Detection of lncRNA-mediated transcription regulation networks in stem cell differentiation

The existence of a crosstalk between transcriptional and post-transcriptional regulation is well established. In recent years, long non-coding RNAs (lncRNAs) have emerged as key players in fine-tuning genetic expression, acting as a bridge between the different steps of the gene expression pathway. In this study we apply high-throughput methodologies, combining protein-centric and RNA-centric approaches to systematically identify lncRNAs that mediate transcriptional regulation networks. 
Starting with a protein-centric approach, we conducted an RNA interactome capture (RIC) experiment in the nuclear fraction of human embryonic stem cells (hESCs). This approach led to the identification of several dozen transcription regulators that directly bind RNA, including the pluripotent marker Oct4 and several DNA methyltransferases (DNMTs). By applying the eCLIP and fRIP methodologies to the epigenetic factor DNMT3b, we found two dozen significant RNA interactors. In parallel, using the RNA-centric approach, we conducted a UPA-seq assay in hESCs and their differentiation to embryoid bodies (EBs). In addition to lncRNAs with known functions in pluripotency and differentiation, this led to the identification of many yet uncharacterized lncRNAs whose protein-binding changes over the course of differentiation. 

Further, we plan to conduct a pool perturbation screen followed by single cell RNA-sequencing to further characterize the role of the identified lncRNAs in the gene expression regulation of hESCs, as well as to detect new lncRNAs involved in the process. This will enable us to uncover transcriptional regulatory circuits that contribute to modulate cell fate decisions in pluripotency and differentiation.

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