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Novel DNA motif binding activity observed in vivo with an estrogen receptor ? mutant mouse.

ABSTRACT: Estrogen receptor ? (ER?) interacts with DNA directly or indirectly via other transcription factors, referred to as "tethering." Evidence for tethering is based on in vitro studies and a widely used "KIKO" mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ER? binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ER? has HRE DNA-binding activity, we evaluated the "EAAE" ER?, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ER? null-like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ER? tethering, because KIKO ER? effectively stimulates transcription using HRE motifs. The EAAE-ER? DNA-binding domain mutant mouse demonstrates that ER? DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ER? tethering may not contribute to estrogen responsiveness in vivo.

SUBMITTER: Hewitt SC

PROVIDER: S-EPMC4042070 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Novel DNA motif binding activity observed in vivo with an estrogen receptor α mutant mouse.

Hewitt Sylvia C SC Li Leping L Grimm Sara A SA Winuthayanon Wipawee W Hamilton Katherine J KJ Pockette Brianna B Rubel Cory A CA Pedersen Lars C LC Fargo David D Lanz Rainer B RB DeMayo Francesco J FJ Schütz Günther G Korach Kenneth S KS

Molecular endocrinology (Baltimore, Md.) 20140408 6

Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as "tethering." Evidence for tethering is based on in vitro studies and a widely used "KIKO" mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the ...[more]

PMID: 24713037

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Project description:Using a chromatin immunoprecipitation-paired end diTag cloning and sequencing strategy, we mapped estrogen receptor alpha (ERalpha) binding sites in MCF-7 breast cancer cells. We identified 1,234 high confidence binding clusters of which 94% are projected to be bona fide ERalpha binding regions. Only 5% of the mapped estrogen receptor binding sites are located within 5 kb upstream of the transcriptional start sites of adjacent genes, regions containing the proximal promoters, whereas vast majority of the sites are mapped to intronic or distal locations (>5 kb from 5' and 3' ends of adjacent transcript), suggesting transcriptional regulatory mechanisms over significant physical distances. Of all the identified sites, 71% harbored putative full estrogen response elements (EREs), 25% bore ERE half sites, and only 4% had no recognizable ERE sequences. Genes in the vicinity of ERalpha binding sites were enriched for regulation by estradiol in MCF-7 cells, and their expression profiles in patient samples segregate ERalpha-positive from ERalpha-negative breast tumors. The expression dynamics of the genes adjacent to ERalpha binding sites suggest a direct induction of gene expression through binding to ERE-like sequences, whereas transcriptional repression by ERalpha appears to be through indirect mechanisms. Our analysis also indicates a number of candidate transcription factor binding sites adjacent to occupied EREs at frequencies much greater than by chance, including the previously reported FOXA1 sites, and demonstrate the potential involvement of one such putative adjacent factor, Sp1, in the global regulation of ERalpha target genes. Unexpectedly, we found that only 22%-24% of the bona fide human ERalpha binding sites were overlapping conserved regions in whole genome vertebrate alignments, which suggest limited conservation of functional binding sites. Taken together, this genome-scale analysis suggests complex but definable rules governing ERalpha binding and gene regulation.

Dataset Information

Novel DNA motif binding activity observed in vivo with an estrogen receptor ? mutant mouse.

Publications

Novel DNA motif binding activity observed in vivo with an estrogen receptor α mutant mouse.

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