A novel antisense long noncoding RNA within the IGF1R gene locus is imprinted in hematopoietic malignancies.
Bottom Line: Using both reverse transcription-associated trap and chromatin conformation capture assays, we demonstrate that this lncRNA interacts with chromatin DNA and is involved in the formation of an intrachromosomal enhancer/promoter loop.In addition, IRAIN was downregulated both in leukemia cell lines and in blood obtained from high-risk AML patients.These data identify IRAIN as a new imprinted lncRNA that is involved in long-range DNA interactions.
Affiliation: Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061, PR China Stanford University Medical School, VA Palo Alto Health Care System, Palo Alto, CA 94304, USA.Show MeSH
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Mentions: As several lncRNA molecules regulate gene function by directly binding to their target chromatin DNAs (28–30,49,50), we determined if IRAIN lncRNA bound to IGF1R chromatin DNA, particularly the promoter region. We developed a ‘RAT’ assay that overcomes the high noise to signal background of existing methodologies to detect RNA/DNA interactions (Figure 4A). Cells were treated with formaldehyde to fix the structure of the lncRNA-chromatin conformations. The chromatin DNA-interacting lncRNA was then reverse transcribed into cDNA using strand-specific oligonucleotides and a thermo-stable reverse transcriptase in the presence of biotin-dCTP. After digestion with restriction enzyme or sonication to shear chromatin DNAs, the biotinylated IRAIN cDNA–DNA complex was then separated from other DNA–DNA products by streptavidin pull-down using paramagnetic Dynabeads (Dynal, Invitrogen) and analyzed by PCR using interacting DNA-specific primers.
Affiliation: Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061, PR China Stanford University Medical School, VA Palo Alto Health Care System, Palo Alto, CA 94304, USA.