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Luminal lncRNAs Regulation by ERα-Controlled Enhancers in a Ligand-Independent Manner in Breast Cancer Cells

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Luminal lncRNAs Regulation by ERα-Controlled Enhancers in a Ligand-Independent Manner in Breast Cancer Cells

Valentina Miano et al. Int J Mol Sci.

Abstract

Estrogen receptor-α (ERα) is a ligand-inducible protein which mediates estrogenic hormones signaling and defines the luminal BC phenotype. Recently, we demonstrated that even in absence of ligands ERα (apoERα) binds chromatin sites where it regulates transcription of several protein-coding and lncRNA genes. Noteworthy, apoERα-regulated lncRNAs marginally overlap estrogen-induced transcripts, thus representing a new signature of luminal BC genes. By the analysis of H3K27ac enrichment in hormone-deprived MCF-7 cells, we defined a set of Super Enhancers (SEs) occupied by apoERα, including one mapped in proximity of the DSCAM-AS1 lncRNA gene. This represents a paradigm of apoERα activity since its expression is largely unaffected by estrogenic treatment, despite the fact that E2 increases ERα binding on DSCAM-AS1 promoter. We validated the enrichment of apoERα, p300, GATA3, FoxM1 and CTCF at both DSCAM-AS1 TSS and at its associated SE by ChIP-qPCR. Furthermore, by analyzing MCF-7 ChIA-PET data and by 3C assays, we confirmed long range chromatin interaction between the SE and the DSCAM-AS1 TSS. Interestingly, CTCF and p300 binding showed an enrichment in hormone-depleted medium and in the presence of ERα, elucidating the dynamics of the estrogen-independent regulation of DSCAM-AS1 expression. The analysis of this lncRNA provides a paradigm of transcriptional regulation of a luminal specific apoERα regulated lncRNA.

Keywords: breast cancer; estrogen receptor; lncRNA; super enhancer.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
ApoERα regulated genes through SE binding. (a,b) Volcano plots reporting the adjusted p-value and the log2 Fold Change (FC) of (a) the protein-coding genes and (b) lncRNAs computed from RNA-seq data comparing MCF-7 cells transfected with siERα and siCTR in hormone-deprived (HD) medium; (c) Bar plot reporting the -log10 adjusted p-value of the most significant Gene Ontology Biological Processes enriched for the genes down-regulated (top) or up-regulated (down) upon ERα silencing in HD; (d) Heat map illustrating differential expression of Super-Enhancer (SE)-associated lncRNAs upon siERα transfection and upon nine-hours E2 treatment; (e) Heat map illustrating expression values of SE-associated lncRNAs as FPKM; (f) Bar plot reporting the log2 relative expression of ERα mRNA and eight candidate SE-associated lncRNAs upon ERα silencing in HD; p-value of three biological replicates by unpaired t-test: * p < 0.05; ** p < 0.01; *** p < 0.001; (g) Bar plot reporting the relative expression of eight candidate SE-associated lncRNAs and GREB1 (positive control) in a time-course experiment of 17β-estradiol (E2) or vehicle (Veh) treatment; SD of three biological replicates; p-value by unpaired t-test: * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 2
Figure 2
Characterization of DSCAM-AS1 SE in MCF-7 cells. (a) WashU genome browser view of the DSCAM-AS1 genomic locus, which is reported in association with apoERα ChIP-Seq and RNA-Seq reads enrichment in siCTR-(red) or siERα-(blue) transfected MCF-7 cells; and with GRO-Seq, ChIP-Seq profiles of histone modifications and DNase hypersensitivity signals (yellow), RNA-Pol II and eight TFs (teal-blue) obtained from vehicle treated MCF-7 cells (see the Materials and Methods section for detailed data source). ERα and CTCF ChIA-PET data performed in MCF-7 grown in full medium from ENCODE project are also included [26]. The coordinates of the predicted SE and aERBSs are reported at the top as black and orange boxes, respectively. ChIP-Seq genomic signal profiles are reported as read count per million sequenced reads. (b,c) Bar plots reporting ERα fold enrichment over IgG signal by ChIP-qPCR at DSCAM-AS1 promoter and E5-E6-enhancers, TFF1 promoter (positive control) and KCNQ1OT1 promoter (negative control), in MCF-7 cells grown in HD medium transfected with siERα or siCTR (b); treated for 45′ with 17β-estradiol (E2) or vehicle (veh) (c). Standard error of four biological replicates; p-value by unpaired t-test: ** p < 0.01; (d) Left. Qualitative PCR reactions with primers detecting DSCAM-AS1 promoter interaction with E5 or E6 enhancer sites; primers detecting DSCAM-AS1 genomic region or non-interacting region; Right. Qualitative PCR reactions with primers detecting P2RY2 promoter-enhancer interaction; primers detecting P2RY2 genomic region or non-interacting region. All reactions were loaded on 2% agarose gel. Undigested and Digested samples represent DNA controls recovered during 3C libraries production.
Figure 3
Figure 3
Analysis of ERα co-factor binding at DSCAM-AS1 locus. (a,b) Bar plots reporting p300, GATA3 and FoxM1 fold enrichment over IgG signal at DSCAM-AS1 promoter (a) and E6-enhancer (b) by ChIP-qPCR in MCF-7 cells grown in HD and transfected with siCTR or siERα; (c,d) Bar plots reporting p300, GATA3 and FoxM1 fold enrichment over IgG signal at DSCAM-AS1 promoter (c) and E6-enhancer (d) by ChIP-qPCR in MCF-7 cells grown in HD and treated for 45′ with 17β-estradiol (E2) or vehicle (veh). Standard error of three biological replicates; p-value by unpaired t-test: * p < 0.05; ** p < 0.01.
Figure 4
Figure 4
DSCAM-AS1 SE characterization in drug-resistant BC cell lines. (a) WashU genome browser view of DSCAM-AS1 genomic locus reporting at top the ChIP-Seq genomic signal profile of ERα and H3K27ac ChIP-Seq experiments performed in wild type MCF-7, MCF-7 resistant to Tamoxifen (MCF-7T), MCF-7 resistant to Fulvestrant (MCF-7F), Long Term Estrogen Deprived (LTED) MCF-7 cells, and LTED MCF-7 resistant to Tamoxifen (LTEDT) [31]. (b) Box plot reporting the normalized number of ERα and H3K4me3 ChIP-Seq reads counted at DSCAM-AS1 E6 enhancer (left) and promoter (right) considering data from patient responsive (Good prognosis, Good) or not (Negative prognosis, Negative) to Aromatase Inhibitors treatment (AI) [32]. Black dots represent outliers.

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