Liu, M.; Mamede, I.; Sofi, S.; Pereira, I.; Dostal, V.; Pashos, A. R. S.; McMahon, C.; Waikar, A.; Stephenson, G.; Cech, T. R.; Rinn, J. L.

Some long non-coding RNAs (lncRNAs) are known to regulate gene expression. However, the underlying temporal dynamics of lncRNAs influencing gene and epigenetic regulation and mechanisms of lncRNA regulation in trans are less understood. To investigate this, we genetically engineered 17 doxycycline-inducible lncRNA transgenes for ectopic expression at the H11 safe harbor locus in human pluripotent stem cells (hiPSCs), and we generated high-density temporal RNA-seq and ATAC-seq profiles. Most lncRNA transgenes were induced at 2 hours and maintained expression through the 96-hour time course. Surprisingly, when we sought to identify gene expression changes due to the lncRNAs, we found that the global transcriptional landscape was dominated by a strong systemic response triggered by doxycycline exposure. We rigorously defined this cohort of genes as a Doxycycline-Responsive Gene Signature (DRGS). The DRGS was also present in at least 28 public datasets from dox-inducible transgene studies involving diverse cell types. Next, we determined which lncRNAs exhibited trans-regulatory events. We identified DANCR, FENDRR, LINC00667, LINC00847, LNCPRESS1, and PNKY as lncRNAs that regulate specific transcript expression in trans. The downstream target genes encoded 53 mRNAs and 10 lncRNAs. None of the target lncRNAs altered gene expression proximal to their own loci (i.e., triggering secondary cis-effects). Surprisingly, the target genes of LINC00847 (transcribed from chromosome 22) were substantially enriched on chromosome 19, with a preponderance of target genes encoding RNA metabolism and RNA splicing factors. Collectively, our study provides a resource to discern artifacts in the doxycycline-inducible system and identifies temporally regulated targets of 6 lncRNAs for future mechanistic studies.