Circular RNA Kat6b-e2 expression safeguards self-renewal and lineage differentiation of mouse embryonic stem cells
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ABSTRACT: Circular RNA has been reported to be dynamically expressed during embryonic development and regulates human embryonic stem cells (hESCs), but the identification and regulation of functional circular RNA in mouse embryonic stem cells (mESC) remains unclear. Here we found over 1,000 circular RNAs in different mESC states. One of these, circular RNA Kat6b-exon2 (circKat6b-e2), was significantly overexpressed in mESCs. Furthermore, short-hair RNA (shRNA) mediated knockdown of circKat6b-e2 expression significantly inhibited mESC self-renewal and EB differentiation. The transcriptome also revealed that circKat6b-e2 is required to ensure the differentiation of the mESC lineage. Finally, we also found that more Alu elements surrounding circKat6b-e2 were necessary factors for its expression. Conclusions: our study shows that Alu element-dependent expression of circKat6b-e2 mediates mESC self-renewal and lineage differentiation.
Project description:The Wnt/β-catenin signalling pathway is a key regulator of embryonic stem cell self-renewal and differentiation. Constitutive activation of this pathway has been shown to significantly increase mouse embryonic stem cell (mESC) self-renewal and pluripotency marker expression. In this study, we generated a novel β-catenin knock-out model in mESCs by using CRISPR/Cas9 technology to delete putatively functional N-terminally truncated isoforms observed in previous knock-out models. While we showed that aberrant N-terminally truncated isoforms are not functional in mESCS, we observed that canonical Wnt signalling is not active in mESCs, as β-catenin ablation does not alter mESC transcriptional profile in LIF-enriched culture conditions; on the other hand, Wnt signalling activation represses mESC spontaneous differentiation. We also showed that transcriptionally silent β-catenin (ΔC) isoforms can rescue β-catenin knock-out self-renewal defects in mESCs, cooperating with TCF1 and LEF1 in the inhibition of mESC spontaneous differentiation in a Gsk3 dependent manner.
Project description:Mouse embryonic stem cells (mESCs) possess remarkable characteristics of unlimited self-renewal and pluripotency, which render them highly valuable for both fundamental research and clinical applications. A comprehensive understanding of the molecular mechanisms underlying mESC function is of utmost importance. The Human Silence Hub (HUSH) complex, comprising FAM208A, MPP8, and periphilin, constitutes an epigenetic silencing complex involved in suppressing retroviruses and transposons during early embryonic development. However, its precise role in regulating mESC pluripotency and differentiation remains elusive. In this study, we generated homogenous miniIAA7 tagged Mpp8 mouse ES cell lines. Upon induction of MPP8 protein degradation, we observed impaired proliferation and reduced colony formation ability of mESCs. Furthermore, this study unveils the involvement of MPP8 in regulating the activity of the LIF/STAT3 signaling pathway and Nanog expression in mESCs. Finally, we provide compelling evidence that degradation of the MPP8 protein impairs the differentiation of mESC.
Project description:Purpose:The goals of this study is to analyze the candidate genes motified by CID755674 to promote the mouse embryonic stem cell (ESC) self-renewal through RNA-seq approach. Previously, we found that MEK inhbitior PD0323901 (PD) coporates with protein kinase D inhibitor CID755673 (CID) is able to maitain mouse embryonic stem cell (mESC) self-renewal, we named this conditon is "PC". In order to find out why CID755673 can promote mouse embryonic stem cell self-renewal, we performed RNA-sequence in mESCs treated with PD or PC, and tried to identify the key mechanisms under CID
Project description:Nuclear pore complexes (NPCs) are established players in cell division and differentiation. However studies on the contribution of individual NPC subunits to these processes are still scarce. Here we have used mouse embryonic stem cells (mESCs) to characterize the role of structural components of the NPCs, focusing on the short arm of the Y-complex that comprises Nup85, Seh1 and Nup43. We show that Seh1 and Nup43, although dispensable at the pluripotent stage, are required for normal cell growth rates at that stage and for mESC viability upon differentiation. Lack of Seh1 or Nup43 in mESCs is associated with a mild reduction of NPC density that is also observed when Seh1 interaction with Nup85 is impaired. Nevertheless, mESC proliferation and differentiation are not altered in these ∆E2-GFP-Nup85 mutants, indicating that it is the integrity of the Y-complex, rather than the number of NPCs, that is critical to ensure these processes.
Project description:LONG SUMMARY: In this study, we aimed to characterize the function of Tip110 in embryonic development and in mESC survival and self-renewal using a Tip110+/- mouse model. Mating these mice produces wild-type, Tip110+/-, and Tip110-/- offspring. Tip110-/- offspring die early in mouse postimplantation development and mESCs cannot be derived from Tip110-/- blastocysts. Array analysis was performed to gain a better understanding of the underlying molecular mechanisms at play and to appreciate the biological role of Tip110 in embryo and ESC development. SHORT SUMMARY: We used microarray analysis to detail the global program of gene expression in Tip110-/- outgrowths compared to their wild-type counterparts to gain a better understanding of the biological role of the Tip110 protein in embryonic development and mESC survival and self-renewal.
Project description:Analysis of genes involved in the mESC maintainance between different conditions of growth. The hypothesis tested in the present study was that Myc can sustain mESC self renewal and pluripotency. Results provide important information on the mechanism by which Myc supports mESC self renewal, in comparison with the routinely used LIF+serum culture condition, such as a Myc-dependent specific transcriptional program, which involves induction of an alternative Core Regulatory Network, modulation of signallign pathways (Wnt/bcat) and repression of developmental genes. Total RNA obtained from mESC growth 3 days in the indicated condition compared to the routinely used LIF+serum culture condition. Total RNA obtained from EpiSC growth 3 days in standard condition were also used as control for primed stem cell state.
Project description:Calcineurin-NFAT signaling is associated with a wide range of biological processes and diseases. Our previous study showed that this pathway plays a critical role in mouse embryonic stem cell (mESC) differentiation. However, its function in human ESCs (hESCs) remains unclear. Here, we report that expression of PPP3CC, the gene encoding the catalytic subunit of calcineurin, increases along with the process of hESC differentiation, and its knockdown (KD) enhances the self-renewal ability of hESCs with a simultaneous reduction in the expression of differentiation-associated markers regardless of culture conditions. Moreover, we observed that NFATC3 translocates from the cytoplasm to the nucleus when hESCs exit from a self-renewal state. These results indicate that calcineurin-NFAT signaling is activated and required during hESC differentiation. Mechanistically, we also found that in hESCs NFATC3 interacts with JUN, one of AP-1 complex subunit, and co-expression of exogenous NFATC3 and JUN upregulates lineage markers remarkably even under a self-renewal culture condition. Additionally, inhibition of this cascade represses MAPK signaling rapidly, including ERK1/2, JNK and P38. Taken together, this study delineates the importance of the calcineurin-NFATC3/JUN signaling cascade for the pluripotency maintenance of hESCs.
Project description:The transcriptional repressors Polycomb Repressive Complex 1 (PRC1) and PRC2 are required to maintain cell fate during embryonic development. PRC1 and PRC2 catalyse distinct histone modifications, establishing repressive chromatin at shared targets. Loss of PRC1, but not PRC2, from mouse embryonic stem cells (mESCs) triggers exit from pluripotency. How PRC2 and PRC1, which consists of distinct “variant PRC1” (vPRC1) and “canonical PRC1” (cPRC1) complexes, cooperate to silence genes and support mESC self-renewal is unclear. Here, we report that independent pathways composed of vPRC1 and cPRC1/PRC2 repress shared target genes. Individual loss of vPRC1, cPRC1, or PRC2 disrupts only one pathway and does not impair mESC pluripotency. However, loss of both pathways leads to mESC differentiation and activation of a subset of poised target genes co-occupied by relatively high levels of PRC1/PRC2. Thus, parallel pathways explains the differential requirements for PRC1 and PRC2, and provides robust silencing of poised, lineage-specific genes.
Project description:Embryonic stem cells (ESC) are derived from blastocyst-stage embryos and are thought to be functionally equivalent to the inner cell mass in their developmental potential. ESCs pluripotency is maintained through a complex interplay of different signaling pathways and a network of transcription factors, which is centered around Oct3/4, Sox2 and Nanog. Although, in general, much is known about this pluripotency self-renewal circuitry, the molecular events that lead ESC to exit from pluripotency and begin differentiation are currently less known. Retinoic acid, an active metabolite of the vitamin A (retinol), plays important and pleiotropic roles in vertebrate embryonic development and ESC differentiation. Here we demonstrate that RA promotes early steps of ESC differentiation, and that ESC increase their capacity to synthesize RA during spontaneous differentiation as embryoid bodies, up-regulating the RA biosynthetic pathway components RDH1, RDH10, ADH3, RALDH2, and CRABP2. Microarray derived from total RNA of mESC not treated or treated with all-trans retinoic acid (ATRA) for 2 hours.