LncRNA Functions as a New Emerging Epigenetic Factor in Determining the Fate of Stem Cells

Abstract

Pluripotent stem cells have broad applications in regenerative medicine and offer ideal models for understanding the biological process of embryonic development and specific diseases. Studies suggest that the self-renewal and multi-lineage differentiation of stem cells are regulated by a complex network consisting of transcription factors, chromatin regulators, signaling factors, and non-coding RNAs. It is of great interest to identify RNA regulatory factors that determine the fate of stem cells. Long non-coding RNA (lncRNA), a class of non-coding RNAs with more than 200 bp in length, has been shown to act as essential epigenetic regulators of stem cell pluripotency and specific lineage commitment. In this review, we focus on recent research progress related to the function and epigenetic mechanisms of lncRNA in determining the fate of stem cells, particularly pluripotency maintenance and lineage-specific differentiation. We discuss the role of the Oct4 and Sox2 promoter-interacting lncRNA as identified by Chromatin RNA In Situ reverse Transcription sequencing (CRIST-seq). Further understanding of their potential actions will provide a basis for the development of regenerative medicine for clinical application. This work offers comprehensive details and better understanding of the role of lncRNA in determining the fate of stem cells and paves the way for clinical stem cell applications.

Keywords: cell differentiation; epigenetics; long non-coding RNA; pluripotency; promoter-interacting lncRNA network; reprograming; stem cell.

FIGURE 1

Mechanisms of lncRNA in regulating stem cell pluripotency. Nuclear lncRNAs regulate the expression of pluripotent genes via chromatin remodeling. While lncRNAs in cytosol mainly act as post-transcriptional regulators. They work as miRNA sponges to affect gene expression or signaling pathway.

FIGURE 2

Mapping stemness gene promoter-interacting lncRNA by CRIST-seq. The CRIST-seq assay combines the specificity of Cas9 gene targeting with the simplicity of biotin-lncRNA labeling. Target cells are first transfected with lentiviruses carrying dCas9-FLAG/gRNA that target the promoter of stemness genes, like Oct4 and Sox2, that are two core stem cell factors essential for the establishment and maintenance of pluripotency. The cells are crosslinked to fix the promoter chromatin DNA-lncRNA complex. Following lysis of the cell membrane, the promoter-interacting RNAs are reverse transcribed in situ into biotin-cDNAs (cDNAs) in the isolated nuclei with biotin-dCTP. The promoter chromatin-cDNA complex is immunoprecipitated by a Cas9-FLAG antibody and the promoter-interacting biotin-cDNAs are separated by the biotin-streptavidin bead purification. The CRIST-captured cDNAs are used for library construction and Illumina sequencing is performed for identifying the lncRNAs that interact with the promoter of stemness genes. Integration of the Oct4-Sox2 CRIST-seq data with the RNA-seq data allows the identification of lncRNAs that not only interact with core factor promoters of stem cells, but are also differentially expressed during reprograming. The lncRNA-promoter interaction is subsequently validated by other tools, including RNA-DNA FISH, RAT, ChIRP, and CHOP assays. Gain- and loss-of-function assays are then used to characterize the role of identified lncRNAs in stem cells. The CRIST-seq method offers a more efficient strategy for exploring the lncRNA network in the regulation of reprograming and pluripotency.

FIGURE 3

An overview of lncRNA in regulating stem cell fate. Letters in red: positive regulation; Letters in green: negative regulation.