Myc-regulated microRNAs attenuate embryonic stem cell differentiation.
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ABSTRACT:
SUBMITTER: Lin CH
PROVIDER: S-EPMC2744176 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Multiple studies show that tumor suppressor p53 is a barrier to dedifferentiation; whether this is strictly due to repression of proliferation remains a subject of debate. Here, we show that p53 plays an active role in promoting differentiation of human embryonic stem cells (hESCs) and opposing self-renewal by regulation of specific target genes and microRNAs. In contrast to mouse embryonic stem cells, p53 in hESCs is maintained at low levels in the nucleus, albeit in a deacetylated, inactive state. In response to retinoic acid, CBP/p300 acetylates p53 at lysine 373, which leads to dissociation from E3-ubiquitin ligases HDM2 and TRIM24. Stabilized p53 binds CDKN1A to establish a G(1) phase of cell cycle without activation of cell death pathways. In parallel, p53 activates expression of miR-34a and miR-145, which in turn repress stem cell factors OCT4, KLF4, LIN28A, and SOX2 and prevent backsliding to pluripotency. Induction of p53 levels is a key step: RNA-interference-mediated knockdown of p53 delays differentiation, whereas depletion of negative regulators of p53 or ectopic expression of p53 yields spontaneous differentiation of hESCs, independently of retinoic acid. Ectopic expression of p53R175H, a mutated form of p53 that does not bind DNA or regulate transcription, failed to induce differentiation. These studies underscore the importance of a p53-regulated network in determining the human stem cell state.
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Project description:microRNAs (miRNAs) regulate numerous physiological processes such as cell division and differentiation in many tissue types including stem cells. To probe the role that miRNAs play in regulating processes relevant to embryonic stem cell biology, we used RNA interference to silence DICER and DROSHA, the two main miRNA processing enzymes. Consistent with a role for miRNAs in maintaining normal stem cell division and renewal, we found that perturbation of miRNA pathway function in human embryonic stem cells (hESCs) attenuates cell proliferation. Normal cell growth can be partially restored by introduction of the mature miRNAs miR-195 and miR-372. These miRNAs regulate two tumor suppressor genes, respectively: WEE1, which encodes a negative G2/M kinase modulator of the CycB/CDK complex and CDKN1A, which encodes p21, a CycE/CDK cyclin dependent kinase inhibitor that regulates the G1/S transition. We show that in wild-type hESCs, WEE 1 levels control the rate of hESC division, whereas p21 levels must be maintained at a low level for hESC division to proceed. These data support a model for hESC cell cycle control in which miRNAs regulate negative cell cycle modulators at two phases of the cell cycle to ensure proper replenishment of the stem cell population.
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Project description:The findings that microRNAs (miRNAs) are essential for early development in many species and that embryonic miRNAs can reprogram somatic cells into induced pluripotent stem cells suggest that these miRNAs act directly on transcriptional and chromatin regulators of pluripotency. To elucidate the transcription regulatory networks immediately downstream of embryonic miRNAs, we extended the motif activity response analysis approach that infers the regulatory impact of both transcription factors (TFs) and miRNAs from genome-wide expression states. Applying this approach to multiple experimental data sets generated from mouse embryonic stem cells (ESCs) that did or did not express miRNAs of the ESC-specific miR-290-295 cluster, we identified multiple TFs that are direct miRNA targets, some of which are known to be active during cell differentiation. Our results provide new insights into the transcription regulatory network downstream of ESC-specific miRNAs, indicating that these miRNAs act on cell cycle and chromatin regulators at several levels and downregulate TFs that are involved in the innate immune response.
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Project description:This report demonstrates that introduction of microRNAs (miRNAs) specific to embryonic stem cells enhances the production of mouse induced pluripotent stem (iPS) cells. The miRNAs miR-291-3p, miR-294 and miR-295 increase the efficiency of reprogramming by Oct4, Sox2 and Klf4, but not by these factors plus cMyc. cMyc binds the promoter of the miRNAs, suggesting that they are downstream effectors of cMyc during reprogramming. However, unlike cMyc, the miRNAs induce a homogeneous population of iPS cell colonies.
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Project description:Embryonic stem (ES) cells have been shown to recapitulate normal developmental stages. They are therefore a highly useful tool in the study of developmental biology. Profiling of ES cell-derived cells has yielded important information about the characteristics of differentiated cells, and allowed the identification of novel marker genes and pathways of differentiation. In this review, we focus on recent results from profiling studies of mouse embryos, human islets, and human ES cell-derived differentiated cells from several research groups. Global gene expression data from mouse embryos have been used to identify novel genes or pathways involved in the developmental process, and to search for transcription factors that regulate direct reprogramming. We introduce gene expression databases of human pancreas cells (Beta Cell Gene Atlas, EuroDia database), and summarize profiling studies of islet- or human ES cell-derived pancreatic cells, with a focus on gene expression, microRNAs, epigenetics, and protein expression. Then, we describe our gene expression profile analyses and our search for novel endoderm, or pancreatic, progenitor marker genes. We differentiated mouse ES cells into mesendoderm, definitive endoderm (DE), mesoderm, ectoderm, and Pdx1-expressing pancreatic lineages, and performed DNA microarray analyses. Genes specifically expressed in DE, and/or in Pdx1-expressing cells, were extracted and their expression patterns in normal embryonic development were studied by in situ hybridization. Out of 54 genes examined, 27 were expressed in the DE of E8.5 mouse embryos, and 15 genes were expressed in distinct domains in the pancreatic buds of E14.5 mouse embryos. Akr1c19, Aebp2, Pbxip1, and Creb3l1 were all novel, and none has been described as being expressed, either in the DE, or in the pancreas. By introducing the profiling results of ES cell-derived cells, the benefits of using ES cells to study early embryonic development will be discussed.
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Project description:While endogenous Myc (c-myc) and Mycn (N-myc) have been reported to be separately dispensable for murine embryonic stem cell (mESC) function, myc greatly enhances induced pluripotent stem (iPS) cell formation and overexpressed c-myc confers LIF-independence upon mESC. To address the role of myc genes in ESC and in pluripotency generally, we conditionally knocked out both c- and N-myc using myc doubly homozygously floxed mESC lines (cDKO). Both lines of myc cDKO mESC exhibited severely disrupted self-renewal, pluripotency, and survival along with enhanced differentiation. Chimeric embryos injected with DKO mESC most often completely failed to develop or in rare cases survived but with severe defects. The essential nature of myc for self-renewal and pluripotency is at least in part mediated through orchestrating pluripotency-related cell cycle and metabolic programs. This study demonstrates that endogenous myc genes are essential for mESC pluripotency and self-renewal as well as providing the first evidence that myc genes are required for early embryogenesis, suggesting potential mechanisms of myc contribution to iPS cell formation.
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Project description:The multifunctional histone chaperone, SET, is essential for embryonic development in the mouse. Previously, we identified SET as a factor that is rapidly downregulated during embryonic stem cell (ESC) differentiation, suggesting a possible role in the maintenance of pluripotency. Here, we explore SET's function in early differentiation. Using immunoprecipitation coupled with protein quantitation by LC-MS/MS, we uncover factors and complexes, including P53 and β-catenin, by which SET regulates lineage specification. Knockdown for P53 in SET-knockout (KO) ESCs partially rescues lineage marker misregulation during differentiation. Paradoxically, SET-KO ESCs show increased expression of several Wnt target genes despite reduced levels of active β-catenin. Further analysis of RNA sequencing datasets hints at a co-regulatory relationship between SET and TCF proteins, terminal effectors of Wnt signaling. Overall, we discover a role for both P53 and β-catenin in SET-regulated early differentiation and raise a hypothesis for SET function at the β-catenin-TCF regulatory axis.
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Project description:MicroRNAs (miRs) play an important role in cell differentiation and maintenance of cell identity, but relatively little is known of their functional role in modulating human hematopoietic lineage differentiation. Human embryonic stem cells (hESCs) provide a model system to study early human hematopoiesis. We differentiated hESCs by embryoid body (EB) formation and compared the miR expression profile of undifferentiated hESCs to CD34(+) EB cells. miRs-126/126* were the most enriched of the 7 miRs that were up-regulated in CD34(+) cells, and their expression paralleled the kinetics of hematopoietic transcription factors RUNX1, SCL, and PU.1. To define the role of miRs-126/126* in hematopoiesis, we created hESCs overexpressing doxycycline-regulated miRs-126/126* and analyzed their hematopoietic differentiation. Induction of miRs-126/126* during both EB differentiation and colony formation reduced the number of erythroid colonies, suggesting an inhibitory role of miRs-126/126* in erythropoiesis. Protein tyrosine phosphatase, nonreceptor type 9 (PTPN9), a protein tyrosine phosphatase that is required for growth and expansion of erythroid cells, is one target of miR-126. PTPN9 restoration partially relieved the suppressed erythropoiesis caused by miRs-126/126*. Our results define an important function of miRs-126/126* in negative regulation of erythropoiesis, providing the first evidence for a role of miR in hematopoietic differentiation of hESCs.
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Project description:Cell differentiation typically occurs with concomitant shape transitions to enable specialized functions. To adopt a different shape, cells need to change the mechanical properties of their surface. However, whether cell surface mechanics control the process of differentiation has been relatively unexplored. Here we show that membrane mechanics gate exit from naive pluripotency of mouse embryonic stem cells. By measuring membrane tension during early differentiation, we find that naive stem cells release their plasma membrane from the underlying actin cortex when transitioning to a primed state. By mechanically tethering the plasma membrane to the cortex by enhancing Ezrin activity or expressing a synthetic signaling-inert linker, we demonstrate that preventing this detachment forces stem cells to retain their naive pluripotent identity. We thus identify a decrease in membrane-to-cortex attachment as a new cell-intrinsic mechanism that is essential for stem cells to exit pluripotency.
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Project description:Studies of embryonic stem cells (ESCs) reveal that these cell lines can be derived from differing stages of embryonic development. We analyzed common changes in the expression of microRNAs (miRNAs) and mRNAs in 9 different human ESC (hESC) lines during early commitment and further examined the expression of key ESCenriched miRNAs in earlier developmental states in several species. We show that several previously defined hESC-enriched miRNA groups (the miR-302, -17, and -515 families, and the miR-371-373 cluster) and several other hESC-enriched miRNAs are down-regulated rapidly in response to differentiation. We further found that mRNAs up-regulated upon differentiation are enriched in potential target sites for these hESC-enriched miRNAs. Interestingly, we also observed that the expression of ESC-enriched miRNAs bearing identical seed sequences changed dynamically while the cells transitioned through early embryonic states. In human and monkey ESCs, as well as human-induced pluripotent stem cells (iPSCs), the miR-371-373 cluster was consistently up-regulated, while the miR-302 family was mildly down-regulated when the cells were chemically treated to regress to an earlier developmental state. Similarly, miR-302b, but not mmu-miR-295, was expressed at higher levels in murine epiblast stem cells (mEpiSC) as compared with an earlier developmental state, mouse ESCs. These results raise the possibility that the relative expression of related miRNAs might serve as diagnostic indicators in defining the developmental state of embryonic cells and other stem cell lines, such as iPSCs. These data also raise the possibility that miRNAs bearing identical seed sequences could have specific functions during separable stages of early embryonic development.
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