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Keywords:

  • Mbd3/NuRD;
  • Induced pluripotent stem cells;
  • Epigenetic regulation;
  • Nanog;
  • Reprogramming efficiency

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by overexpression of a defined set of transcription factors requires epigenetic changes in pluripotency genes. Nuclear reprogramming is an inefficient process and the molecular mechanisms that reset the epigenetic state during iPSC generation are largely unknown. Here, we show that downregulation of the nucleosome remodeling and deacetylation (NuRD) complex is required for efficient reprogramming. Overexpression of Mbd3, a subunit of NuRD, inhibits induction of iPSCs by establishing heterochromatic features and silencing embryonic stem cell-specific marker genes, including Oct4 and Nanog. Depletion of Mbd3, on the other hand, improves reprogramming efficiency and facilitates the formation of pluripotent stem cells that are capable of generating viable chimeric mice, even in the absence of c-Myc or Sox2. The results establish Mbd3/NuRD as an important epigenetic regulator that restricts the expression of key pluripotency genes, suggesting that drug-induced downregulation of Mbd3/NuRD may be a powerful means to improve the efficiency and fidelity of reprogramming. STEM Cells2013;31:1278–1286


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Induced pluripotent stem cells (iPSCs) closely resemble embryonic stem cells (ESCs) with regard to epigenetic marks and the pattern of gene expression [1–3]. During iPSC induction, the genome undergoes major epigenetic alterations to reacquire the euchromatic chromatin structure that characterizes pluripotent cells [4–6]. Consistent with epigenetic reprogramming playing a key role in cell-fate conversion, small molecules that target chromatin-modifying enzymes greatly enhance reprogramming efficiency, yielding iPSCs that are similar to ESCs [7–9]. An ESC-specific chromatin remodeling complex, esBAF (embryonic stem cell specific Brg/Brahma-associated factor), has been shown to be important for the maintenance of pluripotency and the differentiation capacity of ESCs [10, 11]. However, the epigenetic pathways that orchestrate chromatin remodeling of specific target genes remain largely unknown.

Changes in chromatin structure are brought about by multiprotein complexes that move or disrupt nucleosomes and recruit chromatin modifying enzymes to specific genes. The nucleosome remodeling and deacetylation (NuRD) complex contains at least seven polypeptides, including the ATPase Mi-2, the histone deacetylases HDAC1&2, Mta1&2 (Metastasis-associated protein1&2), and the methyl-binding domain proteins Mbd2&3. NuRD represses transcription by binding to methylated DNA, mediating heterochromatin formation, and transcriptional silencing [12, 13]. Moreover, Mbd3/NuRD directly regulates expression of pluripotency genes in ESCs to modulate transcriptional heterogeneity and maintain ESC lineage commitment [14]. ESCs lacking Mbd3/NuRD retain Oct4 expression in the absence of the cytokine leukemia inhibitory factor (LIF) and have a restricted potential to differentiate [15, 16], suggesting an important function of Mbd3/NuRD in the establishment or maintenance of pluripotency. Conversely, inhibitors of histone deacetylases or DNA methyltransferases enhance reprogramming, suggesting that the complete erasure of epigenetic marks is required to overcome the barrier to pluripotency [7–9].

In this study, we show that Mbd3/NuRD suppresses iPSC induction by establishing heterochromatic features and silencing ESC-specific marker genes. Depletion of Mbd3 leads to generation of iPSCs even in the absence of c-Myc or Sox2. The results demonstrate a novel epigenetic pathway that controls somatic cell reprogramming, suggesting that Mbd3/NuRD may be a potential target of small molecules that facilitate iPSCs generation.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Plasmids and Antibodies

Retroviral small hairpin RNA (shRNA) vectors are based on pQsupR (Addgene, Cambridge, MA) in which the green fluorescent protein (GFP) coding region was removed. The shRNA sequences are listed in supporting information Table S1. pZXN-Mbd3 was a gift from H. Stunnenberg (Radboud University) and plasmids encoding mouse Oct4, Sox2, Klf4, and c-Myc were from Addgene. The following antibodies were used: anti-SSEA1 (480, Santa Cruz, Dallas, TX), anti-Nanog (30329, Santa Cruz), anti-Oct4 (9081X, Santa Cruz), anti-Sox2 (17320X, Santa Cruz), anti-Mi-2 (11378, Santa Cruz), anti-Mta2 (8106, Abcam, Cambridge, U.K.), anti-Mbd2 (3754, Abcam), anti-Mbd3 (3755, Abcam), anti-HDAC1 (7872, Santa Cruz), anti-HDAC2 (7029, Abcam), anti-Gapdh (48166, Santa Cruz), anti-H3K9me3 (8898, Abcam), anti-H3K4me3 (8580, Abcam), anti-H3K27me3 (6002, Abcam), anti-H4ac (09-866, Upstate), anti-α-tubulin (T9026, Sigma, St. Louis, MO), anti-smooth muscle actin (N1584, Dako), anti-α-fetoprotein (8108, Santa Cruz), anti-nestin (21249, Santa Cruz), anti-5-methylcytosine (anti-5mC; BI-MECY-0100, Eurogentec, Seraing, Belgium), and anti-5-hydroxymethylcytosine (anti-5hmC; 39767, Active Motif, Carlsbad, CA).

Cell Culture and iPSC Generation

Plat-E cells, mouse embryonic fibroblasts (MEFs), and Oct4-GFP MEFs derived from CBA-Tg(Pou5f1-EGFP)2Mnn/J mice [8] were cultured in Dulbecco's modified Eagle's medium (DMEM) medium containing 10% fetal bovine serum (FBS), 50 U/ml penicillin, and 50 mg/ml streptomycin. Murine embryonic stem cells (mESCs) and reprogrammed iPSCs were maintained in mESC culture medium (DMEM/F12, 20% FBS, 1% nonessential amino acids, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 50 U/ml penicillin, 50 mg/ml streptomycin, 1,000 U/ml (leukemia inhibitory factor)) on feeder layers of mitomycin C-treated MEFs. For iPSC induction, retroviruses expressing Oct4, Sox2, Klf4, c-Myc, Nanog, Mbd3, or Mbd3-shRNA were produced by transfecting retroviral vectors into Plat-E cells using the calcium phosphate method. For iPSC induction, MEFs were infected with the respective retroviruses supplemented with 4 μg/ml polybrene (Sigma) and cultured in mESC medium or iSF1 medium (DMEM/high glucose, 10% knockout serum replacement (KSR), 1/200 N2, 1 mM L-glutamine, 1% NEAA, 50 U/ml penicillin, 50 mg/ml streptomycin, 1,000 U/ml LIF, 5 ng/ml fibroblast growth factor (bFGF)). For OSKM (Oct4, Sox2, Klf4, c-Myc)-mediated reprogramming, iPSC generation was monitored 16 days post infection in mESC medium and 12 days post infection in iSF1 medium. For OSK-mediated reprogramming, iPSC generation was monitored 18 or 28 days after infection.

Gene Expression Analysis, Chromatin Immunoprecipitation, and Bisulfite Sequencing

cDNA was synthesized using 1 μg of cellular RNA, random dN6 primers, and Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega). Quantitative polymerase chain reaction (qPCR) was performed using the SYBR Premix Ex Taq II with primers listed in supporting information Table S1. For microarray assays, RNA was transcribed into cDNA and labeled according to the manufacturer's instructions (Affymetrix, Santa Clara, CA). Dual-channel SmartArray (CapitalBio, Beijing) chips containing about 32,000 mouse genes were hybridized to labeled cDNA probes on GenePix Pro 4.0. Data were normalized according to Lowess (locally weighted linear regression) methodology. Cross-linked chromatin was sonicated, precleared, and incubated with specific antibodies. The ratio of DNA in the immunoprecipitates versus input chromatin was assayed by qPCR and normalized to control reactions with IgGs. To monitor DNA methylation of the Oct4 and Nanog promoter, genomic DNA was modified by bisulfite using the EpiTect Bisulfite Kit (Qiagen, Hilden, Germany), amplified by PCR and sequenced.

Methylated DNA Immunoprecipitation

Genomic DNA was sonicated to produce 300–500 bp fragment, denatured for 10 min at 95°C, and immunoprecipitated for 2 h at 4°C with 5 μl of antibodies recognizing 5mC or 5hmC in 500 μl IP buffer containing 10 mM sodium phosphate (pH 7.0), 140 mM NaCl, 0.05% Triton X-100. After incubation for another 2 h at 4°C with 30 μl protein A/G (2:1) sepharose and washing with IP buffer, DNA was extracted and analyzed by real-time PCR.

Cell Characterization

Alkaline phosphatase (AP) staining was performed using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP) substrates (Promega, Fitchburg, WI). For in vitro differentiation, mESCs or iPSCs were harvested by trypsinization and transferred to bacterial culture dishes containing mESC medium without LIF. After 3 days, cells were plated onto gelatin-coated culture dishes and incubated for another 3 days. Differentiation was determined by immunostaining with antibodies against smooth muscle actin, α-fetoprotein, or nestin. For teratoma assays, 1 × 106 mESCs or iPSCs were subcutaneously injected into nude mice. Tumors were surgically dissected after 4 weeks, fixed, embedded in paraffin, and sections were stained with hematoxylin and eosin. To generate chimeras, 15–20 iPSCs were injected into ICR-derived blastocysts, which were transplanted into the uteri of pseudopregnant mice.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Mbd3/NuRD Blocks Reprogramming of Somatic Cells

To investigate the role of NuRD in iPSC generation, we monitored the level of mRNA in two MEF-derived pluripotent iPSC clones and in mESCs. The level of mRNAs encoding the NuRD components Mbd3, Mi-2, and HDAC2 decreased by 50% in both iPSCs and mESCs compared with MEFs (Fig. 1A; supporting information Fig. S1A). The inverse correlation between NuRD and pluripotency suggested that Mbd3/NuRD impairs somatic reprogramming. Exogenous expression of Oct4, Klf4, Sox2, and c-Myc is known to cause global changes in gene expression that are necessary for triggering the initial steps of reprogramming that will eventually lead to pluripotency [1]. To gain insight into the function of Mbd3 in iPSC induction, we infected MEFs expressing GFP under the control of the Oct4 promoter (Oct4-GFP) with retroviruses encoding Oct4/Sox2/Klf4/c-Myc (OSKM) together with expression vectors encoding Mbd3 or shRNA against Mbd3 (shMbd3). The number of AP- and Oct4-GFP-positive clones was markedly reduced if Mbd3 was overexpressed, suggesting that Mbd3 impairs reprogramming (Fig. 1C–1E). In accord with this view, depletion of Mbd3 with different shRNAs led to an approximately 10-fold increase in GFP-positive clones (Fig. 1B; supporting information Fig. S1B–S1E), emphasizing that Mbd3 exerts a negative effect in reprogramming somatic cells to the pluripotent state characteristic of iPSCs. iPSCs were capable of generating teratomas and viable chimeric mice (Fig. 1F, 1G; supporting information Fig. S1F). Moreover, depletion of Mbd3 did not affect cell cycle progression and hence proliferation, indicating that inhibition of reprogramming was not caused by cell cycle arrest (supporting information Fig. S2).

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Figure 1. Mbd3/NuRD suppresses somatic reprogramming. (A): The level of Mbd3 mRNA is decreased in induced pluripotent stem cells (iPSCs) and murine embryonic stem cells (mESCs). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) showing the steady state level of Mbd3 mRNA in MEFs, mESCs, and two iPSC lines (iPS-5# and iPS-14#) induced by ectopic Oct4/Sox2/Klf4/c-Myc (OSKM). Error bars denote SD (n = 3). (B): Western blot with anti-Mbd3 antibody (top) and RT-qPCR (bottom) showing depletion of Mbd3 in MEFs infected with retroviruses expressing different small hairpin RNAs against Mbd3. (C): Depletion of Mbd3 improves Oct4/Sox2/Klf4/c-Myc (OSKM)-mediated iPSC generation. MEFs overexpressing GFP-Oct4/Sox2/Klf4/c-Myc (OSKM) were infected with retroviruses encoding Mbd3 or shMbd3. Cells were stained for AP 16 days after infection. Scale bar = 5 mm. (D): Same experiment as in (C) showing DIC and fluorescence images of Oct4-GFP positive cells. Scale bar = 300 μm. (E): Same experiment as in (C) showing GFP- and AP-positive clones from 1.5 × 104 starting cells (left) as well as the percentage of Oct4-GFP positive cells analyzed by fluorescence-activated cells sorting (right). The mean value of three independent experiments is shown. Error bars denote SD (n = 3). (F): Hematoxylin and eosin staining showing teratoma formation of OSKM-mediated iPSCs generated from Mbd3-deficient MEFs. (G): Chimera formation (left) and germline transmission (middle and right) of iPSCs derived from OSKM-transfected Mbd3-deficient MEFs. Scale bars = 200 μm. Abbreviations: AP, alkaline phosphatase; DIC, differential interference contrast; GFP, green fluorescent protein; iPSCs, induced pluripotent stem cells; MEF, mouse embryonic fibroblast; mESCs, mouse embryonic stem cells; OSKM, Oct4, Sox2, Klf4, c-Myc; pZXN, empty retroviral vector.

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Depletion of Mbd3 Facilitates Reprogramming in the Absence of c-Myc or Sox2

As depletion of Mbd3 enhanced reprogramming efficiency, we examined whether Mbd3 knockdown could replace any of the factors used for somatic cell reprogramming. First, we monitored the effect of Mbd3-shRNA on reprogramming efficiency in the absence of c-Myc. As shown in Figure 2A, overexpression of Mbd3 inhibited OSK-mediated iPSC induction, whereas knockdown of Mbd3 increased iPSC generation, leading to a 10-fold increase in the number of GFP-positive cells in OSK conditions. This indicates that depletion of Mbd3 facilitates somatic reprogramming in the absence of c-Myc.

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Figure 2. Depletion of Mbd3 replaces Sox2 in somatic reprogramming. (A): Knockdown of Mbd3 promotes somatic reprogramming in the absence of c-Myc. Left: Number of AP- and Oct4-GFP-positive clones 28 days after infection of 5 × 104 MEFs with Oct4/Sox2/Klf4 (OSK) and Mbd3 or shMbd3 in mESC medium. Right: Percentage of Oct4-GFP positive cells monitored by fluorescence-activated cells sorting. The mean value of three independent experiments is shown. (B): shMbd3 replaces the need for Sox2 in iSF1 medium. Oct4-GFP-positive MEFs grown in iSF1 medium were induced for reprogramming by the indicated combinations of pluripotency factors. The number of Oct4-GFP+ clones was counted 28 days after infection. The mean value of three independent experiments is shown. (C): Image showing Oct4-GFP-positive MEFs infected with viruses encoding shMbd3 and Oct4/Sox2/Klf4 (OSK), cultured in iSF1 medium. Scale bar = 300 μm. (D): Bisulfite sequencing showing loss of CpG methylation at the Nanog and Oct4 promoter in iPSCs generated by Oct4/Klf4/c-Myc (OKM) in the presence of shMbd3 (sriPS). (E): Immunofluorescence showing expression of the mESC marker proteins SSEA-1 and Nanog in sriPSCs. Scale bars = 100 μm. (F): Immunostaining demonstrating differentiation of induced pluripotent stem cells (iPSCs) into all three germ layers. Primary antibodies against actin, AFP, and nestin, and Cy3-labeled secondary antibodies were used. Scale bars = 100 μm. (G): Hematoxylin and eosin staining of neural tissue, muscle, and epithelium showing teratoma formation by sriPSCs. Scale bars = 100 μm. (H): Image of chimeric mice and GFP fluorescence in the genital ridge showing the capability of sriPSCs in germline transmission. Scale bars = 200 μm. Abbreviations: AFP, α-fetoprotein; AP, alkaline phosphatase; DIC, differential interference contrast; GFP, green fluorescent protein; iPSCs, induced pluripotent stem cells; MEF, mouse embryonic fibroblast; mESCs, mouse embryonic stem cells; OKM, Oct4, Klf4, c-Myc; OSK, Oct4, Sox2 and Klf4; OSKM, Oct4, Sox2, Klf4, c-Myc; SM-actin, smooth muscle actin.

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iPSC formation requires Sox2 both in ESC culture medium and in iSF1 medium containing vitamin C and bFGF [17, 18]. Knockdown of Mbd3 in iSF1 medium did not only enhance OSKM-mediated iPSC formation, but also mediated the generation of Oct4-GFP-positive clones in the absence of Sox2 (Fig. 2B, 2C; supporting information Fig. S3A, S3B). Although reprogramming efficiency was low, Oct4-GFP-positive cells, termed sriPSCs (for Sox2 replacement derived iPSCs), resemble mESCs in morphology, expression of pluripotent marker genes and demethylation of the Oct4 and Nanog promoters (Fig. 2D, 2E; supporting information Fig. S3C). Moreover, sriPSCs exhibited the same proliferation rate as normal mESCs (supporting information Fig. S3D), were capable of forming embryonic bodies (EB), gave rise to teratomas, and differentiated into tissues originating from all three germ layers (Fig. 2F, 2G). Significantly, pluripotent cells induced by Mbd3 depletion were capable of generating germline-transmitting chimeric mice, even in the absence of c-Myc or Sox2. When introduced into early embryos, sriPSCs developed into live chimeras, Oct4-GFP positive cells being present in the genital ridge (Fig. 2H). The observation that downregulation of Mbd3 can substitute for c-Myc or Sox2 in in iPSC generation (Oct4-GFP+ colonies) highlights the essential role of Mbd3 in controlling somatic cell reprogramming and cell fate decision [14, 19]. Of note, reprogramming did not occur with Klf4 and c-Myc alone, indicating that depletion of Mbd3/NuRD does not replace Oct4 in iPSC formation.

Mbd3 Depletion Triggers Upregulation of iPSC Promoting Factors

To get insight into the mechanism by which Mbd3/NuRD regulates reprogramming of somatic cells, we analyzed gene expression in Mbd3-depleted MEFs. Of 32,000 genes analyzed on microarrays, 2,039 were at least twofold up- or downregulated after Mbd3 knockdown, 214 genes being related to transcription, and 33 to chromatin structure. A comparison with recent ChIP-seq data [20] revealed that 55% of these genes are direct targets of Mbd3 and 19% are targets of Mbd3-containing complexes (Fig. 3A). Notably, a significant number of genes that were affected in Mbd3-depleted MEFs was similarly up- or downregulated in mESCs, suggesting that Mbd3 knockdown improved iPSC generation by triggering changes in gene expression toward an ESC-like pattern (supporting information Fig. S4A, Table S2). Similar to Mbd3 depletion, inhibition of the histone deacetylases HDAC1/2, essential components the Mbd3/NuRD complex, by valproic acid (VPA) led to upregulation of ESC-specific genes and downregulation of MEF-specific genes (Fig. 3B). Although changes in ESC- and MEF-specific gene expression were more pronounced in VPA-treated than in Mbd3-depleted cells, the ESC/iPSC-like gene expression pattern was more specific in Mbd3-deficient MEFs (Fig. 3C). Among 29 ESC/iPSC-specific genes, 10 were upregulated by shMbd3, including Nanog, Oct4, Sox2, Lin28, Sall4, and Zfp296 (Fig. 3C; supporting information Table S3), all being involved in iPSC generation [1, 21, 22]. Upregulation of Nanog, Oct4, and Sox2 was validated by reverse transcription qPCR (RT-qPCR) and immunoblots, the level of Nanog mRNA increasing 20-fold (Fig. 3D; supporting information Fig. S4B). Gene ontology analysis showed that the expression pattern of transcription activators and genes that activate MAP kinase (MAPK) pathways was altered upon Mbd3 depletion, most of them being direct targets of Mbd3 or Mbd3-containing protein complexes (supporting information Fig. S4C, S5). Given that inhibition of MAPK pathways promotes somatic reprogramming [5], the finding that depletion of Mbd3 downregulates genes that activate MAPK suggests that inhibition of MAPK may be invoved in iPSC formation. Together, these results reinforce that Mbd3 represses transcription of essential ESC-specific genes and depletion of Mbd3 improves reprogramming efficiency by upregulating the expression of pluripotency genes.

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Figure 3. Mbd3 represses transcription of pluripotency genes. (A): Comparison of microarray data with ChIP-seq data (from ref. [19]) classifying genes that are targeted by Mbd3 and Mbd3-complexes, for example, P400-Mbd3, Tip60-Mbd3, and Brg1-Mbd3. A total of 2039 genes regulated by Mbd3 were grouped according to their association with Mbd3 only (blue) or Mbd3-containing complexes (red). (B): Scatter plots showing the percentage of ESC- or MEF-specific gene expression in Mbd3-deficient and VPA-treated MEFs. Genes that are at least fivefold up- or downregulated in ESCs compared with MEFs were selected. (C): Changes in gene expression (left) and promoter binding by Mbd3/Mbd3-complex (right) of 24 iPS factors and 5 ESC marker genes in mES, Mbd3-deficient, and VPA-treated MEFs. (D): Reverse transcription quantitative polymerase chain reaction showing relative mRNA levels of selected ESC-specific genes in MEFs transfected with small hairpin RNA (shRNA) against Mbd3 (shMbd3) or control shRNA (shCtrl). Error bars represent SD. Abbreviations: iPSCs, induced pluripotent stem cells; MEFs, mouse embryonic fibroblasts; mESCs, mouse embryonic stem cells, VPA, valproic acid.

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Downregulation of Mbd3/NuRD Triggers Establishment of Euchromatic Features at the Promoter of Pluripotency Genes

As Oct4, Sox2, and Nanog form the core regulatory circuit of pluripotency [21], we examined how Mbd3 regulates the expression of these three master genes. Chromatin immunoprecipitation (ChIP) experiments with antibodies against the NuRD subunits Mbd3, Mi-2, and Mta2, revealed that NuRD is associated with the promoter of Nanog and Oct4, but not Sox2 (Fig. 4A). This suggests that binding of Mbd3/NuRD may repress Nanog and Oct4 transcription, disabling the molecular mechanisms necessary to acquire induced pluripotency. In support of this view, depletion of Mbd3 led to decreased NuRD occupancy at the Oct4 and Nanog promoter (Fig. 4B), increased euchromatic histone modifications, for example, acetylation of histone H4 (H4ac) and trimethylation of lysine four at histone H3 (H3K4me3), and decreased trimethylation of histone H3 at lysine nine (H3K9me3) and at lysine 27 (H3K27me3) (Fig. 4C). Moreover, consistent with studies showing that Mbd3 regulates the expression of 5hmC marked genes in ESCs [21], both methylation (5mC) and 5-hydroxymethylation (5hmC) at the Oct4 and Nanog promoter was decreased in Mbd3-deficient MEFs (Fig. 4D). Together, these results reveal that downregulation of Mbd3/NuRD leads triggers the establishment of euchromatic features that facilitate transcription of Oct4 and Nanog, a finding that underscores the role of Mbd3/NuRD as a roadblock to somatic cell reprogramming.

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Figure 4. Knockdown of Mbd3 establishes euchromatic features at the Oct4 and Nanog promoter. (A): Subunits of NuRD are associated with the promoter of Oct4 and Nanog. ChIP assays monitoring Mi-2, Mta2, and Mbd3 occupancy at the promoter of Sox2, Oct4, and Nanog in MEFs. Mouse IgGs were used as negative control. (B): ChIP assays showing decreased occupancy of Mi-2, Mta2, and Mbd3 at the promoter of Oct4 and Nanog in MEFs infected with retroviruses expressing control small hairpin RNA (shRNA) or shMbd3. (C): ChIP assay showing the levels of acetylated histone H4 (H4ac), H3K4me3, H3K9me3, and H3K27me3 at the Oct4 or Nanog promoter after infection of MEFs with retroviruses expressing control shRNA or shMbd3. Data were normalized to histone H3 ChIPs. (D): Methylated DNA immunoprecipitation using anti-5mC and anti-5hmC to determine alterations of 5mC and 5hmC levels at the promoter of Nanog and Oct4 in MEFs infected with viruses expressing control shRNA or shMbd3. Data are normalized to input DNA. Abbreviations: ChIP, chromatin immunoprecipitation; 5mC, 5-methylcytosine; 5hmC, 5-hydroxymethylcytosine; MEFs, mouse embryonic fibroblasts.

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Mbd3/NuRD Blocks Somatic Reprogramming by Repressing Nanog

ESC differentiation is accompanied by downregulation of Nanog and enforced expression of Nanog relieves ESCs from LIF requirement [22–26]. Given that Mbd3 depletion caused pronounced epigenetic changes and transcriptional alterations at the Nanog promoter, we reasoned that Mbd3/NuRD may block somatic cell reprogramming by repressing Nanog. To decipher the role of Nanog in shMbd3-mediated iPSC induction, we transfected MEFs with iPSC-inducing factors (OSKM or OSK) together with an expression vector encoding Nanog and with control shRNA or shMbd3. As shown in Figure 5, ectopic Nanog strongly enhanced reprogramming of MEFs, regardless whether or not c-Myc was present. Notably, knockdown of Mbd3 did not further improve the efficiency of reprogramming if Nanog was overexpressed (Fig. 5A, 5B), indicating that shMbd3 acts upstream of Nanog to improve OSKM- and OSK-mediated reprogramming. As Mbd3 depletion is more efficient in generating fully reprogrammed iPSCs (GFP-Oct4-positive) than partially reprogrammed (GFP-Oct4-negative, AP-positive) cells (Fig. 1E, 1A), Mbd3 may block the transition from partially to fully reprogrammed iPSCs. To test this, we depleted Mbd3 in partially reprogrammed iPS clones (Oct4-GFP-negative, AP-positive) in the absence or presence of ectopic Nanog. In the absence of Nanog, the percentage of Oct4-GFP-positive cells increased 2-3 fold in Mbd3 depleted cells, whereas in the presence of Nanog shMbd3 did not promote the transition to fully reprogrammed iPSCs (Fig. 5C). This demonstrates that, consistent with a recent report [19], Mbd3 acts as a barrier during the late stage of somatic cell reprogramming by silencing Nanog.

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Figure 5. Mbd3/NuRD acts upstream of Nanog in somatic cell reprogramming. (A): MEFs infected with Oct4/Sox2/Klf4/c-Myc (OSKM) alone, together with Nanog or with Nanog plus small hairpin RNA (shRNA)-Mbd3 were cultured in mESC medium, and the average number of AP- and Oct4-GFP-positive clones from 1.5 × 104 MEFs was measured 16 days after infection. Error bars represent SD. (B): MEFs expressing Oct4/Sox2/Klf4 (OSK) alone, together with Nanog or with Nanog plus shRNA-Mbd3 were cultured in mESC medium. The average number of AP- and Oct4-GFP-positive clones generated from 5 × 104 MEFs was measured 28 days after infection. Error bars represent SD. (C): Partially reprogrammed induced pluripotent stem cells (Oct4-GFP-/AP+) were infected with retroviruses encoding shMbd3, Nanog, or Nanog plus shMbd3 and Oct4-GFP-positive cells were monitored after 10 days by fluorescence-activated cells sorting. Representative plots from three independent experiments are shown. Abbreviations: AP, alkaline phosphatase; GFP, green fluorescent protein; MEFs, mouse embryonic fibroblasts; mESCs, mouse embryonic stem cells; OSK, Oct4, Sox2, Klf4; OSKM, Oct4, Sox2, Klf4, c-Myc.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Reprogramming of somatic cells into pluripotent stem cells requires changes in chromatin structure that mediate silencing of lineage-specific genes and activation of pluripotency genes [27, 28]. Chromatin remodelers play active, regulatory roles during the reprogramming process, facilitating binding of transcription activators to regulatory gene sequences and upregulation of reprogramming factors [29]. For example, the SWI/SNF remodeling complex BAF (BRG1-associated factor) promotes iPSC generation by increasing binding of reprogramming factors to the promoters of pluripotent genes [11]. Homozygous knockout or knockdown of any of several BAF subunits results in defects in ESC self-renewal and differentiation, highlighting their critical roles in maintaining the ESC gene expression pattern [10]. Here, we show that NuRD, a well-characterized corepressor complex that represses transcription in a variety of developmental contexts, blocks epigenetic resetting during the transition of somatic cells into iPSCs by silencing some pluripotency-associated genes. Depletion of Mbd3 leads to increased acetylation of histone H4, trimethylation of H3K4, decreased trimethylation of H3K9 at the Nanog and Oct4 promoter, indicating that NuRD impairs somatic reprogramming by establishing heterochromatic features and repressing transcription of key pluripotency genes. Consistent with a recent study showing that NuRD regulates expression of pluripotency genes by establishing defined levels of 5hmC [20], we found that enhanced iPSCs induction by knockdown of Mbd3/NuRD was accompanied by decreased CpG methylation and 5-hydroxymethylation of the Nanog and Oct4 promoter. Thus, depletion of Mbd3/NuRD upregulates the expression of key iPSC promoting factors, facilitating the generation of pluripotent cells. Apparently, modulatory activity of NuRD constrains differentiation of ESCs thereby directly controlling their immediate differentiation potential [14–16, 19]. Depletion of other core components of the NuRD complex, such as Mbd2 or Mi2/CHD4, improved iPSC generation to the same extent as knockdown of Mbd3 (unpublished results). This raises the possibility that the efficiency of somatic reprogramming may be further improved if several components of the NuRD complex are inhibited.

Nanog, the hallmark of pluripotency, has been demonstrated to be decisive for attaining a pluripotent ground state and to serve a role in the late stage of iPSC generation [22–26]. Overexpression of Nanog is sufficient for maintaining the pluripotency of mESCs in the absence of LIF and ensures direct reprogramming of somatic cells to the pluripotent ground state [15, 16]. Therefore, fine-tuning of Nanog levels is necessary for balancing differentiation and self-renewal of ESCs. In this study, we provided data showing that Mbd3 deficiency facilitates iPSC induction by upregulation of Nanog. NuRD has been shown to be recruited to Nanog by the transcriptional repressor Zfp281 and to dampen expression of genes encoding key pluripotency transcription factors, such as Klf4, Klf5, Zfp42, and Tbx3, in ESCs by opposing transcriptional activators [19]. Wild-type ESCs exhibit bimodal expression of these factors, and this phenotypic variation requires Mbd3. During development, ESCs silence these critical pluripotency transcription factors in an Mbd3-dependent manner. Silencing involves NuRD-dependent deacetylation of H3K27 that is required for binding of PRC2, the polycomb repressive complex two [30]. Together, these results reveal that somatic cells face a barrier that prohibits de-differentiation by NuRD-dependent silencing of pluripotency genes. In the absence of Mbd3, NuRD is disassembled, this epigenetic barrier is lowered and pluripotency genes are activated. Low reprogramming efficiency and the potential tumor risk of iPSCs generated by overexpression of the oncogenes c-Myc and Klf4 greatly limit the exploration of somatic reprogramming mechanisms and the clinical use of iPSCs. However, both limitations might be overcome by small molecules that mimic the function of reprogramming factors and thus are capable to replace potentially tumorigenic transcription factors [7, 8, 31]. The NuRD complex contains histone deacetylase (HDAC) activity whose inhibition by butyrate, valproic acid (VPA), suberoylanilide hydroxamic acid, or trichostatin A greatly improves reprogramming efficiency [9]. Inhibitors of DNA methyltransferase, such as azacytidine, or inhibition of histone methyltransferase activity have also been reported to increase reprogramming efficiency [9, 31]. Finally, overexpression of miR302/367, a cluster of microRNA, can replace the requirement of OSKM for both human and mouse iPSC generation [32]. Improved reprogramming efficiency relied on suppression of HDAC activity, suggesting that NuRD acts downstream of miR302/367 to regulate iPSC induction. Ultimately, the goal is to design a cocktail of reprogramming factors and compounds that efficiently and reliably reprogram somatic cells to pluripotent stem cells. Given the importance of chromatin-based mechanisms in the reprogramming process, it seems likely that additional chromatin remodelers and other mechanisms of actions are involved in iPSC generation that will emerge in the years to come.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Chromatin plays a fundamental role in pluripotency and stem cell biology. Although still sparse, current studies indicate the importance of chromatin remodeling proteins in obtaining the pluripotent state by promoting the open chromatin structure of the pluripotent genome. By artificially reducing the expression of Mbd3, we were able to show that NuRD blocks somatic cell reprogramming by suppressing transcription of key pluripotent genes required for cell fate determination. The results provide insights into the mechanisms that trigger reprogramming of somatic cells into pluripotent cells and identify NuRD as a potential target for drug screening aimed to improve iPSC induction.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

We thank Jinsong Li (Shanghai Institute for Biological Sciences), Xin Guo (Peking University) for valuable discussion and technical support. This work was supported by the National Natural Science Foundation of China (grants 30971453, 31171255, 3107116, 30700411, and 91219101, The National Basic Research Program of China (973 Program, Grant No. 2013CB530700), Shenzhen Bureau of Science Technology and Information (grant JC201005260239A), and the “Strategic Priority Research Program” of the Chinese Academy of Sciences (grant XDA01010405), as well as the Deutsche Forschungsgemeinschaft, an ERC Advanced Grant, and the BMBF-Network ‘EpiSys’ (to I.G.).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
sc-12-0890_sm_SupplFigure1.pdf209KFigure S1 Knockdown of Mbd3 increases the efficiency of iPSC generation. (A) Western blot showing the expression of Mi-2, HDAC1&2 and Mbd2&3 in MEFs, iPSCs and mESCs. (B-D) MEFs infected with Oct4/Sox2/Klf4/c-Myc (OSKM) in the presence of different Mbd3-specific RNAs were cultured in mESC medium and assayed for reprogramming 16 days after infection. (B) Left: Average numbers of AP+ and Oct4-GFP+ clones from 1.5×104 MEFs. Data are from three independent experiments; error bars represent standard deviation. Right: FACS analyses showing the percentage of Oct4-GFP positive cells. (C) Representative experiments showing differential interference contrast (DIC) and fluorescent images of MEFs, scale bars = 300 μm. (D) RT-PCR analysis of mESC marker genes. (E-F) Characterization of iPSCs derived from MEFs expressing OSKM and shMbd3. (E) AP staining and immunofluorescence of mES marker genes (SSEA-1 and Nanog). (F) Immunofluorescence showing embryoid body (EB)-mediated differentiation of iPSCs into ectoderm, mesoderm and endoderm using antibodies against Nestin, SM-actin and ARF. Scale bars = 100 μm. Abbreviations: MEF, mouse embryonic fibroblast; iPSCs, induced pluripotent stem cells; mESCs, mouse embryonic stem cells; RT-qPCR, reverse transcription quantitative polymerase chain reaction; OSKM, Oct4, Sox2, Klf4, c-Myc; DIC, differential interference contrast; GFP, green fluorescent protein; FACS, fluorescence-activated cell sorting; AP, alkaline phosphatase; SM-Actin, smooth muscle actin; AFP, α-fetoprotein.
sc-12-0890_sm_SupplFigure2.pdf74KFigure S2 Depletion of Mbd3 does not alter cell cycle progression. (A) Cell proliferation assay. MEF cells were infected with retroviruses encoding control (shCtrl) or Mbd3-specific shRNAs (shMbd3-1, 2&3). Cell numbers were counted in 24 h intervals 5-11 days after infection. (B) MEFs were infected with retroviruses encoding control (shCtrl) or Mbd3-specific shRNAs and subjected to FACS analysis 10 days after infection. Abbreviations: MEF, mouse embryonic fibroblast; FACS, fluorescence-activated cell sorting.
sc-12-0890_sm_SupplFigure3.pdf125KFigure S3 Depletion of Mbd3/NuRD substitutes for Sox2 in iSF1 medium. (A) Strategy for iPSC induction in iSF1 culture medium. (B) Knockdown of Mbd3 replaces Sox2 in iSF1 medium. Top: 2,500 MEFs infected with Oct4/Sox2/Klf4/c-Myc (OSKM) alone or together with Mbd3 or shMbd3 were cultured in iSF1 medium and assayed for reprogramming 12 days after infection. AP- and Oct4-GFP-positive clones were counted (top left) and the percentage of Oct4-GFP positive cells was analyzed FACS (top right). Bottom: 1.5×104 MEFs infected with Oct4/Sox2/Klf4 (OSK) alone or together with Mbd3 or Mbd3 shRNA were cultured in iSF1 medium and assayed for reprogramming 18 days after infection. AP- and Oct4-GFP-positive clones were counted (bottom left) and the percentage of Oct4-GFP-positive cells was monitored by FACS (bottom right). Error bars represent standard deviation. (C) RT-PCR analysis of showing expression of mESC marker genes in Oct4/ Klf4/c-Myc (OKM) plus shMbd3 derived iPS cells (sriPS). (D) 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) based assay monitoring viability and proliferation of sriPS cells. Abbreviations: MEFs, mouse embryonic fibroblasts; iPSC, induced pluripotent stem cell; mESC, mouse embryonic stem cell; RT-PCR, reverse transcription polymerase chain reaction; OSKM, Oct4, Sox2, Klf4 and c-Myc; OSK, Oct4, Sox2 and Klf4; OKM, Oct4, Klf4 and c-Myc; GFP, green fluorescent protein; AP, alkaline phosphatase; FACS, fluorescence-activated cell sorting; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide.
sc-12-0890_sm_SupplFigure4.pdf140KFigure S4 Microarray analysis of gene expression in control and Mbd3-depleted MEFs. (A) Cluster analysis of Mbd3 regulated genes in mES cells, Mbd3-depleted and VPA treated MEFs. Individual genes are listed in Table S2. (B) Immunoblots showing the expression of iPSC promoting factors upon depletion of Mbd3. MEFs were infected with retroviruses encoding control (shCtrl) or Mbd3-specific shRNAs (shMbd3-1&2) and the level of Nanog, Oct4 and Sox2 was monitored 10 days after infection. (C) Cluster analysis of Mbd3 target genes that are involved in activation of MAPK. Left: Gene expression in mESC, Mbd3-depleted and VPA-treated MEFs. Right: Binding of Mbd3 or Mbd3-containing complexes to gene promoters Individual genes are listed in Table S3. Abbreviations: VPA, valproic acid; MEF, mouse embryonic fibroblast; iPSC, induced pluripotent stem cell; mESC, mouse embryonic stem cell; MAPK, mitogen-activated protein kinase.
sc-12-0890_sm_SupplFigure5.pdf94KFigure S5 Gene Ontology clustering of Mbd3 regulated genes. Gene Ontology analysis of Mbd3 regulated genes based on the GO categories (A) biological process and (B) molecular function.
sc-12-0890_sm_SupplInfor.pdf8KSupplemental Data
sc-12-0890_sm_SupplTable1.pdf15KSupplemental Table 1
sc-12-0890_sm_SupplTable2.pdf2350KSupplemental Table 2
sc-12-0890_sm_SupplTable3.pdf53KSupplemental Table 3

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