SummaryOestrogen receptor-α (ER) is the defining and driving transcription factor in the majority of breast cancers and its target genes dictate cell growth and endocrine response, yet genomic understanding of ER function has been restricted to model systems1-3. We now map genome-wide ER binding events, by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), in primary breast cancers from patients with different clinical outcome and in distant ER positive (ER+) metastases. We find that drug resistant cancers still have ER-chromatin occupancy, but that ER binding is a dynamic process, with the acquisition of unique ER binding regions in tumours from patients that are likely to relapse. The acquired, poor outcome ER regulatory regions observed in primary tumours reveal gene signatures that predict clinical outcome in ER+ disease exclusively. We find that the differential ER binding programme observed in tumours from patients with poor outcome is not due to the selection of a rare subpopulation of cells, but is due to the FoxA1-mediated reprogramming of ER binding on a rapid time scale. The parallel redistribution of ER and FoxA1 cis-regulatory elements in drug resistant cellular contexts is supported by histological co-expression of ER and FoxA1 in metastatic samples. By establishing transcription factor mapping in primary tumour material, we show that there is plasticity in ER binding capacity, with distinct combinations of cis-regulatory elements linked with the different clinical outcomes.

Breast cancer risk is influenced by rare coding variants in susceptibility genes such as BRCA1 and many common, mainly non-coding variants. However, much of the genetic contribution to breast cancer risk remains unknown. We report results from a genome-wide association study (GWAS) of breast cancer in 122,977 cases and 105,974 controls of European ancestry and 14,068 cases and 13,104 controls of East Asian ancestry1. We identified 65 new loci associated with overall breast cancer at p<5x10-8. The majority of credible risk SNPs in the new loci fall in distal regulatory elements, and by integrating in-silico data to predict target genes in breast cells at each locus, we demonstrate a strong overlap between candidate target genes and somatic driver genes in breast tumours. We also find that heritability of breast cancer due to all SNPs in regulatory features was 2-5-fold enriched relative to the genome-wide average, with strong enrichment for particular transcription factor binding sites. These results provide further insight into genetic susceptibility to breast cancer and will improve the utility of genetic risk scores for individualized screening and prevention.

The estrogen receptor is the master transcriptional regulator of breast cancer phenotype and the archetype of a molecular therapeutic target. We mapped all estrogen receptor and RNA polymerase II binding sites on a genome-wide scale, identifying the authentic cis binding sites and target genes, in breast cancer cells. Combining this unique resource with gene expression data demonstrates distinct temporal mechanisms of estrogen-mediated gene regulation, particularly in the case of estrogen-suppressed genes. Furthermore, this resource has allowed the identification of cis-regulatory sites in previously unexplored regions of the genome and the cooperating transcription factors underlying estrogen signaling in breast cancer.

Transcription factors are adaptor molecules that detect regulatory sequences in the DNA and target the assembly of protein complexes that control gene expression. Yet much of the DNA in the eukaryotic cell is in nucleosomes and thereby occluded by histones, and can be further occluded by higher-order chromatin structures and repressor complexes. Indeed, genome-wide location analyses have revealed that, for all transcription factors tested, the vast majority of potential DNA-binding sites are unoccupied, demonstrating the inaccessibility of most of the nuclear DNA. This raises the question of how target sites at silent genes become bound de novo by transcription factors, thereby initiating regulatory events in chromatin. Binding cooperativity can be sufficient for many kinds of factors to simultaneously engage a target site in chromatin and activate gene expression. However, in cases in which the binding of a series of factors is sequential in time and thus not initially cooperative, special “pioneer transcription factors” can be the first to engage target sites in chromatin. Such initial binding can passively enhance transcription by reducing the number of additional factors that are needed to bind the DNA, culminating in activation. In addition, pioneer factor binding can actively open up the local chromatin and directly make it competent for other factors to bind. Passive and active roles for the pioneer factor FoxA occur in embryonic development, steroid hormone induction, and human cancers. Herein we review the field and describe how pioneer factors may enable cellular reprogramming.

Estrogen plays an essential physiologic role in reproduction and a pathologic one in breast cancer. The completion of the human genome has allowed the identification of the expressed regions of protein-coding genes; however, little is known concerning the organization of their cis-regulatory elements. We have mapped the association of the estrogen receptor (ER) with the complete nonrepetitive sequence of human chromosomes 21 and 22 by combining chromatin immunoprecipitation (ChIP) with tiled microarrays. ER binds selectively to a limited number of sites, the majority of which are distant from the transcription start sites of regulated genes. The unbiased sequence interrogation of the genuine chromatin binding sites suggests that direct ER binding requires the presence of Forkhead factor binding in close proximity. Furthermore, knockdown of FoxA1 expression blocks the association of ER with chromatin and estrogen-induced gene expression demonstrating the necessity of FoxA1 in mediating an estrogen response in breast cancer cells.

Complex organisms require tissue-specific transcriptional programs, yet little is known about how these are established. The transcription factor FoxA1 is thought to contribute to gene regulation through its ability to act as a pioneer factor binding to nucleosomal DNA. Through genome-wide positional analyses, we demonstrate that FoxA1 cell type-specific functions rely primarily on differential recruitment to chromatin predominantly at distant enhancers rather than proximal promoters. This differential recruitment leads to cell type-specific changes in chromatin structure and functional collaboration with lineage-specific transcription factors. Despite the ability of FoxA1 to bind nucleosomes, its differential binding to chromatin sites is dependent on the distribution of histone H3 lysine 4 dimethylation. Together, our results suggest that methylation of histone H3 lysine 4 is part of the epigenetic signature that defines lineage-specific FoxA1 recruitment sites in chromatin. FoxA1 translates this epigenetic signature into changes in chromatin structure thereby establishing lineage-specific transcriptional enhancers and programs.

Estrogen Receptor-α (ER) is the key feature in the majority of breast cancers and ER binding to the genome correlates with the Forkhead protein FOXA1 (HNF3α). We now show that FOXA1 is a critical determinant that can influence differential ER-chromatin interactions. We show that almost all ER-chromatin interactions and gene expression changes are dependent on the presence of FOXA1 and that FOXA1 influences genome-wide chromatin accessibility. Furthermore, we show that CTCF is an upstream negative regulator of FOXA1-chromatin interactions. In ER responsive breast cancer cells, the dependency on FOXA1 for tamoxifen-ER activity is absolute and in tamoxifen resistant cells, ER binding occurs independently of ligand, but in a FOXA1 dependent manner. Importantly, expression of FOXA1 in non-breast cancer cells can alter ER binding and function. As such, FOXA1 is a major determinant of estrogen-ER activity and endocrine response in breast cancer cells.

SUMMARY The evolution of prostate cancer from an androgen-dependent state (ADPCa) to one that is androgen-independent (AIPCa) marks its lethal progression. The androgen receptor (AR) is essential in both, though its function in AIPCa is poorly understood. We have defined the direct AR-dependent target genes in both AIPCa and ADPCa by generating AR-dependent gene expression profiles and AR cistromes. In contrast to ADPCa, AR selectively up-regulates M-phase cell cycle genes in AIPCa including UBE2C, a gene that inactivates the M-phase checkpoint. Selective epigenetic marks and collaborating transcription factor occupancy at UBE2C enhancers leads to increased AR recruitment and UBE2C over-expression in AIPCa cell lines and clinical cases. Silencing of UBE2C blocks AIPCa but not ADPCa growth. Thus the role of AR in AIPCa is not to direct the androgen-dependent gene expression program without androgen, but rather to execute a distinct program resulting in androgen-independent growth.