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. 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) .