SUMMARY Once considered junk, non-coding regions of the genome have emerged as central components of evolution, development, and disease. The most common non-coding regulatory elements in the human genome are enhancers, which ensure expression of target genes at the right time in the right cells by controlling their activation. Perturbation to enhancer function is widely accepted as a major, but still poorly understood, component of human brain evolution and disease. There have been major and continuing advances in annotating enhancers and predicting activity of these elements in cells and tissues, including the brain. Despite these advances, predicting the sequence-encoded function of enhancers remains a major challenge. Further, the dynamic and context-dependent chromosomal interactions, epigenetic modifications, and transcription factor activity that ultimately determine enhancer-mediated gene regulation generally remain poorly understood. This represents a significant barrier in understanding the function of enhancers and in interpreting the effect of enhancer sequence variation on human brain development, evolution and disease. As such, there is critical need to determine the relationship between sequence and function for regulatory DNA, and to define the determinants of enhancer activity and gene regulation in the brain. In the initial early stage investigator MIRA funding, we established a productive research program focused elucidating enhancer- mediated gene regulatory wiring in the mammalian brain. We paired functional assays with genetic and genomic approaches to model the function of enhancers, transcription factors, and chromatin remodeling proteins in normal and pathogenic brain development. The overarching goals of our MIRA research program are to: 1) Extend and apply methods to define sequence-encoded enhancer activity in the mammalian brain, 2) Determine the molecular mechanisms of enhancer-mediated gene regulation and transcriptional programming in the brain, and 3) Characterize the consequences of regulatory sequence variation to understand the role of enhancer DNA in the development, evolution, and disorders of the mammalian brain. In the renewal period, we will apply integrative genetic, genomic, and neuroscience methods to address key gaps in the understanding of sequence-encoded enhancer function and to answer fundamental questions regarding gene regulation in the brain. Our work will address basic and translationally-relevant questions regarding the sufficiency and necessity of enhancers for neurodevelopmental gene regulation, and will advance the emerging field of enhancer-based tools for labeling and manipulation of cell types in the brain. Overall, our contributions will help to decipher how transcriptional control is encoded at the genetic and epigenetic level and to illuminate the gene regulatory circuitry of the mammalian brain.