Bony De Kumar
Genomic approaches to understanding the activation of Hox genes and identifying their down stream target loci.
Homeotic genes (HOM/HOX genes) encode homeodomain-transcription factors that regulate patterning along the primary body axis. These proteins are implicated in the elaboration of the body plan of all bilaterally symmetrical animals and their basic functions are well conserved both in invertebrates and in vertebrates. Genetic and regulatory analyses in many model systems have demonstrated that the Hox family of transcription factors provides a highly conserved molecular framework or code for specifying distinct regional properties in many tissues. In vertebrates, mechanisms of regionalization of the hindbrain and spinal cord are highly conserved and coupled to ordered patterns of Hox gene expression and function. Therefore, understanding the regulatory mechanisms which underlie the generation of restricted patterns of Hox expression during neural development provides an important model system for understanding Hox gene regulatory networks involved in the control of regional identity and morphogenesis in diverse developmental contexts.
Though the function of Hox proteins is critical for regulation of diverse developmental processes, very little is known about which downstream target loci they regulate and how specific Hox protein complexes are recruited to these loci. One challenge is that all 39 mammalian Hox proteins are highly similar. Hence, their individual specificity is likely to be modulated by subtle differences in cofactors, interacting proteins, target binding sites, modification or other unknown processes. Therefore identifying in vivo relevant Hox response elements should provide important insight into how Hox cofactors, binding partners, co-activators or co-repressors and properties of binding sites serve to potentiate their functional roles.
The development of approaches for studying epigenetic changes in chromatin modification and binding of proteins to DNA across the entire genome provides a means for systematically investigating the activation of Hox clusters and identifying downstream target loci. Using these approaches in embryos can be hampered by technical challenges due to the limiting availability of materials for experimentation. However, the availability of embryonic stem (ES) cells, which can mimic many in vivo aspects of tissue development by differentiation in a lineage specific manner provide a good model for addressing these fundamental issues.
The overall goal of my thesis project is to characterize and utilize programmed neural differentiation of ES cells in combination with genomic technologies as a model system to investigate coordinated activation and cis-regulation of Hox genes and to identify in vivo Hox response elements and their associated target genes. Towards these aims, I am using ES cells and transgenic mice carrying epitope-tagged versions of different Hox proteins to characterize chromatin changes and identify binding sites by assaying chromatin immune precipitation (ChIP) products on microarrays (ChIP-chip) or by sequencing (ChIP-seq). These studies will contribute to and extend current understanding of Hox regulatory networks and target genes in animal development, disease and evolution.