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GATA3 acts upstream of FOXA1 in mediating ESR1 binding by shaping enhancer accessibility.

ABSTRACT: Estrogen receptor (ESR1) drives growth in the majority of human breast cancers by binding to regulatory elements and inducing transcription events that promote tumor growth. Differences in enhancer occupancy by ESR1 contribute to the diverse expression profiles and clinical outcome observed in breast cancer patients. GATA3 is an ESR1-cooperating transcription factor mutated in breast tumors; however, its genomic properties are not fully defined. In order to investigate the composition of enhancers involved in estrogen-induced transcription and the potential role of GATA3, we performed extensive ChIP-sequencing in unstimulated breast cancer cells and following estrogen treatment. We find that GATA3 is pivotal in mediating enhancer accessibility at regulatory regions involved in ESR1-mediated transcription. GATA3 silencing resulted in a global redistribution of cofactors and active histone marks prior to estrogen stimulation. These global genomic changes altered the ESR1-binding profile that subsequently occurred following estrogen, with events exhibiting both loss and gain in binding affinity, implying a GATA3-mediated redistribution of ESR1 binding. The GATA3-mediated redistributed ESR1 profile correlated with changes in gene expression, suggestive of its functionality. Chromatin loops at the TFF locus involving ESR1-bound enhancers occurred independently of ESR1 when GATA3 was silenced, indicating that GATA3, when present on the chromatin, may serve as a licensing factor for estrogen-ESR1-mediated interactions between cis-regulatory elements. Together, these experiments suggest that GATA3 directly impacts ESR1 enhancer accessibility, and may potentially explain the contribution of mutant-GATA3 in the heterogeneity of ESR1+ breast cancer.

SUBMITTER: Theodorou V

PROVIDER: S-EPMC3530671 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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GATA3 acts upstream of FOXA1 in mediating ESR1 binding by shaping enhancer accessibility.

Theodorou Vasiliki V Stark Rory R Menon Suraj S Carroll Jason S JS

Genome research 20121121 1

Estrogen receptor (ESR1) drives growth in the majority of human breast cancers by binding to regulatory elements and inducing transcription events that promote tumor growth. Differences in enhancer occupancy by ESR1 contribute to the diverse expression profiles and clinical outcome observed in breast cancer patients. GATA3 is an ESR1-cooperating transcription factor mutated in breast tumors; however, its genomic properties are not fully defined. In order to investigate the composition of enhance ...[more]

PMID: 23172872

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Project description:Estrogen Receptor (ESR1) drives growth in the majority of human breast cancers by binding to regulatory elements and inducing transcription events that promote tumor growth. Differences in enhancer occupancy by ESR1, contribute to the diverse expression profiles and clinical outcome observed in breast cancer patients. GATA3 is an ESR1 co-operating transcription factor mutated in breast tumors, however its genomic properties are not fully defined. In order to investigate the composition of enhancers involved in estrogen-induced transcription and the potential role of GATA3, we performed extensive ChIP-sequencing in unstimulated breast cancer cells and following estrogen treatment. We find that GATA3 is pivotal in mediating enhancer accessibility at regulatory regions involved in ESR1-mediated transcription. GATA3 silencing resulted in a global redistribution of co-factors and active histone marks prior to estrogen stimulation. These global genomic changes altered the ESR1 binding profile that subsequently occurred following estrogen, with events exhibiting both loss and gain in binding affinity, implying a GATA3 mediated re-distribution of ESR1 binding. The GATA3-mediated re-distributed ESR1 profile correlated with changes in gene expression, suggestive of its functionality. Chromatin loops at the TFF locus involving ESR1 bound enhancers occurred independently of ESR1 when GATA3 was silenced, indicating that GATA3, when present on the chromatin, may serve as a licensing factor for estrogen- ESR1 mediated interactions between cis-regulatory elements. Together these experiments suggest that GATA3 directly impacts ESR1 enhancer accessibility and may potentially explain the contribution of mutant-GATA3 in the heterogeneity of ESR1+ breast cancer. MCF7 cells were transfected with siRNA and cultured in hormone deprived conditions for a further 3 days. Cells were subsequently treated with 100nM estrogen (E2) or control (Veh) for 6 hrs. We performed two independent biological experiments each using three different siRNAs against GATA3 individually (six siGATA3 replicates in total). For siControl we used RNA from five biological replicates.

Project description:Breast cancer is the most common neoplasm among females. Estrogen receptor (ESR) signaling has a prominent impact in the pathogenesis of breast cancer. Among the transcription factors associated with ESR signaling, FOXM1, GATA3, FOXA1 and ESR1 have been suggested as a candidate in the pathogenesis of this neoplasm. In the current project, we have designed an in silico approach to find long non-coding RNAs (lncRNAs) that regulate these transcription factors. Then, we used clinical samples to carry out validation of our in silico findings. Our systems biology method led to the identification of APTR, AC144450.1, linc00663, ZNF337.AS1, and RAMP2.AS1 lncRNAs. Subsequently, we assessed the expression of these genes in breast cancer tissues compared with the adjacent non-cancerous tissues (ANCTs). Expression of GATA3 was significantly higher in breast cancer tissues compared with ANCTs (Ratio of mean expressions (RME) = 4.99, P value = 3.12E-04). Moreover, expression levels of APTR, AC144450.1, and ZNF337.AS1 were elevated in breast cancer tissues compared with control tissues (RME = 2.27, P value = 5.40E-03; Ratio of mean expressions = 615.95, P value = 7.39E-19 and RME = 1.78, P value = 3.40E-02, respectively). On the other hand, the expression of RAMP2.AS1 was lower in breast cancer tissues than controls (RME = 0.31, P value = 1.87E-03). Expression levels of FOXA1, ESR1, and FOXM1 and linc00663 were not significantly different between the two sets of samples. Expression of GATA3 was significantly associated with stage (P value = 4.77E-02). Moreover, expressions of FOXA1 and RAMP2.AS1 were associated with the mitotic rate (P values = 2.18E-02 and 1.77E-02, respectively). Finally, expressions of FOXM1 and ZNF337.AS1 were associated with breastfeeding duration (P values = 3.88E-02 and 4.33E-02, respectively). Based on the area under receiver operating characteristics curves, AC144450.1 had the optimal diagnostic power in differentiating between cancerous and non-cancerous tissues (AUC = 0.95, Sensitivity = 0.90, Specificity = 0.96). The combination of expression levels of all genes slightly increased the diagnostic power (AUC = 0.96). While there were several significant pairwise correlations between expression levels of genes in non-tumoral tissues, the most robust correlation was identified between linc00663 and RAMP2.AS1 (r = 0.61, P value = 3.08E-8). In the breast cancer tissues, the strongest correlations were reported between FOXM1/ZNF337.AS1 and FOXM1/RAMP2.AS1 pairs (r = 0.51, P value = 4.79E-5 and r = 0.51, P value = 6.39E-5, respectively). The current investigation suggests future assessment of the functional role of APTR, AC144450.1 and ZNF337.AS1 in the development of breast neoplasms.

Project description:Abstract Polycystic ovary syndrome (PCOS) is the most common cause of female infertility and is associated with an increased risk of type 2 diabetes and cardiovascular disease. Understanding the etiology of PCOS is crucial to relieve this tremendous health burden. Twin studies estimate that 70% of PCOS etiology can be explained by genetic factors, motivating studies to identify these factors. Recent genome wide association studies identified a PCOS risk locus that encompasses the gene encoding the follicle-stimulating hormone beta (FSHβ) subunit. The most significant single nucleotide polymorphism (SNP) within this locus, rs11031006 (rs06), resides within a region of DNA (~450 base pairs) that is highly conserved among mammals. As FSH supplementation can restore ovulation in some PCOS patients, deficient FSH signaling may be a causative factor of anovulation and potentially other facets of PCOS. FSH transcription and release is stimulated by gonadotropin-releasing hormone (GnRH) produced in the hypothalamus, as well as activin produced by the gonads and pituitary. We previously hypothesized that the conserved region containing rs06 acts as an enhancer of FSHβ transcription, and we determined through luciferase reporter assays that this highly conserved region enhances transcription ~2 fold compared to the minimal FSHβ promoter alone for human and 2.5 fold for mouse. The rs06 SNP minor allele further increased both mouse and human FSHβ luciferase expression. In the present study, we sought to identify interactions between the enhancer and the hormones, GnRH and activin. With DNA constructs from both human and mouse, we identified synergistic interactions between the enhancer and GnRH- or activin-induction of FSHβ. These interactions were maintained after mutation of the rs06 site to the minor allele, suggesting that this SNP alters basal and not hormone-induced FSHβ transcription. We are currently investigating the effect of the enhancer and rs06 on activin/GnRH synergy. Overall, our findings provide evidence that the enhancer functions as a novel mediator of activin and GnRH regulation of FSHβ transcription in both human and mouse.

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GATA3 acts upstream of FOXA1 in mediating ESR1 binding by shaping enhancer accessibility.

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GATA3 acts upstream of FOXA1 in mediating ESR1 binding by shaping enhancer accessibility.

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