Fig. 2
From: The Eleanor ncRNAs activate the topological domain of the ESR1 locus to balance against apoptosis
4C-Seq (chromosome conformation capture combined with high-throughput sequencing) analysis revealed a long-range interaction between ESR1 and FOXO3 genes in long-term estrogen deprivation (LTED) cells. a Alignment of 4C-Seq and A/B compartment profiles along the entirety of human chromosome 6 (UCSC genome browser, hg19 assembly). Top: 4C-Seq data showing intra-chromosomal interactions with the ESR1 promoter used as bait (arrowhead). Middle: Distribution of A (red) and B (green) compartments in MCF7 cells29. Note the positive correlation relationship between black peaks from 4C-Seq and red peaks representing A compartments. Bottom: distribution of all genes (blue) and genes upregulated in LTED cells (red)27. Thick blue bars represent the positions of 3C-qPCR (chromosome conformation capture–quantitative polymerase chain reaction) primers (a–d and the ESR1 promoter) used in this study. Green and red bars represent BAC DNA used as DNA fluorescence in situ hybridization (FISH) probes in b, c. Bottom: RefSeq genes at the b site. It should be noted that the 4C-Seq profiles showed interaction peaks between b site at 6q21 and the ESR1 promoter (bait for 4C). b Representative DNA FISH images of the ESR1 locus (red, using ESR1p-BAC probe) and b and d sites (green, using b-BAC and d-BAC probes, respectively). The position of each probe is shown in a (green and red bars). Scale bar, 5 µm. c Quantification of the DNA FISH analysis in b. Nuclei with overlapping signals at the ESR1-b site or ESR1-d site were measured. The frequencies of interaction between the ESR1 and b site increased in LTED cells. P values were calculated using two-tailed Fisher’s exact test. d 3Cq-PCR analysis shows interaction frequencies between the ESR1 promoter and a–d sites in MCF7 and LTED cells. Positions of sites a–d are shown in a (blue bars). e Relative expression levels of genes at 6q21 in MCF7 and LTED cells. The MCF7 expression level was set to 1. Data presented in d, e are representative of three independent experiments (mean ± s.e.m.). P values were calculated using unpaired, two-tailed, Student’s t test (***P < 0.001, n.d. = not detected in at least one experiment)
