EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS
a
School of Life Sciences, Beijing b Normal University, Beijing, China; National Institute of Biological Sci‐ ences (NIBS), Beijing, China; c School of Life Sciences and Tech‐ nology, Tongji University, Shanghai, China Correspondence: Shaorong Gao, Ph.D., 1239 Siping Road, Shanghai 200092, China. Telephone: 86‐21‐ 65985182; Fax: 86‐21‐65985182; e‐ mail: [email protected] Received December 15, 2013; ac‐ cepted for publication May 23, 2014; ©AlphaMed Press 1066‐5099/2014/$30.00/0 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typeset‐ ting, pagination and proofreading process which may lead to differ‐ ences between this version and the Version of Record. Please cite this article as doi: 10.1002/stem.1775
Xist Repression Shows Time‐dependent Effects on the Reprogramming of Female Somatic Cells to iPSCs QI CHEN a,b, SHUAI GAO b, WENTENG HE b,c, XIAOCHEN KOU b,c, YANHONG ZHAO b,c, HONG WANG c, SHAORONG GAO b,c Key words. Xist • iPS • Pre‐iPS • X chromosome reactivation • H3K27me3 • macroH2A ABSTRACT Although the reactivation of silenced X chromosomes has been observed as part of the process of reprogramming female somatic cells into induced pluripotent stem cells (iPSCs), it remains unknown whether repression of the X‐inactive specific transcript (Xist) can greatly enhance female iPSC induction similar to that observed in somatic cell nuclear transfer (SCNT) studies. In the present study, we discovered that the repression of Xist plays opposite roles in the early and late phases of female iPSCs induction. Our results demonstrate that the down‐regulation of Xist by an IPTG‐ inducible shRNA system can greatly impair the mesenchymal‐to‐epithelial transition (MET) in the early phase of iPSC induction but can significantly promote the transition of pre‐iPSCs to iPSCs in the late phase. Further‐ more, we demonstrate that although the knockdown of Xist did not affect the H3K27me3 modification on the X chromosome, macroH2A was re‐ leased from the inactivated X chromosome (Xi). This enables the X chro‐ mosome silencing to be a reversible event. Moreover, we demonstrate that the supplementation of vitamin C (Vc) can augment and stabilize the reversible X chromosome by preventing the relocalization of macroH2A to the Xi. Therefore, our study reveals an opposite role of Xist repression in the early and late stages of reprogramming female somatic cells to pluripotency and demonstrates that the release of macroH2A by Xist re‐ pression enables the transition from pre‐iPSCs to iPSCs. STEM CELLS 2014; 00:000–000
INTRODUCTION In mammals, two X chromosomes are both active in the early stages of female pre‐implantation embryos and in the subsequently established fully pluripotent female embryonic stem cells (ESCs). Following embryo devel‐ opment and ESC differentiation, one X chromosome undergoes inactivation. The inactivation event is initiat‐ ed by the long noncoding RNA Xist [1, 2] followed by the Polycomb repressive complex 2 (PRC2)‐mediated H3K27me3 modification and the deposition of histone H2A variant macroH2A [3‐5]. Xist depletion in the early initiation stages of X chromosome inactivation can lead to the reversal of X chromosome silencing and hetero‐ STEM CELLS 2014;00:00‐00 www.StemCells.com
chromatin formation [6, 7]. This Xist‐dependent, re‐ versible inactivation status switches to Xist‐independent silencing after macroH2A deposition and the recruit‐ ment of other related proteins [3‐5, 8]. Xist then acts synergistically with DNA methylation and histone hypoacetylation to maintain the X chromosome inacti‐ vation status in somatic cells [9, 10]. Although Xist dele‐ tion has only minor effects on X chromosome reactiva‐ tion in somatic cells [6, 11], as the remaining DNA‐ methylation and histone hypoacetylation can maintain the Xi [9], it disrupts long‐term maintenance of the H3K27me3 mark [12] and histone macroH2A localiza‐ tion on the Xi [13‐15]. The localization of macroH2A on the Xi can distinguish between irreversible and reversi‐ ©AlphaMed Press 2014
2 ble X chromosome inactivation, and the Xi of murine epiblast stem cells (EpiSCs) without macroH2A can be reactivated by SCNT through injecting mouse somatic nuclei into Xenopus laevis oocytes, which is much easier than the reactivation of Xi of somatic cells with macroH2A deposition [16, 17]. Epigenetic reprogramming has been considered as the major force driving cell fate changes during repro‐ gramming and transdifferentiation. Many epigenetic modifications and factors have been discovered that play critical roles in the induction of iPSCs. For example, results from our lab and others have shown that the recently discovered DNA hydroxylase TET proteins play important roles in the reprogramming of iPSCs [18‐20]. Previous studies have also shown that numerous his‐ tone modification enzymes function either positively or negatively in somatic cell reprogramming. As one im‐ portant epigenetic modification in female cells, X chro‐ mosome inactivation and reactivation are involved in the development of somatic cloned embryos. Previous SCNT studies showed that the Xi is not properly reac‐ tivated and rather retains the epigenetic memory of the initially inactivated X chromosome, which is preferen‐ tially silenced in the extraembryonic tissues [21‐24]. Full X chromosome reactivation in SCNT embryos only oc‐ curs in the inner cell mass as it does during normal em‐ bryo development [25, 26], erasing the memory of the preimplantation Xi for subsequent random X‐ inactivation in the embryonic tissues. However, little attention has been focused on the role of X chromo‐ some inactivation or reactivation during female iPSC induction because most iPSC lines are derived from males. This prompts us to re‐evaluate the role of the X chromosome in female somatic cell reprogramming. The reactivation of silenced X chromosome has been observed during female somatic cell reprogramming mediated by defined transcription factors [27], and Xist disappearance has been observed in this process [28, 29]. In early stage, the reprogramming of female somat‐ ic cells to partially reprogrammed pre‐iPSCs does not involve the reactivation of the Xi [30], which is similar to the reprogramming of somatic cells to EpiSCs [31, 32]. X chromosome reactivation occurs when pre‐iPSCs are converted to iPSCs following treatments that enhance the late phase of reprogramming, such as Vc [30, 31, 33]. To elucidate the molecular mechanisms of female iPSC induction, we focused on X‐related Xist which is downregulated during reprogramming [30], along with H3K27me3 modification [34] and macroH2A deposition [35], both of which are barriers during the reprogram‐ ming of somatic cells into iPSCs. Our observations sug‐ gest a time‐dependent effect of Xist repression on fe‐ male somatic cell reprogramming. In the early stages of reprogramming, Xist is required for MET progression. However, during the transition from pre‐iPSC to iPSC in the late phase of reprogramming, Xist needs to be re‐ pressed for fully pluripotent iPSC induction. Moreover, we also uncovered a transition from an irreversible Xi www.StemCells.com
Xist repression promotes pre‐iPSCs to iPSCs transition status to a reversible Xi status, which is related to Xist expression and macroH2A releasing, and can be aug‐ mented and stabilized by Vc.
MATERIALS AND METHODS Mice and Cell Culture Pathogen‐free mice were housed in the animal facility of the National Institute of Biological Sciences, Beijing. All of our study procedures were consistent with the guide for the care and use of laboratory animals. Mouse embryonic fibroblasts (MEFs) were derived from individual 13.5‐dpc embryos collected from female Oct4‐green fluorescent protein (OG2) transgenic mice [36] that had been previously mated with male Rosa26‐ M2rtTA mice. Genomic DNA was isolated from the MEFs using a standard protocol and the gender was confirmed using the sex‐determining region Y (Sry) gene PCR (primer listed in Table S1). Control female ESCs and iPSCs were cultured on mitomycin C (MMC)‐treated MEFs in ES medium containing Dulbecco’s modified Eagle’s medium (DMEM) (Gibco Invitrogen, Carlsbad, CA) supplemented with 15% (vol/vol) fetal bovine se‐ rum (FBS), 1 mM L‐glutamine, 0.1 mM mercaptoethanol, 1% nonessential amino acid stock, and 1,000 U/ml LIF (all from Chemicon, Temecula, CA) [37]. Other reagents used in the experiments were doxycycline (DOX, Sigma‐Aldrich, St. Louis, MO, 1 μg/ml), IPTG (Sigma‐Aldrich, St. Louis, MO, 4 mM) and Vc (Sigma‐Aldrich, St. Louis, MO, 50 μg/mL).
Plasmid and iPS Cells Induction pFUW‐tetO‐Oct4, Sox2, Klf4 and c‐Myc (TetO‐OSKM) were generously provided by Dr. Rudolf Jaenisch’s la‐ boratory at the Whitehead Institute for Biomedical Re‐ search. The control vectors used were pLKO‐based IPTG‐inducible non‐target shRNA (SHC332, Sigma‐ Aldrich, St. Louis, MO) and pSicoR (11579, Addgene, Cambridge, MA). The Xist shRNA sequence was cloned into the IPTG‐inducible PLKO vector and the pSicoR vec‐ tor under the U6 promoter. All oligos designed for Xist knockdown are listed in Table S1. The iPSCs derivation procedure was performed ac‐ cording to the methods described previously [33, 37, 38]. MEFs were first infected with TetO‐OSKM lentiviruses, along with the pLKO‐based IPTG‐inducible Xist shRNA, the non‐target control shRNA lentivirus, the pSicoR‐based Xist shRNA, or the control empty‐pSicoR lentivirus. The infected MEFs were then cultured in ES medium supplemented with DOX, and IPTG was added to the culture medium according to the experimental design. Oct4‐GFP‐positive iPSCs (GFP‐iPSCs) were picked and digested for subsequent culture on MMC‐treated MEFs. To derive the pre‐iPSCs, colonies were picked and cultured on MEFs. Oct4‐GFP‐negative colonies that re‐ mained GFP negative during passage were selected. To derive the GFP‐iPSCs from the pre‐iPSCs, the pre‐iPSCs ©AlphaMed Press 2014
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Xist repression promotes pre‐iPSCs to iPSCs transition
were treated with DOX, Vc and IPTG as described. Oct4‐ GFP‐positive colonies were then selected and passaged.
CA, www.bdbiosciences.com) according to standard proce‐ dures.
Experimental Design
AP Staining
To investigate the effects of Xist repression from the beginning of iPSC induction, MEFs (50000‐100000 cells / 35 mm dish) were infected at passage 3 with TetO‐ OSKM lentiviruses, along with either pSicoR‐based Xist shRNA or control empty‐pSicoR lentivirus, and then treated with either DOX or DOX plus IPTG. The efficien‐ cy of iPSC generation was calculated from the number of colonies that were both alkaline phosphatase (AP)‐ positive and morphologically similar to the ESCs at day 15. The same experiments were performed with IPTG‐ inducible pLKO‐based Xist shRNA and non‐target control shRNA lentivirus as described above. To investigate the temporal effects of Xist repres‐ sion on iPSC induction, MEFs were infected with TetO‐ OSKM and pLKO‐based IPTG‐inducible Xist shRNA lentivirus and then treated with DOX. IPTG was added to the induction medium at different time points. The efficiency of the resulting iPSC generation was calculat‐ ed by counting the number of AP‐positive colonies at day 18. The same experiments were performed with the pLKO‐based non‐target control shRNA lentivirus to con‐ trol for the effects of IPTG treatment. To investigate the effects of Xist repression and Vc on the transition from pre‐iPSCs to iPSCs, pre‐iPSCs car‐ rying the pLKO‐based IPTG‐inducible Xist shRNA were cultured in ES medium with DOX and then treated with different combinations of IPTG and Vc as designed. The efficiency of iPSC generation was calculated using the number of Oct4‐GFP‐positive colonies. Each treatment group of the above experiments contained three replicates.
AP staining was performed with the Leukocyte Alkaline Phosphatase Kit (Sigma‐Aldrich, St. Louis, MO) accord‐ ing to the manufacturer’s recommendations.
Real‐time PCR (qPCR) The total RNA was purified with Trizol reagent (Invitrogen, Grand Island, NY) and reverse transcribed using M‐MLV Re‐ verse Transcriptase (Promega, Madison, WI) and Rnasein Rnase Inhibitor (Promega, Madison, WI) according to the manufacturer’s recommendations. The qPCR was performed with SYBR Premix Ex Taq (Takara, Tokyo, Japan) and the ABI7500 Real‐Time PCR System (Applied Biosystems, Grand Island, NY, www.lifetechnologies.com). All reactions were performed in triplicate using a 1/10 concentration of the cDNA obtained as described above. The expression level of the gene transcript in each sample was normalized to glycer‐ aldehyde‐3‐phosphate dehydrogenase (GAPDH), and the relative expression level was estimated using the comparative CT method. Primers used are listed in Table S1.
Fluorescence Activated Cell Sorting (FACS) Analysis Pluripotent cells were digested and removed from the MMC‐treated MEFs using differential adhesion. FACS was performed to differentiate the GFP‐positive and GFP‐negative cells, using a FACS LSRII instrument (BD biosciences, San Jose, www.StemCells.com
RNA Fluorescence In Situ Hybridization (FISH) and Immunofluorescence Staining RNA FISH for Xist was performed with a Type 1 Probe Set (Cy3) according to the user manual for the QuantiGene ViewRNA ISH Cell Assay (Affymetrix, Santa Clara, CA). For the detection of the MET or pluripotent mark‐ ers, iPSCs grown on the gelatin coated cover slides were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton‐X, blocked with 5 BSA and incubated with the first antibody against Nanog (1:500, Cosmo Bio Co., Ltd, Tokyo, Japan), SSEA1 (1:200, Millipore, Darmstadt, Germany) and beta‐catenin (1:500, Santa Cruz Biotech‐ nology, Inc., Texas, U.S.A.) according to a protocol de‐ scribed elsewhere [39]. For the X chromosome related protein, immunofluorescence staining was performed as previously described [35], first using an antibody against H3K27me3 (1:250, Millipore, Darmstadt, Ger‐ many) and macroH2A (1:100, Millipore, Darmstadt, Germany). The samples were incubated with the ap‐ propriate secondary antibodies after three washes. The following fluorochrome‐conjugated secondary antibod‐ ies were applied: Alexa Fluor 594 goat anti‐rabbit IgG (Molecular Probes, Grand Island, NY), Alexa Fluor 594 goat anti‐mouse IgM (Molecular Probes, Grand Island, NY), Alexa Fluor 594 goat anti‐mouse IgG (Molecular Probes, Grand Island, NY) and Alexa Fluor 594 rabbit anti‐goat IgG (Molecular Probes, Grand Island, NY). The DNA was labeled with DAPI, and the stained cells were mounted on slides. The slides were observed with a NIKON ECLIPSE 80i microscope (Nikon Instruments Inc., Melville, NY, www.nikoninstruments.com) using the Plan Fluor 40×/ Oil 100× objective. Selected layers were focused on using the Volocity Demo 5.5 software (PerkinElmer, Massachusetts, www.perkinelmer.com).
Teratoma Formation and Chimeric Mouse Generation
For the teratoma formation, 106 female iPSCs or pre‐ iPSCs were injected subcutaneously into each flank of nude BALB/C mice. Paraffin sections of the formalin‐ fixed teratoma specimens were prepared 3–5 weeks after the injections, and an analysis of the H&E‐stained tissue sections was performed for each specimen. Chimeric mice were generated as previously de‐ scribed [37]. In brief, approximately 10 iPS cells were microinjected into the ICR eight‐cell embryos using a piezo‐actuated microinjection pipette. After culturing for 1 day, the embryos were transplanted into the uter‐ us of pseudopregnant mice. When the chimeric mice ©AlphaMed Press 2014
Xist repression promotes pre‐iPSCs to iPSCs transition
4 reached adulthood, they were mated with ICR mice to test for germ‐line transmission.
RESULTS
Knockdown of Xist Showed Opposite Effects in the Early and Late Stages of the Repro‐ gramming of Female Somatic Cells to iPSCs To explore the potential role of Xist repression in iPSC induction, two shRNA expression systems were used in our studies of Xist repression. The first was a pSicoR‐ based system that could induce shRNA expression con‐ stitutively; the second was a pLKO‐based IPTG‐inducible system that could control shRNA expression at different time points (Figure. S1A, S1B). We first investigated the effects of Xist repression from the beginning of iPSC induction. Mixtures of fe‐ male and male OG2 MEFs were infected with the TetO‐ OSKM lentiviruses and either the pSicoR‐based Xist shRNA virus or the control empty‐pSicoR virus. We no‐ ticed that the AP‐positive colony numbers (0.32% com‐ pared to 0.54%) significantly decreased in the MEFs that had been induced with Oct4/Sox2/Klf4/c‐Myc (OSKM) and Xist shRNA compared to the empty‐pSicoR control group (Figure. S1C, S1D). Because Xist is expressed only in female MEFs, we then derived MEFs from female and male embryos separately to investigate the role of Xist repression in the different genders. For the females, the number of AP‐positive colonies produced from the MEFs induced with OSKM and Xist shRNA was approxi‐ mately 30 percent of that from the empty‐pSicoR con‐ trol group (Figure. 1A, 1B). However, no significant dif‐ ference was observed between the TetO‐OSKM and the Xist shRNA infected male MEFs and the empty‐pSicoR control group (Figure. 1A, 1B). The number of GFP‐ positive colonies was consistent with the AP staining results (Figure. 1B). Taken together, these results sug‐ gest that Xist repression in the early stages of repro‐ gramming decreased the reprogramming efficiency of female MEFs while only slightly affecting male iPSC generation. This was further demonstrated using adult female tail tip fibroblasts (TTFs) (Figure. S1E, S1F). Simi‐ lar results were abtained from experiments using an‐ other Xist shRNA (Figure. S1G S1H). We also repeated our experiments using the IPTG‐inducible Xist shRNA and non‐target shRNA control and obtained similar re‐ sults (Figure. S1I). The above data showed that early Xist knockdown decreased the reprogramming efficiency of female iPSC induction. Considering that the silenced X chromosome might be reactivated during the late stage of repro‐ gramming, we hypothesized that Xist repression might function differently during the early and late stages of reprogramming. To test our hypothesis, female MEFs were infected with the TetO‐OSKM virus, along with the IPTG‐inducible Xist shRNA virus, in order to control Xist repression at different time points. The Xist shRNA could successfully down‐regulate Xist expression to www.StemCells.com
25.97% (Figure. S1B), and colonies appeared at around day (D) 9 in the OSKM induced reprogramming. Xist expression could be observed for as long as 12 days in some cells undergoing reprogramming but disappeared in the derived GFP‐iPSCs (Figure. S1J). When Xist shRNA was induced by IPTG starting at D0 of reprogramming, the number of AP‐positive colonies was only 50% of that of the pLKO‐non‐target‐shRNA control group, 58% of non‐IPTG treated Xist shRNA infected group, further confirming our previous observations of the pSicoR sys‐ tem (Figure. 1C, 1D). Moreover, IPTG treatment during the first 8 days resulted in the formation of significantly fewer AP‐positive colonies. However, IPTG treatment after D9 resulted in the formation of more AP‐positive colonies, producing a 1.5‐fold increase over the non‐ target shRNA control group, and IPTG treatment after D12 also resulted in the formation of around 2 fold in‐ crease of AP‐positive colonies (Figure. 1C, 1D, 1E, 1F). We further confirmed that this was not due to the IPTG treatment itself, as female MEFs infected with the TetO‐ OSKM and the non‐target control shRNA had no signifi‐ cant effects on the formation of AP‐positive colonies when IPTG was added at different time points (Figure. 1E). Taken together, the above results demonstrate that Xist repression during the first 8 days of reprogramming decreases the efficiency of female somatic cell repro‐ gramming, while Xist repression after D9 can actually improve the efficiency of reprogramming.
The Knockdown of Xist Impaired MET in the Early Stage of Reprogramming To investigate the dynamic roles of Xist repression in decreasing reprogramming efficiency, we first hypothe‐ sized that Xist depletion could cause cell death or re‐ duce cell proliferation. We infected female MEFs with the TetO‐OSKM virus along with either Xist shRNA or an empty‐pSicoR control virus. However, no significant differences in cell proliferation were noticed during first 4 days between the OSKM and Xist shRNA treated group and the empty‐pSicoR control group (Figure. 2A). The cell numbers began to show differences after D6 (Figure. 2A), indicating that some epigenetic changes accumulated during the early reprogramming stage that were not apparent from the cell proliferation. The second hypothesis was that Xist repression im‐ pairs MET during the initiation phase of reprogramming [40, 41]. We harvested the mRNA of female MEFs in‐ duced with OSKM and either Xist shRNA or empty‐ pSicoR control shRNA during reprogramming. We then analyzed the expression levels of mesenchymal and epithelial marker genes. Compared to the empty‐pSicoR control group, mesenchymal genes (Slug, Bmp2, Tgfb1 and Zeb1) showed a delayed downregulation in the Xist shRNA treated group (Figure. 2B), while epithelial genes (Epcam, Cdh1, Tgfb3 and Tgfbr3) showed an inhibited upregulation (Figure. 2C). As a consequence of aberrant MET, the expression of pluripotent markers Esrrb, SSEA1 and Sox2 appeared much lower in the Xist shRNA ©AlphaMed Press 2014
5 treated group (Figure. 2D). In addition, a delayed mor‐ phological transformation and fewer cell aggregations were observed in the Xist shRNA group compared with non‐target shRNA treated group (Figure. S2A). Further‐ more, cells with beta‐catenin‐positive epithelial‐like intercellular junctions appeared less in Xist shRNA‐ treated group (Figure. S2B). These results suggest that Xist repression at the beginning of female iPSC induc‐ tion decreased reprogramming efficiency by interfering with MET process.
The Knockdown of Xist Facilitates the Transi‐ tion of pre‐iPSCs to iPSCs Our observation that Xist repression increased repro‐ gramming efficiency after colony formation, when the cells were in the pre‐iPSC stage, suggested a functional relationship between Xist repression and the transition from pre‐iPSCs to iPSCs. We next sought to investigate whether Xist repression played an important role in promoting pre‐iPSC reprogramming. In our system, the removal of exogenous OSKM dur‐ ing the first 8 days caused cells to return to fibroblast‐ like phenotype and resulted in no colony formation at D12, regardless of whether Xist shRNA was induced after exogenous OSKM withdrawal (data not shown). This indicated that Xist repression could not compen‐ sate for exogenous OSKM in reprogramming. To observe the pre‐iPSC to iPSC transition, pre‐iPSCs were derived by infecting female OG2 MEFs with the TetO‐OSKM and the IPTG‐inducible Xist shRNA. Oct4‐ GFP‐negative colonies were then selected around D12 (Figure. 3A). These Oct4‐GFP‐negative pre‐iPSCs lacked endogenous Oct4 expression and lost their colony mor‐ phology after DOX withdrawal (Figure. 3B). Subsequently, Xist could be knocked down in the derived pre‐iPSCs when treated with IPTG, making it possible to observe a relationship between Xist repres‐ sion and the pre‐iPSC to iPSC transition. For some un‐ stable pre‐iPSCs that could easily turn into GFP‐iPSCs themselves, IPTG treatment enhanced the Oct4‐GFP‐ positive colony formation, although this increase was less than 2‐fold in magnitude (Figure. S3A). For the sta‐ ble pre‐iPSCs that remained Oct4‐GFP‐negative for a relatively long time, the IPTG treatment significantly increased the formation of Oct4‐GFP‐positive colonies by approximately 4‐ to 6‐fold over the no‐IPTG control group, especially when accompanied with Vc (Figure. S3B). To investigate whether this increase was a conse‐ quence of accelerated reprogramming kinetics, we di‐ vided the same stable pre‐iPSCs into four groups, each of which received different treatments in a 24‐format experiment. One group was treated with only DOX as a control, the second group was treated with IPTG for Xist repression, the third group was treated with Vc to facili‐ tate pre‐iPSC reprogramming, and the fourth group was treated with both IPTG and Vc. The number of Oct4‐ GFP‐positive colonies was counted during iPSC induc‐ tion. We noticed that Oct4‐GFP‐positive colonies ap‐ peared at about D5 in all groups and grew in number www.StemCells.com
Xist repression promotes pre‐iPSCs to iPSCs transition until about D12 (Figure. 3C), indicating that Xist repres‐ sion did not promote iPSC induction by accelerating the reprogramming kinetics. Furthermore, FACS analysis showed that approximately 10% of the pre‐iPSCs treat‐ ed with IPTG and Vc became Oct4‐GFP positive, com‐ pared with 1.62% of the pre‐iPSCs treated with only Vc (an approximately 6‐fold change) (Figure. 3D, 3E). The Oct4‐GFP‐positive colonies produced after the pre‐iPSCs were treated with IPTG and Vc appeared larger and brighter than those produced with only Vc (Figure. 3F). These results demonstrate that Xist repression pro‐ motes the pre‐iPSC to iPSC transition and that the com‐ bination of IPTG and Vc treatment is the most efficient.
X Chromosome Inactivation Status and Pluripotency Differs in Female pre‐iPSCs and GFP‐iPSCs We next sought to compare the pluripotent marker expression and X chromosome inactivation pattern be‐ tween the pre‐iPSCs and GFP‐iPSCs that were derived from pre‐iPSCs after IPTG and Vc treatment. The pre‐ iPSCs were quite different from the GFP‐iPSCs, as they express Xist and stain positively for H3K27me3 on the Xi (Figure. 4A, 4B, 5A, S4A, S4B). Moreover, unlike the GFP‐iPSCs, the pre‐iPSCs were DOX dependent, endog‐ enous Oct4‐GFP negative, and lacked expression of Nanog and other pluripotent genes, such as endoge‐ nous Sox2, Dppa4 and Rex1 (Figure. 3B, 4C, S4C). In ad‐ dition, the pre‐iPSCs and GFP‐iPSCs both stain positively for SSEA1 (Figure. 4D). Taken together, these results demonstrate that the pre‐iPSCs had a status similar to EpiSCs, which also have Xist expression and H3K27me3 on the Xi [42‐44], making them more easily differentiat‐ ed than the GFP‐iPSCs and ESCs. We then sought to evaluate the capability of the pre‐iPSCs and GFP‐iPSCs to differentiate into three germ layers. As a consequence of their lower pluripotency, the pre‐iPSCs were incapable of inducing teratoma for‐ mation when injected into BALB/C mice. In contrast, most GFP‐iPSCs contributed to teratomas with three germ layers (Figure. 4E). Next, we performed blastocyst injection to further confirm the pluripotency of the GFP‐ iPSCs. Chimeric mice with germline transmission ability could be efficiently produced from the GFP‐iPSCs cell lines while the pre‐iPSCs could not (Figure. 4F, Table. S2). These results demonstrated that the pre‐iPSCs lacked full pluripotency compared to the GFP‐iPSCs.
The Knockdown of Xist Converted the Xi From an Irreversible to Reversible State Our previous data suggested that pre‐iPSCs exist in an EpiSC‐like state. However, when we focused on some X chromosome related proteins, we found that pre‐iPSCs had higher macroH2A expression than the GFP‐iPSCs and stained positive for macroH2A on the Xi (Figure. 5A, 5B). Considering that the EpiSCs were observed to have Xist expression without macroH2A binding on the Xi [17, 42‐44], our pre‐iPSCs were in a more differentiated ©AlphaMed Press 2014
6 state than the EpiSCs, with irreversible Xi indicated by the deposition of macroH2A. Based on the characteris‐ tics of reversible and irreversible Xi and the relationship between Xist and macroH2A, we hypothesized that Xist knockdown could help in releasing macroH2A from the Xi to transit the Xi of pre‐iPSCs from an irreversible to a reversible state. To address this hypothesis, pre‐iPSCs were first treated with IPTG to induce Xist shRNA, and Vc was added as described earlier. IPTG was then withdrawn from the medium at different time points, and the Oct4‐GFP‐postive cells were measured by FACS at D12. The percentage of Oct4‐GFP‐positive cells among the pre‐iPSCs that were never treated with IPTG was used as a control and for normalization purposes. Without the addition of Vc, a slight increase in the generation of Oct4‐GFP‐positive cells was observed (Figure. 5C, left). Meanwhile, dramatic increase (of approximately 4‐fold) in the generation of Oct4‐GFP‐positive cells was noticed when the pre‐iPSCs were treated with both IPTG and Vc (Figure. 5C, right). However, when IPTG was withdrawn in the first 2 days, leaving only Vc treatment, the per‐ centage of Oct4‐GFP‐positive cells achieved was nearly the same as in control group treated with only Vc (Fig‐ ure. 5C, right). A significant increase (of more than 2‐ fold) was achieved when IPTG was withdrawn after D3, and this increase was maximized when IPTG was with‐ drawn after D5 (Figure. 5C, right). These observations suggest a gradual process for Xist repression to remodel the Xi in the first 5 days with the help of Vc. To further investigate Xist expression, H3K27me3 and macroH2A deposition on the Xi during the transi‐ tion from pre‐iPSCs to iPSCs, we divided the same pre‐ iPSCs into four groups and treated them separately with DOX, DOX plus IPTG, DOX plus Vc, or DOX plus both Vc and IPTG. We found that cells with Xist expression, H3K27me3 and macroH2A deposition on the Xi were gradually reduced during pre‐iPSC reprogramming, but the downregulation of Xist occurred first and H3K27me3 disappeared finally (Figure 5D, 5E, 5F). Therefore, we propose that Xist repression might occur early during X chromosome reactivation followed by macroH2A release and loss of H3K27me3. As expected, we confirmed that the pre‐iPSCs in all groups had H3K27me3 on the Xi in first 3 days of culture, but lost macroH2A localization in the Xi when Xist was re‐ pressed in IPTG treated groups (Figure. 5B, 5D, 5E, 5F). The pre‐iPSCs treated or not treated with IPTG differed significantly in the percentage of cells containing macroH2A on the Xi but not H3K27me3 during first 8 days of culture (Figure. 5D, 5E). These results demon‐ strate that Xist repression mainly affects macroH2A deposition, but not H3K27me3, on the Xi of pre‐iPSCs. We further investigated the role of Vc on Xist ex‐ pression, H3K27me3 modification and macroH2A depo‐ sition. Vc was found to accelerate H3K27me3 elimina‐ tion from the Xi, as Vc treated groups were found to have a lower percentage of cells with H3K27me3 on the Xi (Figure 5D). In contrast, when comparing Vc‐only and www.StemCells.com
Xist repression promotes pre‐iPSCs to iPSCs transition DOX‐only group, Vc itself had no significant effects on macroH2A release and Xist repression (Figure. 5E, 5F).
Vc Helps in Maintaining the Reversible X Chromosome State According to the above findings, Xist repression pro‐ motes macroH2A release from the Xi during pre‐iPSC reprogramming. We then further tested whether Xist is re‐expressed and macroH2A is recruited to the Xi after IPTG withdrawal. When pre‐iPSCs were first treated with IPTG for 3 days and then cultured without IPTG for another 3 days, re‐expression of Xist was observed and most cells showed macroH2A relocalization to the Xi, similar to those that were never treated with IPTG (Fig‐ ure. 6A, 6B left, 6C left). However, for pre‐iPSCs treated continually with Vc, although the re‐expression of Xist could be observed, the cells lost macroH2A from the Xi and did not get macroH2A back after IPTG withdrawal (Figure. 6B right, 6C right, 6D). These observations indi‐ cate that cells losing Xist were not in a stable state and some unknown factors can induce Xist re‐expression and macroH2A recruitment. But Vc can help to prevent macroH2A relocalization. Furthermore, when pre‐iPSCs were first treated with IPTG for 5 days and then cul‐ tured without IPTG for another 3 days, the re‐ expression of Xist was no longer observed (Figure 6A, 6B, 6D). However, the relocalization of macroH2A to the Xi still occurred in non‐Vc treated group (Figure 6A, 6C, 6D), indicating that macroH2A was released but not completely eliminated at this time, and may be recruit‐ ed to the Xi by remaining Xist. We also examined whether H3K27me3 on the Xi can be affected by IPTG and Vc treatment. In fact, Vc accelerated H3K27me3 elimination from the Xi in pre‐iPSCs and the withdrawal of IPTG did not block this process (Figure. 6A, 6D, 6E). Our results demonstrate that the effect of Xist repres‐ sion on the X chromosome is reversible during the early stage of female pre‐iPSCs reprogramming but becomes irreversible at the late stage before X chromosome was completely reactivated. Xist repression mainly released macroH2A from the Xi to convert the irreversible Xi of the pre‐iPSCs to a reversible Xi status. Furthermore, Vc had certain advantage on H3K27me3 elimination from the Xi and prevented macroH2A relocalization to the Xi. We also investigated the effects of Xist repression on H3K27me3 mark and macroH2A deposition at the early stage of reprogramming. We found that most cells showed Xist expression, macroH2A deposition and H3K27me3 mark on the Xi during the early stage of re‐ programming. Xist repression could release macroH2A from the Xi but H3K27me3 was not affected (Figure S5A, S5B, S5C). Moreover, we observed a reversible Xist repression process in female MEFs induced with Tet‐ OSKM and IPTG for first 3 days and then cultured with‐ out IPTG for another 3 days. After IPTG withdraw, Xist re‐expressed and macroH2A relocalized to the X chro‐ mosome (Figure S5D, S5E), similar to that observed in the early stage of reprogramming of pre‐iPSCs. The X
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7 chromosome was not reactivated during this process as H3K27me3 persisted on the Xi (Figure S5F).
A Model for the X Chromosome Reactivation in Female iPSCs Induction Taken all together, this study demonstrated that Xist repression shows time‐dependent effects on female somatic cell reprogramming. The knockdown of Xist impairs MET in the early phase of iPSC induction but plays an important and beneficial role in the late phase of reprogramming. Moreover, Xist repression facilitates the pre‐iPSC to iPSC transition by releasing macroH2A from the Xi. The Xi in pre‐iPSC undergoes a transition from Xist shRNA dependent reversible stage to a Xist shRNA independent stage before complete reactivation. Vc helps in eliminating H3K27me3 from the Xi, prevent‐ ing macroH2A relocalization to the Xi and stabilizing the reversible Xi (Figure 6F). DISCUSSION In the present study, we demonstrate that Xist repres‐ sion has a time‐dependent effect on female iPSC induc‐ tion, which is different from the results obtained in pre‐ vious SCNT studies. Xist repression in the early stage of female somatic cell reprogramming can greatly de‐ crease iPSC induction by suppressing MET progression. In contrast, Xist repression in the late stage of repro‐ gramming can greatly facilitate the pre‐iPSC to iPSC transition. For the early stage of reprogramming, the MET has been considered to be one of the most well‐defined molecular events [40, 41]. Our observations that Xist expression impairs MET progression in the early stage of female somatic cell reprogramming indicate a corre‐ lation between the MET and the Xist expression. As Xist is reported to function in maintaining genomic stability and X chromosome silencing in somatic cells, it may be required for preventing aberrant genomic and epigenet‐ ic changes in early reprogramming stage. Consequently, the knockdown of Xist in the early stage of reprogram‐ ming may cause some aberrant epigenetic changes ac‐ cumulated which triggers impaired MET and delayed cell proliferation. Following the built‐up of a preliminary pluripotent network, Xist functions negatively in the late stage of reprogramming. At this point, the repres‐ sion of Xist can facilitate reprogramming. However, it remains unknown how the repression of Xist, a Xi‐ related RNA transcript but not a movable protein, af‐ fects the expression of MET‐related autosomal genes. The female iPSC induction system might provide a use‐ ful tool for dissecting the connection between Xist re‐ pression and autosomal genes regulation. The beneficial effect of Xist repression on the pre‐ iPSC to iPSC transition attracted significant interest with respect to understanding the process of X chromosome reactivation in reprogramming. According to previous finding, loss of silencing marks on the X chromosome in www.StemCells.com
Xist repression promotes pre‐iPSCs to iPSCs transition female cells is a diagnostic feature of fully pluripotent state and provides unambiguous demonstration of ma‐ jor epigenetic erasure[45]. Despite acquiring some simi‐ larities to ES cells, partially reprogrammed iPSCs with X‐ inactivation and Xist expression are in a status similar to EpiSCs, which are inefficient for teratoma formation and chimeric mice generation. Therefore, X chromo‐ some reactivation is important for attaining true pluripotency during reprogramming. In addition, Xist can recruit X chromosome silencing factors and thus antagonize the X chromosome reactivation process. Failing to repress Xist completely in the partially repro‐ grammed iPSCs is linked to a more differentiated state. Taken together, Xist and X‐inactivation might function as the barriers for female somatic cell reprogramming. To elucidate the mechanism of the X chromosome reactivation, we focused particularly on Xist expression and the Xi‐enriched histone variant macroH2A and his‐ tone modification H3K27me3, and found that Xist re‐ pression can successfully release macroH2A from the Xi. Interestingly, the relocalization of macroH2A onto the X chromosome could be detected when IPTG was with‐ drawn from the pre‐iPSC culture (which stops the Xist repression), indicating that some unknown factors per‐ sist to maintaining the Xi. Therefore, other factors facili‐ tating the reprogramming are required for complete reactivation of the X chromosome after Xist repression. We have found that Vc has certain advantage on H3K27me3 elimination from the Xi and prevent the relocalization of macroH2A to the Xi. Taken together, the combination of Xist repression and Vc treatment is an efficient combination for X chromosome reactivation during the pre‐iPSC to iPSC transition in female somatic cell reprogramming. Reprogramming can be considered as the reversal of differentiation in many aspects [46]. According to pre‐ viously reported Xist‐dependent reversible Xi transition to Xist‐independent irreversible Xi, macroH2A produced an irreversible Xi status during differentiation [5]. We raised the hypothesis that during the reprogramming of somatic cells to pre‐iPSCs, the X chromosome remains in an irreversible silencing state with Xist expression and the macroH2A deposition, then the irreversible Xi turns into a reversible status without Xist expression and macroH2A deposition and is then completely reac‐ tivated in the final stage of reprogramming. We indeed noticed that the effect of Xist shRNA changed from a reversible to irreversible state during the reactivation of the Xi in reprogramming of pre‐iPSCs to iPSCs. Previous SCNT studies showed that the Xi can not be completely reactivated and rather retains the epigenet‐ ic memory of the inactive X chromosome [21, 22, 24]. Similarly, the similar epigenetic memory might also function during iPSCs induction. Taking into account of some aberrant reactivated X chromosomes that had eliminated H3K27me3 but retained macroH2A, they are in an unstable status that can be silenced by X chromo‐ some inactivation factors recruited by macroH2A. Alt‐ hough we focused on macroH2A in the present study, ©AlphaMed Press 2014
Xist repression promotes pre‐iPSCs to iPSCs transition
8 other proteins acting in X chromosome inactivation may also aberrantly remain on the Xi after X chromosome reactivation. This prompts us to re‐consider the charac‐ ter of a truly reactivated X chromosome, which should not be defined based solely on the absence of H3K27me3. Therefore, it deserves to further dissect other factors that might involve in antagonizing the X chromosome reactivation in female somatic cells in the future. CONCLUSION In conclusion, we have demonstrated for the first time that the repression of Xist plays a time‐dependent role in female iPSC induction, which is distinct from that observed in somatic cloned embryos. Xist repression impairs MET in the early phase of reprogramming but promotes the pre‐iPSC to iPSC transition in the late phase. Most importantly, we discovered a Xist‐ repression‐dependent reversible Xi stage in the X chro‐ mosome reactivation process in female iPSCs induction. During this process, the release of macroH2A from the Xi through Xist depletion can cause the transition from irreversible Xi to a reversible Xi. Meanwhile, Vc aug‐ ments and stabilizes the reversible Xi status by acceler‐ ating H3K27me3 elimination and preventing macroH2A relocalization to the Xi.
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ACKNOWLEDGMENTS We are grateful to our colleagues in the laboratory for their assistance with the experiments and in the prepa‐ ration of this manuscript. We are also grateful to Jiayu Chen for some primers and Kai Miao for instruction on Xist RNA FISH. This project was supported by the Minis‐ try of Science and Technology (Grants 2010CB944900 and 2012CBA01308) and the National Natural Science Foundation of China (31325019 and 91319306). DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST The authors have no potential conflicts of interest.
AUTHOR CONTRIBUTIONS Q.C.: conception and design, collection and/or assembly of the data, data analysis and interpretation, and manu‐ script writing; S.G., W.H., X.K., Y.Z., H.W.: provision of study material and collection and/or assembly of the data; S.G.: conception and design, financial support, data analysis and interpretation, manuscript writing, and final approval of the manuscript.
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Xist repression promotes pre‐iPSCs to iPSCs transition
Figure 1. Time‐dependent effects of Xist repression on female somatic cells reprogramming. A. The number of Alkaline phosphatase (AP)‐positive colonies achieved in the female or male mouse embryonic fi‐ broblasts (MEF) induced with Oct4/Sox2/Klf4/cMyc (OSKM) and Xist shRNA compared to the empty‐pSicoR‐treated control group and the mock‐treated group, collected at day 15. The number of initially plated fibroblasts was 50000 per well in a six‐well plate. B. AP staining results and fluorescence images of Oct4‐GFP‐positive colonies derived from MEFs that were induced with OSKM and Xist shRNA compared to the empty‐pSicoR‐treated control (Ctrl) group, collected at day 15. Scale bars represent 100 μm. C. The relative number of AP‐positive colonies obtained following IPTG treatment on different days during OSKM in‐ duction, collected at day 18. Female MEFs were infected with pFUW‐tetO‐Oct4, Sox2, Klf4 and c‐Myc (TetO‐OSKM) and IPTG‐inducible Xist shRNA viruses (Green) or non‐target‐shRNA viruses (Red). ‘No IPTG’ stands for MEFs infected but never treated with IPTG. D. The Xist shRNA treated part in (C). Ctrl stands for MEFs never treated with IPTG. An asterisk above a column indi‐ cates a significant difference between this data point and Ctrl. E. The non‐target shRNA treated part in (C). Ctrl stands for MEFs never treated with IPTG. F. AP staining results representative for (C). The data shown represent three independent replicates. The statistical tests used are t‐test, error bars stand for standard deviation. * indicates p value
