Abstract
SummaryBoth 3D chromatin architecture and long non-coding RNAs (lncRNAs) play essential roles in pluripotency maintenance. However, whether lncRNAs are involved in organizing 3D chromatin structure remains largely unexplored. We identified 39 lncRNAs bound by Klf4, among which we further revealed the 5430416N02Rik promoter is a chromatin interaction hub. Knockout of the 5430416N02Rik locus reduces the proliferation rate of embryonic stem cells (ESCs). Moreover, deleting both the promoter and the gene body of 5430416N02Rik causes a more severe proliferation defect and has a more profound impact on the transcriptome than deleting the gene body alone. The reduced proliferation of the 5430416N02Rik locus knockout ESCs is mainly due to the downregulation of Mid1, the expression of which requires the inter-chromosomal interaction between Mid1 and 5430416N02Rik loci. In summary, our data demonstrated that the lncRNA 5430416N02Rik gene locus maintains the fast proliferation of ESCs by activating the expression of Mid1 through chromatin interaction.
Highlights
Embryonic stem cells (ESCs), derived from the inner cell mass of pre-implantation blastocysts, are able to self-renew indefinitely, while maintaining the differentiation potential into all types of cells in the body (Evans and Kaufman, 1981)
To test the possibility that long non-coding RNAs (lncRNAs) serve as scaffold molecules, and collaborate with Klf4 and other Chromatin architectural proteins (CAPs) to form large RNA/protein complexes and organize 3D chromatin architecture, RNA-binding protein immunoprecipitation coupled with sequencing (RIP-seq) was carried out to detect whether Klf4 binds to any lncRNAs
To circumvent the specificity issue of the Klf4 antibody, two additional RIP experiments (Klf4_3F and Flag_3F) were conducted in 3F ESCs, in which 33Flag tag is fused to the N terminus of endogenous Klf4 (Figures S1A–S1D), with Klf4 and Flag antibodies, respectively
Summary
Embryonic stem cells (ESCs), derived from the inner cell mass of pre-implantation blastocysts, are able to self-renew indefinitely, while maintaining the differentiation potential into all types of cells in the body (Evans and Kaufman, 1981). Understanding the molecular mechanisms of pluripotency maintenance is beneficial for the potential application ESCs in regenerative medicine. It has become clear that 3D chromatin architecture plays an essential role in the establishment and maintenance of pluripotency (Apostolou et al, 2013; Wei et al, 2013a, 2013b; Zhang et al, 2013). Extensive chromatin reorganization, altering chromatin interactions both within and between TADs, occurs during lineage specification of ESCs (Dixon et al, 2015; PhillipsCremins et al, 2013). Unique pluripotency-specific chromatin interactomes around the key pluripotency gene loci, Nanog and Oct, are maintained in ESCs, and are functionally important for pluripotency (Apostolou et al, 2013; Wei et al, 2013a)
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