Fig. 5: ARSD is regulated by FOXA1/GATA3/ ERα mediated chromatin looping at the transcriptional level. | Cell Death & Disease

Fig. 5: ARSD is regulated by FOXA1/GATA3/ ERα mediated chromatin looping at the transcriptional level.

From: ARSD, a novel ERα downstream target gene, inhibits proliferation and migration of breast cancer cells via activating Hippo/YAP pathway

Fig. 5

A The predicted binding sites of FOXA1, GATA3, and ESR1 in the ARSD enhancer/promoter by Jaspar online software. B The pattern diagram shows the scores, CpG islands, and the corresponding locations of FOXA1, GATA3, and ERα binding site in ARSD enhancer/promoter. C The fragment of ARSD enhancer/promoter containing FOXA1, GATA3, and ESR1 binding site (−2786bp~−2050bp) was inserted into the luciferase reporter vectors by two restricted endonuclease (Sac I (CGAGCTCG) and Sma I (TCCCCCGGGGGA)). HEK293T-FOXA1, GATA3 or ERα overexpression cells and HEK293T-NC control cells were co-transfected pGL3-enhancer ARSD-promoter reporter vector, pGL3-enhancer vector or pGL3-control vector with Renilla luciferase reporter vector, respectively. Luciferase activity was normalized to Renilla. *p < 0.05, **p < 0.01 and ***p < 0.001 (Student’s t-test) as compared to control cells. Data are presented as mean ± SD (n = 3). D The ChIP-PCR assay used normal IgG (IgG) or anti-FOXA1, GATA3 or ERα antibodies to determine whether FOXA1, GATA3 or ERα can bind the corresponding binding site in the ARSD enhancer/promoter in MCF-7 cells. Input was used to be as positive control, and IgG was used to be as negative control. E The experimental strategies of chromosome conformation capture (3 C). This assay was applied to determine higher-order chromatinic interactions at the ARSD enhancer/promoter locus upon activation in MCF-7 cells. F The PCR is performed with 3 C template and primers. Analysis of 3 C data by gel electrophoresis. Verification that premixing primers from the ARSD enhancer/promoter locus. G qRT-PCR with 3 C template and primers. H Schematic illustration of the ARSD expression regulated by GATA3/FOXA1/ERα network via mediating chromatin looping. H-a The chromatin loop formation mediated by the pioneer factors, GATA3 and FOXA1, which open the compressed chromatin conformation and bind to the ERα/ERE complex to enhance the ARSD expression in MCF7 cells. H-b Epigenetic modification of ARSD promoter/enhancer in MDA-MB-231 cells by DNA methylation and EZH2, an enzymatic catalytic subunit of polycomb repressive complex 2 (PRC2) that can alter downstream target genes expression by trimethylation of Lys-27 in histone 3 (H3K27me3).

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