Prolactin Drives a Dynamic STAT5A/HDAC6/HMGN2 Cis-Regulatory Landscape Exploitable in ER+ Breast Cancer

The hormone, prolactin, has been implicated in breast cancer pathogenesis and regulates chromatin engagement by the transcription factor, STAT5A. STAT5A is known to inducibly bind promoters and cis-regulatory elements genome wide, though the mechanisms by which it exerts specificity and regulation of target gene expression remain enigmatic. We previously identified HDAC6 and HMGN2 as cofactors that facilitate prolactin induced, STAT5A mediated gene expression. Here, multi-condition STAT5A, HDAC6, and HMGN2 ChIP-seq with parallel condition RNA-seq are utilized to reveal the cis-regulatory landscape and cofactor dynamics underlying prolactin stimulated gene expression in breast cancer. We find that prolactin regulated genes are significantly enriched for cis-regulatory elements bound by HDAC6 and HMGN2, and that inducible STAT5A binding at enhancers, rather than promoters, conveys specificity for prolactin regulated genes. The selective HDAC6 inhibitor, ACY-241, blocks prolactin induced STAT5A chromatin engagement at cis-regulatory elements as well as a significant proportion of prolactin stimulated gene expression. We identify functional pathways known to contribute to the development and/or progression of breast cancer that are activated by prolactin and inhibited by ACY-241. Additionally, we find that the DNA sequences underlying shared STAT5A and HDAC6 binding-sites at enhancers are differentially enriched for estrogen response elements (ESR1 and ESR2 motifs) relative to enhancers bound by STAT5A alone. Gene set enrichment analysis identifies significant overlap of ERα regulated genes with genes regulated by prolactin, particularly prolactin regulated genes with promoters or enhancers co-occupied by both STAT5A and HDAC6. High quality STAT5A, HDAC6, and HMGN2 ChIP-seq datasets were generated in full accordance with guidelines and quality standards established by the ENCODE consortia. Of primary importance, the antibodies utilized here were rigorously tested for specificity, confirmed by knockdown of each targeted protein. To investigate hormone stimulated binding dynamics and the influence of pharmacological HDAC6 inhibition, the chromatin of MCF-7 human breast cancer cells was harvested for ChIP before and after exposure to prolactin (30 minutes; 250 ng/mL) and/or ACY-241 [Citarinostat] (120 minutes; 2 µM). To enable the most informative comparisons across ChIP targets, for each experimental condition (and biological replicate), pooled chromatin was aliquoted for incubation with each antibody immediately prior to immunoprecipitation, ensuring sampling from an identical cell population. Sequencing depth, read quality, and read duplication rates were all well within ideal specifications for mammalian point-source transcription factors. Reads mapping to ENCODE blacklist regions were removed before union peaksets of non-overlapping, robust (MACS2 FDR<0.01) peaks were identified for each factor (88,709 STAT5A peaks; 12,815 HDAC6 peaks; 102,661 HMGN2 peaks). In parallel experiments, we generated matching condition RNA-seq expression data identifying significant (FDR<0.05), differentially expressed genes following prolactin stimulation and/or ACY-241 treatment.