallelic expression of Igf2 is associated with severe developmental disorders and with several cancers in humans. Also, the activity of the insulator at this locus is subject to epigenetic modifications. The objectives of the project are to (i) analyse the mechanisms by which Igf2 insulator organizes chromatin structure at the endogenous locus to prevent the enhancer promoter interaction, (ii) evaluate the ability of the Igf2 insulator to organize chromatin at a heterologous locus and interfere in VDJ recombination at the TCR-b and identify new cis and trans acting factors responsible for chromatin organization

BACs carrying the mouse gDNA from the TCR-b locus were characterized further to avoid the possibility of chimerism and internal rearrangements. One of these was subsequently chosen for sub-cloning the DNA fragments relevant for creating the targeting construct for the knock-in. More specifically, we created a mini library, in a pSP72 derived vector, of the BAC DNA digested with BamHI and HindIII. The library was screened to obtain about 10.5kb region to be used for making the targeting construct (Figure-1). Also, the BAC DNA was used to obtain probes that are to be used for screening the correctly targeted ES clones (Figure-1). Through several sequential steps of cloning, we have obtained a targeting construct that will be used for homologous recombination in mouse ES cells. The construct carries DNA from the TCR-b locus, 2.5 kb insulator region from the Igf2 locus, neomycin resistance gene for selection of recombinants and DTA cassette for negative selection. We have also obtained primary embryonic fibroblasts from neomycin resistant mouse embryos that will be used during ES cell culture.

Figure-1:   TCR-b locus of mouse. (a) Schematic diagram of the locus showing relative positions of V, D, J and C segments along with Eb and the proposed position of 2.5kb Igf2 insulator insertion. (b) Detailed restriction map of the region being manipulated. Probes used for screening the BAC (hatched lines) and probes to be used for screening targeted ES cell clones (dotted lines) are also shown. Restriction enzymes relevant for manipulation are shown. Ahd, AhdI; H, HindIII; Bg, BglII; B, BamHI; Sac, SacI

Creating the knock-in of the insulator in the mouse genome will provide us useful understanding about nature of insulator function, its mechanism of action, ability to acquire epigenetic modifications (imprinting) and VDJ recombination. To complement our in vivo approach, we have initiated experiments that rely on understanding the role of transcriptional insulator in VDJ recombination in TCR miniloci originally designed by Sleckman and Alt. We are currently making stable transfectants of the manipulated miniloci in 5B3 cells. 5B3 cells have an inducible RAG system and have been successfully used to understand VDJ recombination ex vivo.

At its endogenous location, the mechanism of 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. We propose to use primary fibroblasts and/or nuclei derived from liver cells of appropriate genotypes and assay the various histone tail modifications in the region as a read out of heterochromatinized or euchromatinized region and determining the presence of a chromatin boundary. We have established primary fibroblast lines from +/DMRdel13, DMRdel13/+ and DMRdel13/DMRdel13 mice. Experiments are in progress to optimise the Chromatin Immunoprecipitation (ChIP) assays.