Enhancers are essential cis-regulatory elements that orchestrate cell-type-specific gene expression. While conceptually simple, their mechanisms of action are remarkably complex. At the level of individual elements, active enhancers typically consist of a central transcription factor binding region flanked by a pair of divergent core promoters. On a broader genomic scale, individual enhancers frequently collaborate with other regulatory elements to achieve precise and robust transcriptional control. In this thesis, I aim to bridge these two layers of enhancer regulation through a combination of genome engineering, high-throughput screening, and functional genomics. I first dissected the sequence–function relationship of a model long-range human enhancer, “eNMU,” which drives an extraordinary ~10,000-fold activation of its target gene NMU from 94 kb away. Systematic dissection guided by the divergent transcription model revealed extensive transcription factor synergy at this enhancer and uncovered a complex interplay between the core divergently transcribed enhancer unit and surrounding cis-regulatory elements. Notably, these include intrinsically inactive facilitators that augment and buffer enhancer output, as well as an adjacent retroviral long terminal repeat (LTR) promoter that acts to repress enhancer activity. These two emerging modes of cis-acting logic may be broadly utilized across the genome, suggesting that the complexity of enhancer regulation has been significantly underestimated. In a parallel line of investigation, I identified a heat shock–induced, HSF1-bound distal enhancer and systematically characterized ~200 HSF1-bound distal elements using a massively parallel episomal reporter assay and the sensitive PRO-cap assay. This analysis revealed that heat-induced enhancer transcription is a prerequisite for heat-induced enhancer activity. Together, these findings offer new insights into the multilayered mechanisms by which enhancers function and are themselves regulated.