Discovering lncRNAs that Mediate Transcriptional Circuits in Human Pluripotency

The existence of a crosstalk between transcriptional and post-transcriptional regulation is well established. Recently, long non-coding RNAs (lncRNAs) have emerged as key players in fine-tuning gene regulation, bridging different steps of the gene expression pathway.
Applying high-throughput methodologies and combining protein-centric and RNA-centric approaches, we aim to identify lncRNAs that mediate transcriptional regulatory networks.

Employing a protein-centric approach, we conducted an RNA interactome capture experiment in the nuclear fraction of human embryonic stem cells (hESCs). This 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 methodology, we identified a putative interaction between DNMT3b and the lncRNA DANT1. In parallel, we took an RNA-centric approach. We carried out the UPA-seq assay which led to the discovery of many protein-binding RNAs in hESCs. Among these RNAs were many well-studied lncRNAs, such as MALAT1 and MIAT, as well as lncRNAs with previously unknown functions, including DANT1. Furthermore, by applying a guilt-by-association analysis, we found that DNMT3b and DANT1 are significantly co-expressed during differentiation of hESCs to embryoid bodies.

Following these results, we plan to conduct a pooled perturbation screen followed by single cell RNA sequencing to study the role of the identified lncRNAs in the gene expression regulation of hESCs, as well as to detect new functional lncRNAs. This will enable us to gain insights into the role of lncRNAs as modulators of the transcription regulatory network in pluripotency and differentiation.