Multiplex Enhancer Interference Reveals Collaborative Control of Gene Regulation by Estrogen Receptor α-Bound Enhancers

• Published in: Cell systems Vol. 5; no. 4; pp. 333 - 344.e5
• Main Authors: Carleton, Julia B., Berrett, Kristofer C., Gertz, Jason
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 25.10.2017
• Subjects: Cell Line; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; CRISPR/Cas9; enhancer; Enhancer Elements, Genetic - genetics; enhancer interference; estrogen receptor alpha; Estrogen Receptor alpha - genetics; Gene Expression Regulation - genetics; gene regulation; Humans; Promoter Regions, Genetic - genetics; Protein Binding - genetics; Transcription Factors - genetics; Transcription, Genetic - genetics; enhancer interference; enhancer; CRISPR/Cas9; gene regulation; estrogen receptor alpha
• ISSN: 2405-4712; 2405-4720
• DOI: 10.1016/j.cels.2017.08.011

Multiple regulatory regions have the potential to regulate a single gene, yet how these elements combine to affect gene expression remains unclear. To uncover the combinatorial relationships between enhancers, we developed Enhancer-interference (Enhancer-i), a CRISPR interference-based approach that uses 2 different repressive domains, KRAB and SID, to prevent enhancer activation simultaneously at multiple regulatory regions. We applied Enhancer-i to promoter-distal estrogen receptor α binding sites (ERBS), which cluster around estradiol-responsive genes and therefore may collaborate to regulate gene expression. Targeting individual sites revealed predominant ERBS that are completely required for the transcriptional response, indicating a lack of redundancy. Simultaneous interference of different ERBS combinations identified supportive ERBS that contribute only when predominant sites are active. Using mathematical modeling, we find strong evidence for collaboration between predominant and supportive ERBS. Overall, our findings expose a complex functional hierarchy of enhancers, where multiple loci bound by the same transcription factor combine to fine-tune the expression of target genes. [Display omitted] •Multiple ER binding sites (ERBS) cluster near genes up-upregulated by estrogen•Multiplex Enhancer-i facilitates dissection of ERBS contribution to gene expression•Distance to target gene and strength of ERE motif predict ERBS necessity•Predominant sites and supportive sites collaborate to produce the estrogen response Carleton et al. have developed a CRISPR-based technique, Enhancer-i, which enables simultaneous deactivation of multiple enhancers and allows for the functional dissection of how enhancers work together to regulate gene expression. They used Enhancer-i to identify estrogen receptor α bound enhancers required for the transcriptional response to estrogens and discovered that specific combinations of enhancers collaborate to produce the estrogen response.