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Regulation of somatic cell reprogramming through inducible mir-302 expression

Regulation of somatic cell reprogramming through inducible mir-302 expression 1054–1065 Nucleic Acids Research, 2011, Vol. 39, No. 3 Published online 24 September 2010 doi:10.1093/nar/gkq850 Regulation of somatic cell reprogramming through inducible mir-302 expression 1, 1 2 3 Shi-Lung Lin *, Donald C. Chang , Chun-Hung Lin , Shao-Yao Ying , 1 1 Davey Leu and David T. S. Wu 1 2 WJWU and LYNN Institute for Stem Cell Research, Santa Fe Springs, CA 90670, USA, Taiwan Adventist Hospital, Taipei 10556, Taiwan, ROC and Department of Cell and Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA Received June 22, 2010; Revised August 10, 2010; Accepted September 12, 2010 understanding is limited by co-transfection of three or ABSTRACT four specific transcription factors, either Oct3/4–Sox2– Global demethylation is required for early zygote Klf4–cMyc or Oct3/4–Sox2–Nanog–Lin28, to promote development to establish stem cell pluripotency, induced pluripotent stem (iPS) cell formation (1–3). yet our findings reiterate this epigenetic reprogram- Among these reprogramming factors, POU class ming event in somatic cells through ectopic intro- 5 homeobox 1 (Oct3/4) and sex determining region duction of mir-302 function. Here, we report that Y-box 2 (Sox2) are essential. Recent studies found that both Oct4 and Sox2 are also crucial for expressing induced mir-302 expression beyond 1.3-fold of the mir-302 in human embryonic stem (hES) cells (4,5). concentration in human embryonic stem (hES) H1 Mir-302 belongs to a class of small, non-coding RNAs and H9 cells led to reprogramming of human hair known as microRNAs (miRNA) that function as cyto- follicle cells (hHFCs) to induced pluripotent stem plasmic gene silencers by suppressing translation of (iPS) cells. This reprogramming mechanism func- targeted messenger RNAs (mRNA). The majority of tioned through mir-302-targeted co-suppression of mir-302-targeted genes are transcripts of developmental four epigenetic regulators, AOF2 (also known as signals and oncogenes (6); nevertheless, their interactions KDM1 or LSD1), AOF1, MECP1-p66 and MECP2. and overall functions remain unknown. The genomic Silencing AOF2 also caused DNMT1 deficiency and sequence encoding mir-302 is located in the 4q25 locus further enhanced global demethylation during of human chromosome 4, a conserved region frequently somatic cell reprogramming (SCR) of hHFCs. associated with longevity (7). In humans, mir-302 is pre- Re-supplementing AOF2 in iPS cells disrupted dominantly expressed in hES and iPS cells, but not in differentiated cells (8,9). Loss of mir-302 has been such global demethylation and induced cell differ- observed prior to hES cell differentiation and proliferation entiation. Given that both hES and iPS cells highly during early embryonic development (8). Analogously in express mir-302, our findings suggest a novel link mice, its homologous mir-291/294/295 family presents a between zygotic reprogramming and SCR, providing similar expression profile (10). Therefore, it is conceivable a regulatory mechanism responsible for global that embryonic stem cell-specific miRNAs such as mir-302 demethylation in both events. As the mechanism and mir-291/294/295 play a pivotal role in regulating cell of conventional iPS cell induction methods stemness and pluripotency, whose functions may be remains largely unknown, understanding this applied to enhance the efficiency of SCR for iPS cell microRNA (miRNA)-mediated SCR mechanism may generation. shed light on the improvements of iPS cell The initiation of SCR involves a highly coordinated DNA demethylation and histone methylation mechanism generation. that is able to alter a genome-wide scale of chromatin struc- ture and gene activity. To this, mir-302 may silence certain INTRODUCTION epigenetic regulators to affect the status of genomic DNA methylation. Utilizing high throughput analysis with Somatic cell reprogramming (SCR) requires global DNA online miRNA-target prediction programs demethylation to reset cell stemness, yet the mechanism TARGETSCAN (http://www.targetscan.org/) and underlying this epigenetic event is unclear. Current *To whom correspondence should be addressed. Tel: +1 323 442 1856; Fax: +1 323 442 3158; Email: shilungl@mirps.org The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2011, Vol. 39, No. 3 1055 PICTAR-VERT (http://pictar.mdc-berlin.de/), we found mechanism, we investigated the functional role of that lysine-specific histone demethylases (namely AOF, mir-302 in human SCR. KDM or LSD) and methyl CpG-binding proteins (MECP) are two major groups of the epigenetic regula- tors targeted by mir-302. AOF contains two familial MATERIALS AND METHODS members AOF1 and AOF2, both of which function to Cell culture and electroporation repress gene transcription by demethylating histone 3 on lysine 4 (H3K4) (11–13). Inhibition of AOF2 by its an- hHFCs were isolated and dissociated from a minimum of tagonist tranylcypromine augments H3K4 methylation and two hair dermal papillae by 4 mg/ml collagenase I diges- stimulates Oct3/4 expression in embryonal carcinoma cells tion for 45 min in fresh RPMI 1640 medium supplemented (11,12). In transgenic knockout mice, loss of either AOF1 with 20% FBS. For culturing melanocytes, the isolated or AOF2 substantially increases H3K4 methylation cells were cultivated in Medium 254 with the addition of (13,14). AOF1-knockout mice demonstrate normal body human melanocytes growth supplement-2 (HMGS-2, development but fail to set up de novo DNA methylation Invitrogen, Carlsbad, CA, USA) in the absence of anti- imprints during oogenesis (13), while AOF2 deficiency biotics at 37 C under 5% CO . For culturing fibroblasts causes embryonic lethality due to a progressive loss of (hFB), the isolated cells were cultivated in Medium 106 genomic DNA methylation and lack of global cell differ- with the addition of low serum growth supplement (LSGS, entiation (14). As a result, silencing of both AOF1 and Invitrogen) in the absence of antibiotics at 37 C under 5% AOF2 is likely to be sufficient in inducing global DNA CO . Cultures were passaged at 70–80% confluency by demethylation. Our recent studies further showed that exposing cells to trypsin/EDTA solution for 1 min and ectopic expression of the whole mir-302 familial cluster rinsing once with phenol red-free DMEM medium induced not only global demethylation via silencing (Invitrogen), and the detached cells were replated at 1:10 MECP1-p66 and MECP2 but also the co-expression of dilution in fresh Medium 254 with HMGS-2 or Medium Oct3/4–Sox2–Nanog genes, which led to the 106 with LSGS, respectively. For electroporation, a reprogramming of both normal and cancerous human mixture of pTet-On-tTS-mir302s (10 mg) and pTet-On- skin cells into a hES-like pluripotent state (6,15). A Adv-Neo() (50 mg) was added with the isolated hHFCs similar mir-302 transfection approach was also shown to (20 000–50 000) in a hypoosmolar buffer (200 ml; increase Oct3/4–Nanog co-expression by 2-fold in hES cells Eppendorf, Westbury, NY, USA) and electroporation (16). Taken together, these findings suggest that mir-302 was performed using Eppendorf Multiporator at 300– may concurrently suppress AOF1/2 and MECP1/2 to 400 V for 150 ms. The electroporated cells were first induce global demethylation and to activate the grown in phenol red-free DMEM medium (Invitrogen) co-expression of hES-specific genes required for SCR. supplemented with 20% knockout serum, 1% MEM However, a critical link between mir-302 and AOF1/2 is non-essential amino acids, 10 ng/ml bFGF, 1 mM still missing. GlutaMax and 1 mM sodium pyruvate, for 24 h at 37 C MiRNA concentration determines the efficiency of its under 5% CO . Then, 850 mg/ml G418 and >3.75 mg/ml gene targeting. To assess this dose-dependent mir-302 doxycycline (Dox) were added into the medium. The effect on AOF1/2 silencing and global demethylation, we medium and antibiotics were refreshed daily for 3–5 adopted a novel inducible pTet-On-tTS-miR302s days till the cells expressed strong red fluorescent RGFP. expression vector (Figure 1A) in conjunction with electro- Next, the individual red fluorescent cell (mirPS) was moni- poration delivery to reprogram normal human hair follicle tored under a TE2000 inverted microscopic system cells (hHFCs) isolated from the dermal papilla region. (Nikon, Melville, NY, USA) and separately collected Skin hHFCs were chosen due to their accessibility and into a 96 well, using MO-188NE 3D micromanipulators abundance in differentiated mesenchymal lineage cells, (Nikon). In the absence of Dox, the mirPS cells were such as keratinocytes, melanocytes and fibroblasts. We grown and passaged in knockout DMEM/F-12 medium also mimicked the natural mir-302 expression pattern by (Invitrogen) supplemented with 20% knockout serum, expressing all four native mir-302 familial members, 1% MEM non-essential amino acids, 100 mM mir-302a, b, c and d, in one intact intronic cluster ß-mercaptoethanol, 1 mM GlutaMax, 1 mM sodium (mir-302s; Supplementary Figure S1A) (8). Upon doxycyc- pyruvate, 10 ng/ml bFGF, 100 IU/ml penicillin/ line (Dox) stimulation, the biogenesis of mir-302 followed 100 mg/ml streptomycin/ 250 mg/ml G418, 0.1 mM A83-01 the natural intronic miRNA pathway, in which mir-302 and 0.1 mM valproic acid (Stemgent, San Diego, CA, was transcribed with a gene encoded for red fluorescent USA), at 37 C under 5% CO . Alternatively, in the protein (RGFP) and then further spliced into individual presence of Dox (2.5–5 mg/ml; Sigma-Aldrich, St. Louis, mir-302 members by spliceosomal components and cyto- MO, USA), the mirPS cells were cultivated and passaged plasmic RNaseIII Dicers (Figure 1B) (6). MiRNA micro- in the same feeder-free cultural condition with addition of array analysis revealed that all mir-302 members except 0.05 mM GSK inhibitor SB216763 (Stemgent). Addition of mir-302b* were efficiently expressed in transfected hHFCs GSK inhibitor could facilitate mirPS cell proliferation but after Dox stimulation (Supplementary Figure 1B). The with a slight tendency to cause neural differentiation. For procedure for generating mir-302-induced pluripotent neural cell induction, the mirPS cells were grown in the stem (mirPS) cells is summarized in Supplementary above feeder-free cultural condition with 0.05 mM Figure 2. Through this inducible mir-302 expression SB216763 but no Dox. 1056 Nucleic Acids Research, 2011, Vol. 39, No. 3 Figure 1. Inducible mir-302 expression and its effect on hHFC reprogramming. (A) Construct of the Dox-inducible pTet-On-tTS-miR302s vector. (B) Mechanism of intronic mir-302 biogenesis. (C) Northern and western blot analyses of the dose-dependent mir-302 effect on the expression of core reprogramming factors and melanocytic marker genes (n=5, P< 0.01). (D) Northern blot analysis of the time-course mir-302 effect on Oct3/4– Sox2–Nanog co-activation and SCR (n=5, P< 0.01). (E) Northern blot analysis of hES marker gene expression induced by a high mir-302 concentration in mirPS cells compared to those in hES WA01-H1 (H1) and WA09-H9 (H9) cells (n=5, P< 0.01). (F) FACS flow cytometry sorting of positively reprogrammed cells (mirPS-hHFC) with dual expression of mir-302 maker RGFP and hES cell marker Oct3/4. Construction of the RGFP transgene encoding mir-302s mixture with MluI/Pvu1 restriction enzymes at 37 C for (RGFP-mir-302s) 4 h. The digested mixture was collected with a gel extrac- tion filter in 30 ml of ddH O and ligated together with T 2 4 The mir-302 familial cluster (mir-302s) was generated as DNA ligase at 8 C for 16 h. This formed the RGFP-mir- reported earlier (6). The mir-302s consists of four parts, 302s transgene, which could be further cleaved out of the including precursor miRNAs (pre-miRNAs) of mir-302a, vector with XhoI/HindIII digestion. b, c and d. Synthetic oligonucleotides (Sigma-Genosys, St Louis, MO, USA) were listed in Supplementary Table S1. Construction of the inducible pTet-On-tTS-mir302s vector For expression, we mixed an equal amount (1:1) of the mir-302s cluster and a pre-made SpRNAi-RGFP transgene We first modified a Dox-inducible pSingle-tTS-shRNA vector from our previous study (6), and then digested the vector (Clontech, Palo Alto, CA, USA) by replacing its Nucleic Acids Research, 2011, Vol. 39, No. 3 1057 U6 promoter with a TRE-CMV promoter isolated from a (Abcam), DNMT1 (Abcam), HDAC2 (Abcam), pTRE-Tight plasmid (Clontech). Then, the modified H3K4me2/3 (Abcam), AID/AICDA (Santa Cruz), vector was digested with XhoI/HindIII restriction AOF1 (Abcam), AOF2 (Sigma), MECP1-p66 (Millipore, enzymes at 37 C for 4 h, purified by a gel extraction Billerica, MA), MECP2 (Abcam), TRP1 (Santa Cruz), filter in 30 ml of ddH O, and then mixed and ligated with keratin 16 (Abcam), b-actin (Chemicon, Temecula, CA, the XhoI/HindIII-cleaved RGFP-mir-302s transgene (1:1) USA) and RGFP (Clontech). with T DNA ligase at 8 C for 16 h. This formed the in- ducible pTet-On-tTS-mir302s vector. Luciferase 3 -UTR reporter assay Luciferase assays were performed using the pMir-Report Immunostaining assay miRNA Expression Reporter Vector System (Ambion), Embedding, sectioning and immunostaining tissue according to the manufacturer’s instruction. The mir-302 samples were performed as reported (6). Primary target sites (normal and/or mutant) were inserted in the antibodies included Tuj1 (Abcam Inc., Cambridge, MA, 3 -UTR cloning site of the pMir-Report Luciferase USA), ABCA2 (Santa Cruz Biotechnology, Santa Cruz, Reporter vector. The two target sites were synthesized CA, USA), COL1A1 (Santa Cruz), COL2A1 (Santa and separated by 12-CAGT-repeats. Another pMir- Cruz), M-cadherin (Abcam), MUC2 (Abcam), MUC5B Report ß-gal Control vector was used as a no reporter (Abcam), CD13 (Santa Cruz), CD105 (Abcam), keratin control. We transfected 200 ng of the reporter vector 14 (Abcam), keratin 16 (Abcam), Mitf (Abcam), TRP1 into 50 000 mirPS cells in the absence or presence of (Santa Cruz), a-SMA (Abcam), ED-A fibronectin (Santa Dox treatment, using a FuGene HD reagent (Roche) fol- Cruz), GAD67 (Abcam), EN4 (Abcam), SC (Abcam), lowing the manufacturer’s suggestion. Cell lysates were BMP11 (Abcam), cTnT (Abcam), myosin HC (Abcam) harvested 48 h after transfection, and the knockdown and RGFP (Clontech). Fluorescent dye-labeled goat levels of luciferase were normalized and shown by ratios anti-rabbit or horse anti-mouse antibody was used as the of relative luciferase activity (RFA), which was calculated secondary antibody (Invitrogen, Molecular Probes). by the level of luciferase activity in Dox-treated (Dox-on) Positive results were examined and analyzed at 100 or mirPS cells divided by that of untreated (Dox-off) 200 magnification under a fluorescent 80i microscopic mirPS cells. Mir-434-expressing cells generated by quantitation system with a Metamorph imaging program electroporating hHFCs with pTet-On-tTS-miR434-5p (Nikon). were served as a negative control. Bisulfite DNA sequencing Northern blot analysis TM Genomic DNAs were isolated from about two million Total RNAs (10 mg) were isolated with a mirVana cells using a DNA isolation kit (Roche) and 1 mg of the miRNA isolation kit (Ambion, Austin, TX, USA), isolated DNAs were further treated with bisulfite fractionated by either 15% TBE-urea polyacrylamide gel (CpGenome DNA modification kit, Chemicon, or 3.5% low melting point agarose gel electrophoresis, and Temecula, CA, USA), according to the manufacturers’ electroblotted onto a nylon membrane. Detection of suggestions. Meanwhile, 2 mg of untreated DNAs were mir-302 was performed with a locked nucleic acid digested with a CCGG-cutting restriction enzyme HpaII [LNA]-DNA probe (5 -[TCACTGAAAC]ATGGAAGC and then analyzed by 1% agarose gel electrophoresis to ACTTA-3 ), while probes for other gene detection were determine genome-wide demethylation. The treatment synthesized and listed in Supplementary Table S2. All with bisulfite converted all unmethylated cytosine to probes were purified by high-performance liquid chroma- uracil, while methylated cytosine remained as cytosine. tography and tail labeled with terminal transferase (20 U) For bisulfite DNA sequencing analyses, we amplified the for 20 min in the presence of [ P]-dATP (>3000 Ci/mM, promoter regions of Oct3/4 and Nanog with PCR. Primers Amersham International, Arlington Heights, IL, USA). included 5 -GAGGCTGGAGCAGAAGGATTGCTTTG 0 0 G-3 and 5 -CCCTCCTGAC CCATCACCTCCACCAC Anti-miRNA oligonucleotide transfection 0 0 C-3 for Oct3/4, and 5 -TGGTTAGGTTGGTTTTAAA For mir-302 knockdown assay, we encapsulated the above 0 0 T TTTTG-3 and 5 -AACCCACCCTTATAAATTCTC anti-mir-302 [LNA]-DNA probe (2–10 mg/ml) with a AATTA-3 for Nanog (1). The bisulfite-modified DNAs FuGENE HD reagent (Roche, Indianapolis, IA, USA) (50 ng) were first mixed with the primers (total 100 pmol) and applied to cell cultures at 30–40% confluency. Cell in 1 PCR buffer, heated to 94 C for 2 min, and immedi- culture medium was refreshed 12-h post-treatment. The ately cooled on ice. Next, 25 cycles of PCR were per- mir-302-knockdown rate was determined by northern formed as follows: 94 C for 1 min and 70 C for 3 min, blot analysis at 24-h post-treatment. using an Expand High Fidelity PCR kit (Roche). The amplified DNA product with a correct size was further Western blot analysis fractionized by 3% agarose gel electrophoresis, purified Western blotting was performed as reported (6). Major with a gel extraction filter (Qiagen), and then used in primary antibodies in this study included Oct3/4 (Santa DNA sequencing. A detailed profile of the DNA methy- Cruz), SSEA-1 (Santa Cruz), SSEA-3 (Santa Cruz), lation sites was generated by comparing the unchanged SSEA-4 (Santa Cruz), Sox2 (Santa Cruz), Nanog (Santa cytosine in the converted DNA sequence to the unconvert- Cruz), Klf4 (Santa Cruz), Lin28 (Abcam), UTF1 ed one. 1058 Nucleic Acids Research, 2011, Vol. 39, No. 3 Microarray analysis cells were cultivated in the feeder-free mirPS cell cultural condition with addition of 0.05 mM SB216763. Microarray analyses were performed as reported earlier (6). Human genome GeneChip U133 plus 2.0 arrays Nuclear transfer (Affymetrix, Santa Clara, CA, USA) were used to detect MirPS cell cytoplasm with different concentrations of the alterations of over 47 000 human gene expression mir-302 was prepared by varying in Dox treatments patterns in tested cells, while miRNA microarray (from 1.0, 5.0, 10.0 to 20.0 mg/ml). The concentrations of analyses were done by LC Sciences (San Diego, CA, cytoplasmic mir-302 were confirmed by northern blot USA). Total RNAs from each tested sample were TM analysis. Hybrids [mirPS-nuclear transfer (NT) cells] of isolated using a mirVana miRNA isolation kit mirPS cell cytoplasm and hFB nuclei were generated (Ambion), following the manufacturer’s suggestion. The using a set of MO-188NE 3D hydraulic fine/coarse purity and quantity of isolated RNAs were assessed using micromanipulators and micro-injectors/holders under a 1% formaldehyde–agarose gel electrophoresis and spec- TE2000 inverted microscopic system (Nikon). The trophotometer measurement (Bio-Rad). The sample mirPS-NT cells were then grown in knockout DMEM/ signals were normalized using the total average difference F-12 medium (Invitrogen) supplemented with 20% between perfectly matched probes and mismatched knockout serum, 1% MEM non-essential amino acids, probes. Alterations of genome-wide gene expression 100 mM b-mercaptoethanol, 1 mM GlutaMax, 1 mM patterns were analyzed using Affymetrix Microarray TM sodium pyruvate, 10 ng/ml bFGF, 100 IU/ml penicillin/ Suite version 5.0, Expression Console version 1.1.1 100 mg/ml streptomycin, 0.1 mM A83-01 and 0.1 mM (Affymetrix) and Genesprings (Silicon Genetics) soft- valproic acid, at 37 C under 5% CO . wares. Changes in gene expression rates >1-fold were con- sidered as positive differential genes. For gene clustering, a DNA fingerprinting plug-in program Genetrix (Epicenter Software) was used in conjunction with the Affymetrix softwares. Signals of PCR was used to amplify the variable numbers of tandem the sample were normalized with the internal housekeep- repeats (VNTR) of the D1S80 locus on human chromo- ing control average in each microarray. some 1. Genomic DNAs were isolated with a DNA isola- tion kit (Roche) and 50 ng of the DNAs were amplified by PCR with primers 5 -GAAACTGGCCTCCAAACACT Implantation and teratoma formation 0 0 GCCCGCCG-3 and 5 -GTCTTGTTGGAGATGCACG Approximately 5–10 mirPS or mirPS-NT cell-derived T GCCCCTTGC-3 (50 pmol each). PCR was performed embryoid bodies (4- to 16-cell-stage) were suspended in as follows: one cycle at 94 C for 4 min and then 30 cycles 50 ml of a mixture of DMEM and Matrigel (2:1), through 94 C for 30 s, 68 C for 30 s and 72 C for 2 min, followed by implantation into the uterus of a 6-week-old using the Expand High Fidelity PCR kit (Roche). The female pseudopregnant immunocompromised SCID-beige amplified DNAs were further fractionized by 2.5% mouse. The pseudopregnant mice were prepared by agarose gel electrophoresis. intraperitoneal injection of 1 IU human menopausal go- nadotrophin for 2 days and then human chorionic go- Statistic analysis nadotrophin (hCG) for one more day. The cells and Any change over 75% of signal intensity in the analyses of mice were not treated with Dox before or after implant- immunostaining, western blotting and northern blotting ation. The mice were anesthetized with 2.5% Avertin was considered as a positive result, which in turn was solution, 0.4 ml per mouse during implantation. analyzed and presented as mean ± SE. Statistical Xenografted masses were monitored 3–4 weeks after the analysis of data was performed by one-way ANOVA. implantation or when the sizes were grown to >100 mm . When main effects were significant, the Dunnett’s Cysts/teratomas were dissected and the volumes were post-hoc test was used to identify the groups that calculated using the formula (length  width )/2. Cyst/ differed significantly from the controls. For pairwise com- teratoma lesions were counted, weighed and subjected to parison between two treatment groups, the two-tailed further histological analysis. Formation of teratoma-like student t-test was used. For experiments involving more tissue cysts was usually observed at 2.5-week than two treatment groups, ANOVA was performed post-implantation. followed by a post hoc multiple range test. Probability values of P< 0.05 was considered significant. All P-values were determined from two-tailed tests. Microinjection Microinjection was performed on the nuclei of cells plated 8–12 h before on a collagen-coated glass coverslip. Cells RESULTS were microinjected using a set of Eppendorf microinjector Mir-302-induced SCR is a dose-dependent mechanism and MO-188NE 3D hydraulic micromanipulators involving AOF2 suppression and Oct3/4–Sox2–Nanog mounted on a Nikon TE2000 microscope system with co-activation duration and pressure settings of 0.5 s and 30 hectopascals, respectively. The microinjection buffer contained 10 mg/ml Mir-302-mediated gene silencing is a dose-dependent AOF2, 2 mg/ml fluorescein (FITC)-labeled dextran dye, reaction due to its mismatched targeting. Following an 10 mMNaH PO and 70 mM KCl, pH 7.2. After injection, increase of Dox concentration up to 10 mM, we observed 2 4 Nucleic Acids Research, 2011, Vol. 39, No. 3 1059 that transcription of mir-302 and its RGFP marker gene AOF2, MECP1-p66 and MECP2 (Figure 1C). Western was proportionally elevated, while expressions of blot analysis showed that hHFCs expressed abundant melanocytic markers tyrosinase-related protein 1 (TRP1) AOF2 and MECP1/2 but scarce AOF1, suggesting that and keratin 16 were reduced in all transfected cells silencing of AOF2 plays a major role in hHFC (Figure 1C). Accordingly, core reprogramming factors reprogramming. Deficiency of either AOF1 or AOF2 is Oct3/4, Sox2 and Nanog were all strongly stimulated by sufficient to cause global demethylation (13,14). Our a threshold of Dox 7.5 mM, indicating a dose-dependent previous studies have also demonstrated that mir-302 correlation between mir-302 concentration and Oct3/4– mediated MECP1/2 silencing enhances global Sox2–Nanog co-activation. This concurrent Oct3/4– demethylation (6,15). Therefore, mir-302 induces SCR Sox2–Nanog gene activation is an essential step for iPS through a combinatory mechanism involving both Oct3/ cell induction (2,3). Time-course measurement of 4–Sox2–Nanog co-activation and AOF1/2 suppression, reprogramming hHFCs to mirPS cells further showed while further MECP1/2 silencing enhances the result of that the Oct3/4–Sox2–Nanog co-activation was most AOF1/2-associated global demethylation. prominent after Days 5–6, the time frame required for initiating SCR (Figure 1D). Almost no cell division was Mir-302 silences AOF2 to trigger DNMT1 deficiency detected during the first 3–4 days after treatment of and global demethylation 7.5 mM Dox. In this dose-dependent SCR process To validate the interactions between mir-302 and its epi- (Figure 1C), we discovered three critical mir-302 concen- genetic targets, we used a luciferase 3 -untranslated region trations. First, at the treatment of 5 mM Dox, the induced (3 -UTR) reporter assay (Figure 3A), which showed that mir-302 level was closely similar to that found in hES H1 treatments with various mir-302 concentrations resulted in and H9 cells, but not sufficient to reprogram hHFCs. different epigenetic patterns in mirPS cells. In the presence Second, at 7.5 mM Dox, mir-302 concentration was of 10 mM Dox, mir-302 effectively bound to the target sites raised to over 30% higher than that of H1 and H9 cells of AOF1, AOF2, MECP1-p66 and MECP2 mRNAs and and began inducing co-expression of the core successfully silenced over 80% of the reporter luciferase reprogramming factors Oct3/4–Sox2–Nanog and Lin28 expression in all targets (Figure 3B). Suppression of the (Figure 1E). Last, at 10 mM Dox, the stimulated mir-302 real target genes in mirPS cells was also confirmed concentration was optimal for reprogramming hHFCs to by western blot analyses, consistent with the results of mirPS cells and forming embryoid bodies. Treatment of the luciferase 3 -UTR reporter assay (Figure 3C). >12 mM Dox caused severe cell cycle arrest at the G0/G1 Accordingly, we detected a significant decrease of phase, which ultimately hindered mirPS cell formation. DNMT1 (loss over 87 ± 3% of the level in hHFCs) and Fluorescence-activated cell sorter (FACS) analysis increase of H3K4 di-/tri-methylation (H3K4me2/3; gain further confirmed that 91% of the transfected cells 103 ± 17%) in response to the silencing of AOF2 (loss treated with 10 mM Dox expressed over twice more 93 ± 2%) by mir-302. Previous studies have demonstrated Oct3/4 than those treated with only 5 mM Dox that AOF2 is required for stabilizing DNA (Figure 1F). For the first time, this study established the methyltransferase 1 (DNMT1) and preserving its activity threshold concentration of a hES-specific miRNA for on the maintenance of global DNA methylation (14), SCR. The mechanism underlying this SCR process is con- whereas active global demethylation can promote Oct3/ sistent with the reported iPS cell induction using ectopic 4–Nanog activation in early mouse embryos and mouse– transfection of Oct3/4–Sox2–Nanog–Lin28 factors (2). human fused heterokaryons (17,18). Conceivably, the Following the increase of Dox-induced mir-302 expres- deficiency of DNMT1 caused mirPS cell genomes to be sion, we also observed a significant change of mirPS cell susceptible to a certain demethylation activity. This colony morphology from hES-cell-like 2D aggregations demethylation effect was further enhanced by (at 7.5 mM Dox) to embryoid body (EB)-like three- co-suppression of MECP1/2 (loss 96 ± 1% and dimensional colonies (at 10 mM Dox), which highly 81 ± 5%, respectively) (6) and eventually led to global resembled early mammalian zygotes (Figure 2A). After demethylation and Oct3/4–Nanog activation (Figure 4A cultivated on a low attachment (overhanging) surface, and B). On the flipside, a lower mir-302 concentration these EB-like colonies formed sphere-like EBs strongly induced by 5 mM Dox failed to trigger any significant and homogeneously expressing pluripotent Oct3/4–Sox2– Nanog markers as well as other standard hES markers silencing effect on either the target sites of the reporter (Figure 2B and C), indicating that their undifferentiated gene or the targeted epigenetic genes, except status was well maintained by the induced mir-302. A MECP1-p66, indicating that mir-302-induced global similar undifferentiated status is often observed in mam- demethylation is dose dependent and requires malian zygotes before the morula stage, but rarely in co-suppression of AOF1/2 and MECP1/2 (Figure 3B three/four-factor-induced iPS cell colonies. This is and D). probably due to a lower mir-302 level expressed in both Methylation site-sensitive HpaII digestion assays con- human iPS and late blastocyst-derived hES cells (9). From firmed that mirPS cell genomes isolated from the group morphology and hES-specific gene expression, there was treated with 10 mM Dox underwent global demethylation no significant difference between mirPS cell colonies and (Figure 4A). When further assessing the methylation mirPS cell-derived EBs. Accompanying the Oct3/4–Sox2– patterns of Oct3/4 and Nanog promoters with bisulfite Nanog co-activation, we also found a proportional loss of DNA sequencing, we observed that both promoters were multiple epigenetic regulator expression, including AOF1, almost completely demethylated in a fashion resembling 1060 Nucleic Acids Research, 2011, Vol. 39, No. 3 Figure 2. Morphological and genetic changes of mirPS cell properties in response to different mir-302 concentrations. (A) Morphological changes of mirPS cell colonies in response to Dox-induced mir-302 expression. Mir-434-expressing cells generated by electroporating hHFCs with pTet-On-tTS-miR434-5p were served as a negative control. A morphological comparison between a morula-staged rat embryo and a mirPS cell colony at 32–64 cell stage was shown in the lower panel. (B) Fluorescent microscope examination showing strong and homogeneous expression of the core pluripotent stem cell markers Oct3/4, Sox2 and Nanog in a mirPS-derived embryoid body. BF-DIC, bright field with differential interference contrast. (C) Western blotting confirming the expression patterns of major hES-specific marker proteins in completely (10 mM Dox) and partially (5 mM Dox) reprogrammed mirPS cells compared to those found in hES H1 and H9 cells (n=4, P< 0.01). Mir-302 expression was proportional to Dox concentration. hES H1 and H9 cells (Figure 4B). Similar global of red fluorescent RGFP protein (Supplementary Figure demethylation patterns have also been found in iPS cells S3A), and further selected by G418 antibiotics to ensure (2,3). In contrast, neither global demethylation nor SCR their purity. Consistent with our previous reports (6,15), was observed in the transfected cells treated with only each individual mirPS cell could grow into a homogeneous 5 mM of Dox (Figures 1C, 4A and B). We subsequently EB (Supplementary Figure S3B) and form teratoma-like evaluated this global demethylation effect in over 47 000 tissue cysts (Figure 5C) in immunocompromised human gene expression patterns using microarray analyses SCID-beige mice, containing various differentiated and revealed that approximately half of the transcriptome tissues derived from all three embryonic germ layers, expression in mirPS cells was changed from a somatic ectoderm, mesoderm and definitive endoderm hHFC mode to a uniform hES-like expression pattern (Figure 5D). These results confirmed the hES cell-like sharing over 91% similarity to that of H1/H9 cells pluripotency of mirPS cells. (Figure 4C). Hierarchical clustering of the top 30 most Next, we evaluated the role of mir-302–targeted AOF2 differentially expressed hES-specific genes and epigenetic silencing in the process of SCR using recombinant AOF2 regulators in microarrays further showed an extremely protein and its inhibitor tranylcypromine (Figure 5A). high correlation between reprogrammed mirPS and hES Under conditions of 10 mM Dox stimulation, the success H1/H9 cells (Figure 4D). Thus, we conclude that mir-302 rate of complete SCR approached 100% (477/480). After regulates the epigenetic reprogramming of genomic that, the reprogrammed mirPS cells could be cultivated to methylation patterns through co-suppression of AOF2 over 26–28 passages under our feeder-free cultural condi- and DNMT1 during SCR. tion in the absence of Dox and GSK inhibitor (‘Materials and Methods’ section), indicating the completion of SCR. However, when a GSK inhibitor was presented in the Mir-302-induced SCR is reversible and dependent on cultural medium without sufficient Dox stimulation, AOF2–DNMT1 suppression majority of mirPS cells differentiated into neuron-like cells. Given that glycogen synthase kinase 3 (GSK3) is a After SCR completion (5–6 days post-transfection of mir-302), reprogrammed mirPS cells were visually distin- major gatekeeper for embryonic neural induction (19), this guished by their sphere-shape morphology and expression result suggests that GSK inhibitor can induce the Nucleic Acids Research, 2011, Vol. 39, No. 3 1061 Figure 3. Analysis of mir-302-induced silencing effects on targeted epigenetic genes and global demethylation. (A) Constructs of the luciferase 3 -UTR reporter genes, which carry either two normal (T1+T2) or two mutant (M1+M2), or a mixture of both (T1+M2 or M1+T2), mir-302 0 0 target sites in the 3 -UTR. The mutant sites contained a mismatched TCC motif in place of the uniform 3 -CTT end of the normal target sites. (B) Effects of Dox-induced mir-302 on the luciferase expression (n=5, P< 0.01). (C) and (D) Western blot analyses showing the changes of major mir-302-targeted epigenetic gene expression induced by high (10 mM Dox) and low (5 mM Dox) mir-302 concentrations in mirPS cells compared to those found in hES H1 and H9 cells (n=4, P< 0.01). ectodermic differentiation of mirPS cells. Notably, these not cause any effect in all tests. Therefore, SCR is a re- differentiated cells could be reprogrammed back to mirPS versible mechanism dependent on mir-302–mediated cells after re-supplementation of 7.5 mM Dox in the AOF2 silencing. cultural medium. The same neuronal differentiation Following this reversible SCR process, we measured the could also be triggered by treatment of 10 mg/ml corresponding changes of global demethylation, AOF2– DNMT1 co-suppression and Oct3/4–Sox2–Nanog anti-mir-302 LNA–DNA oligonucleotides in mirPS cells (Figure 5A and B), indicating the critical role of mir-302 co-activation in all tested mirPS and differentiated cells. concentration in maintaining pluripotent cell stemness. Bisulfite DNA sequencing assays revealed that, under the Alternatively, while being microinjected with recombinant GSK inhibitor influence, the global demethylation of AOF2 into the cell nuclei, the differentiated cells failed to Oct3/4 and Nanog promoters were observed only in mirPS cells treated with 7.5 mM Dox (mirPS+10 mM be reprogrammed back to mirPS cells even after 10 mM Dox stimulation, demonstrating the inhibitory role of Dox) and the re-reprogrammed cells co-treated with AOF2 in the mechanism of mir-302-induced SCR. 10 mM Dox, AOF2 and tranylcypromine (mirPS+10 mM Further treatment of tranylcypromine removed the Dox–AOF2), whereas the differentiated cells treated blockade of AOF2 in the process of SCR and, in conjunc- with 10 mM Dox and AOF2 (mirPS+10 mM Dox+AOF2) tion with the stimulation of 10 mM Dox, could again re- failed to complete the demethylation (Figure 5E). program the differentiated cells to mirPS cells with all These resulting global demethylation patterns per- hES-like properties. Microinjection of blank buffer did fectly matched the morphological changes observed in 1062 Nucleic Acids Research, 2011, Vol. 39, No. 3 Figure 4. Mir-302-induced genomic DNA demethylation and global gene expression alteration. (A) HpaII cleavage showing the vast loss of global CpG methylation, identified by increased presence of smaller DNA fragments, at a genome-wide scale in mirPS cells treated with 10 mM but not 5 mM Dox. (B) Bisulfite DNA sequencing in the promoter regions of Oct3/4 and Nanog. Methylation maps show the most frequently observed patterns. Black and white circles indicate the methylated and unmethylated cytosine sites, respectively. (C) Analysis of global gene expression patterns before and after global demethylation, using human genome GeneChip U133 plus 2.0 arrays (Affymetrix; n=3, P< 0.01–0.05). (D) Microarray hierarchical clustering of the top 30 most differentially expressed hES-specific genes and epigenetic regulators between somatic hHFCs, reprogrammed mirPS-hHFCs (mirPS+10 mM Dox) and hES H1/H9 cells. Figure 5A. In addition, western blot assays also cells (Figure 5F), confirming that mir-302–targeted showed that the simultaneous AOF2–DNMT1 co- AOF2 silencing is required for global demethylation suppression and Oct3/4–Sox2–Nanog co-activation only during SCR. In accordance to this counteraction occurred in the mirPS+10 mM Dox and mirPS+10 mM between mir-302 and AOF2, we identified that SCR is a Dox–AOF2 cells but not in mirPS+10 mM Dox+AOF2 reversible process. Nucleic Acids Research, 2011, Vol. 39, No. 3 1063 Figure 5. Reversible SCR process regulated by mir-302–mediated AOF2 silencing. (A) Reversible interchange between hES-like mirPS and neuron-like differentiated cells as a result of mir-302 and AOF2 counteraction. (B) Northern blots showing the mir-302 knockdown (65%) by 10 mg/ml anti-mir-302 LNA–DNA oligonucleotides. (C) Typical morphology of a mirPS EB-derived teratoma-like tissue cyst (2.5 weeks post-implantation). (D) Pluripotent differentiation of mirPS-hHFCs into teratoma-like cysts containing various tissues derived from all three em- bryonic germ layers. Immunostaining the cross-sections of mirPS EB-derived tissue cysts showed the positive results of specific differentiation markers, including neuronal b-tubulin isotype-III (Tuj1; green) and/or ATP-binding cassette sub-family A member 2 (ABCA2; blue), cartilage collagen type II a-1 (COL2A1; green), muscle-associated M-cadherin (green), gut-epithelium-related mucin 2 (MUC2; green), and glandular epithelium-related CD13 (green). Photographs were taken with Nikon TE2000 microscopic system at 200 magnification. (E) Global demethylation patterns in Oct3/4 and Nanog promoters corresponding to the result of (A). (F) Changes of Oct3/4, Sox2, Nanog, AOF2 and DNMT1 expression in response to the results of (A) and (E). Global genomic DNA demethylation does not require any developed. Conversely, transfer of mirPS nuclei into nuclear effector hFB cytoplasm also failed to form any viable cells. Thus, the reprogramming ability of mirPS cells is Mir-302 is a cytoplasmic effector. To further rule out the preserved in the cytoplasm rather than nucleus. These possible involvement of any nuclear factor from the hFB nucleus-transferred mirPS (mirPS-NT) cells mir-302–mediated global demethylation, we transferred preserved all the same traits as mirPS cells in terms of human adult fibroblast (hFB) nuclei into the cytoplasm global demethylation (Figure 6B and C), Oct3/4–Sox2– of mirPS cells pre-treated with 10 mM Dox. Most (93%) Nanog co-activation and AOF2–DNMT1 co-suppression of the hybrid cells successfully formed mirPS-like iPS cells (Figure 6D), and in vivo pluripotency (Figure 6F and and embryoid bodies (Figure 6A). When cytoplasm Supplementary Figure 4). Using DNA fingerprinting in derived from the mirPS cells pre-treated with <7.5 mM human D1S80 alleles, the mirPS-NT-derived teratoma Dox, neither mirPS-like cell nor embryoid body was cysts were confirmed to be originated from human cell 1064 Nucleic Acids Research, 2011, Vol. 39, No. 3 Figure 6. Assessment of the reprogramming ability preserved in mirPS cytoplasm. (A) Embryoid body derived from a single mirPS–hFB hybrid (mirPS-NT) cell after limiting dilution. Scale bars = 100 mm. (B) HpaII cleavage showing global demethylation (smaller DNA fragments) in reprogrammed mirPS-NT cells compared to that in the somatic hHFC and hFB cells. (C) Bisulfite DNA sequencing in the Oct3/4 and Nanog promoters, showing CpG methylation maps. Black and white circles indicate the methylated and unmethylated cytosine sites, respectively. (D) Northern and western blot analyses of mir-302, AOF2–DNMT1 and Oct3/4–Sox2–Nanog expression patterns in mirPS-NT cells compared to those in the somatic hFB and reprogrammed mirPS (mirPS+Dox) cells. (E) DNA fingerprinting of the variable numbers of tandem repeats (VNTRs) in the D1S80 locus of human genome, confirming that mirPS-NT, mirPS-hHFC and hFB cells all originated from the same human source not mouse. (F) Pluripotent tissue types found in teratoma-like cysts derived from mirPS-NT cells. Markers (green): keratin 14, actin a-2 in smooth muscle aorta (a-SMS, ACTA2), CD105 and MUC2. H&E, histological staining with hematoxylin and eosin. BF, bright field. (G) Proposed mechanism of mir-302– induced SCR. sources rather than mouse cells (Figure 6E). In view of the stemness and pluripotency of the reprogrammed these hES-like properties inherent in mirPS-NT cells, we somatic cells (6,15). In this study, we further revealed conclude that mir-302 is able to not only induce SCR but the mechanism involved (Figure 6G), showing that also preserve this reprogramming ability in the cytoplasm mir-302 significantly decreases AOF2 and DNMT1 of mirPS cells. activities and, in conjunction with the co-suppression of MECP1/2, results in global genomic DNA demethylation and H3K4 modification. Subsequently, these epigenetic DISCUSSION reprogramming events induce hES-specific gene expres- Our previous study established that mir-302 functions not sion, in particular Oct3/4, Sox2 and Nanog, which in only to improve the efficiency of SCR but also to enhance turn further stimulates mir-302 expression, and so on to Nucleic Acids Research, 2011, Vol. 39, No. 3 1065 et al. (2007) Induced pluripotent stem cell lines derived from form a positive feedback cycle essential for maintaining human somatic cells. Science, 318, 1917–1920. SCR. When compared to the previous three/four-factor 3. Wernig,M., Meissner,A., Foreman,R., Brambrink,T., Ku,M., reprogramming methods, our approach adopts a novel Hochedlinger,K., Bernstein,B.E. and Jaenisch,R. (2007) In vitro entry point in the same mechanistic cycle to complete reprogramming of fibroblasts into a pluripotent ES-cell-like state. SCR. Also, without introducing the oncogenic Klf4 and Nature, 448, 318–324. 4. Marson,A., Levine,S.S., Cole,M.F., Frampton,G.M., c-Myc genes, this new approach likely provides safer Brambrink,T., Johnstone,S., Guenther,M.G., Johnston,W.K., in vivo applications. From this novel SCR mechanism, Wernig,M., Newman,J. et al. (2008) Connecting microRNA genes we learned that the effector(s) responsible for to the core transcriptional regulatory circuitry of embryonic stem reprogramming genomic epigenetics resides in the mirPS cells. Cell, 134, 521–533. cytoplasm and can enter somatic cell nuclei after NT 5. Card,D.A., Hebbar,P.B., Li,L., Trotter,K.W., Komatsu,Y., Mishina,Y. and Archer,T.K. (2008) Oct4/Sox2-regulated miR-302 because mir-302 is a cytoplasmic effector. Thus, our targets cyclin D1 in human embryonic stem cells. Mol. Cell. Biol., findings may also help in clarifying the mechanism of 28, 6426–6438. somatic cell NT, addressing the possible role of mir-302 6. Lin,S.L., Chang,D., Chang-Lin,S., Lin,C.H., Wu,D.T.S., in nuclear reprogramming. Chen,D.T. and Ying,S.Y. (2008) Mir-302 reprograms human skin Through deciphering the interactions between mir-302 cancer cells into a pluripotent ES-cell-like state. RNA, 14, 2115–2124. and its targeted epigenetic genes, we elucidate the intricate 7. Puca,A.A., Daly,M.J., Brewster,S.J., Matise,T.C., Barrett,J., gene regulation circuitry involved in epigenetic Shea-Drinkwater,M., Kang,S., Joyce,E., Nicoli,J., Benson,E. et al. reprogramming during SCR. 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Judson,R.L., Babiarz,J.E., Venere,M. and Blelloch,R. (2009) deficiency of AOF2 and DNMT1 activities occurs most Embryonic stem cell-specific microRNAs promote induced significantly during the same embryogenesis period of pluripotency. Nat. Biotechnol., 27, 459–461. time (14). To this, our finding of mir-302-targeted AOF2 11. Lee,M.G., Wynder,C., Cooch,N. and Shiekhattar,R. (2005) An silencing connects the relationship of all these events and essential role for CoREST in nucleosomal histone 3 lysine 4 demethylation. Nature, 437, 432–435. further extends the presence of this epigenetic 12. Lee,M.G., Wynder,C., Schmidt,D.M., McCafferty,D.G. and reprogramming mechanism in iPS cells during SCR. In Shiekhattar,R. (2006) Histone H3 lysine 4 demethylation is a this extended model, cell stemness, analogous to a target of nonselective antidepressive medications. Chem. Biol., 13, fountain of youth, is inherent in our somatic cells and 563–567. 13. Ciccone,D.N., Su,H., Hevi,S., Gay,F., Lei,H., Bajko,J., Xu,G., can be activated and regulated by mir-302, providing a Li,E. and Chen,T. (2009) KDM1B is a histone H3K4 feasible means to rejuvenate cell stemness and pluripotency demethylase required to establish maternal genomic imprints. through mimicking the natural reprogramming pathway. Nature, 461, 415–418. However, our current work only reveals its partial func- 14. Wang,J., Hevi,S., Kurash,J.K., Lei,H., Gay,F., Bajko,J., Su,H., tionality. To fully utilize this profound mechanism of Sun,W., Chang,H. and Xu,G. (2009) The lysine demethylase LSD1 (KDM1) is required for maintenance of global DNA mir-302, further studies are still needed to realize its poten- methylation. Nat. Genet., 41, 125–129. tial in stem cell research and regenerative medicine. 15. Lin,S.L. and Ying,S.Y. (2008) Role of mir-302 microRNA family in stem cell pluripotency and renewal. In Ying,S.Y. (ed.), Current Perspectives in MicroRNAs. 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(2009) p66(ShcA) adaptor molecule accelerates ES cell neural induction. Mol. Cell. Neurosci., 41, 74–84. REFERENCES 20. Reik,W., Dean,W. and Walter,J. (2001) Epigenetic reprogramming in mammalian development. Science, 293, 1089–1093. 1. Takahashi,K. and Yamanaka,S. (2006) Induction of pluripotent 21. Tang,F., Kaneda,M., O’Carroll,D., Hajkova,P., Barton,S.C., stem cells from mouse embryonic and adult fibroblast cultures by Sun,Y.A., Lee,C., Tarakhovsky,A., Lao,K. and Surani,M.A. defined factors. Cell, 126, 663–676. (2007) Maternal microRNAs are essential for mouse zygotic 2. Yu,J., Vodyanik,M.A., Smuga-Otto,K., Antosiewicz-Bourget,J., development. Genes Dev., 21, 644–648. Frane,J.L., Tian,S., Nie,J., Jonsdottir,G.A., Ruotti,V., Stewart,R. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

Regulation of somatic cell reprogramming through inducible mir-302 expression

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Abstract

1054–1065 Nucleic Acids Research, 2011, Vol. 39, No. 3 Published online 24 September 2010 doi:10.1093/nar/gkq850 Regulation of somatic cell reprogramming through inducible mir-302 expression 1, 1 2 3 Shi-Lung Lin *, Donald C. Chang , Chun-Hung Lin , Shao-Yao Ying , 1 1 Davey Leu and David T. S. Wu 1 2 WJWU and LYNN Institute for Stem Cell Research, Santa Fe Springs, CA 90670, USA, Taiwan Adventist Hospital, Taipei 10556, Taiwan, ROC and Department of Cell and Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA Received June 22, 2010; Revised August 10, 2010; Accepted September 12, 2010 understanding is limited by co-transfection of three or ABSTRACT four specific transcription factors, either Oct3/4–Sox2– Global demethylation is required for early zygote Klf4–cMyc or Oct3/4–Sox2–Nanog–Lin28, to promote development to establish stem cell pluripotency, induced pluripotent stem (iPS) cell formation (1–3). yet our findings reiterate this epigenetic reprogram- Among these reprogramming factors, POU class ming event in somatic cells through ectopic intro- 5 homeobox 1 (Oct3/4) and sex determining region duction of mir-302 function. Here, we report that Y-box 2 (Sox2) are essential. Recent studies found that both Oct4 and Sox2 are also crucial for expressing induced mir-302 expression beyond 1.3-fold of the mir-302 in human embryonic stem (hES) cells (4,5). concentration in human embryonic stem (hES) H1 Mir-302 belongs to a class of small, non-coding RNAs and H9 cells led to reprogramming of human hair known as microRNAs (miRNA) that function as cyto- follicle cells (hHFCs) to induced pluripotent stem plasmic gene silencers by suppressing translation of (iPS) cells. This reprogramming mechanism func- targeted messenger RNAs (mRNA). The majority of tioned through mir-302-targeted co-suppression of mir-302-targeted genes are transcripts of developmental four epigenetic regulators, AOF2 (also known as signals and oncogenes (6); nevertheless, their interactions KDM1 or LSD1), AOF1, MECP1-p66 and MECP2. and overall functions remain unknown. The genomic Silencing AOF2 also caused DNMT1 deficiency and sequence encoding mir-302 is located in the 4q25 locus further enhanced global demethylation during of human chromosome 4, a conserved region frequently somatic cell reprogramming (SCR) of hHFCs. associated with longevity (7). In humans, mir-302 is pre- Re-supplementing AOF2 in iPS cells disrupted dominantly expressed in hES and iPS cells, but not in differentiated cells (8,9). Loss of mir-302 has been such global demethylation and induced cell differ- observed prior to hES cell differentiation and proliferation entiation. Given that both hES and iPS cells highly during early embryonic development (8). Analogously in express mir-302, our findings suggest a novel link mice, its homologous mir-291/294/295 family presents a between zygotic reprogramming and SCR, providing similar expression profile (10). Therefore, it is conceivable a regulatory mechanism responsible for global that embryonic stem cell-specific miRNAs such as mir-302 demethylation in both events. As the mechanism and mir-291/294/295 play a pivotal role in regulating cell of conventional iPS cell induction methods stemness and pluripotency, whose functions may be remains largely unknown, understanding this applied to enhance the efficiency of SCR for iPS cell microRNA (miRNA)-mediated SCR mechanism may generation. shed light on the improvements of iPS cell The initiation of SCR involves a highly coordinated DNA demethylation and histone methylation mechanism generation. that is able to alter a genome-wide scale of chromatin struc- ture and gene activity. To this, mir-302 may silence certain INTRODUCTION epigenetic regulators to affect the status of genomic DNA methylation. Utilizing high throughput analysis with Somatic cell reprogramming (SCR) requires global DNA online miRNA-target prediction programs demethylation to reset cell stemness, yet the mechanism TARGETSCAN (http://www.targetscan.org/) and underlying this epigenetic event is unclear. Current *To whom correspondence should be addressed. Tel: +1 323 442 1856; Fax: +1 323 442 3158; Email: shilungl@mirps.org The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2011, Vol. 39, No. 3 1055 PICTAR-VERT (http://pictar.mdc-berlin.de/), we found mechanism, we investigated the functional role of that lysine-specific histone demethylases (namely AOF, mir-302 in human SCR. KDM or LSD) and methyl CpG-binding proteins (MECP) are two major groups of the epigenetic regula- tors targeted by mir-302. AOF contains two familial MATERIALS AND METHODS members AOF1 and AOF2, both of which function to Cell culture and electroporation repress gene transcription by demethylating histone 3 on lysine 4 (H3K4) (11–13). Inhibition of AOF2 by its an- hHFCs were isolated and dissociated from a minimum of tagonist tranylcypromine augments H3K4 methylation and two hair dermal papillae by 4 mg/ml collagenase I diges- stimulates Oct3/4 expression in embryonal carcinoma cells tion for 45 min in fresh RPMI 1640 medium supplemented (11,12). In transgenic knockout mice, loss of either AOF1 with 20% FBS. For culturing melanocytes, the isolated or AOF2 substantially increases H3K4 methylation cells were cultivated in Medium 254 with the addition of (13,14). AOF1-knockout mice demonstrate normal body human melanocytes growth supplement-2 (HMGS-2, development but fail to set up de novo DNA methylation Invitrogen, Carlsbad, CA, USA) in the absence of anti- imprints during oogenesis (13), while AOF2 deficiency biotics at 37 C under 5% CO . For culturing fibroblasts causes embryonic lethality due to a progressive loss of (hFB), the isolated cells were cultivated in Medium 106 genomic DNA methylation and lack of global cell differ- with the addition of low serum growth supplement (LSGS, entiation (14). As a result, silencing of both AOF1 and Invitrogen) in the absence of antibiotics at 37 C under 5% AOF2 is likely to be sufficient in inducing global DNA CO . Cultures were passaged at 70–80% confluency by demethylation. Our recent studies further showed that exposing cells to trypsin/EDTA solution for 1 min and ectopic expression of the whole mir-302 familial cluster rinsing once with phenol red-free DMEM medium induced not only global demethylation via silencing (Invitrogen), and the detached cells were replated at 1:10 MECP1-p66 and MECP2 but also the co-expression of dilution in fresh Medium 254 with HMGS-2 or Medium Oct3/4–Sox2–Nanog genes, which led to the 106 with LSGS, respectively. For electroporation, a reprogramming of both normal and cancerous human mixture of pTet-On-tTS-mir302s (10 mg) and pTet-On- skin cells into a hES-like pluripotent state (6,15). A Adv-Neo() (50 mg) was added with the isolated hHFCs similar mir-302 transfection approach was also shown to (20 000–50 000) in a hypoosmolar buffer (200 ml; increase Oct3/4–Nanog co-expression by 2-fold in hES cells Eppendorf, Westbury, NY, USA) and electroporation (16). Taken together, these findings suggest that mir-302 was performed using Eppendorf Multiporator at 300– may concurrently suppress AOF1/2 and MECP1/2 to 400 V for 150 ms. The electroporated cells were first induce global demethylation and to activate the grown in phenol red-free DMEM medium (Invitrogen) co-expression of hES-specific genes required for SCR. supplemented with 20% knockout serum, 1% MEM However, a critical link between mir-302 and AOF1/2 is non-essential amino acids, 10 ng/ml bFGF, 1 mM still missing. GlutaMax and 1 mM sodium pyruvate, for 24 h at 37 C MiRNA concentration determines the efficiency of its under 5% CO . Then, 850 mg/ml G418 and >3.75 mg/ml gene targeting. To assess this dose-dependent mir-302 doxycycline (Dox) were added into the medium. The effect on AOF1/2 silencing and global demethylation, we medium and antibiotics were refreshed daily for 3–5 adopted a novel inducible pTet-On-tTS-miR302s days till the cells expressed strong red fluorescent RGFP. expression vector (Figure 1A) in conjunction with electro- Next, the individual red fluorescent cell (mirPS) was moni- poration delivery to reprogram normal human hair follicle tored under a TE2000 inverted microscopic system cells (hHFCs) isolated from the dermal papilla region. (Nikon, Melville, NY, USA) and separately collected Skin hHFCs were chosen due to their accessibility and into a 96 well, using MO-188NE 3D micromanipulators abundance in differentiated mesenchymal lineage cells, (Nikon). In the absence of Dox, the mirPS cells were such as keratinocytes, melanocytes and fibroblasts. We grown and passaged in knockout DMEM/F-12 medium also mimicked the natural mir-302 expression pattern by (Invitrogen) supplemented with 20% knockout serum, expressing all four native mir-302 familial members, 1% MEM non-essential amino acids, 100 mM mir-302a, b, c and d, in one intact intronic cluster ß-mercaptoethanol, 1 mM GlutaMax, 1 mM sodium (mir-302s; Supplementary Figure S1A) (8). Upon doxycyc- pyruvate, 10 ng/ml bFGF, 100 IU/ml penicillin/ line (Dox) stimulation, the biogenesis of mir-302 followed 100 mg/ml streptomycin/ 250 mg/ml G418, 0.1 mM A83-01 the natural intronic miRNA pathway, in which mir-302 and 0.1 mM valproic acid (Stemgent, San Diego, CA, was transcribed with a gene encoded for red fluorescent USA), at 37 C under 5% CO . Alternatively, in the protein (RGFP) and then further spliced into individual presence of Dox (2.5–5 mg/ml; Sigma-Aldrich, St. Louis, mir-302 members by spliceosomal components and cyto- MO, USA), the mirPS cells were cultivated and passaged plasmic RNaseIII Dicers (Figure 1B) (6). MiRNA micro- in the same feeder-free cultural condition with addition of array analysis revealed that all mir-302 members except 0.05 mM GSK inhibitor SB216763 (Stemgent). Addition of mir-302b* were efficiently expressed in transfected hHFCs GSK inhibitor could facilitate mirPS cell proliferation but after Dox stimulation (Supplementary Figure 1B). The with a slight tendency to cause neural differentiation. For procedure for generating mir-302-induced pluripotent neural cell induction, the mirPS cells were grown in the stem (mirPS) cells is summarized in Supplementary above feeder-free cultural condition with 0.05 mM Figure 2. Through this inducible mir-302 expression SB216763 but no Dox. 1056 Nucleic Acids Research, 2011, Vol. 39, No. 3 Figure 1. Inducible mir-302 expression and its effect on hHFC reprogramming. (A) Construct of the Dox-inducible pTet-On-tTS-miR302s vector. (B) Mechanism of intronic mir-302 biogenesis. (C) Northern and western blot analyses of the dose-dependent mir-302 effect on the expression of core reprogramming factors and melanocytic marker genes (n=5, P< 0.01). (D) Northern blot analysis of the time-course mir-302 effect on Oct3/4– Sox2–Nanog co-activation and SCR (n=5, P< 0.01). (E) Northern blot analysis of hES marker gene expression induced by a high mir-302 concentration in mirPS cells compared to those in hES WA01-H1 (H1) and WA09-H9 (H9) cells (n=5, P< 0.01). (F) FACS flow cytometry sorting of positively reprogrammed cells (mirPS-hHFC) with dual expression of mir-302 maker RGFP and hES cell marker Oct3/4. Construction of the RGFP transgene encoding mir-302s mixture with MluI/Pvu1 restriction enzymes at 37 C for (RGFP-mir-302s) 4 h. The digested mixture was collected with a gel extrac- tion filter in 30 ml of ddH O and ligated together with T 2 4 The mir-302 familial cluster (mir-302s) was generated as DNA ligase at 8 C for 16 h. This formed the RGFP-mir- reported earlier (6). The mir-302s consists of four parts, 302s transgene, which could be further cleaved out of the including precursor miRNAs (pre-miRNAs) of mir-302a, vector with XhoI/HindIII digestion. b, c and d. Synthetic oligonucleotides (Sigma-Genosys, St Louis, MO, USA) were listed in Supplementary Table S1. Construction of the inducible pTet-On-tTS-mir302s vector For expression, we mixed an equal amount (1:1) of the mir-302s cluster and a pre-made SpRNAi-RGFP transgene We first modified a Dox-inducible pSingle-tTS-shRNA vector from our previous study (6), and then digested the vector (Clontech, Palo Alto, CA, USA) by replacing its Nucleic Acids Research, 2011, Vol. 39, No. 3 1057 U6 promoter with a TRE-CMV promoter isolated from a (Abcam), DNMT1 (Abcam), HDAC2 (Abcam), pTRE-Tight plasmid (Clontech). Then, the modified H3K4me2/3 (Abcam), AID/AICDA (Santa Cruz), vector was digested with XhoI/HindIII restriction AOF1 (Abcam), AOF2 (Sigma), MECP1-p66 (Millipore, enzymes at 37 C for 4 h, purified by a gel extraction Billerica, MA), MECP2 (Abcam), TRP1 (Santa Cruz), filter in 30 ml of ddH O, and then mixed and ligated with keratin 16 (Abcam), b-actin (Chemicon, Temecula, CA, the XhoI/HindIII-cleaved RGFP-mir-302s transgene (1:1) USA) and RGFP (Clontech). with T DNA ligase at 8 C for 16 h. This formed the in- ducible pTet-On-tTS-mir302s vector. Luciferase 3 -UTR reporter assay Luciferase assays were performed using the pMir-Report Immunostaining assay miRNA Expression Reporter Vector System (Ambion), Embedding, sectioning and immunostaining tissue according to the manufacturer’s instruction. The mir-302 samples were performed as reported (6). Primary target sites (normal and/or mutant) were inserted in the antibodies included Tuj1 (Abcam Inc., Cambridge, MA, 3 -UTR cloning site of the pMir-Report Luciferase USA), ABCA2 (Santa Cruz Biotechnology, Santa Cruz, Reporter vector. The two target sites were synthesized CA, USA), COL1A1 (Santa Cruz), COL2A1 (Santa and separated by 12-CAGT-repeats. Another pMir- Cruz), M-cadherin (Abcam), MUC2 (Abcam), MUC5B Report ß-gal Control vector was used as a no reporter (Abcam), CD13 (Santa Cruz), CD105 (Abcam), keratin control. We transfected 200 ng of the reporter vector 14 (Abcam), keratin 16 (Abcam), Mitf (Abcam), TRP1 into 50 000 mirPS cells in the absence or presence of (Santa Cruz), a-SMA (Abcam), ED-A fibronectin (Santa Dox treatment, using a FuGene HD reagent (Roche) fol- Cruz), GAD67 (Abcam), EN4 (Abcam), SC (Abcam), lowing the manufacturer’s suggestion. Cell lysates were BMP11 (Abcam), cTnT (Abcam), myosin HC (Abcam) harvested 48 h after transfection, and the knockdown and RGFP (Clontech). Fluorescent dye-labeled goat levels of luciferase were normalized and shown by ratios anti-rabbit or horse anti-mouse antibody was used as the of relative luciferase activity (RFA), which was calculated secondary antibody (Invitrogen, Molecular Probes). by the level of luciferase activity in Dox-treated (Dox-on) Positive results were examined and analyzed at 100 or mirPS cells divided by that of untreated (Dox-off) 200 magnification under a fluorescent 80i microscopic mirPS cells. Mir-434-expressing cells generated by quantitation system with a Metamorph imaging program electroporating hHFCs with pTet-On-tTS-miR434-5p (Nikon). were served as a negative control. Bisulfite DNA sequencing Northern blot analysis TM Genomic DNAs were isolated from about two million Total RNAs (10 mg) were isolated with a mirVana cells using a DNA isolation kit (Roche) and 1 mg of the miRNA isolation kit (Ambion, Austin, TX, USA), isolated DNAs were further treated with bisulfite fractionated by either 15% TBE-urea polyacrylamide gel (CpGenome DNA modification kit, Chemicon, or 3.5% low melting point agarose gel electrophoresis, and Temecula, CA, USA), according to the manufacturers’ electroblotted onto a nylon membrane. Detection of suggestions. Meanwhile, 2 mg of untreated DNAs were mir-302 was performed with a locked nucleic acid digested with a CCGG-cutting restriction enzyme HpaII [LNA]-DNA probe (5 -[TCACTGAAAC]ATGGAAGC and then analyzed by 1% agarose gel electrophoresis to ACTTA-3 ), while probes for other gene detection were determine genome-wide demethylation. The treatment synthesized and listed in Supplementary Table S2. All with bisulfite converted all unmethylated cytosine to probes were purified by high-performance liquid chroma- uracil, while methylated cytosine remained as cytosine. tography and tail labeled with terminal transferase (20 U) For bisulfite DNA sequencing analyses, we amplified the for 20 min in the presence of [ P]-dATP (>3000 Ci/mM, promoter regions of Oct3/4 and Nanog with PCR. Primers Amersham International, Arlington Heights, IL, USA). included 5 -GAGGCTGGAGCAGAAGGATTGCTTTG 0 0 G-3 and 5 -CCCTCCTGAC CCATCACCTCCACCAC Anti-miRNA oligonucleotide transfection 0 0 C-3 for Oct3/4, and 5 -TGGTTAGGTTGGTTTTAAA For mir-302 knockdown assay, we encapsulated the above 0 0 T TTTTG-3 and 5 -AACCCACCCTTATAAATTCTC anti-mir-302 [LNA]-DNA probe (2–10 mg/ml) with a AATTA-3 for Nanog (1). The bisulfite-modified DNAs FuGENE HD reagent (Roche, Indianapolis, IA, USA) (50 ng) were first mixed with the primers (total 100 pmol) and applied to cell cultures at 30–40% confluency. Cell in 1 PCR buffer, heated to 94 C for 2 min, and immedi- culture medium was refreshed 12-h post-treatment. The ately cooled on ice. Next, 25 cycles of PCR were per- mir-302-knockdown rate was determined by northern formed as follows: 94 C for 1 min and 70 C for 3 min, blot analysis at 24-h post-treatment. using an Expand High Fidelity PCR kit (Roche). The amplified DNA product with a correct size was further Western blot analysis fractionized by 3% agarose gel electrophoresis, purified Western blotting was performed as reported (6). Major with a gel extraction filter (Qiagen), and then used in primary antibodies in this study included Oct3/4 (Santa DNA sequencing. A detailed profile of the DNA methy- Cruz), SSEA-1 (Santa Cruz), SSEA-3 (Santa Cruz), lation sites was generated by comparing the unchanged SSEA-4 (Santa Cruz), Sox2 (Santa Cruz), Nanog (Santa cytosine in the converted DNA sequence to the unconvert- Cruz), Klf4 (Santa Cruz), Lin28 (Abcam), UTF1 ed one. 1058 Nucleic Acids Research, 2011, Vol. 39, No. 3 Microarray analysis cells were cultivated in the feeder-free mirPS cell cultural condition with addition of 0.05 mM SB216763. Microarray analyses were performed as reported earlier (6). Human genome GeneChip U133 plus 2.0 arrays Nuclear transfer (Affymetrix, Santa Clara, CA, USA) were used to detect MirPS cell cytoplasm with different concentrations of the alterations of over 47 000 human gene expression mir-302 was prepared by varying in Dox treatments patterns in tested cells, while miRNA microarray (from 1.0, 5.0, 10.0 to 20.0 mg/ml). The concentrations of analyses were done by LC Sciences (San Diego, CA, cytoplasmic mir-302 were confirmed by northern blot USA). Total RNAs from each tested sample were TM analysis. Hybrids [mirPS-nuclear transfer (NT) cells] of isolated using a mirVana miRNA isolation kit mirPS cell cytoplasm and hFB nuclei were generated (Ambion), following the manufacturer’s suggestion. The using a set of MO-188NE 3D hydraulic fine/coarse purity and quantity of isolated RNAs were assessed using micromanipulators and micro-injectors/holders under a 1% formaldehyde–agarose gel electrophoresis and spec- TE2000 inverted microscopic system (Nikon). The trophotometer measurement (Bio-Rad). The sample mirPS-NT cells were then grown in knockout DMEM/ signals were normalized using the total average difference F-12 medium (Invitrogen) supplemented with 20% between perfectly matched probes and mismatched knockout serum, 1% MEM non-essential amino acids, probes. Alterations of genome-wide gene expression 100 mM b-mercaptoethanol, 1 mM GlutaMax, 1 mM patterns were analyzed using Affymetrix Microarray TM sodium pyruvate, 10 ng/ml bFGF, 100 IU/ml penicillin/ Suite version 5.0, Expression Console version 1.1.1 100 mg/ml streptomycin, 0.1 mM A83-01 and 0.1 mM (Affymetrix) and Genesprings (Silicon Genetics) soft- valproic acid, at 37 C under 5% CO . wares. Changes in gene expression rates >1-fold were con- sidered as positive differential genes. For gene clustering, a DNA fingerprinting plug-in program Genetrix (Epicenter Software) was used in conjunction with the Affymetrix softwares. Signals of PCR was used to amplify the variable numbers of tandem the sample were normalized with the internal housekeep- repeats (VNTR) of the D1S80 locus on human chromo- ing control average in each microarray. some 1. Genomic DNAs were isolated with a DNA isola- tion kit (Roche) and 50 ng of the DNAs were amplified by PCR with primers 5 -GAAACTGGCCTCCAAACACT Implantation and teratoma formation 0 0 GCCCGCCG-3 and 5 -GTCTTGTTGGAGATGCACG Approximately 5–10 mirPS or mirPS-NT cell-derived T GCCCCTTGC-3 (50 pmol each). PCR was performed embryoid bodies (4- to 16-cell-stage) were suspended in as follows: one cycle at 94 C for 4 min and then 30 cycles 50 ml of a mixture of DMEM and Matrigel (2:1), through 94 C for 30 s, 68 C for 30 s and 72 C for 2 min, followed by implantation into the uterus of a 6-week-old using the Expand High Fidelity PCR kit (Roche). The female pseudopregnant immunocompromised SCID-beige amplified DNAs were further fractionized by 2.5% mouse. The pseudopregnant mice were prepared by agarose gel electrophoresis. intraperitoneal injection of 1 IU human menopausal go- nadotrophin for 2 days and then human chorionic go- Statistic analysis nadotrophin (hCG) for one more day. The cells and Any change over 75% of signal intensity in the analyses of mice were not treated with Dox before or after implant- immunostaining, western blotting and northern blotting ation. The mice were anesthetized with 2.5% Avertin was considered as a positive result, which in turn was solution, 0.4 ml per mouse during implantation. analyzed and presented as mean ± SE. Statistical Xenografted masses were monitored 3–4 weeks after the analysis of data was performed by one-way ANOVA. implantation or when the sizes were grown to >100 mm . When main effects were significant, the Dunnett’s Cysts/teratomas were dissected and the volumes were post-hoc test was used to identify the groups that calculated using the formula (length  width )/2. Cyst/ differed significantly from the controls. For pairwise com- teratoma lesions were counted, weighed and subjected to parison between two treatment groups, the two-tailed further histological analysis. Formation of teratoma-like student t-test was used. For experiments involving more tissue cysts was usually observed at 2.5-week than two treatment groups, ANOVA was performed post-implantation. followed by a post hoc multiple range test. Probability values of P< 0.05 was considered significant. All P-values were determined from two-tailed tests. Microinjection Microinjection was performed on the nuclei of cells plated 8–12 h before on a collagen-coated glass coverslip. Cells RESULTS were microinjected using a set of Eppendorf microinjector Mir-302-induced SCR is a dose-dependent mechanism and MO-188NE 3D hydraulic micromanipulators involving AOF2 suppression and Oct3/4–Sox2–Nanog mounted on a Nikon TE2000 microscope system with co-activation duration and pressure settings of 0.5 s and 30 hectopascals, respectively. The microinjection buffer contained 10 mg/ml Mir-302-mediated gene silencing is a dose-dependent AOF2, 2 mg/ml fluorescein (FITC)-labeled dextran dye, reaction due to its mismatched targeting. Following an 10 mMNaH PO and 70 mM KCl, pH 7.2. After injection, increase of Dox concentration up to 10 mM, we observed 2 4 Nucleic Acids Research, 2011, Vol. 39, No. 3 1059 that transcription of mir-302 and its RGFP marker gene AOF2, MECP1-p66 and MECP2 (Figure 1C). Western was proportionally elevated, while expressions of blot analysis showed that hHFCs expressed abundant melanocytic markers tyrosinase-related protein 1 (TRP1) AOF2 and MECP1/2 but scarce AOF1, suggesting that and keratin 16 were reduced in all transfected cells silencing of AOF2 plays a major role in hHFC (Figure 1C). Accordingly, core reprogramming factors reprogramming. Deficiency of either AOF1 or AOF2 is Oct3/4, Sox2 and Nanog were all strongly stimulated by sufficient to cause global demethylation (13,14). Our a threshold of Dox 7.5 mM, indicating a dose-dependent previous studies have also demonstrated that mir-302 correlation between mir-302 concentration and Oct3/4– mediated MECP1/2 silencing enhances global Sox2–Nanog co-activation. This concurrent Oct3/4– demethylation (6,15). Therefore, mir-302 induces SCR Sox2–Nanog gene activation is an essential step for iPS through a combinatory mechanism involving both Oct3/ cell induction (2,3). Time-course measurement of 4–Sox2–Nanog co-activation and AOF1/2 suppression, reprogramming hHFCs to mirPS cells further showed while further MECP1/2 silencing enhances the result of that the Oct3/4–Sox2–Nanog co-activation was most AOF1/2-associated global demethylation. prominent after Days 5–6, the time frame required for initiating SCR (Figure 1D). Almost no cell division was Mir-302 silences AOF2 to trigger DNMT1 deficiency detected during the first 3–4 days after treatment of and global demethylation 7.5 mM Dox. In this dose-dependent SCR process To validate the interactions between mir-302 and its epi- (Figure 1C), we discovered three critical mir-302 concen- genetic targets, we used a luciferase 3 -untranslated region trations. First, at the treatment of 5 mM Dox, the induced (3 -UTR) reporter assay (Figure 3A), which showed that mir-302 level was closely similar to that found in hES H1 treatments with various mir-302 concentrations resulted in and H9 cells, but not sufficient to reprogram hHFCs. different epigenetic patterns in mirPS cells. In the presence Second, at 7.5 mM Dox, mir-302 concentration was of 10 mM Dox, mir-302 effectively bound to the target sites raised to over 30% higher than that of H1 and H9 cells of AOF1, AOF2, MECP1-p66 and MECP2 mRNAs and and began inducing co-expression of the core successfully silenced over 80% of the reporter luciferase reprogramming factors Oct3/4–Sox2–Nanog and Lin28 expression in all targets (Figure 3B). Suppression of the (Figure 1E). Last, at 10 mM Dox, the stimulated mir-302 real target genes in mirPS cells was also confirmed concentration was optimal for reprogramming hHFCs to by western blot analyses, consistent with the results of mirPS cells and forming embryoid bodies. Treatment of the luciferase 3 -UTR reporter assay (Figure 3C). >12 mM Dox caused severe cell cycle arrest at the G0/G1 Accordingly, we detected a significant decrease of phase, which ultimately hindered mirPS cell formation. DNMT1 (loss over 87 ± 3% of the level in hHFCs) and Fluorescence-activated cell sorter (FACS) analysis increase of H3K4 di-/tri-methylation (H3K4me2/3; gain further confirmed that 91% of the transfected cells 103 ± 17%) in response to the silencing of AOF2 (loss treated with 10 mM Dox expressed over twice more 93 ± 2%) by mir-302. Previous studies have demonstrated Oct3/4 than those treated with only 5 mM Dox that AOF2 is required for stabilizing DNA (Figure 1F). For the first time, this study established the methyltransferase 1 (DNMT1) and preserving its activity threshold concentration of a hES-specific miRNA for on the maintenance of global DNA methylation (14), SCR. The mechanism underlying this SCR process is con- whereas active global demethylation can promote Oct3/ sistent with the reported iPS cell induction using ectopic 4–Nanog activation in early mouse embryos and mouse– transfection of Oct3/4–Sox2–Nanog–Lin28 factors (2). human fused heterokaryons (17,18). Conceivably, the Following the increase of Dox-induced mir-302 expres- deficiency of DNMT1 caused mirPS cell genomes to be sion, we also observed a significant change of mirPS cell susceptible to a certain demethylation activity. This colony morphology from hES-cell-like 2D aggregations demethylation effect was further enhanced by (at 7.5 mM Dox) to embryoid body (EB)-like three- co-suppression of MECP1/2 (loss 96 ± 1% and dimensional colonies (at 10 mM Dox), which highly 81 ± 5%, respectively) (6) and eventually led to global resembled early mammalian zygotes (Figure 2A). After demethylation and Oct3/4–Nanog activation (Figure 4A cultivated on a low attachment (overhanging) surface, and B). On the flipside, a lower mir-302 concentration these EB-like colonies formed sphere-like EBs strongly induced by 5 mM Dox failed to trigger any significant and homogeneously expressing pluripotent Oct3/4–Sox2– Nanog markers as well as other standard hES markers silencing effect on either the target sites of the reporter (Figure 2B and C), indicating that their undifferentiated gene or the targeted epigenetic genes, except status was well maintained by the induced mir-302. A MECP1-p66, indicating that mir-302-induced global similar undifferentiated status is often observed in mam- demethylation is dose dependent and requires malian zygotes before the morula stage, but rarely in co-suppression of AOF1/2 and MECP1/2 (Figure 3B three/four-factor-induced iPS cell colonies. This is and D). probably due to a lower mir-302 level expressed in both Methylation site-sensitive HpaII digestion assays con- human iPS and late blastocyst-derived hES cells (9). From firmed that mirPS cell genomes isolated from the group morphology and hES-specific gene expression, there was treated with 10 mM Dox underwent global demethylation no significant difference between mirPS cell colonies and (Figure 4A). When further assessing the methylation mirPS cell-derived EBs. Accompanying the Oct3/4–Sox2– patterns of Oct3/4 and Nanog promoters with bisulfite Nanog co-activation, we also found a proportional loss of DNA sequencing, we observed that both promoters were multiple epigenetic regulator expression, including AOF1, almost completely demethylated in a fashion resembling 1060 Nucleic Acids Research, 2011, Vol. 39, No. 3 Figure 2. Morphological and genetic changes of mirPS cell properties in response to different mir-302 concentrations. (A) Morphological changes of mirPS cell colonies in response to Dox-induced mir-302 expression. Mir-434-expressing cells generated by electroporating hHFCs with pTet-On-tTS-miR434-5p were served as a negative control. A morphological comparison between a morula-staged rat embryo and a mirPS cell colony at 32–64 cell stage was shown in the lower panel. (B) Fluorescent microscope examination showing strong and homogeneous expression of the core pluripotent stem cell markers Oct3/4, Sox2 and Nanog in a mirPS-derived embryoid body. BF-DIC, bright field with differential interference contrast. (C) Western blotting confirming the expression patterns of major hES-specific marker proteins in completely (10 mM Dox) and partially (5 mM Dox) reprogrammed mirPS cells compared to those found in hES H1 and H9 cells (n=4, P< 0.01). Mir-302 expression was proportional to Dox concentration. hES H1 and H9 cells (Figure 4B). Similar global of red fluorescent RGFP protein (Supplementary Figure demethylation patterns have also been found in iPS cells S3A), and further selected by G418 antibiotics to ensure (2,3). In contrast, neither global demethylation nor SCR their purity. Consistent with our previous reports (6,15), was observed in the transfected cells treated with only each individual mirPS cell could grow into a homogeneous 5 mM of Dox (Figures 1C, 4A and B). We subsequently EB (Supplementary Figure S3B) and form teratoma-like evaluated this global demethylation effect in over 47 000 tissue cysts (Figure 5C) in immunocompromised human gene expression patterns using microarray analyses SCID-beige mice, containing various differentiated and revealed that approximately half of the transcriptome tissues derived from all three embryonic germ layers, expression in mirPS cells was changed from a somatic ectoderm, mesoderm and definitive endoderm hHFC mode to a uniform hES-like expression pattern (Figure 5D). These results confirmed the hES cell-like sharing over 91% similarity to that of H1/H9 cells pluripotency of mirPS cells. (Figure 4C). Hierarchical clustering of the top 30 most Next, we evaluated the role of mir-302–targeted AOF2 differentially expressed hES-specific genes and epigenetic silencing in the process of SCR using recombinant AOF2 regulators in microarrays further showed an extremely protein and its inhibitor tranylcypromine (Figure 5A). high correlation between reprogrammed mirPS and hES Under conditions of 10 mM Dox stimulation, the success H1/H9 cells (Figure 4D). Thus, we conclude that mir-302 rate of complete SCR approached 100% (477/480). After regulates the epigenetic reprogramming of genomic that, the reprogrammed mirPS cells could be cultivated to methylation patterns through co-suppression of AOF2 over 26–28 passages under our feeder-free cultural condi- and DNMT1 during SCR. tion in the absence of Dox and GSK inhibitor (‘Materials and Methods’ section), indicating the completion of SCR. However, when a GSK inhibitor was presented in the Mir-302-induced SCR is reversible and dependent on cultural medium without sufficient Dox stimulation, AOF2–DNMT1 suppression majority of mirPS cells differentiated into neuron-like cells. Given that glycogen synthase kinase 3 (GSK3) is a After SCR completion (5–6 days post-transfection of mir-302), reprogrammed mirPS cells were visually distin- major gatekeeper for embryonic neural induction (19), this guished by their sphere-shape morphology and expression result suggests that GSK inhibitor can induce the Nucleic Acids Research, 2011, Vol. 39, No. 3 1061 Figure 3. Analysis of mir-302-induced silencing effects on targeted epigenetic genes and global demethylation. (A) Constructs of the luciferase 3 -UTR reporter genes, which carry either two normal (T1+T2) or two mutant (M1+M2), or a mixture of both (T1+M2 or M1+T2), mir-302 0 0 target sites in the 3 -UTR. The mutant sites contained a mismatched TCC motif in place of the uniform 3 -CTT end of the normal target sites. (B) Effects of Dox-induced mir-302 on the luciferase expression (n=5, P< 0.01). (C) and (D) Western blot analyses showing the changes of major mir-302-targeted epigenetic gene expression induced by high (10 mM Dox) and low (5 mM Dox) mir-302 concentrations in mirPS cells compared to those found in hES H1 and H9 cells (n=4, P< 0.01). ectodermic differentiation of mirPS cells. Notably, these not cause any effect in all tests. Therefore, SCR is a re- differentiated cells could be reprogrammed back to mirPS versible mechanism dependent on mir-302–mediated cells after re-supplementation of 7.5 mM Dox in the AOF2 silencing. cultural medium. The same neuronal differentiation Following this reversible SCR process, we measured the could also be triggered by treatment of 10 mg/ml corresponding changes of global demethylation, AOF2– DNMT1 co-suppression and Oct3/4–Sox2–Nanog anti-mir-302 LNA–DNA oligonucleotides in mirPS cells (Figure 5A and B), indicating the critical role of mir-302 co-activation in all tested mirPS and differentiated cells. concentration in maintaining pluripotent cell stemness. Bisulfite DNA sequencing assays revealed that, under the Alternatively, while being microinjected with recombinant GSK inhibitor influence, the global demethylation of AOF2 into the cell nuclei, the differentiated cells failed to Oct3/4 and Nanog promoters were observed only in mirPS cells treated with 7.5 mM Dox (mirPS+10 mM be reprogrammed back to mirPS cells even after 10 mM Dox stimulation, demonstrating the inhibitory role of Dox) and the re-reprogrammed cells co-treated with AOF2 in the mechanism of mir-302-induced SCR. 10 mM Dox, AOF2 and tranylcypromine (mirPS+10 mM Further treatment of tranylcypromine removed the Dox–AOF2), whereas the differentiated cells treated blockade of AOF2 in the process of SCR and, in conjunc- with 10 mM Dox and AOF2 (mirPS+10 mM Dox+AOF2) tion with the stimulation of 10 mM Dox, could again re- failed to complete the demethylation (Figure 5E). program the differentiated cells to mirPS cells with all These resulting global demethylation patterns per- hES-like properties. Microinjection of blank buffer did fectly matched the morphological changes observed in 1062 Nucleic Acids Research, 2011, Vol. 39, No. 3 Figure 4. Mir-302-induced genomic DNA demethylation and global gene expression alteration. (A) HpaII cleavage showing the vast loss of global CpG methylation, identified by increased presence of smaller DNA fragments, at a genome-wide scale in mirPS cells treated with 10 mM but not 5 mM Dox. (B) Bisulfite DNA sequencing in the promoter regions of Oct3/4 and Nanog. Methylation maps show the most frequently observed patterns. Black and white circles indicate the methylated and unmethylated cytosine sites, respectively. (C) Analysis of global gene expression patterns before and after global demethylation, using human genome GeneChip U133 plus 2.0 arrays (Affymetrix; n=3, P< 0.01–0.05). (D) Microarray hierarchical clustering of the top 30 most differentially expressed hES-specific genes and epigenetic regulators between somatic hHFCs, reprogrammed mirPS-hHFCs (mirPS+10 mM Dox) and hES H1/H9 cells. Figure 5A. In addition, western blot assays also cells (Figure 5F), confirming that mir-302–targeted showed that the simultaneous AOF2–DNMT1 co- AOF2 silencing is required for global demethylation suppression and Oct3/4–Sox2–Nanog co-activation only during SCR. In accordance to this counteraction occurred in the mirPS+10 mM Dox and mirPS+10 mM between mir-302 and AOF2, we identified that SCR is a Dox–AOF2 cells but not in mirPS+10 mM Dox+AOF2 reversible process. Nucleic Acids Research, 2011, Vol. 39, No. 3 1063 Figure 5. Reversible SCR process regulated by mir-302–mediated AOF2 silencing. (A) Reversible interchange between hES-like mirPS and neuron-like differentiated cells as a result of mir-302 and AOF2 counteraction. (B) Northern blots showing the mir-302 knockdown (65%) by 10 mg/ml anti-mir-302 LNA–DNA oligonucleotides. (C) Typical morphology of a mirPS EB-derived teratoma-like tissue cyst (2.5 weeks post-implantation). (D) Pluripotent differentiation of mirPS-hHFCs into teratoma-like cysts containing various tissues derived from all three em- bryonic germ layers. Immunostaining the cross-sections of mirPS EB-derived tissue cysts showed the positive results of specific differentiation markers, including neuronal b-tubulin isotype-III (Tuj1; green) and/or ATP-binding cassette sub-family A member 2 (ABCA2; blue), cartilage collagen type II a-1 (COL2A1; green), muscle-associated M-cadherin (green), gut-epithelium-related mucin 2 (MUC2; green), and glandular epithelium-related CD13 (green). Photographs were taken with Nikon TE2000 microscopic system at 200 magnification. (E) Global demethylation patterns in Oct3/4 and Nanog promoters corresponding to the result of (A). (F) Changes of Oct3/4, Sox2, Nanog, AOF2 and DNMT1 expression in response to the results of (A) and (E). Global genomic DNA demethylation does not require any developed. Conversely, transfer of mirPS nuclei into nuclear effector hFB cytoplasm also failed to form any viable cells. Thus, the reprogramming ability of mirPS cells is Mir-302 is a cytoplasmic effector. To further rule out the preserved in the cytoplasm rather than nucleus. These possible involvement of any nuclear factor from the hFB nucleus-transferred mirPS (mirPS-NT) cells mir-302–mediated global demethylation, we transferred preserved all the same traits as mirPS cells in terms of human adult fibroblast (hFB) nuclei into the cytoplasm global demethylation (Figure 6B and C), Oct3/4–Sox2– of mirPS cells pre-treated with 10 mM Dox. Most (93%) Nanog co-activation and AOF2–DNMT1 co-suppression of the hybrid cells successfully formed mirPS-like iPS cells (Figure 6D), and in vivo pluripotency (Figure 6F and and embryoid bodies (Figure 6A). When cytoplasm Supplementary Figure 4). Using DNA fingerprinting in derived from the mirPS cells pre-treated with <7.5 mM human D1S80 alleles, the mirPS-NT-derived teratoma Dox, neither mirPS-like cell nor embryoid body was cysts were confirmed to be originated from human cell 1064 Nucleic Acids Research, 2011, Vol. 39, No. 3 Figure 6. Assessment of the reprogramming ability preserved in mirPS cytoplasm. (A) Embryoid body derived from a single mirPS–hFB hybrid (mirPS-NT) cell after limiting dilution. Scale bars = 100 mm. (B) HpaII cleavage showing global demethylation (smaller DNA fragments) in reprogrammed mirPS-NT cells compared to that in the somatic hHFC and hFB cells. (C) Bisulfite DNA sequencing in the Oct3/4 and Nanog promoters, showing CpG methylation maps. Black and white circles indicate the methylated and unmethylated cytosine sites, respectively. (D) Northern and western blot analyses of mir-302, AOF2–DNMT1 and Oct3/4–Sox2–Nanog expression patterns in mirPS-NT cells compared to those in the somatic hFB and reprogrammed mirPS (mirPS+Dox) cells. (E) DNA fingerprinting of the variable numbers of tandem repeats (VNTRs) in the D1S80 locus of human genome, confirming that mirPS-NT, mirPS-hHFC and hFB cells all originated from the same human source not mouse. (F) Pluripotent tissue types found in teratoma-like cysts derived from mirPS-NT cells. Markers (green): keratin 14, actin a-2 in smooth muscle aorta (a-SMS, ACTA2), CD105 and MUC2. H&E, histological staining with hematoxylin and eosin. BF, bright field. (G) Proposed mechanism of mir-302– induced SCR. sources rather than mouse cells (Figure 6E). In view of the stemness and pluripotency of the reprogrammed these hES-like properties inherent in mirPS-NT cells, we somatic cells (6,15). In this study, we further revealed conclude that mir-302 is able to not only induce SCR but the mechanism involved (Figure 6G), showing that also preserve this reprogramming ability in the cytoplasm mir-302 significantly decreases AOF2 and DNMT1 of mirPS cells. activities and, in conjunction with the co-suppression of MECP1/2, results in global genomic DNA demethylation and H3K4 modification. Subsequently, these epigenetic DISCUSSION reprogramming events induce hES-specific gene expres- Our previous study established that mir-302 functions not sion, in particular Oct3/4, Sox2 and Nanog, which in only to improve the efficiency of SCR but also to enhance turn further stimulates mir-302 expression, and so on to Nucleic Acids Research, 2011, Vol. 39, No. 3 1065 et al. (2007) Induced pluripotent stem cell lines derived from form a positive feedback cycle essential for maintaining human somatic cells. Science, 318, 1917–1920. SCR. When compared to the previous three/four-factor 3. Wernig,M., Meissner,A., Foreman,R., Brambrink,T., Ku,M., reprogramming methods, our approach adopts a novel Hochedlinger,K., Bernstein,B.E. and Jaenisch,R. (2007) In vitro entry point in the same mechanistic cycle to complete reprogramming of fibroblasts into a pluripotent ES-cell-like state. SCR. Also, without introducing the oncogenic Klf4 and Nature, 448, 318–324. 4. Marson,A., Levine,S.S., Cole,M.F., Frampton,G.M., c-Myc genes, this new approach likely provides safer Brambrink,T., Johnstone,S., Guenther,M.G., Johnston,W.K., in vivo applications. From this novel SCR mechanism, Wernig,M., Newman,J. et al. (2008) Connecting microRNA genes we learned that the effector(s) responsible for to the core transcriptional regulatory circuitry of embryonic stem reprogramming genomic epigenetics resides in the mirPS cells. Cell, 134, 521–533. cytoplasm and can enter somatic cell nuclei after NT 5. Card,D.A., Hebbar,P.B., Li,L., Trotter,K.W., Komatsu,Y., Mishina,Y. and Archer,T.K. (2008) Oct4/Sox2-regulated miR-302 because mir-302 is a cytoplasmic effector. Thus, our targets cyclin D1 in human embryonic stem cells. Mol. Cell. Biol., findings may also help in clarifying the mechanism of 28, 6426–6438. somatic cell NT, addressing the possible role of mir-302 6. Lin,S.L., Chang,D., Chang-Lin,S., Lin,C.H., Wu,D.T.S., in nuclear reprogramming. Chen,D.T. and Ying,S.Y. (2008) Mir-302 reprograms human skin Through deciphering the interactions between mir-302 cancer cells into a pluripotent ES-cell-like state. RNA, 14, 2115–2124. and its targeted epigenetic genes, we elucidate the intricate 7. Puca,A.A., Daly,M.J., Brewster,S.J., Matise,T.C., Barrett,J., gene regulation circuitry involved in epigenetic Shea-Drinkwater,M., Kang,S., Joyce,E., Nicoli,J., Benson,E. et al. reprogramming during SCR. 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Nucleic Acids Research – Oxford University Press

Published: Feb 24, 2011

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