SRA

Estrogen receptor-a (ERa) is a ligand-inducible protein which mediates estrogenic hormones signaling and defines the luminal breast cancer phenotype. Recently, we demonstrated that ERa binds chromatin in absence of ligand (apoERa) regulating transcription of protein-coding genes and several lncRNAs. Noteworthy, apoERa-regulated lncRNAs marginally overlap estrogen-induced transcripts representing a signature of luminal breast cancer genes. DSCAM-AS1 is a paradigmatic example of apoERa activity since its expression is largely unaffected by estrogenic treatment despite an E2-induced increment of ERa binding on its promoter. Analysing H3K27ac ChIP-Seq performed in hormone-deprived MCF-7, we identified a set of Super Enhancers (SEs) occupied by apoERa including one mapped in proximity of DSCAM-AS1. Using ChIP-qPCR, we validated ChIP-Seq signal of apoERa, p300 and CTCF at both DSCAM-AS1 TSS and at its associated SE. Furthermore, analysing MCF-7 ChIA-PET data and performing a 3C experiment, we confirmed a long range chromatin interaction between the SE and the DSCAM-AS1 TSS. Interestingly, CTCF binding downstream to DSCAM-AS1 shows an enrichment in hormone-depleted medium as compared to other experimental conditions, indicating that CTCF demarcates enhancer actions at DSCAM-AS1 locus. The analysis of this lncRNA provides a paradigm of transcriptional regulation of a luminal specific apoERa regulated lncRNA. Overall design: The RNA-seq datasets were obtained from libraries generated with the TruSeq stranded library preparation kit (Illumina) using as input material poly(A+) RNA depleted of ribosomal RNAs fractions. Pool of 6 libraries (pooled at equimolar concentration) was generated, quantified and run on the HiSeq2000 (Illumina) sequencer in 75 nts paired-end sequencing mode following manufacturer instruction. A total of 6 datasets, with an average depth of 91.5 million paired-end reads, were obtained, composed of triplicates of two MCF-7 culture conditions: i) hormone-deprived cells treated with ESR1 mRNA (siRNA) (48h) or hormone-deprived cells treated with control siRNA (siCTR).