Mammary-Enriched Transcription Factors Synergize to Activate the Wap Super-Enhancer for Mammary Gland Development

Super-enhancers are large clusters of enhancers critical for cell-type-specific development. In a previous study, 440 mammary-specific super-enhancers, highly enriched for an active enhancer mark H3K27ac; a mediator MED1; and the mammary-enriched transcription factors ELF5, NFIB, STAT5A, and GR, were identified in the genome of the mammary epithelium of lactating mice. However, the triggering mechanism for mammary-specific super-enhancers and the molecular interactions between key transcription factors have not been clearly elucidated. In this study, we investigated in vivo protein–protein interactions between major transcription factors that activate mammary-specific super-enhancers. In mammary epithelial cells, ELF5 strongly interacted with NFIB while weakly interacting with STAT5A, and it showed modest interactions with MED1 and GR, a pattern unlike that in non-mammary cells. We further investigated the role of key transcription factors in the initial activation of the mammary-specific Wap super-enhancer, using CRISPR-Cas9 genome editing to introduce single or combined mutations at transcription factor binding sites in the pioneer enhancer of the Wap super-enhancer in mice. ELF5 and STAT5A played key roles in igniting Wap super-enhancer activity, but an intact transcription factor complex was required for the full function of the super-enhancer. Our study demonstrates that mammary-enriched transcription factors within a protein complex interact with different intensities and synergize to activate the Wap super-enhancer. These findings provide an important framework for understanding the regulation of cell-type-specific development.