Sensitive ChIP-DSL technology reveals an extensive estrogen receptor alpha-binding program on human gene promoters

Abstract

ChIP coupled with microarray provides a powerful tool to determine in vivo binding profiling of transcription factors to deduce regulatory circuitries in mammalian cells. Aiming at improving the specificity and sensitivity of such analysis, we developed a new technology called ChIP-DSL using the DNA selection and ligation (DSL) strategy, permitting robust analysis with much reduced materials compared with standard procedures. We profiled general and sequence-specific DNA binding transcription factors using a full human genome promoter array based on the ChIP-DSL technology, revealing an unprecedented number of the estrogen receptor (ERalpha) target genes in MCF-7 cells. Coupled with gene expression profiling, we found that only a fraction of these direct ERalpha target genes were highly responsive to estrogen and that the expression of those ERalpha-bound, estrogen-inducible genes was associated with breast cancer progression in humans. This study demonstrates the power of the ChIP-DSL technology in revealing regulatory gene expression programs that have been previously invisible in the human genome.

Fig. 1.

The ChIP-DSL scheme. A key feature of the technology is oligonucleotide ligation templated by chromatin immunoprecipitated (ChIP) DNA followed by DSL. This permits high-throughput analysis of target genes with much improved specificity and sensitivity.

Fig. 2.

Global analysis of promoter occupancy by ChIP-DSL. (A) Global analysis of Pol II-bound promoters in E₂-stimulated MCF-7 cells. A set of tiled genomic loci (yellow) served as internal negative controls because most genomic sequences are not expected to interact with general and sequence-specific transcription factors. Pol II-positive (red) and -negative (black) promoters were identified based on the single-array error model at P < 0.001, and the percentages of Pol II-positive and -negative promoters are shown in Inset. (B) ChIP/qPCR validation of the ChIP-DSL results. (C) Promoter profiling of modified histones in E₂-treated MCF-7 cells. (Left) Percentages of positive promoters. (Right) Overlap of positive promoters with Pol II. The overlap between Pol II binding and individual histone modification events is shown in individual Venn diagrams. (D) Correlation of gene expression with promoter occupancy by Pol II and histone modification markers. Gene expression profiling in E₂-induced MCF-7 cells was carried out on Illumina gene expression arrays. Approximately 10,000 genes common to both promoter and expression profiling arrays and reliably scored in all measurements were used to construct the binary map by unsupervised hierarchical clustering analysis.

Fig. 3.

Promoter profiling of ERα in E₂-induced MCF-7 cells. (A and B) ERα-bound promoters were identified (red) at P < 0.0001. The percentage of ERα-bound promoters scored positively by all three analytical methods is shown in A Inset, and additional promoters scored positively by one or two methods are indicated in the peripheries of the Venn diagram in B. (C) Listed are the newly identified ERα-positive promoters on chromosomes 21 and 22. Ratios were deduced from array measurements. Selected promoters were validated by ChIP/qPCR (Right). (D) Motif analysis of anti-ERα-enriched promoters. The first motif appears common to gene promoters in general, but the protein(s) recognizing this motif is unknown. When this motif is masked, the most enriched motif corresponds to full- or half-consensus estrogen responsive element. Allowing one base mismatch, the percentage of promoters containing a full- or half-consensus estrogen responsive element among total ERα-bound promoters was calculated and shown in Right.

Fig. 4.

Locus-specific tiling array analysis of ERα binding and histone modifications in E₂-induced MCF-7 cells. Individual genes and scales are shown at the top, and probe positions and gene structure are indicated at the bottom. Individual transcription factors and chromatin remodeling markers profiled are indicated on the left. Transcription starts and known or putative enhancers are designated by filled and open arrowheads, respectively, at the bottom.

Fig. 5.

E₂-induced gene expression and the biological relevance of direct ERα target genes. (A) Relationship between ERα binding and histone modifications. To directly compare ERα binding and E₂-induced gene expression, 467 of 578 ERα-bound promoters common between our promoter array and the Illumina gene expression array were analyzed. The majority of ERα-positive promoters was also marked by Pol II and modified histones associated with gene activation. (B) Venn diagram showing the overlap between ERα-bound promoters and E₂-induced genes. (C) Gene expression profiling in response to E₂ treatment. ERα-bound and E₂-regulated genes are grouped into four distinct classes. Among up-regulated genes, 29 were rapidly induced, and the level remained relatively constant afterward; eight were induced in a time-dependent manner; and 12 were induced followed by a rapid decay. E₂-induced, genes represent 10.5% of total ERα-bound genes in the promoter-proximal region. Only five ERα-bound genes were down-regulated by E₂, which represent 1.1% of total ERα-bound genes in the promoter-proximal region. (D) Segregation of ER expression and breast tumor grade (both indicated at the top by blue bars) based on ERα-bound and E₂-induced genes in MCF-7 cells. (E) Kaplan–Meier plots of patient survival in different groups segregated based on ERα-bound and E₂-inducted genes in MCF-7 cells. Statistical significance was determined by the χ² test.