Promoter and enhancer RNAs regulate chromatin reorganization and activation of miR-10b/HOXD locus and neoplastic transformation in glioma [4C-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: We used 4C-Seq technique with HOXD3/HOXD4/miR10b_gene_body as a viewpoint (NlaIII) to characterize the differences in 3D chromatin architecture between tumorigenic adherent glioma cell line (LN229, U251), low-passage glioma-initiating stem-like cells grown as spheroids (GBM8, GBM6, GBM4) and fetal human astrocytes, neuroprogenitors (NPC) in 500 kb HOXD/miR10b TAD domain. Moreover we examined changes in HOXD/miR10b TAD architecture after HOXD-AS2 (promoter) OR LINC01116 (enhancer) KD in glioma and 'transformed' astrocytes with active LINC01116 (enhancer). Additionally, HOXD/miR10b chromatin architecture was analyzed after the activation of LINC01116 (enhancer RNA) in fetal human astrocytes.