Long non-coding RNAs (lncRNAs) are known players in the regulatory circuitry of the self-renewal in human embryonic stem cells (hESCs). However, most hESC-specific lncRNAs remain uncharacterized. Here we demonstrate that growth-arrest-specific transcript 5 (GAS5), a known tumour suppressor and growth arrest-related lncRNA, is highly expressed and directly regulated by pluripotency factors OCT4 and SOX2 in hESCs. Phenotypic analysis shows that GAS5 knockdown significantly impairs hESC self-renewal, but its overexpression significantly promotes hESC self-renewal. Using RNA sequencing and functional analysis, we demonstrate that GAS5 maintains NODAL signalling by protecting NODAL expression from miRNA-mediated degradation. Therefore, we propose that the above pluripotency factors, GAS5 and NODAL form a feed-forward signalling loop that maintains hESC self-renewal. As this regulatory function of GAS5 is stem cell specific, our findings also indicate that the functions of lncRNAs may vary in different cell types due to competing endogenous mechanisms.

As a novel epigenetic mechanism, histone H3 methylation at R17 and R26, which is mainly catalyzed by coactivator-associated protein arginine methyltransferase 1 (CARM1), has been reported to modulate the transcription of key pluripotency factors and to regulate pluripotency in mouse embryos and mouse embryonic stem cells (mESCs) in previous studies. However, the role of CARM1 in human embryonic stem cells (hESCs) and the regulatory mechanism that controls CARM1 expression during ESCs differentiation are presently unknown. Here, we demonstrate that CARM1 plays an active role in the resistance to differentiation in hESCs by regulating pluripotency genes in response to BMP4. In a functional screen, we identified the miR-181 family as a regulator of CARM1 that is induced during ESC differentiation and show that endogenous miR-181c represses the expression of CARM1. Depletion of CARM1 or enforced expression of miR-181c inhibits the expression of pluripotency genes and induces differentiation independent of BMP4, whereas overexpression of CARM1 or miR-181c inhibitor elevates Nanog and impedes differentiation. Furthermore, expression of CARM1 rescue constructs inhibits the effect of miR-181c overexpression in promoting differentiation. Taken together, our findings demonstrate the importance of a miR-181c-CARM1 pathway in regulating the differentiation of hESCs.

Chromosomal translocation is the most common form of chromosomal abnormality and is often associated with congenital genetic disorders, infertility and cancers. The lack of cellular and animal models for chromosomal translocations, however, has hampered our ability to understand the underlying disease mechanisms and to develop new therapies. Here, we show that site-specific chromosomal translocations can be generated in mouse embryonic stem cells (mESCs) via CRISPR/Cas9. Mouse ESCs carrying translocated chromosomes can be isolated and expanded to establish stable cell lines. Furthermore, chimeric mice can be generated by injecting these mESCs into host blastocysts. The establishment of ESC-based cellular and animal models of chromosomal translocation by CRISPR/Cas9 provides a powerful platform for understanding the effect of chromosomal translocation and for the development of new therapeutic strategies.

BACKGROUND: The proliferation and migration of vascular smooth muscle cells (VSMCs) significantly contribute to vascular remodeling. Recent studies have suggested that enhancer-associated long noncoding RNAs (elncRNAs) play crucial roles in regulating gene expression and cell fate. However, the specific elncRNAs implicated in VSMC dysfunction and their regulatory mechanisms remain poorly understood. METHODS: This study used multiomics profiling techniques, including cleavage under targets and tagmentation (CUT&Tag), promoter capture high-throughput chromosome conformation capture, and microarray analysis, to identify long noncoding RNA (LncRNA)-ITGA2 (integrin α2) as a novel elncRNA involved in VSMC dysfunction. A carotid artery wire injury model was used to study its role in vascular remodeling in vivo. RNA sequencing and CRISPR-Cas9 gene-editing technology were employed to explore downstream targets of LncRNA-ITGA2. Chromatin immunoprecipitation sequencing, chromatin isolation by RNA purification, and RNA immunoprecipitation were performed to investigate its mechanistic role in VSMC proliferation and migration. RESULTS: LncRNA-ITGA2 was highly expressed in PDGF-BB (platelet-derived growth factor BB)-induced proliferative human VSMCs and was elevated in coronary atherosclerotic tissues from coronary artery disease patients compared to controls. Gain-of-function and loss-of-function studies suggested that LncRNA-ITGA2 markedly enhanced PDGF-BB-induced VSMC proliferation and migration. In vivo, the overexpression of LncRNA-ITGA2 promoted neointimal hyperplasia, indicating its partial functional conservation. Mechanistically, we identified ITGA2 as a downstream target of LncRNA-ITGA2. Promoter capture high-throughput chromosome conformation capture detected promoter‒enhancer interactions at the ITGA2 locus following PDGF-BB treatment. LncRNA-ITGA2 could directly bind to the enhancer-ITGA2 and led to an increase in H3K27 acetylation in both the enhancer-ITGA2 and the promoter-ITGA2. Additionally, we found that LncRNA-ITGA2 interacted with the DNA-binding protein NONO (non-pou domain containing octamer-binding protein), which also bound to the ITGA2 promoter. Ultimately, the use of knockout cell lines for NONO, LncRNA-ITGA2, and its promoter validated the proposed regulatory mechanism. CONCLUSIONS: This study identifies a novel mechanism by which elncRNA (LncRNA-ITGA2), in a NONO-dependent manner, mediates interactions between enhancer-ITGA2 and promoter-ITGA2, resulting in increased ITGA2 expression and subsequently promoting VSMC proliferation and migration. These findings highlight LncRNA-ITGA2 as an attractive diagnostic and therapeutic target for human proliferative vascular diseases.