RCOR2 Is a Subunit of the LSD1 Complex That Regulates ESC Property and Substitutes for SOX2 in Reprogramming Somatic Cells to Pluripotency§

Authors


  • Author contributions: P.Y. and Y.W.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; J.C., H.L., L.K., Y.Z., and S.C.: provision of study material, data analysis and interpretation; B.Z. and S.G.: conception and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript. P.Y. and Y.W. contributed equally to this article.

  • First published online in STEM CELLS EXPRESS March 23, 2011.

  • §

    Disclosure of potential conflicts of interest is found at the end of this article.

Abstract

Histone demethylase LSD1 can form complex with different Rcor family corepressors in different cell types. It remains unknown if cell-specific Rcor proteins function specifically in distinct cell types. Here, we report that Rcor2 is predominantly expressed in ESCs and forms a complex with LSD1 and facilitates its nucleosomal demethylation activity. Knockdown of Rcor2 in ESCs inhibited ESC proliferation and severely impaired the pluripotency. Moreover, knockdown of Rcor2 greatly impaired the formation of induced pluripotent stem (iPS) cells. In contrast, ectopic expression of Rcor2 in somatic cells together with Oct4, Sox2, and Klf4 promoted the formation of iPS cells. Most interestingly, ectopic expression of Rcor2 in both mouse and human somatic cells effectively substituted the requirement for exogenous Sox2 expression in somatic cell reprogramming. STEM CELLS 2011;

INTRODUCTION

ESCs, which are derived from the inner cell mass of a blastocyst, possess two distinguishing properties, that is, self-renewal ability and pluripotency [1, 2]. Genetic studies have revealed that core transcription factors Oct4, Sox2, and Nanog play essential roles in maintaining ESCs pluripotency [3–5]. These master pluripotency regulators not only target active pluripotency-related genes to promote ESCs pluripotency but also target many developmental genes collaboratively with polycomb group (PcG) proteins to silence these developmental genes [6]. In addition to the core transcription factors, several chromatin-modifying factors have been recently shown to be important for maintaining ESCs pluripotency and reprogramming somatic cells to pluripotency. The histone demethylase JMJD2C and one novel component of Polycomb repressive complex 2 in ESCs, JARID2, were reported to play critical roles in maintaining ESCs pluripotency [7, 8–12]. More recently, a study demonstrated that the ATP-dependent BAF chromatin remodeling components could enhance somatic cell reprogramming [13]. Interestingly, Oct4-interaction protein networks recently identified have revealed that many chromatin-modifying factors, including LSD1, might be involved in regulating either ESC pluripotency and/or somatic cell reprogramming [14, 15].

LSD1/KDM1a (BHC110) is an amine oxidase that demethylates histone H3 lysine 4 (both monomethylated and dimethylated lysine 4) by a flavin adenine dinucleotide–dependent oxidative reaction [16]. LSD1 is a component of various protein complexes that contain several transcription corepressors, including the RE1-silencing transcription factor (REST) corepressor CoREST, BHC80, HDAC1/2, CtBP, BRAF35, and several zinc finger proteins [17–20]. Although LSD1 alone can demethylate H3K4 in peptides or bulk histones, only RCOR1-LSD1 complex possesses the nucleosome demethylase activity [18, 19]. The RCOR1-LSD1 complex also interacts with REST to regulate neuronal genes expression in neural tissues [21]. In mammalian genomes, there are three Rcor family genes (Rcor1/CoREST, Rcor2, and Rcor3) and their orthologous genes have been found in Xenopus laevis, Drosophilla melanogaster, and Caenorthabditis elegans [22]. The function of RCOR1 has been extensively investigated previously, whereas the roles of RCOR2 and RCOR3 remain largely unknown.

Here, we report that RCOR2, a close homologue of CoREST/RCOR1, is predominantly expressed in ESCs and forms a complex with LSD1. Moreover, RCOR2 but not RCOR1 plays important roles in regulating ESCs pluripotency and reprogramming somatic cells to pluripotency. Most interestingly, RCOR2 can substitute for SOX2 in the reprogramming of both mouse and human differentiated somatic cells to form induced pluripotent stem (iPS) cells.

MATERIALS AND METHODS

Cell Culture

293T cells, HeLa cells and mouse fibroblast cells (MEF) were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, www.invitrogen.com) supplemented with 10% FBS and 2 mM L-glutamine. Mouse ESCs and iPS cells were maintained on irradiated MEF feeder cells in DMEM (Chemicon, Temecula, CA, www.chemicon.com) supplemented with 15% FBS, 2 mM L-glutamine, 10−4 M nonessential amino acids, 10−4 M β-mercaptoethanol, and 1,000 U/ml mouse leukemia inhibitory factor. Human embryonic stem (ES) and iPS cells were maintained on irradiated MEF feeder cells in DMEM/F12 supplemented with 20% knockout serum replacement, 10 ng/ml bFGF, 10−4 M nonessential amino acids, 10−4 M β-mercaptoethanol, 2 mM L-glutamax and 50 μg/ml penicillin/streptomycin.

Retrovirus Production and Rcor2-Overexpressing Stable Cell Line Construction

Flag-HA tag and multiple cloning site amplified from pOZ-FH-N (from Nakatani) was inserted into BglII-NotI site of pMX–internal ribosome entry site (IRES)–enhanced green fluorescent protein (EGFP) to generate pYS. Human Rcor2 was cloned into XhoI-NotI site of pYS, and then was transiently cotransfected with Pol/Gag and VSVG into 293T cells at a 10:9:1 ratio using Vigofect (Vigorous, Beijing, China, www.vigorousbio.com) to produce retroviruses. Forty-eight hours after transfection, the supernatant of transfectant was collected and filtered through a 0.45-μm pore size cellulose acetate filter (Millipore, Billerica, MA, www.millipore.com). HeLa cells were seeded at 8 × 105 cells per 100 mm dish 1 day before transduction. The medium was replaced with virus-containing supernatant supplemented with 4 μg/ml polybrene and incubated for 24 hours. Rcor2-overexpressing cells were sorted after 48 hours by flow cytometric analysis of green fluorescent protein (GFP) signals. The Flag-Rcor2 stable cell line was established and propagated as suspension cells.

Affinity Purification and Mass Spectrometry

Affinity purification was performed as previously described [19]. Briefly, nuclear extracts were prepared from 500 ml HeLa cell suspensions (∼ 2 × 108 cells) and mixed with M2 anti-Flag agarose (Sigma, St. Louis, MO, www.sigmaaldrich.com) that was equilibrated with the same buffer used in nuclear extract preparation (20 mM Tris-HCl [pH 7.9], 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM phenylmethylsulfonyl fluoride) overnight at 4°C. The resin was washed with an excess amount of buffer containing 20 mM Tris-HCl (pH 7.9), 0.5 M KCl plus 0.1% NP-40, and then eluted with buffer containing 20 mM Tris-HCl (pH 7.9), 0.1 M KCl plus 0.1 mg/ml Flag peptide (Sigma). Samples were loaded onto SDS-polyacrylamide gel electrophoresis (PAGE), silver stained, and bands of interest were cut from gel and subjected to LC-MS/MS sequencing and the data was then analyzed.

Histone Demethylase Assay

Demethylation activity on free histones or nucleosomal histone was carried out as previously reported [18]. Briefly, bulk histones or mononucleosomes were incubated with purified His-LSD1 with or without purified His-RCOR2 in the histone demethylase activity (HDM) assay buffer (50 mM Tris [pH 8.5], 50 mM KCl, 5 mM MgCl, 0.5% bovine serum albumin, and 5% glycerol) from 1 to 3 hours at 37°C. The demethylase activity under various conditions was evaluated by Western blotting using H3K4me2 antibodies.

Lentivirus Production and Rcor2Kd ES Cell Line Construction

pSicoR-mCherry plasmids containing Lsd1shRNA, Rcor1shRNA, Rcor2shRNA, and GFPshRNA were separately cotransfected with pSPAX2 and pMD2G into 293T cells using Vigofect (Vigorous). Viral supernatant fractions were harvested after 48 hours, filtered through a 0.45-μm low protein binding cellulose acetate filter (Millipore), and concentrated by centrifugation (Optima L-100 XP, Beckman Coulter, Brea, CA, www.beckmancoulter.com). A total of 5 × 104 R1 ESCs were incubated with virus for 6 hours and then seeded onto irradiated MEF feed cells. Red fluorescence protein-positive cells were sorted after 48 hours using MoFlo XDP cell sorter (Beckman Coulter) and further cultured in ES medium.

Alkaline Phosphatase Staining and Immunocytochemistry

Alkaline phosphatase staining was performed using the Alkaline Phosphatase Substrate Kit (Sigma) according to the manufacturer's instructions. For immunocytochemistry, cells were fixed with PBS containing 4% paraformaldehyde for 15 minutes at room temperature. After being washed with TBST, the cells were then treated with PBS containing 0.1% Triton X-100 for 15 minutes at room temperature and then incubated with PBS containing 4% normal goat serum (Chemicon) for 30 minutes at room temperature. The following primary antibodies were used in this study: Oct4 (1:50, ES CELL MARKER SAMPLE KIT, Chemicon), Sox2 (1:500, Abcam, Cambridge, UK, www.abcam.com), Nanog (1:500, Abcam), and SSEA1 (1:50, ES CELL MARKER SAMPLE KIT, Chemicon). The following secondary antibodies were used: Alexa 594-conjugated goat anti-mouse IgM (1:500, Invitrogen) and Alexa 594-conjugated goat anti-rabbit IgG (1:500, Invitrogen). Nuclei were stained with 4′,6-diamidino-2-phenylindole. Stained cells mounted on slides were observed on a LSM 510 META confocal microscope (Zeiss, Oberkochen, Germany, www.zeiss.com).

Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Quantitative RT-PCR Analysis

Total RNA was extracted from cells using TriZol reagent (Invitrogen) according to the manufacturer's instructions. Complementary DNA synthesis was performed with the M-MLV Reverse Transcriptase Kit (Promega, Fitchburg, WI, www.promega.com) following the manufacturer's instructions. Quantitative RT-PCR was performed using a SYBR Green-based PCR Master Mix (Applied BioSystems, Foster City, CA, www.appliedbiosystems.com) and signals were detected with ABI7500 Real-Time PCR System (Applied BioSystems). Glyceraldehyde 3-phosphate dehydrogenase was used as endogenous control. All the primers used are listed in Supporting Information Table 1.

Cell Proliferation Assay and Cell Cycle Analysis

To determine the cell population doubling time, 1 × 105 R1 ESCs and Rcor2kd ESCs were plated onto 24-well plates and counted after 48 hours. The cell population doubling time was obtained with the equation Yend = Ystart × 2(t/T), where T is the cell population doubling time, Ystart is the starting number of ESCs plated, and Yend is the ending number of ESCs after growth for a period of time, t. Each cell line was counted a minimum of five times. For cell cycle analysis, cells were labeled with bromodeoxyuridine (BrdU) and 7-amino actinomycin D (7-AAD; BD Pharmingen BrdU Flow Kits) according to the manufacturer's instructions and analyzed by flow cytometry.

Plasmid Construction and iPS Cell Generation

LV-tetO-Oct4, Sox2, Klf4, and c-Myc are generously provided by Dr. Rudolf Jaenisch's laboratory at Whitehead Institute for Biomedical Research. LV-tetO-Rcor2, Rcor1 were constructed using the same vector. Inducible iPS cells were generated as previous described [23, 24]. Briefly, 293T cells were transfected with the LV-tetO vectors together with packaging plasmids psPAX2 and pMD2G. The medium was replaced 12 hours after transfection, and the virus supernatants were harvested after subsequent 24 hours. A total of 1 × 105 MEF cells or tail tip fibroblast cells collected from tetracycline reverse transcriptional activator (rtTA) × 129/SV mice were incubated with filtrated viral supernatants containing 5 μg/ml polybrene. The infection medium was replaced after 12 hours with ES medium supplemented with 1 μg/ml doxycycline. The medium was replaced everyday until the ES-like colonies appeared at around 12 days after infection. The cells were cultured for another 4 days with ES medium without doxycycline, and then the colonies were picked.

For human Oct3/4, Klf4, c-Myc together with Rcor2 (OKMR2)-iPS cell generation, the plasmids pMIG containing human Oct4, Klf4, and c-Myc obtained from Addgene and pYS-RCOR2 was transiently cotransfected with package plasmids into 293T cells using Vigofect (Vigorous) separately. Viral supernatants were harvested after 48 hours, filtered through a 0.45-μm low protein binding cellulose acetate filter and concentrated by centrifugation. A total of 5 × 104 human fibroblast cells were incubated with virus for 24 hours and then seeded onto irradiated MEF feed cells after 5 days. iPS cell culture medium was substituted after 7 days. iPS colonies were manually picked and mechanically dissociated for following passage.

Chimeric Mice Generation and Tetraploid Complementation

To produce chimeric mice, 7–10 iPS cells were microinjected into the ICR 8-cell stage embryos using piezo-actuated microinjection pipette. After culture for 1 day, embryos were transplanted into of 2.5 days postcoitum of pseudopregnant ICR females. The tetraploid embryos were produced by fusion of two-cell stage embryos collected from ICR mice. Tetraploid blastocysts complementated with iPS cells were transplanted into the surrogate mothers to test full pluripotency.

Microarray Analysis

Affymetrix GeneChip Mouse Genome 1.0 Array (Affymetrix) was used for microarray hybridizations to examine the global gene expression of R1 ESCs and Rcor2kd ESCs. Briefly, total RNA was isolated using Trizol reagent (Invitrogen). cDNA was synthesized and amplified with WT cDNA Synthesis and Amplification Kit (Affymetrix), labeled with biotin using WT Terminal Labeling Kit (Affymetrix), hybridized to Affymetrix GeneChip Mouse Genome 1.0 Array and analyzed by GeneChip Scanner 3000 7G3000 (Affymetrix). All samples were prepared in three biological repeats. Data were analyzed using GCOS 1.4 software provided by Affymetrix. Signal values of probes presented in two samples were plotted in a scatter graph. Pearson's correlation coefficient (R) between samples was calculated by Excel.

RESULTS

RCOR2 Is a Novel Component of the LSD1 Complex in ESCs

RCOR family proteins were first identified as corepressors of REST that participate in repressing neuronal genes in non-neuronal tissues [21, 25]. The RCOR proteins can form multiple corepressor protein complexes, including the LSD1 complex [18, 19] and the CtBP complex [20]. However, despite high sequence similarities, RCOR2 is the only protein in the RCOR family that has never been identified in these protein complexes [18–20].

Using a retroviral expression vector carrying an IRES, we established HeLa cells stably coexpressing Flag-RCOR2 and EGFP after sorting for EGFP-positive cells (Supporting Information Fig. S1). The RCOR2 complex was affinity purified using agarose conjugated with anti-Flag antibodies under stringent washing conditions (0.5 M KCl). RCOR2-associated proteins were separated by SDS/PAGE and then identified by mass spectrometry. The identified subunits include LSD1/KDM1, RCOR1/CoREST, HDAC1/2, BHC80, GSE1, and ZMYM2 (Fig. 1A and Supporting Information Table 2), similar to what was previously reported for the LSD1 complex [19]. Our results indicate that RCOR2 is a novel integral subunit of the LSD1 complex. Its absence in previous purifications is likely due to the low level of expression of RCOR2 in HeLa cells.

Figure 1.

RCOR2 regulates LSD1-mediated histone demethylation. (A): Immunoaffinity purification of RCOR2-containing protein complex. The N-terminal FLAG-epitope tag human RCOR2 (e-RCOR2) was stably expressed in HeLa cells by retroviral transduction. The RCOR2-associated proteins were purified using agarose conjugated with anti-FLAG antibodies under stringent washing conditions (0.5 M KCl), separated on 8% SDS-polyacrylamide gel electrophoresis, and visualized by silver staining. The identities of these proteins were revealed by MS/MS on the right. (B): Comparable demethylation of free histones by recombinant His-LSD1 in the presence and absence of recombinant His-RCOR2. Free histones were isolated from HeLa cells using acidic extraction. The demethylation reaction of LSD1 was analyzed by Western blot using an anti-H3K4me2 or anti-H3 control antibodies. (C): Nucleosomal histone demethylase activity by LSD1 in the presence and absence of RCOR2. The nucleosomal histones were purified from HeLa cells using micrococcal nuclease digestion. The demethylation reaction of LSD1 was analyzed by Western blot using an anti-H3K4me2 or anti-H3 control antibodies. IP, immunoprecipitation.

LSD1 is a histone H3K4me1/2 demethylase, and its association with RCOR1/CoREST is essential for LSD1 activity on nucleosomal substrates [18, 19]. Similar to RCOR1, our results showed that the addition of RCOR2 also converted LSD1 from a free histone-specific enzyme (Fig. 1B) to a nucleosomal histone demethylase (Fig. 1C).

These results suggest that RCOR2 has similar biochemical function as RCOR1 in facilitating LSD1's demethylase activity on nucleosomal substrates, and it may function in specific cell types. Subsequently, we further examined the expression pattern of Rcor2 in different cell types and tissues. As shown in Figure 2A, RCOR2 is predominantly expressed in ESCs, whereas the expression of RCOR1 was more ubiquitous (Fig. 2A). Comparing mouse ES and MEF cells, the expression levels of LSD1 and REST did not change much whereas the expression of Rcor2 could only be detected in ESCs (Fig. 2B and Supporting Information Fig. S2). Similarly, expression of Rcor2 is significantly higher in human ESCs than in other cell types tested (Fig. 2C).

Figure 2.

RCOR2 is a novel subunit of LSD1 complex that exclusively expressed in ESCs. (A): Rcor2 is exclusively expressed in ESCs compared with Rcor1. Real-time polymerase chain reaction analysis of Rcor1 (left panel) and Rcor2 (right panel) expression levels in various mouse tissues. Transcript levels were normalized to Gapdh levels. Error bars represent SD (n = 3). (B): Western blot analysis of RCOR1, RCOR2, LSD1, and REST expression in mouse ESCs and MEF cells. ACTIN was used as control. (C): Western blot analysis of RCOR1, RCOR2, REST, and LSD1 expression in HeLa cells, human ESCs, and human fibroblast cells. ACTIN was used as control. (D): Western blot analysis for mouse ESC nuclear extracts (input) or the samples derived from anti-Rcor2, anti-Lsd1, and control IgG immunoprecipitation using Rcor2 and Lsd1 antibodies. Abbreviations: Gapdh, glyceraldehyde 3-phosphate dehydrogenase; hESC, human ESC; IP, immunoprecipitation; MEF, mouse fibroblast cells; mESC, mouse ESC; REST, RE1-silencing transcription factor.

To further validate the interaction between RCOR2 and LSD1 in ESCs, we performed immunoprecipitation using antibodies against RCOR2 or LSD1. Endogenous RCOR2 and LSD1 proteins do indeed form a protein complex in mouse ESCs because they reciprocally immunoprecipitated each other under stringent washing conditions (0.5 M KCl; Fig. 2D). Taken together, we found RCOR2 is a novel subunit of LSD1 complex that is exclusively expressed in mouse ESCs, with similar biochemical function as RCOR1. Given the fact that Lsd1 is universally expressed in many cell types, we speculated that Rcor2, which is specifically and highly expressed in ESCs, may play an important role in ESCs. More recently, RCOR2 and LSD1, but not RCOR1 were identified as OCT4-interacting proteins in ESCs [14, 15], supporting a role of an ESC-specific RCOR2 containing LSD1 complex in pluripotency.

RCOR2 but Not RCOR1 Affect ESC Cell Cycle Progression and Somatic Reprogramming

To investigate the function of Rcor2 in ESCs, we used the pSicoR-mCherry lentivirus system to knockdown Rcor2 expression. Two shRNA constructs targeting different regions of Rcor2 transcripts were used to establish stable Rcor2 knockdown (Rcor2kd) ESC lines. Knockdown efficiency was verified by real-time polymerase chain reaction (RT-qPCR) and Western blot (Fig. 3A). No significant changes of the Rcor1, Lsd1, and Rest mRNA levels were observed between the Rcor2kd cells and the mock-treated cells (Fig. 3B). The Rcor2kd ESC colonies were less compact, and the surface of these colonies appeared much rougher than those of the control group (Supporting Information Fig. S3A), which indicated that Rcor2kd ESCs may partially differentiate. We also used specific Rcor1 siRNAs to validate the role of Rcor1 in ESCs, and RT-qPCR results showed that the expression level of Rcor1 was efficiently downregulated, whereas the mRNA levels of other LSD1 complex components, such as Lsd1, Rcor2, Rest, were similar to the mock group (Supporting Information Fig. S4A).

Figure 3.

Knockdown of Rcor2 impaired mouse ESCs proliferation. (A): Validation of Rcor2 knockdown efficiency by RT-qPCR and Western blot analysis. Two shRNA constructs (1# and 2#) targeted different regions of Rcor2 transcripts were tested. Transcript levels were normalized to the levels of ESCs treated with empty pSicoR vector (mock). Error bars represent SD (n = 3). (B): RT-qPCR analysis to validate Lsd1, Rcor1, and Rest expression levels in Rcor2kd ESCs. The expressions of Lsd1, Rcor1, and Rest were not significantly changed when Rcor2 was knocked down in mouse ESCs. Transcript levels were normalized to Gapdh levels. Error bars represent SD (n = 3). (C): Doubling time counting analysis of control ESCs, Rcor1kd ESCs, Rcor2kd ESCs, and Rcor2kdp16kd ESCs. ESC doubling times were calculated by counting cell numbers 48 hours after seeding same numbers of cells. It took nearly twice as long for Rcor2kd ESCs to double (32.44 hours vs. 16.57 hours) compared with the Rcor1kd and control ESCs. p16 RNAi can partially rescue the progression defect in Rcor2kd ESCs (*, p = .0317). ESCs treated with empty pSicoR vector were used as control. (D): p16 siRNA can partially rescue the impaired proliferation in Rcor2kd mouse ESCs. Fluorescence-activated cell sorting analysis of BrdU incorporation in the control ESCs treated with empty pSicoR vector (upper panel, left), the Rcor2kd ESCs (upper panel, right); the Rcor2kd ESCs transfected with control siRNA (lower panel, left), and the Rcor2kd ESCs transfected with siRNA targeting to p16Ink4a (lower panel, right). For the dot plot, 7-AAD staining for DNA content was shown on x-axis, and BrdU staining was shown on y-axis. In this representative experiment, the percentage of S-phase ESCs (in the frame) was dramatically decreased when Rcor2 knockdown (39.27% to control 67.54%), and the knockdown of p16Ink4a partially rescued the cell cycle defect in Rcor2kd ESCs (48.99% to control 39.1%). (E): RT-qPCR analysis of cell cycle inhibitors including p16Ink4a, p15Ink4b, p21Cip, p53, and p19Arf in both control ESCs (mock), Rcor2kd ESCs, and Rcor2kd ESCs transfected with siRNA targeting to p16Ink4a. ESCs treated with empty pSicoR vector were used as control (mock). Transcript levels were normalized to control group (mock) levels. Error bars represent SD (n = 3). Abbreviations: 7-AAD, 7-amino actinomycin D; BrdU, bromodeoxyuridine; GFPkd, GFP knockdown cells; RT-qPCR, real-time polymerase chain reaction.

Rcor2kd ESCs proliferate much slower than control cells. Cell doubling times were calculated by counting cell numbers 48 hours after seeding the same numbers of cells. It took nearly twice as long for Rcor2kd ESCs to double compared with the control ESCs (Fig. 3C). In addition, Rcor2kd and control ESCs were labeled with BrdU for 45 minutes and then subjected to flow cytometry analysis. Although approximately 67.5% of control ESCs were in S-phase, the percentage of S-phase cells among the Rcor2kd ESCs was dramatically reduced to only about 39% (Fig. 3D). These data collectively indicated that Rcor2 is required for normal ESC proliferation. We also tried to generate Lsd1kd ESC lines using the same protocol as Rcor2 but failed to expand the colonies due to the increased cell death and impaired cell cycle progression (data not shown). Consistent with our data, Lsd1 knockout has also been reported to affect cell proliferation [26].

In contrast, no obvious cell cycle delay was observed in Rcor1kd cells (Fig. 3C). We next examined the expression levels of cell cycle inhibitors, such as p15Ink4b, p16Ink4a, p19Arf, p21Cip, and p53 in both Rcor2kd and Rcor1kd ESCs. Indeed, the expression levels of some cell cycle inhibitors, especially Ink4a and Ink4b, were upregulated in Rcor2kd ESCs but not in Rcor1kd ESCs based on RT-qPCR analysis (Fig. 3E and Supporting Information Fig. S4B). We also tried to rescue expansion defect in Rcor2kd ESCs, and the result showed that reduction of p16Ink4a by specific siRNAs can partially rescue the progression defect in Rcor2kd ESCs (Supporting Information Fig. S5 and Fig. 3C–3E). Taken together, Rcor2 but not Rcor1 is a key component that regulates ESC cycle progression.

Previous reports showed that deletion of cell cycle inhibitor like p16Ink4a, p15Ink4b, which are upregulated in Rcor2kd ESCs but not Rcor1kd ESCs (Fig. 3E and Supporting Information Fig.S4B), can accelerate iPS reprogramming process [27]. So, we reasoned that Rcor2 might be an important factor facilitating mouse somatic cell reprogramming. By using the inducible iPS system established previously [24, 28], 1 × 105 rtTA integrated MEF cells were transduced with the four inducible factors Oct3/4, Sox2, Klf4, and c-Myc (OSKM) in combination with shRNAs targeting Rcor1, Rcor2, or GFP. Alkaline phosphatase (AP)-positive colonies were counted after culturing these cells in ESC medium supplemented with 1 μg/ml doxycycline for 10 days. Downregulation of Rcor1 and Rcor2 mRNA after RNAi were verified by RT-qPCR (Supporting Information Fig. S6). Interestingly, compared with GFP or Rcor1 RNAi groups, knockdown of Rcor2 exhibited far fewer AP-positive colonies (Fig. 4A, upper panel). To exclude variation due to viral infection, tail tip fibroblast cells were collected from the iPS mice generated by tetraploid complementation using iPS cells that were initially reprogrammed with the above-mentioned inducible system [29]. These cells were then transduced with shRNA targeting Rcor1, Rcor2, or GFP and simultaneously induced with 1 μg/ml doxycycline for 10 days. Again, RNAi targeting Rcor2, but not Rcor1, led to a significant reduction of the number of AP-positive colonies (Fig. 4A, lower panel). As we considered that the effect of Rcor2 knockdown in compromising iPS cell formation is likely due to an important role of Rcor2 in ESC self-renewal rather than in impeding the reprogramming process itself, we decided to ectopically express mouse Rcor2 along with the other Yamanaka factors and examine reprogramming efficiency. Induction of Rcor2 expression together with OSKM or inducible factors Oct3/4, Sox2, and Klf4 (OSK) could significantly promote the formation of AP+ colonies during iPS cell production (Fig. 4B, 4C and Supporting Information Fig. S6).

Figure 4.

Rcor2 but not Rcor1 is required for iPS formation. (A): AP staining results on day 10 of iPS cell generation with knockdown of GFP (left), Rcor2 (middle), and Rcor1 (right). rtTA-integrated MEF cells (upper panel) and tail tip fibroblast cells from iPS cell-derived tetraploid mice (lower panel) were both used. Knockdown of Rcor2, but not Rcor1, reduced the number of AP+ colonies. (B): AP staining results on day 10 of iPS cell generation with different combinations of transcriptional factors plus Rcor2. Upper panel showed the AP staining of colonies derived with OSK and OSKM (from left to right); lower panel showed that derived with OSK plus Rcor2, OKM plus Rcor2, and OSKM plus Rcor2 (from left to right). Induction of Rcor2 together with OSKM or OSK increased the number of AP+ colonies. (C): Counting of AP-positive colonies. Three independent experiments were counted for each group. Abbreviations: AP+, alkaline phosphatase positive; GFP, green fluorescent protein; iPS cell, induced pluripotent stem cell; MEF, mouse fibroblast; OSK, inducible factors Oct3/4, Sox2, and Klf4; OSKM, inducible factors Oct3/4, Sox2, Klf4, and c-Myc.

To further confirm the significant role of Rcor2 in iPS generation, we adopted the same inducible iPS system on MEF cells isolated from rtTA-OG2 transgenic mice, in which Oct4 expression could be monitored by GFP expression. GFP+ colonies was counted after culturing these cells in ESC medium supplemented with 1 μg/ml doxycycline for 20 days. Consistent with AP staining results, both knockdown and overexpression of Rcor2 would affect the number of GFP+ colonies, whereas Rcor1 had no effect (Supporting Information Fig. S7). These results further proved the previous prediction that Rcor2 might function as a candidate factor in promoting iPS induction [13].

Rcor2 Is Required for the Correct Expression Pattern of Mouse ESC Self-Renewal and Pluripotency Marker Genes

To gain insight into the state of Rcor2kd ESCs, both self-renewal and differentiation marker genes were examined in this cell line. RT-qPCR results showed that the expression levels of Sox2 and Nanog were significantly reduced in Rcor2kd ESCs, but Oct3/4 was only moderately downregulated comparing with the control group (Fig. 5A). Similar results were obtained with Western blot analysis (Fig. 5B). Immunofluorescent staining also confirmed that Sox2 was nearly undetectable in Rcor2kd ESCs, whereas the expression of Oct3/4 and Nanog was not severely affected (Supporting Information Fig. S3B). Quantitative RT-PCR results indicated upregulation of endoderm and mesoderm markers including Ck8, T/Brachyury, Sox17, and Gata4 in Rcor2kd ESCs (Fig. 5C). However, no significant changes were observed in expression levels of ectoderm markers Nestin and Map2 (Fig. 5C). Moreover, the Rcor2kd ES chimeric mice were generated, which exhibited either very low chimerism or died within 2 days after birth (Supporting Information Fig. S3C), indicating that Rcor2 indeed affected the differentiation or growth ability of chimeric mice to some extent. On the other side, knockdown of Rcor1 in ESCs appeared with no significant changes in expression levels of both pluripotency and differentiation marker genes (Supporting Information Fig. S4C).

Figure 5.

RCOR2 is required for correct expression pattern of mouse embryonic stem (ES) self-renewal and pluripotency markers. (A): In RT-qPCR analysis, Sox2 expression was dramatically reduced in mouse ESCs upon Rcor2 knockdown; Nanog expression is also reduced but to a lesser extent. Transcript levels were normalized to the levels of mock ESCs. Error bars represent SD (n = 3). (B): Western blot analysis of ES-specific transcription factors and LSD1 complex proteins in RCOR2kd and the mock ESCs. (C): RT-qPCR analysis to examine the expression levels of differentiation marker genes in Rcor2kd ESCs. Transcript levels were normalized to the levels of mock ESCs. Error bars represent SD (n = 3). (D): RT-qPCR analysis to examine the expression levels of epithelial genes, mesenchymal genes, and genes involved in different pathways after Rcor2 was knockdown in mouse ESCs. Transcript levels were normalized to the levels of mock ESCs. Error bars represent SD (n = 3). Abbreviations: GFPkd, GFP knockdown cells; REST, RE1-silencing transcription factor; RT-qPCR, real-time polymerase chain reaction; TGF, transforming growth factor.

As Rcor2-Lsd1 complex can negatively regulate transcription by demethylation of active histone marks H3K4 methylation, the target genes of Rcor2 and Lsd1 may be upregulated in Rcor2kd ESCs. In our experiment, the expression level of T/Brachyury, which is reported to be the monitor of primitive streak-like population formation during early ESC differentiation in vitro, increased about twofold in Rcor2kd ESCs comparing with the control group. Moreover, in our chromatin immunoprecipitation and quantitative PCR analyses of selected genes using mESCs, both Rcor2 and Lsd1 enriched in T/Brachyury promoter regions (Supporting Information Fig. S8), suggesting that T/Brachyury may be a direct target gene of Rcor2. We also observed the enrichment of Rcor2 and Lsd1 in the promoter region of p16, which is consistent with the upregulation of p16 in Rcor2kd ESCs.

Rcor2 is particularly expressed in neural tissue during embryogenesis [30, 31]. Knockdown of Rcor2 results in the neural stem cell marker Sox2 suppression in our results, which suggests their functional overlap. Moreover, upregulation of mesoendoderm marker genes was observed in Rcor2kd ESCs. Therefore, we speculated that Rcor2 may attenuate the differentiation potential into mesoderm and endoderm in ESCs.

Two recently published articles reported that mesenchymal to epithelial transition is necessary and important for iPS cell reprogramming process [32, 33]. In our experiments, epithelial genes including E-cadherin, Epcam, Ocln, and Cldn3 were downregulated, whereas mesenchymal genes such as snail, slug, and cdh2 were moderately upregulated after Rcor2 knockdown in ESCs (Fig. 5D), which coincidences with the rough surface and less compacted colonies morphology we observed in Rcor2kd ESCs (Supporting Information Fig. S3A). We also checked the expression of genes involved in transforming growth factor (TGF)-β, Wnt, and Fgf pathways, and found most genes were downregulated after Rcor2 knockdown except bone morphogenetic protein 4 (Bmp4) (Fig. 5D). The expression levels of the genes mentioned above were also checked in Rcor1kd ESCs, and no significant differences were found compared with the control groups (Supporting Information Fig. S4D).

To further investigate the genes affected by Rcor2 knockdown, microarray analysis was performed to compare the global gene expression characteristics between Rcor2kd ESCs and R1 ESCs. The results revealed that Sox2 gene expression level was markedly downregulated in the Rcor2kd ESCs and meanwhile, the mesoderm and endoderm layer marker genes' expression levels were upregulated (Supporting Information Fig. S9).

RCOR2 Substitutes for SOX2 in Reprogramming of Both Mouse and Human Somatic Cells into iPS Cells

Ectopic expression of Sox2 is required for iPS cells formation, such that omitting Sox2 can lead to the pre-iPS cell formation, but these cells fail to eventually become pluripotent [34]. The results presented above have shown that Sox2 expression was dramatically decreased in Rcor2kd ESCs but not Rcor1kd ESCs. Therefore, we postulated that Rcor2 might induce endogenous Sox2 expression during somatic cell reprogramming. Endogenous Sox2 expression levels were monitored by RT-qPCR from day 1 through day 10 in the MEF cells transduced with OKMR2 or Oct3/4, Klf4, c-Myc together with Rcor1 (OKMR1), respectively. Comparing with the control group in which MEF cells were transduced with 4 Yamanaka factors (4F), the OKMR2 group could upregulate endogenous Sox2 at similar time points and to similar expression levels, whereas the OKMR1 group failed to activate endogenous Sox2 expression (Fig. 6A).

Figure 6.

Rcor2 can replace Sox2 in the generation of mouse iPS cells. (A): Rcor2 but not Rcor1 can enhance the expression of Sox2 during reprogramming. Real-time polymerase chain reaction analysis of endogenous Sox2 expression levels during reprogramming process by transduction of Oct3/4, Klf4, c-Myc (OKM), and Sox2 (S) or Rcor2 (R2) or Rcor1 (R1). Transcript levels were normalized to Gapdh expression levels. Error bars represent SD (n = 3). (B): Morphology of mouse R1 ES, 4F-iPS, OKMR2-iPS, and OKR2-iPS cells. Scale bar = 100 μm. (C): Reverse transcription polymerase chain reaction of endogenous and exogenous Oct3/4, Sox2, and Nanog expression in OKR2- and OKMR2-iPS cells. (D): Immunostaining for OCT3/4, SOX2, NANOG, and SSEA1 in OKR2-iPS cells. Scale bar = 20 μm. (E): H&E staining of teratomas derived from OKR2-iPS cells. Scale bar = 50 μm. (F): Photograph of chimeric mouse (left) with germ line transmission mouse (right) generated from OKR2-iPS cells. (G): OKMR2-iPS cells were able to generate a whole mouse by tetraploid complementation. Abbreviations: Gapdh, glyceraldehyde 3-phosphate dehydrogenase; iPS cell, induced pluripotent stem cell; OKMR2, Oct3/4, Klf4, c-Myc with Rcor2; rtTA-MEF, rtTA-integrated mouse fibroblast cells.

As iPS cells could be generated with a three-factor cocktail that does not include c-Myc [35, 36], we tried to use OKMR2 and Oct3/4 and Klf4 with Rcor2 (OKR2) combinations to generate iPS cells separately. Both exogenous OKMR2 and OKR2 treatment of MEF cells were able to generate iPS cells with normal ES characteristics (Fig. 6B). We also used the inducible iPS system on rtTA-OG2 MEF cells mentioned above to determine the reprogramming efficiencies by counting GFP+ after culturing these cells in ESC medium supplemented with 1 μg/ml doxycycline for 20 days. The efficiencies were 0.047% for OKMR2 group and 0.024% for OKR2 group, which was slightly lower than the OSKM group with 0.064% (Supporting Information Fig. S7).The OKMR2- and OKR2-iPS cells showed endogenous expression of ES marker genes including Oct3/4, Sox2, and Nanog based on RT-PCR and immunostaining, and these cells were also positive for the mES-specific surface antigen SSEA1 (Fig. 6C, 6D). Teratomas containing tissues for all three germ layers were also generated after injection of OKR2-iPS cells into severe combined immunodeficiency (SCID) mice (Fig. 6E). Moreover, chimeric mice with germ line transmission ability were also successfully generated from these OKR2-iPSCs (Fig. 6F). To further investigate the full pluripotency of OKMR2- and OKR2-iPS cells, we tried to generate iPSC-derived mice using tetraploid complementation. Each iPS cell line was tested using 300 tetraploid embryos with ICR background, and one live mouse was obtained from OKMR2-iPS cells (Fig. 6G). No live pups were obtained from OKR2-iPS cells.

It has been previously shown that Sox2 could be substituted by small molecule during mouse iPS cells derivation [37, 38], however, not human iPS cells generation. Next, we tried to test if Rcor2 could substitute Sox2 for generating human iPS cells using retrovirus system established previously [39]. We successfully generated several human OKMR2-iPS cell lines with morphology similar to human ESCs (Fig. 7A). ES marker genes expressions detected by RT-qPCR and immunofluorescent staining also confirmed the pluripotency of these iPS cells (Fig. 7B, 7C). Finally, teratomas containing tissues of all three germ layers were successfully generated from the OKMR2-iPS cell lines after injection into SCID mice (Fig. 7D), indicating that the human iPS cells generated by overexpression of Rcor2 together with Oct4, Klf4, and c-Myc are pluripotent. To our knowledge, Rcor2 is the first non-Sox family protein factor that can substitute for Sox2 in the induction of iPS cells from both mouse and human somatic cells.

Figure 7.

Rcor2 can replace Sox2 in the generation of human iPS cells. (A): Morphology of human iPS cells generated with OCT3/4, KLF4, c-MYC, and RCOR2 (OKMR2). Scale bar = 20 μm. (B): RT-qPCR analysis of ESC marker genes expression in human OKMR2-iPS cell lines. Human ESCs were used as control. Transcript levels were normalized to Gapdh expression levels. Error bars represent SD (n = 3). (C): Immunofluorescent staining for OCT3/4, SOX2, NANOG, SSEA4, and TRA1-81 in human OKMR2-iPS cells. Scale bar = 20 μm. (D): Photographs of teratomas containing tissues of all three germ layers generated from human OKMR2-iPS cells. Scale bar = 100 μm. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ESC, embryonic stem cell; OKMR2-iPS, Oct3/4, Klf4, c-Myc with Rcor2-induced pluripotent stem cell.

DISCUSSION

To our knowledge, this is the first study showing that an ES-specific corepressor, RCOR2, which forms a complex with the histone demethylase LSD1, plays important roles in regulating ESC pluripotency and facilitating reprogramming somatic cells to pluripotency. Furthermore, RCOR2 can substitute for SOX2 in the reprogramming of both mouse and human somatic cells to pluripotency.

As the first discovered histone demethylase, LSD1 is ubiquitously expressed in many types of cells, whereas the RCOR family proteins that connect LSD1 to chromatin display cell type-specific expression patterns. RCOR1, the first Rcor family member characterized, is ubiquitously expressed in somatic cells, whereas RCOR2 and RCOR3 were found to be specifically and highly expressed in ESCs and testis (data not shown), respectively. This suggests that the Lsd1 corepressor complex might interact with different repressors via Rcor family proteins to carry out specific functions in different cell types. Through loss of function study, we observed that ES specific-expressed Rcor2 plays important roles in regulating both self-renewal and pluripotency of ESCs. Knockdown of Rcor2 in ESCs greatly inhibited ESC proliferation, which coincides with the similar proliferation defects observed in the Lsd1−/− ESCs [26]. The proliferation defects observed in Rcor2 knockdown ESCs might be in part due to the upregulation of the cell cycle inhibitors such as p16Ink4a.

In addition to the cell proliferation defects observed in Rcor2 knockdown ESCs, the ESC pluripotency was severely impaired. Downregulation of pluripotency transcription factors especially Sox2 was observed in Rcor2 knockdown ESCs. TGF-β family signaling has been reported to be involved in both ESCs self-renewal and differentiation [40]. During in vitro differentiation of mouse ESCs, BMP4 was involved in specification of T/Brachyury-positive primitive streak layer [41] and inhibition of BMP4 during early gastrula stage leads to neural induction [42, 43]. In the present study, we further examined the expression patterns of the secreted TGF-β pathway ligands in Rcor2 knockdown ESCs. Our results indicated that upregulation of BMP4 expression together with impaired expression of TGF-β inhibitor Noggin and Chordin might be correlated with the downregulation of Sox2 expression in Rcor2 knockdown ESCs.

iPS cells, which are generated from differentiated somatic cells through the ectopic expression of four transcription factors, Oct4, Sox2, Klf4, and c-Myc, provide an opportunity to generate patient-specific pluripotent stem cells [34, 44-50]. Searching for other transcription factors that could replace these original four factors will be important not only for expanding our understanding about reprogramming but also for generating safer iPS cells for clinical applications. It has been shown previously that c-Myc is dispensable for iPS cell generation and can be omitted from the process [35, 36]. Moreover, Oct4 and Klf4 can be substituted by the nuclear factors Nr5a2 and Esrrb, respectively, in reprogramming somatic cells [51, 52]. So far, Sox2 can only be substituted by either transcription factors belonging to the Sox family [35] or small molecules that inhibit TGFβ pathway [37, 38]. As Rcor2 plays important roles in regulating both ESC proliferation and pluripotency, we further investigated the role of Rcor2 in reprogramming of somatic cells to pluripotency. Our results demonstrated that knockdown of Rcor2 expression in the process of iPS induction severely reduced the iPS cells generation. Moreover, we found that expression of Rcor2 in somatic cells could activate endogenous Sox2 expression, which in turn could substitute Sox2 in reprogramming somatic cells to pluripotency.

CONCLUSION

In conclusion, our study provides the first evidence showing that the ES-specific corepressor RCOR2, which forms a complex with the histone H3K4 demethylase LSD1, could substitute for Sox2 in reprogramming of both mouse and human fibroblasts into iPS cells.

Acknowledgements

We are grateful to our colleagues in our laboratories for their assistance with experiments and in the preparation of the article. This work was supported by the Chinese Ministry of Science and Technology (2008AA022311, 2010CB944900 and 2011CB964800 to S. G., 2007AA02Z1A6 and 2011CB965300 to B. Z., 2007AA02Z1A3 to S.C.).

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.

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