Abstract 2920: Transcriptional regulation of estrogen response through long-range chromatin interactions

Abstract

Abstract Classical 17β-estradiol (E2) signaling requires the activity of the estrogen receptor at genomic regulatory regions. Differences in DNA sequence, ligand, receptor type and co-factor availability can affect this activity. However, the role of chromatin structure in the transmission of the estrogen signal is relatively unexplored. The 3D interaction between regulatory elements and promoters at long-range chromatin interaction sites presents a novel and dynamic mechanism of transcriptional regulation. Recent work to study such interactomes has identified relationships among histone modifications and loop boundaries [1], as well as among specific proteins, including estrogen receptor alpha (ERα), that appear to mediate the formation of chromatin loops [2-4]. We wish to identify the protein, DNA sequence, and epigenetic characteristics required for chromatin looping at ERα-bound sites. The publication of an ERα-bound interactome in the MCF-7 breast cancer cell line [2] has permitted us to identify the 67-kb loop region upstream of the krüppel-like factor 9 (KLF9) gene as an ideal candidate region for examination of the role of chromatin looping in breast cancer ERα-mediated transcriptional regulation. To study chromatin looping in situ, we have developed a model system which integrates a luciferase gene with the entire native regulatory locus of KLF9 on a single BAC. With this construct we can use reporter assays to screen for the elements required for long-distance chromatin interaction mediated, E2 stimulated transcriptional regulation. We detect early estrogen-induced activation of KLF9 in MCF-7 breast cancer cells and have validated 3/5 previously reported ERα binding sites [2, 5, 6] in this region by quantitative ChIP. Enrichment of ERα at these sites is E2 dependent. Additionally, we observe unique transcriptional responses from proximal and distal ER-bound sites in reporter assays. We hypothesize that the KLF9 loop is responsible for a rapid repression of transcriptional that occurs between 2-4 hours post E2 treatment. Chromatin looping is a regulatory mechanism common among most nuclear hormones and occurs at many estrogen-responsive genes; this process may present a novel mechanism for drug targeting in the treatment of many anti-endocrine resistant cancers and disorders. Future work will test our hypothesis of loop-based repression, identify the elements required for looping at the KLF9 locus and detect patterns in epigenetic modifications that may regulate or be regulated by the loop structure. Unique protein targets involved in looping will be examined for regulatory roles at a genome-wide level. 1. Handoko, L., et al., Nat Genet, 2011: p. 630-8. 2. Fullwood, M.J., et al., Nature, 2009: p. 58-64. 3. Tan, S.K., et al., EMBO J, 2011: p. 2569-81. 4. Kagey, M.H., et al., Nature, 2010: p. 430-435. 5. Carroll, J.S., et al., Nat Genet, 2006: p. 1289-1297. 6. Hua, S., et al., Mol Syst Biol, 2008. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2920. doi:1538-7445.AM2012-2920