Genome binding/occupancy profiling by high throughput sequencing
Summary
The mammary luminal lineage relies on the common cytokine-sensing transcription factor STAT5 to establish super-enhancers during pregnancy and activate mammary genes required for the nutrition of the offspring. Exploiting progressive differentiation during lactation, we investigated how hormonal cues shape an evolving enhancer landscape and impact the biology of mammary cells. Employing ChIP-seq, we uncover a changing transcription factor occupancy at mammary enhancers. Using mouse genetics, we demonstrate changing biological properties of enhancers as lactation progresses, with individual enhancers gaining strength and an abolished need for the Wap seed enhancer. We further investigated whether permissive chromatin facilitates cell-specific transcription factor binding. Wap enhancers translocated into the widely expressed neighboring Ramp3 gene retained their mammary-specificity and failed to activate the receptive Ramp3 gene in non-mammary tissues. Our studies unveil a previously unrecognized progressive enhancer landscape, in which structurally equivalent components serve unique and differentiation-specific functions. While enhancer redundancy has been suggested and demonstrated for many genes, components of the cytokine-responsive mammary tripartite Wap super-enhancer display a remarkable specificity.
Overall design
ChIP-seq for STAT5A, GR, RNA Pol II, MED1 and H3K27ac in the mammary tissues of wild type, △E1, △E3, △E1a/3, △E1a/2/3, E12-R3 and E2-R3 mutant mice at day one and ten of lactation. ChIP-seq for STAT3, GR and H3K27ac in the kidney tissues of wild type and E12-R3 mutant mice.
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