Overlapping functions of Krüppel-like factor family …...Overlapping functions of Krüppel-like factor family members: targeting multiple transcription factors to maintain the naïve

STEM CELLS AND REGENERATION RESEARCH ARTICLE

Overlapping functions of Kruppel-like factor family members:targeting multiple transcription factors to maintain the naïvepluripotency of mouse embryonic stem cellsMariko Yamane1,2,*, Satoshi Ohtsuka1,3,*, Kumi Matsuura1,2, Akira Nakamura4 and Hitoshi Niwa1,2,5,‡

ABSTRACTKruppel-like factors (Klfs) have a pivotal role in maintaining self-renewal of mouse embryonic stem cells (mESCs). The functions ofthree Klf family members (Klf2, Klf4 and Klf5) have been identified, andare suggested to largely overlap. For further dissection of theirfunctions, we applied an inducible knockout system for these Klffamily members and assessed the effects of combinatorial loss offunction. As a result, we confirmed that any one of Klf2, Klf4 and Klf5was sufficient to support self-renewal, whereas the removal of all threecompromised it. The activity of any single transcription factor, exceptfor a Klf family member, was not sufficient to restore self-renewalof triple-knockout mESCs. However, some particular combinationsof transcription factors were capable of the restoration. The triple-knockout mESCs were successfully captured at primed state. Thesedata indicate that the pivotal function of a Klf family member istransduced into the activation of multiple transcription factors in anaïve-state-specific manner.

KEY WORDS: Embryonic stem cells, Pluripotency, Transcriptionfactor

INTRODUCTIONKrüppel-like factors (Klfs) encode zinc-finger transcription factorsand have a pivotal role in maintaining self-renewal in pluripotentstem cells (PSCs). Functions related to pluripotency were identifiedin three of the Klf family members. Klf4 was identified as one ofthe Yamanaka factors to reprogram somatic cells into PSCs incooperation with Oct3/4 (Pou5f1 – Mouse Genome Informatics),Sox2 and Myc (Takahashi and Yamanaka, 2006), as well as thetarget of the leukemia inhibitory factor (LIF) signal in mouseembryonic stem cells (mESCs) (Hall et al., 2009; Niwa et al., 2009).Klf2 was reported to be a target of Oct3/4 (Hall et al., 2009) and tomediate the action of the mitogen-activated protein kinase kinase(MEK) inhibitor in mESCs to support pluripotency in definedculture condition (Yeo et al., 2014). Klf5 was reported to govern the

rapid proliferation and stable self-renewal of mESCs (Ema et al.,2008). The overlapping functions of these three Klf family membersin self-renewal of mESCs were examined by partial loss-of-functionanalyses with small interference RNA (siRNA)-mediatedknockdown (Jiang et al., 2008). Their functions in reprogrammingof somatic cells (Nakagawa et al., 2008) and primed PSCs (Jeonet al., 2016) into naïve PSCs have also been characterized. However,their functions in maintenance of pluripotency have not been wellassessed by complete loss-of-function assays in mESCs. Here, weapplied a combinatorial inducible knockout strategy to Klfs andassessed their chimera contribution abilities, confirming that Klf2,Klf4 andKlf5 share overlapping functions, and thatKlf2 andKlf4 arenot essential to maintain pluripotency in mESCs.

RESULTSKlf2 and Klf4 are not essential to maintain pluripotencyWe generated a series of inducible knockout mESCs by serialmodifications of the endogenous alleles of Klf2, Klf4 and Klf5 withintroduction of loxP sites and a tamoxifen (Tx)-inducible form ofCre recombinase (Fig. S1). Inducible knockout ofKlf2, but notKlf4,resulted in significant reduction of efficiency in stem cell colonyformation (Figs 1A and 2A). However, constitutive knockout ofeach gene in Klf2- or Klf4-null mESCs showed stable self-renewalwith a normal proliferation ratio comparable to that of wild-typemESCs in either conventional culture medium with fetal calf serum(FCS) or very low FCS with knockout serum replacement (KSR)(Figs 1B and 2B). The complete loss of the targeted gene productwas confirmed by western blot analysis (Fig. S2). Quantitative PCRanalysis of reverse-transcribed mRNA (RT-qPCR) revealed thatthey expressed comparable levels of the transcripts of functionallyverified pluripotency-associated transcription factors, such as Oct3/4, Sox2, Tbx3, Nanog, Esrrb, Gbx2, Nr5a2, Tfcp2l1 and Nr0b1(Figs 1C and 2C). They also expressed the remaining Klf familymembers (Klf4 and Klf5 in Klf2-null mESCs, and Klf2 and Klf5 inKlf4-null mESCs) at similar levels to wild-type mESCs, suggestingthat there was no obvious dosage compensation within the three Klffamily members. The expression levels of the floxed alleles werelower than those of wild-type alleles without induction of Creactivity, which might be caused by the leaky activation of Crewithout induction, as well as the faint effect of the deletion for self-renewal of these mESCs. It has been known that the pluripotency-associated transcription factors show distinct expression patterns inself-renewing populations, either homogeneous (Oct3/4 and Sox2)or heterogeneous in various degrees (Nanog, Klf2, Klf4, Klf5 andTbx3) (Niwa et al., 2009). When the expression patterns of thepluripotency-associated transcription factors were examined byimmunostaining, both Klf2- and Klf4-null mESCs showed similarpatterns to those in wild-type mESCs except for the deleted genes(Figs 1D and 2D). When a single Klf2- or Klf4-null mESC carryingReceived 10 December 2017; Accepted 30 April 2018

1Laboratory for Pluripotent Stem Cell Studies, RIKEN Center for DevelopmentalBiology (CDB), 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.2Department of Pluripotent Stem Cell Biology, Institute of Molecular Embryologyand Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811,Japan. 3Department of Life Science, Medical Research Institute, KanazawaMedical University, 1-1 Daigaku, Uchinada kahoku, Ishikawa 920-0293, Japan.4Department of Germline Development, Institute of Molecular Embryology andGenetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811,Japan. 5JST, CREST, Sanbancho, Chiyoda-ku, Tokyo 1020075, Japan.*These authors contributed equally to this work

‡Author for correspondence ([email protected])

H.N., 0000-0002-5280-8031

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© 2018. Published by The Company of Biologists Ltd | Development (2018) 145, dev162404. doi:10.1242/dev.162404

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the ubiquitous EGFP expression vector (CAG-EGFP-IZ) wasinjected into blastocysts followed by transfer into pseudo-pregnant mice, they gave rise to chimeric embryos with highlevels of contributions (Figs 1E and 2E), indicating that both Klf2-and Klf4-null mESCs retain pluripotency.

Klf2/Klf4 double-knockout mESCs retain pluripotencyInducible knockout of both Klf2 and Klf4 simultaneously resultedin dramatic reduction of efficiency in stem cell colony formation(Fig. 3A). However, Klf2/Klf4-null mESCs with constitutiveknockout were able to be established. They continued self-renewal with slower proliferation ratio than wild-type mESCs,with loss of Klf2 and Klf4, irrespective of the culture condition(Fig. 3B, Fig. S2). They showed comparable levels of the transcriptsof pluripotency-associated transcription factors (Fig. 3C). Theexpression ofKlf5was slightly but significantly increased (Fig. 3C),suggesting its compensatory function. The expression patterns ofpluripotency-associated transcription factors in cell populationswere also similar to those of wild-type mESCs (Fig. 3D). When asingle Klf2/Klf4-null mESC carrying the ubiquitous EGFPexpression vector was injected into blastocysts, followed bytransfer into pseudo-pregnant mice, they gave rise to chimericembryos with high levels of contributions (Fig. 3E), indicating thatKlf2/Klf4-null mESCs retain pluripotency. Because these knockoutmESCs retain only Klf5 among the three Klf family membersknown to function in mESCs, Klf5 alone might be sufficient tosupport pluripotency in mESCs.

Combinatorial knockout of Klf2 or Klf4 with Klf5 allow self-renewal of mESCsIt was reported that Klf5-null mESCs showed destabilized self-renewal and slow proliferation (Ema et al., 2008).When knockout ofKlf5was induced using the same strategy, a significant proportion ofthe Klf5-null mESCs formed stem cell colonies (Fig. 4A). Theestablished Klf5-null mESCs showed self-renewal with slightlyslower proliferation ratio than wild-type mESCs (Fig. 4B). Theyshowed comparable levels of the transcripts of pluripotency-associated transcription factors, as well as expression patterns incell populations similar to those of wild-type mESCs, with completeloss of Klf5 protein (Fig. 4C,D). However, when a single Klf5-nullmESC carrying the ubiquitousEGFP expression vector was injectedinto blastocysts, then transferred into pseudo-pregnant mice, theynever gave rise to chimeric embryos, although the parental floxmESCs efficiently produced chimeric embryos. In previous reports,conventional injection strategy with multiple Klf5-null mESCs intoblastocysts gave low contribution to chimeras (Ema et al., 2008),suggesting that loss of Klf5 caused reduction of pluripotency.Reduction of pluripotencymight be detected more sensitively by oursingle mESC injection assay. Alternatively, it could be simplycaused by their proliferation capacity after implantation. Inducibleknockout of Klf2 and Klf5 caused significant reduction of efficiencyin stem cell colony formation (Fig. 4A), but the Klf2:Klf5 and Klf4:Klf5 double-knockout mESCs were able to be established (Fig. 4B).These double-knockout mESCs expressed comparable levels ofthe transcripts of pluripotency-associated transcription factors, and

Fig. 1. Inducible knockout of Klf2.(A) Colony formation of Klf2 knockoutmESCs. The relative colony numbersin the culture with or without Tx areshown (+Tx=1.0). Error bars indicatestandard deviations of triplicateresults. *P<0.01, Student’s t-test(n=3) versus −Tx. (B) Proliferation ofKlf2 knockout mESCs. Cell numbersthat originated from 104 cells afterculture for 5 days are shown. Errorbars indicate standard deviations offive independent results. (C) RT-qPCR analysis of gene expression ininducible and constitutive Klf2knockout mESCs. The relative geneexpression levels [wild-typemESC (wtES)=1.0] in inducible knockoutmESCs cultured with or without Tx for3 days and constitutive knockoutmESCs are indicated. Error barsindicate standard deviations oftriplicate quantifications. *P<0.01,Student’s t-test (n=3) versus −Txd3.(D) Immunostaining of constitutiveKlf2 knockout mESCs. The loss ofimmunoreactivity against anti-Klf2antibody (Ab) confirms a properknockout event. (E) Chimeracontribution of constitutive Klf2knockout mESCs. Three independentclones derived from the sameinducible knockout mESCs weretested. The numbers indicate thenumber of chimeric embryos/numberof recovered embryos/number ofinjected blastocysts.

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displayed similar expression patterns in cell populations to those ofwild-type mESCs (Fig. 4D,E,F), but lost the ability to contribute tochimeric embryos after single-cell injection, as in the case of Klf5-null mESCs. Klf2 expression was slightly elevated in Klf4:Klf5-nullmESCs, whereas Klf4 expression was unchanged in Klf2:Klf5-nullmESCs. These data suggested that the double-knockout mESCs areable to maintain the mESC-like state with sufficient expression ofkey pluripotency-associated transcription factors.

Klf2:Klf4:Klf5 triple knockout compromises self-renewalTo confirm the overlapping function of these three Klf familymembers, we generated inducible triple-knockout mESCs for Klf2,Klf4 and Klf5. Induction of a triple-knockout event with Tx resultedin complete cease of self-renewal. Very few differentiated colonieswere observed after induction of the triple knockout, and increasedapoptosis and cell death were observed by fluorescence-activatedcell sorting (FACS) analysis (Fig. S3); therefore, the removal of thethree Klf family members seemed to induce cell death rather thandifferentiation. Induction of Klf2:Klf4:Klf5 triple knockout resultedin rapid downregulation of naïve-state-specific transcription factors(Nanog, Esrrb, Tbx3, Gbx2, Tfcp2l1, Nr0b1 and Nr5a2), followedby gradual downregulation of general pluripotency-associatedtranscription factors (Oct3/4 and Sox2) (Fig. 5A), suggestingindirect (or parallel) regulation of the genes in the latter category bythe Klf family. These data suggested that the overlapping functionsof the Klfs are essential to maintain self-renewal of mESCs viaactivation of naïve-state-specific genes.

To test the functional specificity of the Klf family members tomaintain the ES-like state in mESCs, we performed phenotypicrescue experiments using the inducible triple-knockout mESCs. Theparental triple-floxed mESCs retained pluripotency as confirmed bytheir chimera formation ability (Fig. 5C). When we assessed therescue ability of various pluripotency-associated transcriptionfactors, we found that Klf2, Klf4 and Klf5 were able to restorestem cell colony formation in the triple-knockout mESCs, whileothers (Oct3/4, Sox2,Nanog, Esrrb, Tbx3,Gbx2,Nr5a2,Nr0b1 andTfcp2l1) were unable to, confirming the functional specificity of theKlf family members (Fig. 5D and data not shown). As none ofthe single naïve-state-specific transcription factors were able tomaintain the ES-like state in the triple-knockout mESCs, it could beinterpreted that the functions of the Klf family do not converge on aparticular single target gene among these candidates.

Klf17 shares functionwithKlf2,Klf4 andKlf5 tomaintain theES-like stateThe data shown above indicated that the three Klf family membersKlf2, Klf4 and Klf5 share a unique function to maintain the ES-likestate. However, the analysis of our previous RNA sequencing(RNA-seq) data of mESCs (Yamane et al., 2015) revealed that manyother Klf family members are also expressed at comparable levels toKlf2, Klf4 and Klf5 in mESCs (Fig. 5B), suggesting a possiblecontribution of other Klf family members. The phenotype of theKlf2:Klf4:Klf5 triple knockout might be caused by the reduction ofthe overlapping functions below the threshold level as a sum, to

Fig. 2. Inducible knockout of Klf4.(A) Colony formation of Klf4 knockoutmESCs. The relative colony numbersin the culture with or without Tx areshown (+Tx=1.0). Error bars indicatestandard deviations of triplicateresults. (B) Proliferation of Klf4knockout mESCs. Cell numbers thatoriginated from 104 cells after culturefor 5 days are shown. Error barsindicate standard deviations of fiveindependent results. (C) RT-qPCRanalysis of gene expression ininducible and constitutive Klf4knockout mESCs. The relative geneexpression levels [wild-type mESC(wt ES)=1.0] in inducible knockoutmESCs cultured with or without Tx for3 days and constitutive knockoutmESCs are indicated. Error barsindicate standard deviations oftriplicate quantifications. *P<0.01,Student’s t-test (n=3) versus −Txd3.(D) Immunostaining of constitutiveKlf4 knockout mESCs. The loss ofimmunoreactivity against anti-Klf4 Abconfirms a proper knockout event.(E) Chimera contribution ofconstitutive Klf4 knockout mESCs.Three independent clones derivedfrom the same inducible knockoutmESCs were tested. The numbersindicate the number of chimericembryos/number of recoveredembryos/number of injectedblastocysts.

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which other Klf family members also contribute. To address thispossibility, we tested the abilities of other Klf family members tomaintain the ES-like state of the triple-knockout mESCs. Themousegenome contains 17 Klf family members that are categorized intothree groups (or unclassified) based on the amino acid sequence oftheir three tandem zinc fingers located at the C-terminus(McConnell and Yang, 2010). Klf2, Klf4 and Klf5 all belong toGroup 2. We chose nine members, excluding Klf2, Klf4 and Klf5,from all categories: Klf3 and Klf8 from Group 1, Klf6 and Klf7 fromGroup 2, Klf9, Klf10 and Klf16 from Group 3; and Klf15 and Klf17from unclassified (Fig. 5E). In addition, we also included Sp1 andSp5 in this assay because of the similarity of their amino acidsequence in the three tandem zinc fingers to that of the Klf family,and their high level of expression in mESCs (Fig. 5B). The codingsequences were inserted into expression vectors with or without afusion of Ty1-tag, and their abilities to maintain the mESC-like stateof the triple-knockout mESCs were assessed. Proper proteinexpression from these vectors was confirmed by western blotanalysis of the protein lysates prepared from the pools of stabletransfectants obtained in the absence of Tx with anti-Ty1 antibody(Fig. S5A). In the culture with Tx, we found that the rescue abilitywas quite specific to Klf2, Klf4 and Klf5 with or without Ty1(Fig. 5F). The sole exception was Klf17, although the number ofrescued clones was lower than those of canonical Klfs. Thesetransfectants continued propagation, and proper rescue events wereconfirmed by western blot and RT-qPCR analyses of the pooledtransfectants grown in the presence of Tx (Fig. S2). It was recentlyreported that KLF17 is expressed in the epiblast of human pre-

implantation embryos (Blakeley et al., 2015) as well as in humannaïve-like PSCs (Takashima et al., 2014). Interestingly, thesehuman PSCs lack the expression of KLF2, suggesting functionalreplacement by KLF17 (Blakeley et al., 2015). The rescue ability ofhuman KLF17 was comparable to that of mouse Klf17 and lowerthan that of human KLF4. These data suggested a possible role ofKLF17 that might share an overlapping function with KLF4 andKLF5 in human naïve PSCs as a replacement for KLF2.

The third zinc finger defines the unique functionTaking advantage of the rescue system, we next addressed themolecular basis that specifies the function of the Klf familymembers in mESCs. The rescue assay with a series of mutant formsof Klf4 indicated that the zinc-finger domain is required for itsspecific function. However, the N-terminal region might alsopossess a specific function because complete deletion resulted inabolishment of the rescue ability, while deletion of the knownfunctional domains, transactivation domain [TAD; amino acids (aa)91-109 of 1-483 aa for Klf4] (Geiman et al., 2000) and serine-richdomain (SRR; aa 124-148) for phosphorylation by ERK1/2(MAPK3/MAPK1) and binding to βTrCP2 (FBXW11) for proteindegradation (Kim et al., 2012) caused partial loss (Fig. 5G). Fromthe domains swapped between Klf4 and other Klf family members[Klf3 of Group 1, Klf7 of Group 2 and Klf9 of Group 3 (Fig. 5I)] wecould see that the zinc-finger domain of Klf7, but not that of Klf3and Klf9, was capable of replacing that of Klf4 (Fig. 5H), indicatingthe conserved function of the zinc-finger domain in Group 2 Klffamily members. This was confirmed by successive replacement

Fig. 3. Inducible knockout of Klf2and Klf4. (A) Colony formation of Klf2:Klf4 knockout mESCs. The relativecolony numbers in the culture with orwithout Tx are shown (+Tx=1.0). Errorbars indicate standard deviations oftriplicate results. *P<0.01, Student’s t-test (n=3) versus −Tx. (B) ProliferationofKlf2 knockoutmESCs. Cell numbersthat originated from 104 cells afterculture for 5 days are shown. Errorbars indicate standard deviations offive independent results. *P<0.01,Student’s t-test (n=5) versus EB5.(C) RT-qPCR analysis of geneexpression in inducible andconstitutive Klf2:Klf4 knockoutmESCs. Relative gene expressionlevels [wild-type mESC (wt ES)=1.0] ininducible knockout mESCs culturedwith or without Tx for 3 days andconstitutive knockout mESCs areindicated. Error bars indicate standarddeviations of triplicate quantifications.*P<0.01, Student’s t-test (n=3) versus−Txd3. (D) Immunostaining ofconstitutive Klf2:Klf4 knockoutmESCs. The loss of immunoreactivityagainst anti-Klf2 and Klf4 Abs confirmsa proper knockout event. (E) Chimeracontribution of constitutive Klf2:Klf4knockout mESCs. Three independentclones derived from the sameinducible knockout mESCs weretested. The numbers indicate thenumber of chimeric embryos/numberof recovered embryos/number ofinjected blastocysts.

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with the zinc-finger domain of Drosophila luna (Fig. 5H) thatshows the highest homology to Group 2 mouse Klf family members(Fig. 5E, Fig. S4).Drosophila luna and dar1, but notKrüppel, seemto be the ancestral genes of the Klf family, considering their highesthomology among the Drosophila genome to the mouse Klf familymembers at the three tandem zinc-finger domains and the conservedexon boundary between the first and second zinc-finger domains(Fig. S4). We confirmed that both wild-type Krüppel and the Klf4mutant carrying the zinc-finger domain of Krüppel lack the rescueability (Fig. 5J). Therefore, Klf is not a Krüppel-like factor in thisrevised homology search. These data also suggested thecontribution of the N-terminal region to the specific function inPSCs because the native form of mouse Klf7 and Drosophila luna

failed to restore the ability to maintain the ES-like state in thetriple-knockout mESCs, indicating insufficient function of theirN-terminal domain.

These data indicated that Klf2, Klf4 and Klf5 share specificfunctions to support pluripotency, mainly based on the specific zinc-finger sequence. To identify the responsible region within the zinc-finger domain, we performed subdomain swapping in the threetandem zinc-finger domains between Klf4 and Klf3 in Klf4/3, achimeric molecule that consists of the N-terminal region of Klf4 andthe zinc-finger domain of Klf3 (Fig. 5I). When a series of theswapping mutants was tested, we found that the mutant carrying thethird zinc finger ofKlf4 restored the ability tomaintain the mESC-likestate in the triple-knockout mESCs (Fig. 5H). These data indicated

Fig. 4. Inducible knockout of Klf5, Klf2:Klf5 and Klf4:Klf5. (A) Colony formation of Klf5, Klf2:Klf5 and Klf4:Klf5 knockout mESCs. The relative colony numbersin culture with or without Tx are shown (+Tx=1.0). Error bars indicate standard deviations of triplicate results. *P<0.01, Student’s t-test (n=3) versus −Tx.(B) Proliferation of Klf5, Klf2:Klf5 and Klf4:Klf5 knockout mESCs. Cell numbers that originated from 104 cells after culture for 5 days are shown. Error bars indicatestandard deviations of five independent results. (C) RT-qPCR analysis of gene expression in inducible and constitutive Klf5 knockout mESCs. The relative geneexpression levels [wild-type mESC (wt ES)=1.0] in inducible knockout mESCs cultured with or without Tx for 3 days and constitutive knockout mESCs areindicated. Error bars indicate standard deviations of triplicate quantifications. *P<0.01, Student’s t-test (n=3) versus −Txd3. (D) Immunostaining of constitutiveKlf5, Klf2:Klf5 and Klf4:Klf5 knockout mESCs. The loss of nuclear signal with anti-Klf5 Ab confirms a proper knockout event. (E) RT-qPCR analysis of geneexpression in inducible and constitutive Klf2:Klf5 knockout mESCs. Relative gene expression levels (wt ES=1.0) in inducible knockout mESCs cultured with orwithout Tx for 3 days and constitutive knockout mESCs are indicated. Error bars indicate standard deviations of triplicate quantifications. *P<0.01, Student’s t-test(n=3) versus −Txd3. (F) RT-qPCR analysis of gene expression in inducible and constitutive Klf4:Klf5 knockout mESCs. Relative gene expression levels(wt ES=1.0) in inducible knockout mESCs cultured with or without Tx for 3 days and constitutive knockout mESCs are indicated. Error bars indicate standarddeviations of triplicate quantifications. *P<0.01, Student’s t-test (n=3) versus −Txd3.

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that the specific function of the zinc-finger domain depends on theunique third zinc finger shared among Group 2 Klf family members.

Overlapping functions of Klf family members, targetingmultiple naïve-specific transcription factorsTriple knockout of Klf2, Klf4 and Klf5 resulted in thedownregulation of multiple naïve-specific transcription factors,while the restoration of any one of them was able to maintain themESC-like state of the triple-knockout mESCs. It is possible thatthe overlapping functions of the Klf family are connected tomultiple targets downstream. To address this, we chose five naïve-specific transcription factors (Nanog, Esrrb, Tbx3, Gbx2 andNr5a2) to test the ability of their combinations to support themESC-like state of the triple-knockout mESCs. When all 32

combinations were tested in the inducible triple-knockout mESCscarrying a Rex1 (Zfp42)-mCherry reporter as a monitor of the naïvestate (Kalkan et al., 2017; Toyooka et al., 2008), we found thatparticular combinations showed the ability to support stem cellcolony formation with Rex1-mCherry expression (Fig. 6A). Thecombination of Nanog and Tbx3 showed significant ability, whichwas further enhanced by the addition of Esrrb. The combination ofNanog, Esrrb and Gbx2 also showed the synergistic potential. Inmost cases, the primary stem cell colonies were able to beexpanded and serially passaged more than ten times while keepingmESC morphology and Rex1-mCherry expression (Fig. 6B),although they showed slower proliferation ratio and higherincidence of differentiation than wild-type mESCs. These dataindicated that forced expression of particular combinations of

Fig. 5. Inducible knockout of Klf2:Klf4:Klf5 and the functional rescue assay. (A) RT-qPCR analysis of the time course of gene expression levels in inducibletriple-knockout mESCs. Relative gene expression levels (day 0=1.0) in inducible triple knockout mESCs cultured with Tx for the indicated periods (day 0 to 4) areshown. Error bars indicate standard deviations of triplicate quantifications. (B) The expression levels of the Klf family members in mESCs. The fragments perkilobase of transcript per million reads (FPKMs) from RNA-seq data [GEO accession number GSE76143] are shown. Error bars indicate standard deviations ofRNA-seq data of biological triplicates of Zscan10fl/fl mESCs. (C) Chimera contribution of inducible triple-knockout mESCs. The inducible knockout mESCscultured without Tx were tested. The numbers indicate the number of chimeric embryos/number of recovered embryos/number of injected blastocysts. (D) Colonystaining in the rescue assay. Inducible triple-knockout mESCs transfected with empty and Klf4 expression vectors were cultured with or without Tx for 7 days. Thecolonies were stained by Leischman staining. (E) Phylogenetic tree of mouse Klf family members. The amino acid sequences encoding triple zinc-finger motifswere aligned and analyzed by the UPGMA method. Group 1, 2 and 3 members are labeled with yellow, blue and green, respectively. (F) Rescue ability ofmouse Klf family members. The ratio of colony numbers with versus without Tx (rescue index) are indicated for three independent experiments. Error bars indicates.d. of triplicate experiments. (G) Schematic of the various mutant forms of Klf4. Asterisks indicate a point mutation. (H) Rescue ability of mutant forms of Klf4.(I) Schematic of the various chimeric mutants of Klf4. (J) Rescue ability of chimeric mutants of Klf4.

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transcription factors were able to maintain the ES-like state in theabsence of the canonical Klf family members.Then, we examined the expression levels of the endogenous

pluripotency-associated genes in the rescued clones by RT-qPCR(Fig. 6C). These rescued clones maintained the expression ofOct3/4,Sox2, Tfcp2l1, Sall4 and Foxd3 at comparable levels to those of wild-type mESCs. In contrast, the expression levels of Utf1, Nr0b1, Tcl1and Dppa3 were reduced, although their expression was maintainedin the triple-knockout mESCs rescued byKlf2. Exceptionally,Dppa3expression level was restored in the rescue clone with all fivetransgenes, suggesting their synergistic effect. These data suggestedthat the former set of genes was indirectly activated by the Klf familyvia at least three naïve transcription factors to maintain the ES-likestate, whereas the latter set was directly activated by Klf familymembers, although dispensable for maintenance of the ES-like state.

Klf function is dispensable in primed PSCsIt was suggested that the functions of the Klf family members inPSCs are specific to the naïve state based on their high level of

expression in the naïve state as well as their potential to reprogramprimed PSCs into the naïve state (Jeon et al., 2016). For strictconfirmation of this hypothesis, we assessed whether the triple-knockout mESCs adapt to conversion to the primed state in vitro.The inducible triple-knockout mESCs were cultured in thecondition for epiblast stem cells (EpiSCs) (Sugimoto et al., 2015),which contained activin, FGF2 and IWP2 (inhibitor of porcupine toblock autocrine Wnt protein signaling) instead of LIF, with orwithout Tx for 5 days, followed by passages in the same condition.After three passages, we found that the inducible triple-knockoutmESCs cultured with Tx showed flattened colony morphology andlost the expression of Rex1-mCherry, whereas those without Txretained a dome-like colony morphology with strong Rex1-mCherrysignal (Fig. 7A). Then, we analyzed the gene expression patterns inthese cells by RT-qPCR (Fig. 7B,C). The triple-knockout mESCscultured without Tx maintained the expression of several naïvemarkers (Nanog, Tbx3, Esrrb, Nr5a2, Tfcp2l1 and Nr0b1) atcomparable levels to those of wild-type mESCs, with faintinduction of primed markers [Fgf5, Otx2, Wnt3, Dnmt3b, Oct6

Fig. 6. Rescue assay ofKlf2:Klf4:Klf5 inducible knockoutmESCswith combinatorial forced expression of five transcription factors. (A) Rescue ability of32 combinations of five transcription factors (Nanog, Esrrb, Tbx3, Gbx2 and Nr5a2). The resulting numbers of stem cell colonies for each combination wereintegrated from five independent experiments (with error bars indicating s.d.). (B) Colonymorphologies of the rescued clones obtained from different combinationsof transcription factors. The combination of transcription factors in each clone is indicated in C. Pairs of phase-contrast images and fluorescent images to detectRex1-mCherry are shown. Clone numbers are also indicated. (C) RT-qPCR analysis of gene expression in the rescued triple-knockout mESCs. The relative geneexpression levels [wild-type mESC (wt ES)=1.0] are indicated. Error bars indicate standard deviations of triplicate quantifications. *P<0.01, Student’s t-test (n=3)versus wt ES.

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(Pou3f1 – Mouse Genome Informatics), Sox3 and Sall2] (Smith,2017; Tesar et al., 2007). In contrast, the triple-knockout mESCscultured with Tx showed a similar expression pattern of naïve andprimed markers to that of embryo-derived EpiSCs (Tesar et al.,2007). These triple-knockout cells continued propagation up to tenpassages. This was in contrast to the triple-knockout mESCscultured in the condition for naïve PSCs with LIF, which suddenlyceased proliferation. These data indicated that the functions of Klf2,Klf4 and Klf5 are dispensable for self-renewal in primed PSCs.

DISCUSSIONThe overlapping function of the three Klf family members Klf2,Klf4 and Klf5 in mESCs to maintain self-renewal was first reportedby Jiang et al., who observed partial loss of function using siRNA-mediated knockdown (Jiang et al., 2008). Among them, thefunctions of Klf2 and Klf4 have been further emphasized inrelation to pluripotency because of their higher potential forreprogramming than that of Klf5 (Jeon et al., 2016; Nakagawa et al.,2008), tight links to the known signal pathways to maintainpluripotency (Niwa et al., 2009; Yeo et al., 2014) and uniquepotential to reset naïve state in human PSCs (Takashima et al.,2014). Here, we employed the conventional strategy of inducibleknockout for these three Klf family members. The cell biologicalcharacterization revealed that both Klf2-null and Klf4-null mESCs

retained pluripotency as evaluated by their abilities to contribute tochimeric embryos after single-cell injection into the blastocyst.Moreover, Klf2:Klf4 double-knockout mESCs maintainedpluripotency, indicating that both Klf2 and Klf4 are not essentialto maintain pluripotency in mESCs. These results are consistentwith the phenotypes of Klf2 and Klf4 knockout embryos reportedpreviously. Klf2-null embryos die between embryonic day (E) 12.5and E14.5, owing to defects in tunica media formation and bloodvessel stabilization (Kuo et al., 1997), whereas Klf4-null mice dieimmediately after birth as a result of the defects in the epithelialbarrier in skin and colon (Katz et al., 2002; Segre et al., 1999). Bothshow no phenotype in pluripotent cell population at pre- and peri-implantation stage. We also demonstrated that the specific functionof the Klf family members in maintaining pluripotency is restrictedto these three Klfs and to Klf17 to a weaker degree. Therefore, Klf5is the sole Klf family member working in Klf2:Klf4-null mESCs forthe maintenance of pluripotency, and its function is sufficientbecause the expression level of Klf17 is negligible in mESCs(Fig. 5B). The overlapping potential of Klf17 with the canonicalKlfs might suggest the significance of human KLF17 in humannaïve PSCs, because in these cells the expression of KLF17 is high,whereas the expression ofKLF2 –which has a significant role in themaintenance of pluripotency in naïve mESCs – is low (Blakeleyet al., 2015). The functional redundancy of these Klf family

Fig. 7. Transition of triple-knockout mESCs to the primed state. (A) Colony morphologies of the inducible triple-knockout mESCs cultured in EpiSC conditionfor three passages with or without Tx. Pairs of phase-contrast images and fluorescent images to detect Rex1-mCherry are shown. (B) RT-qPCR analysisof the expression of naïve marker genes in the triple-knockout mESCs cultured in EpiSC condition for three passages. The relative gene expression levels[wild-type mESC (wt ES)=1.0] are indicated. Error bars indicate standard deviations of triplicate quantifications. *P<0.01, Student’s t-test (n=3) versus EpiSC.(C) RT-qPCR analysis of the expression of primed marker genes in the triple-knockout mESCs cultured in EpiSC condition for three passages. The relativegene expression levels (embryo-derived EpiSC=1.0) are indicated. Error bars indicate standard deviations of triplicate quantifications. *P<0.01, Student’s t-test(n=3) versus EpiSC. (D) Scheme of the function of the Klf family members in mESCs.

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members is a typical example of their overlapping functions (Niwa,2018).The loss of Klf5 resulted in the loss of the ability to contribute to

chimeric embryos in our strict assay system. Highly contributedchimeras might die after implantation because Klf5-null embryosshow lethal phenotype at this timing (Ema et al., 2008). However, itwas previously reported that Klf5-null mESCs were able to give riseto chimeric embryos by the conventional strategy of blastocystinjection, although the contribution was poor (Ema et al., 2008).Here, we did not perform the chimera assay with injection ofmultiple mESCs into blastocysts for our Klf5-null mESCs, so wecould not rule out the possibility that a minor proportion ofKlf5-nullmESCs retain the ability to contribute to chimeric embryos. Thepoor ability might be caused by the slow proliferation of Klf5-nullmESCs that can cause elimination from chimeric embryos afterimplantation when the epiblasts expand very rapidly (Snow, 1977).Alternatively, they might have reduced ability to respond to theexternal signal in the developmental context, which could bemasked in vitro by excess amount of external signals, such as LIF, inthe culture medium, although essential in the developmental contextas found in the Klf5-null embryos (Lin et al., 2010).The domain swapping experiment revealed that the third zinc-

finger domain of the Klfs is crucial for their specific function inmESCs. The comparison of the amino acid sequences of thisdomain between Klf4 and Klf3 elucidated the difference by onlyfour amino acids (64Q, 65K, 69A and 80M in Fig. S6).Interestingly, these four residues cluster out of the third zinc-finger domains when Klf4 binds to the target sequence with the firstand second zinc fingers (Hashimoto et al., 2016), suggesting theirfunction in the interaction with other protein(s) rather than the targetDNA sequence, or with an additional target DNA sequence besidesthe one bound by the three zinc-finger domains of Klf4 (Schuetzet al., 2011). The specific function of the zinc-finger domain in Klf4was shared by that of Drosophila luna, which shows the highestamino acid homology with conserved exon-intron organization intheDrosophila genome, suggesting that the pluripotency-associatedfunction of the zinc-finger domain is a co-optional use of theevolutionarily conserved function as in the case of the evolution ofSox2 (Niwa et al., 2016). Among the genes sharing homology in thethree tandem zinc-finger domains, Drosophila luna and daf, as wellas Caenorhabditis elegans klf2, share the common exon boundarybetween the first and second zinc fingers with most of the mouse Klffamily members (Fig. S4). These genes could be the descendants ofthe ancestral gene of the vertebrate Klf family. They are in contrastto Drosophila Krüppel, which shows lower homology without thisboundary, of which the zinc finger domain does not support self-renewal of mESCs. Therefore, Klf family members are not likeKrüppel in the evolutional sense.The rescue experiment with multiple naïve-specific transcription

factors revealed that the common function of Klf family members ismediated by multiple target genes. The potent targets includeNanog and Tbx3, both of which are well known for their function tosupport LIF-independent self-renewal of mESCs (Mitsui et al.,2003; Niwa et al., 2009). They could be the direct targets activatedby the Klf family, considering their rapid downregulation in thetriple-knockout mESCs. The rescue effect might be based on theirparallel function in activating the core transcription factors, such asOct3/4 and Sox2 (Niwa et al., 2009). In addition to them, Esrrb andGbx2 showed contribution to support the ES-like state in the triple-knockout mESCs. These genes are also known for their function insupporting LIF-independent self-renewal of mESCs (Martello et al.,2012; Tai and Ying, 2013). When these transgenes were introduced

into the triple-knockout mESCs, they supported the expression of aset of pluripotency-associated transcription factors, including Oct3/4, Sox2, Sall4, Foxd3 and Tfcp2l1. These genes, except Tfcp2l1, areknown as core components of the pluripotency-associatedtranscription factors because they are expressed in both naïve andprimed PSCs (Factor et al., 2014). Oct3/4 and Sox2 are absolutelyessential for maintaining self-renewal of mESCs (Masui et al., 2007;Niwa et al., 2000), whereas Sall4 is required for rapid and stableself-renewal (Sakaki-Yumoto et al., 2006) and Foxd3 is known forits function to control transition from naïve to primed state(Respuela et al., 2016; Krishnakumar et al., 2016). Tfcp2l1 isspecifically expressed in naïve PSCs and possesses the ability tosupport LIF-independent self-renewal in mESCs (Martello et al.,2013), suggesting its functional importance in maintaining self-renewal. Because these functionally verified pluripotency-associated genes are activated in the rescued triple-knockoutmESCs without canonical Klfs, the involvement of the canonicalKlfs in activating this set of pluripotency-associated genes isdispensable. In contrast, there is another set of genes that showsdecreased levels of expression in the rescued triple-knockoutmESCs. This set includes Utf1, Nr0b1, Tcl1 and Dppa3, none ofwhich are known for their essentiality to maintain self-renewal.However, their function to modulate stable self-renewal has beenreported. For example, Utf1 is reported to function in executingpluripotency by regulation of the bivalent genes (Jia et al., 2012).We previously demonstrated that Nr0b1-null mESCs showdestabilized self-renewal by de-repression of Zscan4c (Fujii et al.,2015). Tcl1 is known to be involved in rapid proliferation (Miyazakiet al., 2013). The lack of proper activation of these genes might be acause of the slow proliferation of the rescued triple-knockoutmESCs with high incidence of differentiation. Therefore, theoverlapping function of the canonical Klfs could be to upregulatemultiple transcription factors to activate the core transcriptionfactors, as well as to activate unique target genes to coordinate rapidand stable self-renewal (Fig. 7D).

The function of the Klf family members is strictly specific to thenaïve state because the triple-knockout mESCs are successfullycaptured at primed state. These data confirmed that the primed statePSCs possess a transcriptional network to maintain the expressionof core transcription factors, which is completely distinct from thosein the naïve state PSCs, as proposed previously (Weinberger et al.,2016). Rapid and complete exit from the naïve state by removal ofthe Klf family members will provide opportunity to analyze theformative state recently proposed to be a transient state betweenthe naïve and primed states (Smith, 2017). The extended analysis inthe context of the transcription factor network will be required forfurther elucidation of the functions of these Klf family members inmaintaining naïve pluripotency. The cell biological evidence shownhere will provide a solid basis to systemic omics analysis in future.

MATERIALS AND METHODSCell cultureEB5 mESCs (derived from male E14tg2a mESCs, Oct3/4IRES-BSD-pA/+,deposited to RIKEN Cell Bank) and their derivatives were cultured on agelatin-coated surface in Glasgow minimum essential medium (G-MEM)(Wako Pure Chemical, 078-05525) supplemented with 10% KSR (ThermoFisher Scientific, 10826-028), 1% FCS (Hyclone), 1× sodium pyruvate(Nacalai Tesque, 06977-34), 1× nonessential amino acids (Nacalai Tesque,06344-56), 10−4 M 2-mercaptoethanol (Nacalai Tesque, 21417-52) and1000 U/ml LIF (home-made conditioned medium produced by COS cellstransiently transfected with pCAGGS-LIF). For the proliferation assays, themESCs were also cultured in G-MEM supplemented with 10% FCS, 1×sodium pyruvate, 1× nonessential amino acids, 10−4 M 2-mercaptoethanol

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and 1000 U/ml LIF. All mESC lines described in this article will beavailable from RIKEN Cell Bank.

To assess the transition to primed state, the mESCs were cultured inDulbecco’s modified Eagle medium/Ham’s F-12 (Wako Pure Chemical,048-29785), supplemented with 15% KSR, 1× sodium pyruvate, 1×nonessential amino acids, 10−4 M 2-mercaptoethanol, 20 ng/ml activin A(human/mouse/rat recombinant, R&D Systems, 338-AC-010), 12 ng/mlFGF2 (human recombinant, Wako Pure Chemical, 067-04031) and 2 µMIWP2 (AdoQ Bioscience, A12707-5) on mitomycin C-treated mouseembryonic fibroblast cells (Sugimoto et al., 2015).

Plasmid constructionFor generation of the Klf2 knockout (KO) vector, genomic DNA fragmentsfor 5′ and 3′ homology arms (Chr:8, 72317939-72319206 and 72321033-72324220 in GRCm38), as well as the floxed region containing exons 2 and 3(Chr:8, 72319207-72321032 in GRCm38), were amplified from EB5genomic DNA using the primers Klf2-5′, Klf2-3′ and Klf2-flox,respectively, shown in Table S1. The PCR fragments of 5′ and 3′homology arms were assembled in the ClaI-NotI sites of pBR-blue II,resulting in pBR-Klf2 5′+3′. The floxed region was subcloned in theNheI siteof ploxP-BNA. Then, the SpeI fragment carrying the floxed genome wasintroduced into the SpeI site between the 5′ and 3′ homology arms of pBR-Klf2 5′+3′, resulting in pBR-Klf2 5′+flox+3′. Finally, the XhoI fragment oftheFrt-IRES-pac-pA-Frt cassettewas introduced into theBamHI site flankingthe 5′ end of the 3′ loxP sequence of pBR-Klf2 5′+flox+3′ using the partialfill-in method, resulting in pBR-Klf2 floxKO.

For generation of the Klf4KO vector, genomic DNA fragments for 5′ and3′ homology arms (Chr:4, 55532490-55531410 and 55528124-55524067in GRCm38), as well as the floxed region containing exons 3, 4 and 5(Chr:4, 55531419-55528125 in GRCm38), were amplified from EB5genomic DNA using the primers Klf4-5′, Klf4-3′ and Klf4-flox,respectively, shown in Table S1. These PCR fragments of 5′ and 3′homology arms were assembled in the ClaI-NotI sites of pBR-blue II,resulting in pBR-Klf4 5′+3′. The floxed region was subcloned in the NheIsite of ploxPP2272. Then, the SpeI fragment carrying the floxed genomewas introduced into the SpeI site between the 5′ and 3′ homology arms ofpBR-Klf4 5′+3′, resulting in pBR-Klf4 5′+flox+3′. Finally, the XhoIfragment of the Frt-IRES-pac-pA-Frt cassette and Frt-IRES-Hytk [fusion ofhph and HSV-tk (Lupton et al., 1991)]-pA-Frt was introduced into the AgeIsite flanking the 3′ end of the 3′ loxP sequence of pBR-Klf4 5′+flox+3′using adaptor DNA, resulting in pBR-Klf4 floxKO pac and pBR-Klf4 floxKOHytk, respectively.

For generation of the Klf5KO vector, genomic DNA fragments for 5′ and3′ homology arms (Chr:14, 99299127-99300223 and 99303877-99307091in GRCm38), as well as the floxed region containing exons 2 and 3 (Chr:14,99300224-99303885 in GRCm38), were amplified from EB5 genomicDNA using the primers Klf5-5′, Klf5-3′ and Klf5-flox, respectively, shownin Table S1. These PCR fragments were assembled in the SacII-XhoI sites ofpBR-blue II, resulting in pBR-Klf5 5′+3′. The floxed region was subclonedin the BamHI site of ploxP-BNA. Then, the SpeI fragment carrying thefloxed genome was introduced into the AgeI site between the 5′ and3′ homology arms of pBR-Klf5 5′+3′ using adaptor DNA, resulting in pBR-Klf5 5′+flox+3′. Finally, the XhoI fragment of Frt-SA-IRES-neo-pA:PGK-pacΔtk [fusion of pac and HSV tk (Chen and Bradley, 2000)]-pA-Frtcassette was introduced into the NheI site flanking the 3′ end of the 5′ loxPsequence of pBR-Klf5 5′+flox+3′ using the adaptor DNA, resulting in pBR-Klf5 floxKO.

For generation of the Rex1-mCherry knock-in vector, mCherry-IRES-pac-pA cassette was introduced into the SpeI-EcoRI sites of Rex1 5′+3′homology arms as described previously (Toyooka et al., 2008).

The PiggyBac vectors pPBCAG-MerCreMer-IH, pPBCAG-EGFP-IZ,pBRPBCAG-cHA-IN and pBRPBCAG-Ty1linker-cHA-IN were constructedbased on pGG131 (Guo et al., 2009). Ty1 linker consists of 3× Ty1(EVHTNQDPLD) and glycine linker (GGSGGGGSGGGSSSS). Forconstruction of the expression vectors of the Klf family members, theentire coding regions were amplified from the complementary DNA(cDNA) of mESCs using the primers shown in Table S1, and the PCRproducts were digested and inserted into the XhoI-NotI site of either

pBRPBCAG-cHA-IN or pBRPBCAG-Ty1linker-cHA-IN. Drosophila lunaand Krüppel were amplified from the cDNA of Drosophila embryos orDrosophila genomic DNA.

Generation of inducible KO mESCsEB5 mESCs (107) were electroporated with 100 µg linearized KO vectorDNA at 800 V and 3 µF in a 0.4-cm cuvette using a Gene Pulser (Bio-Rad),followed by culture with 1.5 µg/ml puromycin (for Klf2 and Klf4 KO pacvector), 200 µg/ml Hygromycin B (for Klf4 KOHytk vector) or 1.5 µg/ml ofpuromycin and 200 µg/ml of G418 (for Klf5 KO vector) for 8 days. Theresulting stem cell colonies were picked up, expanded and genotyped byPCR using the primer pairs KO-PCR1 and KO-IRES.

For the removal of the Frt cassette, the correctly targeted clones wereseeded in a well of a 48-well plate at 104 cells per well, and transfected with1 µg circular pCAG-FLPe-IP plasmid using Lipofectoamine 2000 (ThermoFisher Scientific, 11668027), followed by culture for 3 days. Then, thesetransfected cells were re-plated and cultured for 8 days at clonal density (forKlf2 KO and Klf4 KO pac) or normal density with 1 µM gancyclovir (forKlf4 KOHytk andKlf5 KO) for 8 days. The resulting stem cell colonies werepicked up, expanded and genotyped by PCR.

In the case of Klf2 KO and Klf4 KO, the selection of the gene conversionevent was applied to isolate homozygous KO clones (Nakatake et al., 2013).Heterozygous mESCs (106) carrying the Frt-IRES-pac-pA-Frt cassette wereseeded on a 90-mm dish with medium containing 6 µg/ml puromycin andcultured for 3 days. Then, themediumwas changed to reduce the concentrationof puromycin to 1.5 µg/ml and the cells were cultured for 8 days. The resultingstem cell colonies were picked up, expanded and genotyped by PCR.

To generate the inducible mESC lines, the clones in which the loxP siteswere correctly introduced in both alleles and the Frt cassette were removed,seeded in a well of a 48-well plate at 104 cells per well, and transfected with0.25 µg circular pPB-CAG-MerCreMer-IH, 0.25 µg circular pPB-EGFP-IZand 0.5 µg circular pCAG-PiggyBac transposase (PBase) plasmids usingLipofectoamine 2000, followed by culture for 2 days. Then, thesetransfected cells were re-plated and cultured with 200 µg/ml HygromycinB and 20 µg/ml Zeocin for 8 days. The resulting stem cell colonies werepicked up, expanded and assessed for the expression of Egfp by fluorescentmicroscopic analysis, and the function ofMerCreMerwas assessed by PCRgenotyping of the cells cultured with 1 µg/ml 4-hydroxy tamoxifen (Tx).

For the establishment of the KO clones, the inducible KO mESCs werecultured with 1 µg/ml Tx for 3 days. Then, these cells were dissociated andseeded at clonal density (1000 cells per 90-mm dish), followed by culture for8 days. The resulting stem cell colonies were picked up, expanded andgenotyped by PCR. A PCR reaction with three primers was applied toensure a proper deletion event.

Production of chimeric embryosA dissociated single mESC was injected into a C57BL6 blastocyst bymicroinjection. The blastocyst was then transferred into the uterus of apseudo-pregnant female ICR mouse. Embryos were collected at 13.5 dayspostcoitum to evaluate the chimera contribution ability of mESCs byanalyzing with fluorescence microscopy. The efficiency of chimeraproduction with wild-type mESCs was shown in our previous reports(Ohtsuka et al., 2012; Ohtsuka and Niwa, 2015). All animal experimentsconformed to Institute Guidelines for the Care and Use of LaboratoryAnimals and were approved by the Institutional Committee for LaboratoryAnimal Experimentation (RIKEN Kobe Institute).

Immunostaining and western blottingMonoclonal antibody against Klf2 was obtained by immunization of amouse with purified fusion protein of GST and full-length Klf2, followed bythe standard method. Cells were fixed with 4% paraformaldehyde in PBS for30 min at 4°C, followed by permeabilization with 0.2% Triton X-100 inPBS for 10 min at room temperature. These cells were incubated with thefollowing primary antibodies overnight at 4°C: mouse monoclonal anti-Oct3/4 (Santa Cruz Biotechnology, sc-5279), 1:1000; rabbit polyclonalanti-Sox2 (antisera), 1:1000; rat monoclonal anti-Nanog (e-Bioscience,MLC-51), 1:1000; mouse monoclonal anti-Klf2 (home-made), 1:1000; goatpolyclonal anti-Klf4 (R&D Systems, AF3640), 1:1000; rabbit polyclonal

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anti-Klf5 (Abcam, ab24331); and rabbit polyclonal anti-Tbx3 (antisera,Toyooka et al., 2008). After washing, cells were incubated with appropriateAlexa Fluor 488-conjugated secondary antibodies for 1 h at roomtemperature with Hoechst 33258, and fluorescent images were taken onan Olympus OX-71 equipped with a CCD camera. Western blotting wasperformed with the antibodies for immunostaining as well as mousemonoclonal anti-Ty1 (Diagnode, MAb-054-050, 1:10,000) and rabbitpolyclonal anti-Cdk2 (Santa Cruz Biotechnology, sc-163, 1:1000) for 8 µgof total cell lysates prepared in TDL buffer [150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 0.5% deoxycholic acid, 1% NP-40, 0.1% SDS]. The signalwas detected by SuperSignal West Femto Maximum Sensitivity Substrate(Thermo Fisher Scientific) using the CCD camera system LAS 4000 Mini(Fuji Film).

Rescue assay in triple-KO mESCsThe inducible triple-KO mESCs (Klf2fl/fl:Klf4fl/fl:Klf5fl/fl: CAG-MCM-IH:CAG-Egfp-IZ) were seeded in a well of a 48-well plate at 104 cells per well,and transfected with 0.5 µg circular expression vector (derived frompBRPBCAG-cHA-IN or pBRPBCAG-Ty1linker-cHA-IN) and 0.5 µg circularpCAG-PiggyBac transposase (PBase) plasmid using Lipofectoamine 2000,followed by culture for 2 days with or without Tx. Then, the transfected cellswere re-plated and cultured with 200 µg/ml G418 with or without Tx for8 days. For evaluation of the rescue ability, the colonies were stained withLeishman staining solution, the numbers of the colonies with compactmorphology were counted under a microscope, and the ratio between thepresence and absence of Txwas calculated. For assessing the rescue event, thepool of colonies was cultured for preparation of RNA for quantitative PCRand protein lysate for western blotting. For the combinatorial transfection offive transcription factors (Nanog, Tbx3, Esrrb, Gbx2 and Nr5a2), thesecDNAs were subcloned into pBRPBCAG-cHA-IN. Then, 0.15 µg of each ofthe expression vectors and 0.25 µg of circular PBase vector were transfected.For the combination of fewer than five factors, the total amount of theexpression vector was adjusted with pBRPBCAG-cHA-IN.

Phylogenetic analysis of Klf family membersThe open reading frame sequences for the members of mouse Klf family(Klf1-17), mouse Sp1 and Sp5, and Drosophila luna were collected fromEnsembl genome database. The amino acid sequences of the zinc-fingerdomains consist of the three tandem zinc fingers shown in Fig. S4, and wereanalyzed with the UPGMA program of GENETYX-MAC to obtain theevolutionary tree.

FACS analysisFor apoptotic assay, Annexin V-APC (BD Biosciences) staining wasperformed according to the manufacturer’s protocol using inducible triple-KO mESCs and wild-type mESCs.

Real-time PCR analysisFirst-strandDNAwas synthesized from500 ng of the total RNAprepared by aQuickGene RNA cultured cell HC kit (Kurabo) in 20 µl of the reactionmixture containing oligo-dT primers using a ReverTra Ace first strandsynthesis kit (Toyobo). Real-time PCR was performed withTHUNDERBIRD SYBR qPCR Mix (Toyobo) using a CFX384 Real-TimeSystem (Bio-Rad). Primer sequences are listed in Table S1.

AcknowledgementsWe thank Dr Futatsugi-Nakai (RIKEN CDB) for critical editing of the manuscript.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: A.N., H.N.; Methodology: M.Y., S.O., K.M., H.N.; Validation:H.N.; Formal analysis: H.N.; Investigation: M.Y., S.O., K.M., H.N.; Resources: A.N.,H.N.; Writing - original draft: H.N.; Writing - review & editing: H.N.; Visualization:H.N.; Supervision: H.N.; Project administration: H.N.; Funding acquisition: H.N.

FundingThis work was supported by RIKEN and the Japan Science and Technology Agency(CREST program to H.N.).

Data availabilityRNA-seq data are available at Gene Expression Omnibus (GEO), under accessionnumber GSE76143.

Supplementary informationSupplementary information available online athttp://dev.biologists.org/lookup/doi/10.1242/dev.162404.supplemental

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