Mathieu Lupien

FoxA1 Translates Epigenetic Signatures into Enhancer-Driven Lineage-Specific Transcription

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.

Androgen Receptor Regulates a Distinct Transcription Program in Androgen-Independent Prostate Cancer

The evolution of prostate cancer from an androgen-dependent state to one that is androgen-independent marks its lethal progression. The androgen receptor (AR) is essential in both, though its function in androgen-independent cancers is poorly understood. We have defined the direct AR-dependent target genes in both androgen-dependent and -independent cancer cells by generating AR-dependent gene expression profiles and AR cistromes. In contrast to what is found in androgen-dependent cells, AR selectively upregulates M-phase cell-cycle genes in androgen-independent cells, including UBE2C, a gene that inactivates the M-phase checkpoint. We find that epigenetic marks at the UBE2C enhancer, notably histone H3K4 methylation and FoxA1 transcription factor binding, are present in androgen-independent cells and direct AR-enhancer binding and UBE2C activation. Thus, the role of AR in androgen-independent cancer cells is not to direct the androgen-dependent gene expression program without androgen, but rather to execute a distinct program resulting in androgen-independent growth.

Nucleosome Dynamics Define Transcriptional Enhancers

Chromatin plays a central role in eukaryotic gene regulation. We performed genome-wide mapping of epigenetically marked nucleosomes to determine their position both near transcription start sites and at distal regulatory elements, including enhancers. In prostate cancer cells, where androgen receptor binds primarily to enhancers, we found that androgen treatment dismisses a central nucleosome present at androgen receptor binding sites that is flanked by a pair of marked nucleosomes. A new quantitative model built on the behavior of such nucleosome pairs correctly identified regions bound by the regulators of the immediate androgen response, including androgen receptor and FOXA1. More importantly, this model also correctly predicted previously unidentified binding sites for other transcription factors present after prolonged androgen stimulation, including OCT1 and NKX3-1. Therefore, quantitative modeling of enhancer structure provides a powerful predictive method to infer the identity of transcription factors involved in cellular responses to specific stimuli.

Estrogen-Dependent Signaling in a Molecularly Distinct Subclass of Aggressive Prostate Cancer

BACKGROUNDnThe majority of prostate cancers harbor gene fusions of the 5-untranslated region of the androgen-regulated transmembrane protease serine 2 (TMPRSS2) promoter with erythroblast transformation-specific transcription factor family members. The common fusion between TMPRESS2 and v-ets erythroblastosis virus E26 oncogene homolog (avian) (ERG) is associated with a more aggressive clinical phenotype, implying the existence of a distinct subclass of prostate cancer defined by this fusion.nnnMETHODSnWe used complementary DNA-mediated annealing, selection, ligation, and extension to determine the expression profiles of 6144 transcriptionally informative genes in archived biopsy samples from 455 prostate cancer patients in the Swedish Watchful Waiting cohort (1987-1999) and the United States-based Physicians() Health Study cohort (1983-2003). A gene expression signature for prostate cancers with the TMPRSS2-ERG fusion was determined using partitioning and classification models and used in computational functional analysis. Cell proliferation and TMPRSS2-ERG expression in androgen receptor-negative (NCI-H660) prostate cancer cells after treatment with vehicle or estrogenic compounds were assessed by viability assays and quantitative polymerase chain reaction, respectively. All statistical tests were two-sided.nnnRESULTSnWe identified an 87-gene expression signature that distinguishes TMPRSS2-ERG fusion prostate cancer as a discrete molecular entity (area under the curve = 0.80, 95% confidence interval [CI] = 0.792 to 0.81; P < .001). Computational analysis suggested that this fusion signature was associated with estrogen receptor (ER) signaling. Viability of NCI-H660 cells decreased after treatment with estrogen (viability normalized to day 0, estrogen vs vehicle at day 8, mean = 2.04 vs 3.40, difference = 1.36, 95% CI = 1.12 to 1.62) or ERbeta agonist (ERbeta agonist vs vehicle at day 8, mean = 1.86 vs 3.40, difference = 1.54, 95% CI = 1.39 to 1.69) but increased after ERalpha agonist treatment (ERalpha agonist vs vehicle at day 8, mean = 4.36 vs 3.40, difference = 0.96, 95% CI = 0.68 to 1.23). Similarly, expression of TMPRSS2-ERG decreased after ERbeta agonist treatment (fold change over internal control, ERbeta agonist vs vehicle at 24 hours, NCI-H660, mean = 0.57- vs 1.0-fold, difference = 0.43-fold, 95% CI = 0.29- to 0.57-fold) and increased after ERalpha agonist treatment (ERalpha agonist vs vehicle at 24 hours, mean = 5.63- vs 1.0-fold, difference = 4.63-fold, 95% CI = 4.34- to 4.92-fold).nnnCONCLUSIONSnTMPRSS2-ERG fusion prostate cancer is a distinct molecular subclass. TMPRSS2-ERG expression is regulated by a novel ER-dependent mechanism.

Combinatorial effects of multiple enhancer variants in linkage disequilibrium dictate levels of gene expression to confer susceptibility to common traits

DNA variants (SNPs) that predispose to common traits often localize within noncoding regulatory elements such as enhancers. Moreover, loci identified by genome-wide association studies (GWAS) often contain multiple SNPs in linkage disequilibrium (LD), any of which may be causal. Thus, determining the effect of these multiple variant SNPs on target transcript levels has been a major challenge. Here, we provide evidence that for six common autoimmune disorders (rheumatoid arthritis, Crohns disease, celiac disease, multiple sclerosis, lupus, and ulcerative colitis), the GWAS association arises from multiple polymorphisms in LD that map to clusters of enhancer elements active in the same cell type. This finding suggests a multiple enhancer variant hypothesis for common traits, where several variants in LD impact multiple enhancers and cooperatively affect gene expression. Using a novel method to delineate enhancer-gene interactions, we show that multiple enhancer variants within a given locus typically target the same gene. Using available data from HapMap and B lymphoblasts as a model system, we provide evidence at numerous loci that multiple enhancer variants cooperatively contribute to altered expression of their gene targets. The effects on target transcript levels tend to be modest and can be either gain- or loss-of-function. Additionally, the genes associated with multiple enhancer variants encode proteins that are often functionally related and enriched in common pathways. Overall, the multiple enhancer variant hypothesis offers a new paradigm by which noncoding variants can confer susceptibility to common traits.

MYC regulation of a "poor-prognosis" metastatic cancer cell state

Gene expression signatures are used in the clinic as prognostic tools to determine the risk of individual patients with localized breast tumors developing distant metastasis. We lack a clear understanding, however, of whether these correlative biomarkers link to a common biological network that regulates metastasis. We find that the c-MYC oncoprotein coordinately regulates the expression of 13 different “poor-outcome” cancer signatures. In addition, functional inactivation of MYC in human breast cancer cells specifically inhibits distant metastasis in vivo and invasive behavior in vitro of these cells. These results suggest that MYC oncogene activity (as marked by “poor-prognosis” signature expression) may be necessary for the translocation of poor-outcome human breast tumors to distant sites.

Unique ERα Cistromes Control Cell Type-Specific Gene Regulation

Estrogens play an important role in normal physiology and in a variety of pathological states involving diverse tissues including breast and bone. The mechanism by which estrogens exert cell type- and disease-specific effects, however, remains to be explained. We have compared the gene expression profile of the MCF7 breast cancer cell line with that of the osteoblast-like cell line U2OS-ERalpha by expression microarrays. We find that fewer than 10% of the 17beta-estradiol (E2)-regulated genes are common to both cell types. We have validated this in primary calvarial osteoblasts. To dissect the mechanism underlying the cell type-specific E2 regulation of gene expression in MCF7 and U2OS-ERalpha cells, we compared the ERalpha binding sites on DNA in the two cell types by performing chromatin immunoprecipitation (ChIP) on genomic tiling arrays (ChIP-on-chip). Consistent with the distinct patterns of E2-regulated gene expression in these two cell lines, we find that the vast majority of ERalpha binding sites are also cell type specific and correlate both in position and number with cell type-specific gene regulation. Interestingly, although the forkhead factor FoxA1 plays a critical role in defining the ERalpha cistrome in MCF7 cells, it is not expressed in U2OS-ERalpha cells, and forkhead motifs are not enriched in the ERalpha cistrome in these cells. Finally, the ERalpha cistromes are correlated with cell type-specific epigenetic histone modifications. These results support a model for the cell type-specific action of E2 being driven primarily through specific ERalpha occupancy of epigenetically marked cis-regulatory regions of target genes.

A Comprehensive View of Nuclear Receptor Cancer Cistromes

Nuclear receptors comprise a superfamily of ligand-activated transcription factors that play important roles in both physiology and diseases including cancer. The technologies of chromatin immunoprecipitation followed by array hybridization (ChIP-chip) or massively parallel sequencing (ChIP-seq) has been used to map, at an unprecedented rate, the in vivo genome-wide binding (cistrome) of nuclear receptors in both normal and cancer cells. We developed a curated database of 88 nuclear receptor cistrome data sets and other associated high-throughput data sets including 121 collaborating factor cistromes, 94 epigenomes, and 319 transcriptomes. Through integrative analysis of the curated nuclear receptor ChIP-chip/seq data sets, we discovered novel factor-specific noncanonical motifs that may have important regulatory roles. We also revealed a common feature of nuclear receptor pioneering factors to recognize relatively short and AT-rich motifs. Most nuclear receptors bind predominantly to introns and distal intergenetic regions, and binding sites closer to transcription start sites were found to be neither stronger nor more evolutionarily conserved. Interestingly, while most nuclear receptors appear to be predominantly transcriptional activators, our analysis suggests that the binding of ESR1, RARA, and RARG has both activating and repressive effects. Through meta-analysis of different omic data of the same cancer cell line model from multiple studies, we generated consensus cistrome and expression profiles. We further made probabilistic predictions of the nuclear receptor target genes by integrating cistrome and transcriptome data and validated the predictions using expression data from tumor samples. The final database, with comprehensive cistrome, epigenome, and transcriptome data sets and downstream analysis results, constitutes a valuable resource for the nuclear receptor and cancer community.

Cistromics of Hormone-Dependent Cancer

Alterations in transcription programs are a fundamental feature of cancer. Nuclear receptors, such as the estrogen receptor alpha (ERalpha) and androgen receptors (ARs), are central in this process as they can directly impact gene expression through interaction with the chromatin and subsequent association with coregulators and the transcriptional machinery. Unbiased genome-wide investigations have demonstrated the predominant recruitment of both ERalpha and AR to distant (non-promoter)-regulatory elements. Furthermore, these studies revealed a clear relationship between sites of transcription factor recruitment and gene regulation. Indeed, expression profiles from AR-positive primary prostate tumors and cell lines directly relate to the AR cistrome in prostate cancer cells, while the ERalpha cistrome in breast cancer cells relates to expression profiles from ERalpha-positive primary breast tumors. Additionally, cell-type-specific ERalpha cistromes are linked to lineage-specific estrogen-induced expression profiles in different cell types, for example osteosarcoma and breast cancer cells. The pioneer factor forkhead box A1 (FoxA1/HNF3alpha) plays a central role in AR and ERalpha signaling. It is recruited in a lineage-specific manner translating the epigenetic signature consisting of mono- and dimethylated histone H3 on lysine 4 (H3K4me1/me2) into functional regulatory elements. Hence, through the interplay between the pioneer factor, namely FoxA1, and epigenetic events, the transcriptional potential of a given cell lineage is predefined. Since this directly impacts signaling through nuclear receptors, these discoveries should significantly impact the development of novel therapeutic strategies directed against multiple types of cancer.

Exploiting tumor epigenetics to improve oncolytic virotherapy

Oncolytic viruses (OVs) comprise a versatile and multi-mechanistic therapeutic platform in the growing arsenal of anticancer biologics. These replicating therapeutics find favorable conditions in the tumor niche, characterized among others by increased metabolism, reduced anti-tumor/antiviral immunity, and disorganized vasculature. Through a self-amplification that is dependent on multiple cancer-specific defects, these agents exhibit remarkable tumor selectivity. With several OVs completing or entering Phase III clinical evaluation, their therapeutic potential as well as the challenges ahead are increasingly clear. One key hurdle is tumor heterogeneity, which results in variations in the ability of tumors to support productive infection by OVs and to induce adaptive anti-tumor immunity. To this end, mounting evidence suggests tumor epigenetics may play a key role. This review will focus on the epigenetic landscape of tumors and how it relates to OV infection. Therapeutic strategies aiming to exploit the epigenetic identity of tumors in order to improve OV therapy are also discussed.