Research resource: whole-genome estrogen receptor α binding in mouse uterine tissue revealed by ChIP-seq

Abstract

To advance understanding of mechanisms leading to biological and transcriptional endpoints related to estrogen action in the mouse uterus, we have mapped ERα and RNA polymerase II (PolII) binding sites using chromatin immunoprecipitation followed by sequencing of enriched chromatin fragments. In the absence of hormone, 5184 ERα-binding sites were apparent in the vehicle-treated ovariectomized uterine chromatin, whereas 17,240 were seen 1 h after estradiol (E₂) treatment, indicating that some sites are occupied by unliganded ERα, and that ERα binding is increased by E₂. Approximately 15% of the uterine ERα-binding sites were adjacent to (<10 kb) annotated transcription start sites, and many sites are found within genes or are found more than 100 kb distal from mapped genes; however, the density (sites per base pair) of ERα-binding sites is significantly greater adjacent to promoters. An increase in quantity of sites but no significant positional differences were seen between vehicle and E₂-treated samples in the overall locations of ERα-binding sites either distal from, adjacent to, or within genes. Analysis of the PolII data revealed the presence of poised promoter-proximal PolII on some highly up-regulated genes. Additionally, corecruitment of PolII and ERα to some distal enhancer regions was observed. A de novo motif analysis of sequences in the ERα-bound chromatin confirmed that estrogen response elements were significantly enriched. Interestingly, in areas of ERα binding without predicted estrogen response element motifs, homeodomain transcription factor-binding motifs were significantly enriched. The integration of the ERα- and PolII-binding sites from our uterine sequencing of enriched chromatin fragments data with transcriptional responses revealed in our uterine microarrays has the potential to greatly enhance our understanding of mechanisms governing estrogen response in uterine and other estrogen target tissues.

Fig. 1.

ERE motifs in WT 1-h E₂ ChIP-seq peaks. Motif discovery: ERE motifs were identified by A Genetic Algorithm Guided Formation of Spaced Dyads Coupled with an EM Algorithm for Motif Discovery (GADEM). A, ERE motif identified from 17,240 1-h E₂ peak sequences. B, ERE motif constructed from 48 experimentally identified ERE. C, Consensus ERE (1).

Fig. 2.

ERα-binding sites near Igf1. A, University of California Santa Cruz Genome Browser representation of analysis of ERα ChIP-seq binding sites of Igf1 gene. Several ERα ChIP-seq peaks from the V or 1-h E₂ sequences were mapped near the Igf1 gene. Two were adjacent to the two Igf1 TSS and were previously studied and validated by ChIP-PCR (9) (lower panel; shown by arrows). Five peaks 50 kb 5′ of the TSS were apparent in our ChIP-seq data set and were further analyzed for ERE motifs. The lower panel has the maximum scale on the ERα ChIP-seq tracks set to emphasize the smaller peaks. Peaks 2 and 4 contain consensus ERE: Peak 2, GGTCAtgaTGACC; Peak 4, GGTCAcctTGACC. B, Validation of ERα binding at Igf1 50 kb 5′-sites by ChIP-PCR. Primers flanking regions in five ChIP-seq peaks in the region 50 kb 5′ from the Igf1 TSS or closer to the TSS (−6215/−8270; −29/−2084) were used to analyze independent ChIP samples. Fold enrichments are calculated as the ratio of ERα IP to IgG IP. WT, Wild type.

Fig. 3.

Hox motifs. A, 17,240 1-h E₂ ERα peaks (consisting of both predicted ERE and non-ERE) were scanned for Hox motifs. Approximately 3700 sites were found in about 3200 sequences (18.6% of peaks). B, Levels of different Hox factor or cofactor transcripts in uterine sample derived from microarray analysis. Log signal intensities for Hox family and cofactor transcripts in V- or E₂-treated (2, 6, or 24 h) are displayed. C, Estrogen regulation of genes with ERα binding and Hox motif. Genes associated with the 3700 Hox sites in the ERα ChIP-seq peaks were examined in our microarray data (V vs. 2-, 6-, or 24-h E₂) and clustered to show those with P < 0.001 and fold change greater than 2. These uterine genes are E₂ regulated at various time points, suggesting a role for Hox in ERα-mediated uterine responses.