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Activation of the Imprinted Dlk1-Dio3 Region Correlates with Pluripotency Levels of Mouse Stem Cells*

  • Lei Liu
    Footnotes
    Affiliations
    From the State Key Laboratory of Reproductive Biology, Institute of Zoology, Beijing 100101, China
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  • Guan-Zheng Luo
    Footnotes
    Affiliations
    State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Beijing 100101, China

    Graduate University of the Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100101, China
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  • Wei Yang
    Footnotes
    Affiliations
    State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Beijing 100101, China

    Graduate University of the Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100101, China
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  • Xiaoyang Zhao
    Footnotes
    Affiliations
    From the State Key Laboratory of Reproductive Biology, Institute of Zoology, Beijing 100101, China

    Graduate University of the Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100101, China
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  • Qinyuan Zheng
    Affiliations
    From the State Key Laboratory of Reproductive Biology, Institute of Zoology, Beijing 100101, China

    Graduate University of the Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100101, China
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  • Zhuo Lv
    Affiliations
    From the State Key Laboratory of Reproductive Biology, Institute of Zoology, Beijing 100101, China

    Graduate University of the Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100101, China
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  • Wei Li
    Affiliations
    From the State Key Laboratory of Reproductive Biology, Institute of Zoology, Beijing 100101, China

    Graduate University of the Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100101, China
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  • Hua-Jun Wu
    Affiliations
    State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Beijing 100101, China

    Graduate University of the Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100101, China
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  • Liu Wang
    Affiliations
    From the State Key Laboratory of Reproductive Biology, Institute of Zoology, Beijing 100101, China
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  • Xiu-Jie Wang
    Correspondence
    To whom correspondence may be addressed: 1 Beichen West Rd., Chaoyang District, Beijing 100101, China. Fax: 86-10-64873428;
    Affiliations
    State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Beijing 100101, China
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  • Qi Zhou
    Correspondence
    To whom correspondence may be addressed: 1 Beichen West Rd., Chaoyang District, Beijing 100101, China. Fax: 86-10-64807306;
    Affiliations
    From the State Key Laboratory of Reproductive Biology, Institute of Zoology, Beijing 100101, China
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  • Author Footnotes
    * This work was in part supported by the National High Technology Research and Development Program of China Grant 2006AA02A101 (to Q. Z.), the Ministry of Science and Technology of China Grants 2006CB701501 (to Q. Z.) and 2007CB946901 to (X.-J. W.), and the National Natural Science Foundation of China Grants 90919060 (to Q. Z.) and 30725014 (to X.-J. W.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1 and 2.
    ♦ This article was selected as a Paper of the Week.
    1 These authors contributed equally to this work.
Open AccessPublished:April 09, 2010DOI:https://doi.org/10.1074/jbc.M110.131995
      Low reprogramming efficiency and reduced pluripotency have been the two major obstacles in induced pluripotent stem (iPS) cell research. An effective and quick method to assess the pluripotency levels of iPS cells at early stages would significantly increase the success rate of iPS cell generation and promote its applications. We have identified a conserved imprinted region of the mouse genome, the Dlk1-Dio3 region, which was activated in fully pluripotent mouse stem cells but repressed in partially pluripotent cells. The degree of activation of this region was positively correlated with the pluripotency levels of stem cells. A mammalian conserved cluster of microRNAs encoded by this region exhibited significant expression differences between full and partial pluripotent stem cells. Several microRNAs from this cluster potentially target components of the polycomb repressive complex 2 (PRC2) and may form a feedback regulatory loop resulting in the expression of all genes and non-coding RNAs encoded by this region in full pluripotent stem cells. No other genomic regions were found to exhibit such clear expression changes between cell lines with different pluripotency levels; therefore, the Dlk1-Dio3 region may serve as a marker to identify fully pluripotent iPS or embryonic stem cells from partial pluripotent cells. These findings also provide a step forward toward understanding the operating mechanisms during reprogramming to produce iPS cells and can potentially promote the application of iPS cells in regenerative medicine and cancer therapy.

      Introduction

      It is well known that embryonic stem (ES)
      The abbreviations used are: ES
      embryonic stem
      iPS
      induced pluripotent stem
      MEF
      mouse embryonic fibroblast
      miRNA
      microRNA.
      cells have two defining properties: self-renewal and pluripotency (
      • Silva J.
      • Barrandon O.
      • Nichols J.
      • Kawaguchi J.
      • Theunissen T.W.
      • Smith A.
      ,
      • Silva J.
      • Smith A.
      ). Induced pluripotent stem (iPS) cells from mice have developmental potential identical to ES cells; recent studies confirmed that both have the ability to support full term development by tetraploid complementation (
      • Zhao X.Y.
      • Li W.
      • Lv Z.
      • Liu L.
      • Tong M.
      • Hai T.
      • Hao J.
      • Guo C.L.
      • Ma Q.W.
      • Wang L.
      • Zeng F.
      • Zhou Q.
      ). However, the success rate for obtaining iPS cells with full pluripotency is extremely low as compared with ES cells, which severely hinders basic iPS cell research and the use of this technology in regenerative medicine and other clinical applications.
      Differences between ES and iPS cells in gene expression and differentiation ability have been identified (
      • Chin M.H.
      • Mason M.J.
      • Xie W.
      • Volinia S.
      • Singer M.
      • Peterson C.
      • Ambartsumyan G.
      • Aimiuwu O.
      • Richter L.
      • Zhang J.
      • Khvorostov I.
      • Ott V.
      • Grunstein M.
      • Lavon N.
      • Benvenisty N.
      • Croce C.M.
      • Clark A.T.
      • Baxter T.
      • Pyle A.D.
      • Teitell M.A.
      • Pelegrini M.
      • Plath K.
      • Lowry W.E.
      ,
      • Hu B.Y.
      • Weick J.P.
      • Yu J.
      • Ma L.X.
      • Zhang X.Q.
      • Thomson J.A.
      • Zhang S.C.
      ). Several reports indicated that iPS and ES cells express the same surface protein markers and are equally able to generate teratomas yet that they differ in their developmental potentials (
      • Takahashi K.
      • Yamanaka S.
      ,
      • Meissner A.
      • Wernig M.
      • Jaenisch R.
      ,
      • Wernig M.
      • Meissner A.
      • Foreman R.
      • Brambrink T.
      • Ku M.
      • Hochedlinger K.
      • Bernstein B.E.
      • Jaenisch R.
      ). As extensive chromatin remodeling and genome-wide epigenetic modifications occur during the reprogramming process, it is possible that the obtained iPS cells vary in their degrees of pluripotency due to different levels of reprogramming. Therefore, a fast and effective method to assess the extent of reprogramming and thereby predict the pluripotency of iPS cells would greatly improve iPS cell selection and applications.
      The iPS process dramatically changes cell fate via a few transcription factors (
      • Takahashi K.
      • Yamanaka S.
      ) and has sparked intense interest in molecular and cellular biology. However, its underlying mechanisms are still poorly understood. Multiple studies have aimed to identify the core molecular circuitry controlling cell pluripotency and self-renewal and identified several signaling pathways downstream of the key reprogramming factors (
      • Niwa H.
      • Ogawa K.
      • Shimosato D.
      • Adachi K.
      ,
      • Yamanaka S.
      ,
      • Hochedlinger K.
      • Plath K.
      ,
      • Sridharan R.
      • Tchieu J.
      • Mason M.J.
      • Yachechko R.
      • Kuoy E.
      • Horvath S.
      • Zhou Q.
      • Plath K.
      ,
      • Loh Y.H.
      • Ng J.H.
      • Ng H.H.
      ). How these signaling networks invoked by the induced transcription factors convert cell fate still remains obscure.
      By comparing gene and small RNA expression patterns in iPS cells with different degrees of pluripotency, we identified an imprinted genomic region, the Dlk1-Dio3 region, which was actively expressed in fully pluripotent stem cells but was repressed in the cells with partial pluripotency. The significant and consistent expression difference at this imprinted genomic region may serve as a marker to assess the pluripotency extent of cells. A large cluster of microRNAs (miRNAs) encoded by this imprinted region potentially form a feedback loop by regulating polycomb repressive complex 2 (PRC2) formation and are predicted to be involved in broad developmental regulatory processes, suggesting their master roles in gene expression regulatory networks. Protein-coding genes within the Dlk1-Dio3 region are highly conserved across species, whereas the miRNA cluster is conserved only in mammals. This indicated that it might have evolved early during selection for mechanisms that regulate stem properties, including those in human.

      DISCUSSION

      The production of healthy cells with full pluripotency is essential for stem cell research and its application in regenerative medicine. However, the low efficiency of iPS cell generation and the reduced pluripotency of these cells have long been obstacles. Therefore, fast and effective methods to adjust the pluripotent properties of cells will greatly accelerate the exploration of reprogramming mechanisms and the use of iPS cells in regenerative medicine.
      Here, we identified an imprinted genomic region in mouse that was actively expressed in fully pluripotent stem cell lines, such as ES and 4n-iPS cells, but was repressed in partially pluripotent cells as represented by two 2n-iPS cell lines. Protein-coding genes, a large cluster of miRNAs, and other small RNAs derived from this Dlk1-Dio3 imprinted region consistently exhibited significant expression repression or depletion in the 2n-iPS cells as compared with the ES and 4n-iPS cells. Regardless of the genetic background and origin of the ES and iPS cell lines, the expression differences between the fully and partially pluripotent stem cells were consistently observed. Furthermore, although genes and miRNAs from this region had low overall expression in the 2n-iPS cell lines, their expression was slightly higher in the germline-transmittable 2n-iPS cells than in those without germline transmission ability. These results indicated that the degree of activation of the Dlk1-Dio3 imprinted region is positively correlated with the pluripotency levels of stem cells. The coordinated expression changes of protein-coding genes and miRNAs encoded by this region, measured in independent assays, supports the conclusion that this region is affected by some regulatory mechanism that may be responsible for or respond to the pluripotency status of a cell.
      Our analysis revealed that the miRNA cluster encoded by the Dlk1-Dio3 region only presented in mammalian genomes and is highly conserved among mammals, indicating its specific and crucial role in regulating mammalian development. We expect that this miRNA cluster should have conserved functions in human and other mammals. As some other genes within the Dlk1-Dio3 region are also conserved in non-mammal species, it is very likely that the pluripotency-correlated expression of the entire Dlk1-Dio3 region is indeed a functional reflection of the miRNA cluster.
      After extensively screening the gene and small RNA expression data used in this study, we were not able to identify any other genomic region exhibiting similar or opposite expression patterns as that of the Dlk1-Dio3 imprinted region. These data indicated that the Dlk1-Dio3 region might be the only long genomic locus that exhibits a clear on-and-off switch in cells with full versus partial pluripotency; thus, the expression state of this region is a strong candidate for a marker of the quality of iPS and stem cells from other sources. Fully pluripotent human stem cells are very difficult to obtain via the iPS technique, so identification of such a marker site may greatly accelerate the selection process for good iPS or ES cells and pave the way for their application in regenerative medicine.
      The putative targets of miRNAs from the Dlk1-Dio3 region include three genes in the PRC2 silencing complex. These genes are all expressed at lower levels in the ES and 4n-iPS cells as compared with the 2n-iPS cells, perhaps as a result of higher miRNA expression in the ES and 4n-iPS cells. We hypothesize that the repression of PRC2 complex formation contributes to the lack of methylation and silencing of the Dlk1-Dio3 region, resulting in the activated expression of its encoded genes as observed in our results. The activated miRNAs may act in a feedback loop to further prevent the formation of PRC2 and maintain the active state of the Dlk1-Dio3 region. A recent study showed that PRC2 is not necessary for stem cell pluripotency maintenance (
      • Chamberlain S.J.
      • Yee D.
      • Magnuson T.
      ). Therefore, down-regulation of PRC2 in the ES and 4n-iPS cell lines should not affect their pluripotency. Many studies have been conducted to investigate the function of the Dlk1-Dio3 imprinted region, but no clear conclusions have been made. Here, we propose that some miRNAs from this region regulate cell pluripotency via gene silencing pathways. It is likely that the activated miRNA clusters are involved in the regulation of many developmentally related biological processes as revealed by gene ontology enrichment analysis.
      The connection of the Dlk1-Dio3 region miRNAs with PRC2 also suggests potential applications of these miRNAs in cancer therapy. As one of the PRC2 components, the histone methyltransferase Ezh2 is frequently observed to be overexpressed in various types of cancers (
      • Simon J.A.
      • Lange C.A.
      ). Modulating the formation of PRC2 by manipulating the Dlk1-Dio3 region miRNAs might serve as an approach to prevent the uncontrolled proliferation of some types of cancer cells.
      The discovery of iPS technology demonstrated that the developmental status of cells can be reverted by the activity of a few transcription factors, but its underlying mechanisms remain unknown. We hypothesize that changes in the methylation status of an imprinted genomic region might be involved in the pluripotency regulation of cells, providing a testable model toward understanding the mechanism of iPS cell formation.
      Scientists have long attempted to identify key components that regulate complex traits but with very little success in protein-coding genes. Our results indicated that cell pluripotency levels might be in partially controlled by a group of miRNAs, suggesting the possibility of miRNAs as master regulators of gene expression networks. The synergetic effects of multiple miRNAs toward the same outcome revealed by our model also furthered our understanding of miRNA function mechanisms and shed light on future studies on the coordinated effects of miRNAs.

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