Hox genes regulate the antero-posterior (AP) body axis in all Bilaterians. Hox genes encode transcription factors which regulate expression of downstream target genes important for morphogenesis during early development. While the conserved role of these proteins is well established little is known about the nature of the downstream targets of Hox genes. Another challenge is that Hox proteins have very similar protein sequences, which makes it difficult to understand the basis for their individual specificities and functional activities. Studies in our group using epitope-tagged versions of Hox proteins and programmed differentiation of mouse ES cells have enabled us to examine binding regions and downstream targets of mammalian Hox proteins on a genome wide basis through ChIP-seq approaches. This work has uncovered both novel and overlapping sets of downstream target genes for Hox proteins. Computational analyses of these mammalian Hox binding sites and co-associated binding motifs is also helping to uncover roles for partners (factors and complexes) and co-factors that may serve to potentiate the activity and regulatory roles of the Hox proteins. The function of Hox genes in AP patterning is conserved from arthropods to humans. Hence, my research objective has been to compare and contrast genome wide binding properties of mouse and Drosophila Hox proteins to understand the general principles and molecular mechanisms underlying Hox protein binding at target loci. Towards this goal, I used specific antibodies against Drosophila Hox proteins and a fly stock having an epitope tagged version of Ubx. Using ChIP-seq and ChIP-nexus I have mapped the downstream targets of Antp, Ubx, Abd-A and Abd-B in fly genome in 4-16 hr embryos. Analysis of the target genes is revealing distinct subsets of common and different target regions. Based on finding in our mammalian Hox studies, we have also generated specific antibodies against the Drosophila REST protein (Charltan-Chn) and Mediator complex subunit Med19and mapped their downstream target genes. Our initial results suggest that Mediator, REST and Hox proteins share a large number of common targets in Drosophila genome. Genetic and biochemical experiments further indicate genetic and physical interactions between Hox, Chn and Mediator complexes. This data implies that regulation of Hox target genes may involve input from Chn/REST and Mediator complexs in both Drosophila and mammals. We are now doing locus specific experiments to validate and understand more precisely the mechanisms of these interactions in gene regulation.