The genetic differences found in gene regulatory regions are the largest contributing factor to the diversity of phenotypes within and between mammalian species. However, the micro-evolutionary mechanisms active in closely-related species remain poorly explored, particularly in mammals.In Aim 1, we will exploit five recently sequenced mouse species to determine how often genetic sequence differences alter both the genome-wide binding and regulatory output of a core set of tissue-specific transcription factors (TFs) known to act combinatorially in liver. This project aim will establish the micro-evolutionary processes that drive interspecies changes in transcriptional regulation.In Aim 2, we will create first-generation intercrosses of a subset of these mouse species to dissect the cis and trans contributions to TF binding differences between species, and to explore the functional implications for nearby chromatin and gene expression. This aim will also establish whether any tissue-specific TF binding sites show parent-of-chromosomal origin effects.In Aim 3, we will use classically generated TF knockout mice to identify a set of functionally enriched TF binding sites, which will be further categorized by their conservation across the five mouse species. Then, using the revolutionary ability to rapidly and precisely delete individual binding sites (as well as combinations of binding sites) in the mouse genome by zinc finger nucleases, we will test whether conservation of these protein-DNA contacts can predict functional activity at target genes.This integrated approach combines the comparison of TF binding in closely-related mammals with powerful new experimental tools to afford a comprehensive understanding of the genetics and mechanisms underlying the micro-evolution of transcriptional regulatory networks.