This dissertation, by Yiren Hu, discusses how enhancer activity is regulated to drive rapid, coordinated transcriptional response to signals like estrogen and inflammatory stimuli. Enhancer elements play a central role in instructing precise spatiotemporal gene expression and are capable of activating target gene transcription from a long distance. How do enhancers achieve these functions? And how are enhancers regulated in response to multiple signals at the same time? To answer these questions, we took a high-throughput approach with the power of next-generation sequencing technologies to study the genome wide transcription landscape changes of human breast cancer cells as well as mouse immune cells in response to stimuli like estradiol and TNFα. We tackled enhancer regulatory mechanisms from three aspects and found that: 1) chromatin structure regulators Condensin Complex I and II play essential roles in activating Estrogen Receptor α-bound enhancers by balancing transcription co-activators versus co-repressors homeostasis (see Chapter 1) 2) histone demethylase JMJD6 activates Estrogen Receptor α-bound enhancers and controls coding gene Pol II pausing release by modulating mediator complex protein MED12 chromatin loading (see Chapter 2) 3) Estrogen Receptor α and histone demethylase KDM2B form a two-layer restriction mechanism to repress NFκB enhancers and corresponding pro-inflammatory genes (see Chapter 3 and 4).