Redundant and non-redundant cytokine-activated enhancers control Csn1s2b expression in the lactating mouse mammary gland

Hye Kyung Lee,Lothar Hennighausen,Chengyu Liu,Tyler Kuhns,Michaela Willi

doi:10.1038/s41467-021-22500-w

Abstract

Enhancers are transcription factor platforms that synergize with promoters to control gene expression. Here, we investigate enhancers that activate gene expression several hundred-fold exclusively in the lactating mouse mammary gland. Using ChIP-seq for activating histone marks and transcription factors, we identify two candidate enhancers and one super-enhancer in the Csn1s2b locus. Through experimental mouse genetics, we dissect the lactation-specific distal enhancer bound by the mammary-enriched transcription factors STAT5 and NFIB and the glucocorticoid receptor. While deletions of canonical binding motifs for NFIB and STAT5, individually or combined, have a limited biological impact, a non-canonical STAT5 site is essential for enhancer activity during lactation. In contrast, the intronic enhancer contributes to gene expression only in late pregnancy and early lactation, possibly by interacting with the distal enhancer. A downstream super-enhancer, which physically interacts with the distal enhancer, is required for the functional establishment of the Csn1s2b promoter and gene activation. Lastly, NFIB binding in the promoter region fine-tunes Csn1s2b expression. Our study provides comprehensive insight into the anatomy and biology of regulatory elements that employ the JAK/STAT signaling pathway and preferentially activate gene expression during lactation.

Highlights

Enhancers are transcription factor platforms that synergize with promoters to control gene expression
Each of the five sites bound by STAT5 coincided with at least one GAS motif supporting a direct protein-DNA interaction
While maximum STAT5 binding at the Csn1s2a sites was already observed at day 18 of pregnancy (p18) and remained high throughout lactation, STAT5 binding at the candidate Csn1s2b enhancer was marginally detectable at p18 and was fully established between L1 and L10 (Fig. 1b and Supplementary Fig. 1a–c)

Summary

Introduction

Enhancers are transcription factor platforms that synergize with promoters to control gene expression. ChIP-seq profiles for STAT5 and H3K27ac and other mammary-enriched TFs suggested the presence of highly complex mammary enhancers[8] Most of these enhancers appear to depend on STAT5 as the anchor for the establishment of larger protein complexes, the stage-specific generation of enhancers remains to be understood. We identified two candidate enhancers and one super-enhancer in the extended Csn1s2b locus and investigated a potential synergy between the prolactininduced TF STAT5 and the mammary-enriched Nuclear Factor I B (NFIB) in the establishment of lactation-specific regulatory elements. We employed experimental mouse genetics and functionally dissected the two enhancers, the super-enhancer and the Csn1s2b promoter This permitted us to define the contributions of individual enhancers and the significance of STAT5 and NFIB in the activating the Csn1s2b gene during pregnancy and lactation

Methods

Results

Conclusion