Promoter and enhancer RNAs regulate chromatin reorganization and activation of miR-10b/HOXD locus and neoplastic transformation in glioma [ChIP-seq]

Using a combination of chromatin capture techniques, ChIP-Seq, single-molecule in situ RNA analysis, and computational analysis of large-scale TCGA datasets, we identified an epigenetic mechanism underlying common activation of HOXD/miR-10b locus in otherwise highly heterogenous glioma brain tumors. We demonstrate for the first time that two remote lncRNAs, one associated with a genomic promoter and another with a newly identified enhancer, cooperatively shape the 3D chromatin structure and looping, leading to the activation of a large gene cluster (12 genes, 110 KB) that includes the regulatory miRNA essential for glioma survival. This work describes a new mechanism contributing to neoplastic transformation in gliomagenesis, and identifies new avenues for therapeutic interventions based on the promoter and enhancer associated regulatory transcripts. Overall design: In order to characterize epigenetic status of HOXD/miR10b genomic we performed ChIP-Seq with H3K27Ac (#4353, Cell Signaling) and H3K27Me3 (#4353, Cell Signaling) in low-passage glioma-initiating stem-like cells grown as spheroids (GBM8) and fetal human astrocytes. To analyse whether CTCF/Rad21 binding changes after HOXD-AS2 or LINC01116 KD in GBM8 and LINC01116 activation in fetal human astrocytes we performed ChIP-Seq CTCF (#3418, Cell Signaling), Rad21 (ab992, Abcam) .