Josée Laganière

Genome-Wide Identification of Direct Target Genes Implicates Estrogen-Related Receptor α as a Determinant of Breast Cancer Heterogeneity

Estrogen-related receptor alpha (ERRalpha) is an orphan nuclear receptor, the expression of which correlates with negative prognosis in breast cancer. ERRalpha shares functional features with the estrogen receptor alpha (ERalpha) and its activity is modulated by the ERBB2 signaling pathway. Using genome-wide binding sites location analyses in ERalpha-positive and ERalpha-negative breast cancer cell lines, we show that ERRalpha and ERalpha display strict binding site specificity and maintain independent mechanisms of transcriptional activation. Nonetheless, ERRalpha and ERalpha coregulate a small subset of common target genes via binding either to a dual-specificity binding site or to distinct cognate binding sites located within the extended promoter region of the gene. Although ERRalpha signaling in breast cancer cells is mostly independent of ERalpha, the small fraction of common ERRalpha/ERalpha targets comprises genes with high relevance to breast tumor biology, including genes located within the ERBB2 amplicon and GATA3. Finally, unsupervised hierarchical clustering based on the expression profiling of ERRalpha direct target genes in human breast tumors revealed four main clusters that recapitulate established tumor subtypes. Taken together, the identification and functional characterization of the ERRalpha transcriptional network implicate ERRalpha signaling as a determinant of breast cancer heterogeneity.

Nuclear receptor target gene discovery using high-throughput chromatin immunoprecipitation.

Publisher Summary Nuclear receptors are master transcription factors that regulate the development, physiology, and homeostasis of whole organisms through the direct control of gene expression in response to diverse ligands and hormonal stimuli. Nuclear receptors regulate the expression of their target genes through association with specific DNA regulatory elements. While a significant number of nuclear receptor target genes have been identified to date, it is believed that these genes represent only a small fraction of the regulatory units likely to be under the control of nuclear receptors. Most nuclear receptor target genes identified so far were characterized through ‘‘gene oriented’’ approaches that study the regulation of one candidate gene at a time, and these studies are usually limited to the promoter region. To understand the complex nuclear receptor-driven transcriptional networks that operate in a living organism, a whole genome approach is required. This chapter describes a powerful ‘‘nuclear receptor/whole genome-oriented’’ approach to identify and more accurately study nuclear receptor regulatory networks. Prior to performing the actual Chromatin Immunoprecipitation (ChIP), it is important to remove and freeze an aliquot of the diluted fragmented chromatin corresponding to 10% of the total amount used for one IP. The development of high-throughput ChIP technology now permits a whole-genome analysis of gene regulation by nuclear receptors. The efficient cloning of new regulatory elements harboring high-affinity-binding sites for specific nuclear receptors can now be efficiently achieved, and allows for the identification of new target genes within a particular cell context.