This competitive renewal proposal would continue our highly productive experiments to identify functional elements that control gene expression in the horse using newly developed molecular and computational techniques. The full equine genome sequence, now freely available to researchers worldwide, has dramatically advanced equine research in many areas. However, although the protein coding genes of the horse are now quite well defined, the non-coding elements that control gene transcription and other aspects of gene expression remain largely uncharacterized and are poorly understood. Such information is crucial to exploiting the full potential of genomics to advance equine medicine. Our study dovetails with efforts of other members of the international Horse Genome Project who are collaborating in the coordinated FAANG program (Functional Annotation of Animal Genomes). We hypothesize that application of global run-on and sequencing (GRO-seq) protocols and recently described variations of those techniques (i.e. ChRO-seq, based on chromatin precipitation) can accelerate progress in defining regulatory elements in the equine genome. In Aim 1 we will elucidate transcriptional pathways that govern development of the invasive phenotype of horse placental trophoblast cells and their subsequent terminal differentiation into hormone secreting endometrial cup cells. In Aim 2 we will compare gene regulation characteristics of equine CD4+ T-cells with comparable data from primates recently published by Co-Investigator Danko. Under this aim we would also determine gene regulatory marks of equine CD8+ T-cells and B-cells. In Aim 3 we will compare our ChRO-seq results determined in equine liver with data from the same samples obtained by other members of the equine FAANG consortium using conventional approaches (e.g. ChIP-seq). As we near the end of the second year of this project we feel confident that the relatively simple ChRO-seq assay and associated computational programs developed here at Cornell are generating the same types of information as the traditional methods being used for the equine ENCODE Project, but more efficiently and at lower cost. The work builds on previous Zweig Fund-supported research from the Baker Institute that has made major contributions to the Horse Genome Project.