Unconventional Transcription Factor-lncRNA Interactions in Human Embryonic Stem Cells

Transcription factors (TFs) play a pivot role in embryonic stem cells as key pluripotent markers. In recent years, it has been shown that long non-coding RNAs (lncRNAs) are involved in activation and repression of pluripotency-related genes via epigenetic and transcriptional regulation. To predict novel interactions between TFs and lncRNAs with regulatory functions in pluripotency and differentiation, we sampled RNA from eleven time points during directed differentiation of human Induced Pluripotent Stem Cells (iPSs) to Cardiomyocytes. Analyzing the differential expression patterns of coding and non-coding RNAs across time revealed pairs of TFs and lncRNAs that are significantly co-expressed, suggesting co-regulatory relationships. To confirm direct interactions between TFs and lncRNAs we performed eCLIP (enhanced crosslinking and immunoprecipitation) followed by sequencing on selected TFs. Computational analysis of the CLIP data revealed a small subset of non-coding RNAs with significantly enriched protein binding peaks. Specifically, the eCLIP results signify a direct association between the STAT3 (signal transducer and activator of transcription 3) TF and the lncRNA NORAD (non-coding RNA activated by DNA damage) in human pluripotent cells. Strikingly, knockdown of NORAD in hESCs significantly impaired STAT3 localization to the nucleus. Based on our findings, we propose that lncRNAs may contribute to stemness by directly interacting with TFs, possibly acting as co-regulators to modulate and fine-tune the transcriptional program of their target genes.