Targeting Multiple Transcription Factors to Maintain the Naïve

Home , FBXW11, Klf4, The KLF

STEM CELLS AND REGENERATION RESEARCH ARTICLE Overlapping functions of Krüppel-like factor family members: targeting multiple transcription factors to maintain the naïve pluripotency of mouse embryonic stem cells Mariko Yamane1,2,*, Satoshi Ohtsuka1,3,*, Kumi Matsuura1,2, Akira Nakamura4 and Hitoshi Niwa1,2,5,‡

ABSTRACT rapid proliferation and stable self-renewal of mESCs (Ema et al., Krüppel-like factors (Klfs) have a pivotal role in maintaining self- 2008). The overlapping functions of these three Klf family members renewal of mouse embryonic stem cells (mESCs). The functions of in self-renewal of mESCs were examined by partial loss-of-function three Klf family members (Klf2, Klf4 and Klf5) have been identified, and analyses with small interference RNA (siRNA)-mediated are suggested to largely overlap. For further dissection of their knockdown (Jiang et al., 2008). Their functions in reprogramming functions, we applied an inducible knockout system for these Klf of somatic cells (Nakagawa et al., 2008) and primed PSCs (Jeon family members and assessed the effects of combinatorial loss of et al., 2016) into naïve PSCs have also been characterized. However, function. As a result, we confirmed that any one of Klf2, Klf4 and Klf5 their functions in maintenance of pluripotency have not been well was sufficient to support self-renewal, whereas the removal of all three assessed by complete loss-of-function assays in mESCs. Here, we compromised it. The activity of any single transcription factor, except applied a combinatorial inducible knockout strategy to Klfs and for a Klf family member, was not sufficient to restore self-renewal assessed their chimera contribution abilities, confirming that Klf2, of triple-knockout mESCs. However, some particular combinations Klf4 and Klf5 share overlapping functions, and that Klf2 and Klf4 are of transcription factors were capable of the restoration. The triple- not essential to maintain pluripotency in mESCs. knockout mESCs were successfully captured at primed state. These data indicate that the pivotal function of a Klf family member is RESULTS Klf2 Klf4 transduced into the activation of multiple transcription factors in a and are not essential to maintain pluripotency naïve-state-specific manner. We generated a series of inducible knockout mESCs by serial modifications of the endogenous alleles of Klf2, Klf4 and Klf5 with KEY WORDS: Embryonic stem cells, Pluripotency, Transcription introduction of loxP sites and a tamoxifen (Tx)-inducible form of factor Cre recombinase (Fig. S1). Inducible knockout of Klf2, but not Klf4, resulted in significant reduction of efficiency in stem cell colony INTRODUCTION formation (Figs 1A and 2A). However, constitutive knockout of Krüppel-like factors (Klfs) encode zinc-finger transcription factors each gene in Klf2-orKlf4-null mESCs showed stable self-renewal and have a pivotal role in maintaining self-renewal in pluripotent with a normal proliferation ratio comparable to that of wild-type stem cells (PSCs). Functions related to pluripotency were identified mESCs in either conventional culture medium with fetal calf serum in three of the Klf family members. Klf4 was identified as one of (FCS) or very low FCS with knockout serum replacement (KSR) the Yamanaka factors to reprogram somatic cells into PSCs in (Figs 1B and 2B). The complete loss of the targeted gene product cooperation with Oct3/4 (Pou5f1 – Mouse Genome Informatics), was confirmed by western blot analysis (Fig. S2). Quantitative PCR Sox2 and Myc (Takahashi and Yamanaka, 2006), as well as the analysis of reverse-transcribed mRNA (RT-qPCR) revealed that target of the leukemia inhibitory factor (LIF) signal in mouse they expressed comparable levels of the transcripts of functionally embryonic stem cells (mESCs) (Hall et al., 2009; Niwa et al., 2009). verified pluripotency-associated transcription factors, such as Oct3/ Klf2 was reported to be a target of Oct3/4 (Hall et al., 2009) and to 4, Sox2, Tbx3, Nanog, Esrrb, Gbx2, Nr5a2, Tfcp2l1 and Nr0b1 mediate the action of the mitogen-activated protein kinase kinase (Figs 1C and 2C). They also expressed the remaining Klf family (MEK) inhibitor in mESCs to support pluripotency in defined members (Klf4 and Klf5 in Klf2-null mESCs, and Klf2 and Klf5 in culture condition (Yeo et al., 2014). Klf5 was reported to govern the Klf4-null mESCs) at similar levels to wild-type mESCs, suggesting that there was no obvious dosage compensation within the three Klf family members. The expression levels of the floxed alleles were 1Laboratory for Pluripotent Stem Cell Studies, RIKEN Center for Developmental lower than those of wild-type alleles without induction of Cre Biology (CDB), 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan. 2Department of Pluripotent Stem Cell Biology, Institute of Molecular Embryology activity, which might be caused by the leaky activation of Cre and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, without induction, as well as the faint effect of the deletion for self- Japan. 3Department of Life Science, Medical Research Institute, Kanazawa Medical University, 1-1 Daigaku, Uchinada kahoku, Ishikawa 920-0293, Japan. renewal of these mESCs. It has been known that the pluripotency- 4Department of Germline Development, Institute of Molecular Embryology and associated transcription factors show distinct expression patterns in Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, self-renewing populations, either homogeneous (Oct3/4 and Sox2) Japan. 5JST, CREST, Sanbancho, Chiyoda-ku, Tokyo 1020075, Japan. *These authors contributed equally to this work or heterogeneous in various degrees (Nanog, Klf2, Klf4, Klf5 and Tbx3) (Niwa et al., 2009). When the expression patterns of the ‡ Author for correspondence ([email protected]) pluripotency-associated transcription factors were examined by H.N., 0000-0002-5280-8031 immunostaining, both Klf2- and Klf4-null mESCs showed similar patterns to those in wild-type mESCs except for the deleted genes

Received 10 December 2017; Accepted 30 April 2018 (Figs 1D and 2D). When a single Klf2-orKlf4-null mESC carrying DEVELOPMENT

1 STEM CELLS AND REGENERATION Development (2018) 145, dev162404. doi:10.1242/dev.162404

Fig. 1. Inducible knockout of Klf2. (A) Colony formation of Klf2 knockout mESCs. The relative colony numbers in the 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 Klf2 knockout mESCs. Cell numbers that originated from 104 cells after culture for 5 days are shown. Error bars indicate standard deviations of five independent results. (C) RT- qPCR analysis of gene expression in inducible and constitutive Klf2 knockout mESCs. The relative gene expression levels [wild-type mESC (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 standard deviations of triplicate quantifications. *P<0.01, Student’s t-test (n=3) versus −Txd3. (D) Immunostaining of constitutive Klf2 knockout mESCs. The loss of immunoreactivity against anti-Klf2 antibody (Ab) confirms a proper knockout event. (E) Chimera contribution of constitutive Klf2 knockout mESCs. Three independent clones derived from the same inducible knockout mESCs were tested. The numbers indicate the number of chimeric embryos/number of recovered embryos/number of injected blastocysts.

the ubiquitous EGFP expression vector (CAG-EGFP-IZ)was Combinatorial knockout of Klf2 or Klf4 with Klf5 allow self- injected into blastocysts followed by transfer into pseudo- renewal of mESCs pregnant mice, they gave rise to chimeric embryos with high It was reported that Klf5-null mESCs showed destabilized self- levels of contributions (Figs 1E and 2E), indicating that both Klf2- renewal and slow proliferation (Ema et al., 2008). When knockout of and Klf4-null mESCs retain pluripotency. Klf5 was induced using the same strategy, a significant proportion of the Klf5-null mESCs formed stem cell colonies (Fig. 4A). The Klf2/Klf4 double-knockout mESCs retain pluripotency established Klf5-null mESCs showed self-renewal with slightly Inducible knockout of both Klf2 and Klf4 simultaneously resulted slower proliferation ratio than wild-type mESCs (Fig. 4B). They in dramatic reduction of efficiency in stem cell colony formation showed comparable levels of the transcripts of pluripotency- (Fig. 3A). However, Klf2/Klf4-null mESCs with constitutive associated transcription factors, as well as expression patterns in knockout were able to be established. They continued self- cell populations similar to those of wild-type mESCs, with complete renewal with slower proliferation ratio than wild-type mESCs, loss of Klf5 protein (Fig. 4C,D). However, when a single Klf5-null with loss of Klf2 and Klf4, irrespective of the culture condition mESC carrying the ubiquitous EGFP expression vector was injected (Fig. 3B, Fig. S2). They showed comparable levels of the transcripts into blastocysts, then transferred into pseudo-pregnant mice, they of pluripotency-associated transcription factors (Fig. 3C). The never gave rise to chimeric embryos, although the parental flox expression of Klf5 was slightly but significantly increased (Fig. 3C), mESCs efficiently produced chimeric embryos. In previous reports, suggesting its compensatory function. The expression patterns of conventional injection strategy with multiple Klf5-null mESCs into pluripotency-associated transcription factors in cell populations blastocysts gave low contribution to chimeras (Ema et al., 2008), were also similar to those of wild-type mESCs (Fig. 3D). When a suggesting that loss of Klf5 caused reduction of pluripotency. single Klf2/Klf4-null mESC carrying the ubiquitous EGFP Reduction of pluripotency might be detected more sensitively by our expression vector was injected into blastocysts, followed by single mESC injection assay. Alternatively, it could be simply transfer into pseudo-pregnant mice, they gave rise to chimeric caused by their proliferation capacity after implantation. Inducible embryos with high levels of contributions (Fig. 3E), indicating that knockout of Klf2 and Klf5 caused significant reduction of efficiency Klf2/Klf4-null mESCs retain pluripotency. Because these knockout in stem cell colony formation (Fig. 4A), but the Klf2:Klf5 and Klf4: mESCs retain only Klf5 among the three Klf family members Klf5 double-knockout mESCs were able to be established (Fig. 4B). known to function in mESCs, Klf5 alone might be sufficient to These double-knockout mESCs expressed comparable levels of support pluripotency in mESCs. the transcripts of pluripotency-associated transcription factors, and DEVELOPMENT

2 STEM CELLS AND REGENERATION Development (2018) 145, dev162404. doi:10.1242/dev.162404

Fig. 2. Inducible knockout of Klf4. (A) Colony formation of Klf4 knockout mESCs. The relative colony numbers in the culture with or without Tx are shown (+Tx=1.0). Error bars indicate standard deviations of triplicate results. (B) Proliferation of Klf4 knockout mESCs. Cell numbers that originated from 104 cells after culture for 5 days are shown. Error bars indicate standard deviations of five independent results. (C) RT-qPCR analysis of gene expression in inducible and constitutive Klf4 knockout mESCs. The relative gene expression levels [wild-type mESC (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 standard deviations of triplicate quantifications. *P<0.01, Student’s t-test (n=3) versus −Txd3. (D) Immunostaining of constitutive Klf4 knockout mESCs. The loss of immunoreactivity against anti-Klf4 Ab confirms a proper knockout event. (E) Chimera contribution of constitutive Klf4 knockout mESCs. Three independent clones derived from the same inducible knockout mESCs were tested. The numbers indicate the number of chimeric embryos/number of recovered embryos/number of injected blastocysts.

displayed similar expression patterns in cell populations to those of To test the functional specificity of the Klf family members to wild-type mESCs (Fig. 4D,E,F), but lost the ability to contribute to maintain the ES-like state in mESCs, we performed phenotypic chimeric embryos after single-cell injection, as in the case of Klf5- rescue experiments using the inducible triple-knockout mESCs. The null mESCs. Klf2 expression was slightly elevated in Klf4:Klf5-null parental triple-floxed mESCs retained pluripotency as confirmed by mESCs, whereas Klf4 expression was unchanged in Klf2:Klf5-null their chimera formation ability (Fig. 5C). When we assessed the mESCs. These data suggested that the double-knockout mESCs are rescue ability of various pluripotency-associated transcription able to maintain the mESC-like state with sufficient expression of factors, we found that Klf2, Klf4 and Klf5 were able to restore key pluripotency-associated transcription factors. stem cell colony formation in the triple-knockout mESCs, while others (Oct3/4, Sox2, Nanog, Esrrb, Tbx3, Gbx2, Nr5a2, Nr0b1 and Klf2:Klf4:Klf5 triple knockout compromises self-renewal Tfcp2l1) were unable to, confirming the functional specificity of the To confirm the overlapping function of these three Klf family Klf family members (Fig. 5D and data not shown). As none of members, we generated inducible triple-knockout mESCs for Klf2, the single naïve-state-specific transcription factors were able to Klf4 and Klf5. Induction of a triple-knockout event with Tx resulted maintain the ES-like state in the triple-knockout mESCs, it could be in complete cease of self-renewal. Very few differentiated colonies interpreted that the functions of the Klf family do not converge on a were observed after induction of the triple knockout, and increased particular single target gene among these candidates. apoptosis and cell death were observed by fluorescence-activated cell sorting (FACS) analysis (Fig. S3); therefore, the removal of the Klf17 shares function with Klf2, Klf4 and Klf5 to maintain the three Klf family members seemed to induce cell death rather than ES-like state differentiation. Induction of Klf2:Klf4:Klf5 triple knockout resulted The data shown above indicated that the three Klf family members in rapid downregulation of naïve-state-specific transcription factors Klf2, Klf4 and Klf5 share a unique function to maintain the ES-like (Nanog, Esrrb, Tbx3, Gbx2, Tfcp2l1, Nr0b1 and Nr5a2), followed state. However, the analysis of our previous RNA sequencing by gradual downregulation of general pluripotency-associated (RNA-seq) data of mESCs (Yamane et al., 2015) revealed that many transcription factors (Oct3/4 and Sox2) (Fig. 5A), suggesting other Klf family members are also expressed at comparable levels to indirect (or parallel) regulation of the genes in the latter category by Klf2, Klf4 and Klf5 in mESCs (Fig. 5B), suggesting a possible the Klf family. These data suggested that the overlapping functions contribution of other Klf family members. The phenotype of the of the Klfs are essential to maintain self-renewal of mESCs via Klf2:Klf4:Klf5 triple knockout might be caused by the reduction of activation of naïve-state-specific genes. the overlapping functions below the threshold level as a sum, to DEVELOPMENT

3 STEM CELLS AND REGENERATION Development (2018) 145, dev162404. doi:10.1242/dev.162404

Fig. 3. Inducible knockout of Klf2 and Klf4. (A) Colony formation of Klf2: Klf4 knockout mESCs. The relative colony numbers in the 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 Klf2 knockout mESCs. Cell numbers that originated from 104 cells after culture for 5 days are shown. Error bars indicate standard deviations of five independent results. *P<0.01, Student’s t-test (n=5) versus EB5. (C) RT-qPCR analysis of gene expression in inducible and constitutive Klf2:Klf4 knockout mESCs. Relative gene expression levels [wild-type mESC (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 standard deviations of triplicate quantifications. *P<0.01, Student’s t-test (n=3) versus −Txd3. (D) Immunostaining of constitutive Klf2:Klf4 knockout mESCs. The loss of immunoreactivity against anti-Klf2 and Klf4 Abs confirms a proper knockout event. (E) Chimera contribution of constitutive Klf2:Klf4 knockout mESCs. Three independent clones derived from the same inducible knockout mESCs were tested. The numbers indicate the number of chimeric embryos/number of recovered embryos/number of injected blastocysts. which other Klf family members also contribute. To address this implantation embryos (Blakeley et al., 2015) as well as in human possibility, we tested the abilities of other Klf family members to naïve-like PSCs (Takashima et al., 2014). Interestingly, these maintain the ES-like state of the triple-knockout mESCs. The mouse human PSCs lack the expression of KLF2, suggesting functional genome contains 17 Klf family members that are categorized into replacement by KLF17 (Blakeley et al., 2015). The rescue ability of three groups (or unclassified) based on the amino acid sequence of human KLF17 was comparable to that of mouse Klf17 and lower their three tandem zinc fingers located at the C-terminus than that of human KLF4. These data suggested a possible role of (McConnell and Yang, 2010). Klf2, Klf4 and Klf5 all belong to KLF17 that might share an overlapping function with KLF4 and Group 2. We chose nine members, excluding Klf2, Klf4 and Klf5, KLF5 in human naïve PSCs as a replacement for KLF2. from all categories: Klf3 and Klf8 from Group 1, Klf6 and Klf7 from Group 2, Klf9, Klf10 and Klf16 from Group 3; and Klf15 and Klf17 The third zinc finger defines the unique function from unclassified (Fig. 5E). In addition, we also included Sp1 and Taking advantage of the rescue system, we next addressed the Sp5 in this assay because of the similarity of their amino acid molecular basis that specifies the function of the Klf family sequence in the three tandem zinc fingers to that of the Klf family, members in mESCs. The rescue assay with a series of mutant forms and their high level of expression in mESCs (Fig. 5B). The coding of Klf4 indicated that the zinc-finger domain is required for its sequences were inserted into expression vectors with or without a specific function. However, the N-terminal region might also fusion of Ty1-tag, and their abilities to maintain the mESC-like state possess a specific function because complete deletion resulted in of the triple-knockout mESCs were assessed. Proper protein abolishment of the rescue ability, while deletion of the known expression from these vectors was confirmed by western blot functional domains, transactivation domain [TAD; amino acids (aa) analysis of the protein lysates prepared from the pools of stable 91-109 of 1-483 aa for Klf4] (Geiman et al., 2000) and serine-rich transfectants obtained in the absence of Tx with anti-Ty1 antibody domain (SRR; aa 124-148) for phosphorylation by ERK1/2 (Fig. S5A). In the culture with Tx, we found that the rescue ability (MAPK3/MAPK1) and binding to βTrCP2 (FBXW11) for protein was quite specific to Klf2, Klf4 and Klf5 with or without Ty1 degradation (Kim et al., 2012) caused partial loss (Fig. 5G). From (Fig. 5F). The sole exception was Klf17, although the number of the domains swapped between Klf4 and other Klf family members rescued clones was lower than those of canonical Klfs. These [Klf3 of Group 1, Klf7 of Group 2 and Klf9 of Group 3 (Fig. 5I)] we transfectants continued propagation, and proper rescue events were could see that the zinc-finger domain of Klf7, but not that of Klf3 confirmed by western blot and RT-qPCR analyses of the pooled and Klf9, was capable of replacing that of Klf4 (Fig. 5H), indicating transfectants grown in the presence of Tx (Fig. S2). It was recently the conserved function of the zinc-finger domain in Group 2 Klf reported that KLF17 is expressed in the epiblast of human pre- family members. This was confirmed by successive replacement DEVELOPMENT

4 STEM CELLS AND REGENERATION Development (2018) 145, dev162404. doi:10.1242/dev.162404

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 numbers in 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 indicate standard deviations of five independent results. (C) RT-qPCR analysis of gene expression in inducible and constitutive Klf5 knockout mESCs. The relative gene expression levels [wild-type mESC (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 standard deviations of triplicate quantifications. *P<0.01, Student’s t-test (n=3) versus −Txd3. (D) Immunostaining of constitutive Klf5, 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 gene expression in inducible and constitutive Klf2: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 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 standard deviations of triplicate quantifications. *P<0.01, Student’s t-test (n=3) versus −Txd3. with the zinc-finger domain of Drosophila luna (Fig. 5H) that failed to restore the ability to maintain the ES-like state in the shows the highest homology to Group 2 mouse Klf family members triple-knockout mESCs, indicating insufficient function of their (Fig. 5E, Fig. S4). Drosophila luna and dar1, but not Krüppel, seem N-terminal domain. to be the ancestral genes of the Klf family, considering their highest These data indicated that Klf2, Klf4 and Klf5 share specific homology among the Drosophila genome to the mouse Klf family functions to support pluripotency, mainly based on the specific zinc- members at the three tandem zinc-finger domains and the conserved finger sequence. To identify the responsible region within the zinc- exon boundary between the first and second zinc-finger domains finger domain, we performed subdomain swapping in the three (Fig. S4). We confirmed that both wild-type Krüppel and the Klf4 tandem zinc-finger domains between Klf4 and Klf3 in Klf4/3,a mutant carrying the zinc-finger domain of Krüppel lack the rescue chimeric molecule that consists of the N-terminal region of Klf4 and ability (Fig. 5J). Therefore, Klf is not a Krüppel-like factor in this the zinc-finger domain of Klf3 (Fig. 5I). When a series of the revised homology search. These data also suggested the swapping mutants was tested, we found that the mutant carrying the contribution of the N-terminal region to the specific function in third zinc finger of Klf4 restored the ability to maintain the mESC-like

PSCs because the native form of mouse Klf7 and Drosophila luna state in the triple-knockout mESCs (Fig. 5H). These data indicated DEVELOPMENT

5 STEM CELLS AND REGENERATION Development (2018) 145, dev162404. doi:10.1242/dev.162404

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 inducible triple-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) are shown. Error bars indicate standard deviations of triplicate quantifications. (B) The expression levels of the Klf family members in mESCs. The fragments per kilobase of transcript per million reads (FPKMs) from RNA-seq data [GEO accession number GSE76143] are shown. Error bars indicate standard deviations of RNA-seq data of biological triplicates of Zscan10fl/fl mESCs. (C) Chimera contribution of inducible triple-knockout mESCs. The inducible knockout mESCs cultured without Tx were tested. The numbers indicate the number of chimeric embryos/number of recovered embryos/number of injected blastocysts. (D) Colony staining in the rescue assay. Inducible triple-knockout mESCs transfected with empty and Klf4 expression vectors were cultured with or without Tx for 7 days. The colonies were stained by Leischman staining. (E) Phylogenetic tree of mouse Klf family members. The amino acid sequences encoding triple zinc-finger motifs were aligned and analyzed by the UPGMA method. Group 1, 2 and 3 members are labeled with yellow, blue and green, respectively. (F) Rescue ability of mouse Klf family members. The ratio of colony numbers with versus without Tx (rescue index) are indicated for three independent experiments. Error bars indicate s.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.

that the specific function of the zinc-finger domain depends on the combinations were tested in the inducible triple-knockout mESCs unique third zinc finger shared among Group 2 Klf family members. carrying a Rex1 (Zfp42)-mCherry reporter as a monitor of the naïve state (Kalkan et al., 2017; Toyooka et al., 2008), we found that Overlapping functions of Klf family members, targeting particular combinations showed the ability to support stem cell multiple naïve-specific transcription factors colony formation with Rex1-mCherry expression (Fig. 6A). The Triple knockout of Klf2, Klf4 and Klf5 resulted in the combination of Nanog and Tbx3 showed significant ability, which downregulation of multiple naïve-specific transcription factors, was further enhanced by the addition of Esrrb. The combination of while the restoration of any one of them was able to maintain the Nanog, Esrrb and Gbx2 also showed the synergistic potential. In mESC-like state of the triple-knockout mESCs. It is possible that most cases, the primary stem cell colonies were able to be the overlapping functions of the Klf family are connected to expanded and serially passaged more than ten times while keeping multiple targets downstream. To address this, we chose five naïve- mESC morphology and Rex1-mCherry expression (Fig. 6B), specific transcription factors (Nanog, Esrrb, Tbx3, Gbx2 and although they showed slower proliferation ratio and higher Nr5a2) to test the ability of their combinations to support the incidence of differentiation than wild-type mESCs. These data mESC-like state of the triple-knockout mESCs. When all 32 indicated that forced expression of particular combinations of DEVELOPMENT

6 STEM CELLS AND REGENERATION Development (2018) 145, dev162404. doi:10.1242/dev.162404

Fig. 6. Rescue assay of Klf2:Klf4:Klf5 inducible knockout mESCs with combinatorial forced expression of five transcription factors. (A) Rescue ability of 32 combinations of five transcription factors (Nanog, Esrrb, Tbx3, Gbx2 and Nr5a2). The resulting numbers of stem cell colonies for each combination were integrated from five independent experiments (with error bars indicating s.d.). (B) Colony morphologies of the rescued clones obtained from different combinations of transcription factors. The combination of transcription factors in each clone is indicated in C. Pairs of phase-contrast images and fluorescent images to detect Rex1-mCherry are shown. Clone numbers are also indicated. (C) RT-qPCR analysis of gene expression in the rescued triple-knockout mESCs. 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 wt ES. transcription factors were able to maintain the ES-like state in the expression in the naïve state as well as their potential to reprogram absence of the canonical Klf family members. primed PSCs into the naïve state (Jeon et al., 2016). For strict Then, we examined the expression levels of the endogenous confirmation of this hypothesis, we assessed whether the triple- pluripotency-associated genes in the rescued clones by RT-qPCR knockout mESCs adapt to conversion to the primed state in vitro. (Fig. 6C). These rescued clones maintained the expression of Oct3/4, The inducible triple-knockout mESCs were cultured in the Sox2, Tfcp2l1, Sall4 and Foxd3 at comparable levels to those of wild- condition for epiblast stem cells (EpiSCs) (Sugimoto et al., 2015), type mESCs. In contrast, the expression levels of Utf1, Nr0b1, Tcl1 which contained activin, FGF2 and IWP2 (inhibitor of porcupine to and Dppa3 were reduced, although their expression was maintained block autocrine Wnt protein signaling) instead of LIF, with or in the triple-knockout mESCs rescued by Klf2. Exceptionally, Dppa3 without Tx for 5 days, followed by passages in the same condition. expression level was restored in the rescue clone with all five After three passages, we found that the inducible triple-knockout transgenes, suggesting their synergistic effect. These data suggested mESCs cultured with Tx showed flattened colony morphology and that the former set of genes was indirectly activated by the Klf family lost the expression of Rex1-mCherry, whereas those without Tx via at least three naïve transcription factors to maintain the ES-like retained a dome-like colony morphology with strong Rex1-mCherry state, whereas the latter set was directly activated by Klf family signal (Fig. 7A). Then, we analyzed the gene expression patterns in members, although dispensable for maintenance of the ES-like state. these cells by RT-qPCR (Fig. 7B,C). The triple-knockout mESCs cultured without Tx maintained the expression of several naïve Klf function is dispensable in primed PSCs markers (Nanog, Tbx3, Esrrb, Nr5a2, Tfcp2l1 and Nr0b1)at It was suggested that the functions of the Klf family members in comparable levels to those of wild-type mESCs, with faint

PSCs are specific to the naïve state based on their high level of induction of primed markers [Fgf5, Otx2, Wnt3, Dnmt3b, Oct6 DEVELOPMENT

7 STEM CELLS AND REGENERATION Development (2018) 145, dev162404. doi:10.1242/dev.162404

Fig. 7. Transition of triple-knockout mESCs to the primed state. (A) Colony morphologies of the inducible triple-knockout mESCs cultured in EpiSC condition for three passages with or without Tx. Pairs of phase-contrast images and fluorescent images to detect Rex1-mCherry are shown. (B) RT-qPCR analysis of 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 relative gene 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.

(Pou3f1 – Mouse Genome Informatics), Sox3 and Sall2] (Smith, retained pluripotency as evaluated by their abilities to contribute to 2017; Tesar et al., 2007). In contrast, the triple-knockout mESCs chimeric embryos after single-cell injection into the blastocyst. cultured with Tx showed a similar expression pattern of naïve and Moreover, Klf2:Klf4 double-knockout mESCs maintained primed markers to that of embryo-derived EpiSCs (Tesar et al., pluripotency, indicating that both Klf2 and Klf4 are not essential 2007). These triple-knockout cells continued propagation up to ten to maintain pluripotency in mESCs. These results are consistent passages. This was in contrast to the triple-knockout mESCs with the phenotypes of Klf2 and Klf4 knockout embryos reported cultured in the condition for naïve PSCs with LIF, which suddenly previously. Klf2-null embryos die between embryonic day (E) 12.5 ceased proliferation. These data indicated that the functions of Klf2, and E14.5, owing to defects in tunica media formation and blood Klf4 and Klf5 are dispensable for self-renewal in primed PSCs. vessel stabilization (Kuo et al., 1997), whereas Klf4-null mice die immediately after birth as a result of the defects in the epithelial DISCUSSION barrier in skin and colon (Katz et al., 2002; Segre et al., 1999). Both The overlapping function of the three Klf family members Klf2, show no phenotype in pluripotent cell population at pre- and peri- Klf4 and Klf5 in mESCs to maintain self-renewal was first reported implantation stage. We also demonstrated that the specific function by Jiang et al., who observed partial loss of function using siRNA- of the Klf family members in maintaining pluripotency is restricted mediated knockdown (Jiang et al., 2008). Among them, the to these three Klfs and to Klf17 to a weaker degree. Therefore, Klf5 functions of Klf2 and Klf4 have been further emphasized in is the sole Klf family member working in Klf2:Klf4-null mESCs for relation to pluripotency because of their higher potential for the maintenance of pluripotency, and its function is sufficient reprogramming than that of Klf5 (Jeon et al., 2016; Nakagawa et al., because the expression level of Klf17 is negligible in mESCs 2008), tight links to the known signal pathways to maintain (Fig. 5B). The overlapping potential of Klf17 with the canonical pluripotency (Niwa et al., 2009; Yeo et al., 2014) and unique Klfs might suggest the significance of human KLF17 in human potential to reset naïve state in human PSCs (Takashima et al., naïve PSCs, because in these cells the expression of KLF17 is high, 2014). Here, we employed the conventional strategy of inducible whereas the expression of KLF2 – which has a significant role in the knockout for these three Klf family members. The cell biological maintenance of pluripotency in naïve mESCs – is low (Blakeley characterization revealed that both Klf2-null and Klf4-null mESCs et al., 2015). The functional redundancy of these Klf family DEVELOPMENT

8 STEM CELLS AND REGENERATION Development (2018) 145, dev162404. doi:10.1242/dev.162404 members is a typical example of their overlapping functions (Niwa, into the triple-knockout mESCs, they supported the expression of a 2018). set of pluripotency-associated transcription factors, including Oct3/ The loss of Klf5 resulted in the loss of the ability to contribute to 4, Sox2, Sall4, Foxd3 and Tfcp2l1. These genes, except Tfcp2l1,are chimeric embryos in our strict assay system. Highly contributed known as core components of the pluripotency-associated chimeras might die after implantation because Klf5-null embryos transcription factors because they are expressed in both naïve and show lethal phenotype at this timing (Ema et al., 2008). However, it primed PSCs (Factor et al., 2014). Oct3/4 and Sox2 are absolutely was previously reported that Klf5-null mESCs were able to give rise essential for maintaining self-renewal of mESCs (Masui et al., 2007; to chimeric embryos by the conventional strategy of blastocyst Niwa et al., 2000), whereas Sall4 is required for rapid and stable injection, although the contribution was poor (Ema et al., 2008). self-renewal (Sakaki-Yumoto et al., 2006) and Foxd3 is known for Here, we did not perform the chimera assay with injection of its function to control transition from naïve to primed state multiple mESCs into blastocysts for our Klf5-null mESCs, so we (Respuela et al., 2016; Krishnakumar et al., 2016). Tfcp2l1 is could not rule out the possibility that a minor proportion of Klf5-null specifically expressed in naïve PSCs and possesses the ability to mESCs retain the ability to contribute to chimeric embryos. The support LIF-independent self-renewal in mESCs (Martello et al., poor ability might be caused by the slow proliferation of Klf5-null 2013), suggesting its functional importance in maintaining self- mESCs that can cause elimination from chimeric embryos after renewal. Because these functionally verified pluripotency- implantation when the epiblasts expand very rapidly (Snow, 1977). associated genes are activated in the rescued triple-knockout Alternatively, they might have reduced ability to respond to the mESCs without canonical Klfs, the involvement of the canonical external signal in the developmental context, which could be Klfs in activating this set of pluripotency-associated genes is masked in vitro by excess amount of external signals, such as LIF, in dispensable. In contrast, there is another set of genes that shows the culture medium, although essential in the developmental context decreased levels of expression in the rescued triple-knockout as found in the Klf5-null embryos (Lin et al., 2010). mESCs. This set includes Utf1, Nr0b1, Tcl1 and Dppa3, none of The domain swapping experiment revealed that the third zinc- which are known for their essentiality to maintain self-renewal. finger domain of the Klfs is crucial for their specific function in However, their function to modulate stable self-renewal has been mESCs. The comparison of the amino acid sequences of this reported. For example, Utf1 is reported to function in executing domain between Klf4 and Klf3 elucidated the difference by only pluripotency by regulation of the bivalent genes (Jia et al., 2012). four amino acids (64Q, 65K, 69A and 80M in Fig. S6). We previously demonstrated that Nr0b1-null mESCs show Interestingly, these four residues cluster out of the third zinc- destabilized self-renewal by de-repression of Zscan4c (Fujii et al., finger domains when Klf4 binds to the target sequence with the first 2015). Tcl1 is known to be involved in rapid proliferation (Miyazaki and second zinc fingers (Hashimoto et al., 2016), suggesting their et al., 2013). The lack of proper activation of these genes might be a function in the interaction with other protein(s) rather than the target cause of the slow proliferation of the rescued triple-knockout DNA sequence, or with an additional target DNA sequence besides mESCs with high incidence of differentiation. Therefore, the the one bound by the three zinc-finger domains of Klf4 (Schuetz overlapping function of the canonical Klfs could be to upregulate et al., 2011). The specific function of the zinc-finger domain in Klf4 multiple transcription factors to activate the core transcription was shared by that of Drosophila luna, which shows the highest factors, as well as to activate unique target genes to coordinate rapid amino acid homology with conserved exon-intron organization in and stable self-renewal (Fig. 7D). the Drosophila genome, suggesting that the pluripotency-associated The function of the Klf family members is strictly specific to the function of the zinc-finger domain is a co-optional use of the naïve state because the triple-knockout mESCs are successfully evolutionarily conserved function as in the case of the evolution of captured at primed state. These data confirmed that the primed state Sox2 (Niwa et al., 2016). Among the genes sharing homology in the PSCs possess a transcriptional network to maintain the expression three tandem zinc-finger domains, Drosophila luna and daf, as well of core transcription factors, which is completely distinct from those as Caenorhabditis elegans klf2, share the common exon boundary in the naïve state PSCs, as proposed previously (Weinberger et al., between the first and second zinc fingers with most of the mouse Klf 2016). Rapid and complete exit from the naïve state by removal of family members (Fig. S4). These genes could be the descendants of the Klf family members will provide opportunity to analyze the the ancestral gene of the vertebrate Klf family. They are in contrast formative state recently proposed to be a transient state between to Drosophila Krüppel, which shows lower homology without this the naïve and primed states (Smith, 2017). The extended analysis in boundary, of which the zinc finger domain does not support self- the context of the transcription factor network will be required for renewal of mESCs. Therefore, Klf family members are not like further elucidation of the functions of these Klf family members in Krüppel in the evolutional sense. maintaining naïve pluripotency. The cell biological evidence shown The rescue experiment with multiple naïve-specific transcription here will provide a solid basis to systemic omics analysis in future. factors revealed that the common function of Klf family members is mediated by multiple target genes. The potent targets include MATERIALS AND METHODS Nanog and Tbx3, both of which are well known for their function to Cell culture support LIF-independent self-renewal of mESCs (Mitsui et al., EB5 mESCs (derived from male E14tg2a mESCs, Oct3/4IRES-BSD-pA/+, 2003; Niwa et al., 2009). They could be the direct targets activated deposited to RIKEN Cell Bank) and their derivatives were cultured on a by the Klf family, considering their rapid downregulation in the gelatin-coated surface in Glasgow minimum essential medium (G-MEM) triple-knockout mESCs. The rescue effect might be based on their (Wako Pure Chemical, 078-05525) supplemented with 10% KSR (Thermo parallel function in activating the core transcription factors, such as Fisher Scientific, 10826-028), 1% FCS (Hyclone), 1× sodium pyruvate (Nacalai Tesque, 06977-34), 1× nonessential amino acids (Nacalai Tesque, Oct3/4 and Sox2 (Niwa et al., 2009). In addition to them, Esrrb and 06344-56), 10−4 M 2-mercaptoethanol (Nacalai Tesque, 21417-52) and Gbx2 showed contribution to support the ES-like state in the triple- 1000 U/ml LIF (home-made conditioned medium produced by COS cells knockout mESCs. These genes are also known for their function in transiently transfected with pCAGGS-LIF). For the proliferation assays, the supporting LIF-independent self-renewal of mESCs (Martello et al., mESCs were also cultured in G-MEM supplemented with 10% FCS, 1× − 2012; Tai and Ying, 2013). When these transgenes were introduced sodium pyruvate, 1× nonessential amino acids, 10 4 M 2-mercaptoethanol DEVELOPMENT

9 STEM CELLS AND REGENERATION Development (2018) 145, dev162404. doi:10.1242/dev.162404 and 1000 U/ml LIF. All mESC lines described in this article will be pBRPBCAG-cHA-IN or pBRPBCAG-Ty1linker-cHA-IN. Drosophila luna available from RIKEN Cell Bank. and Krüppel were amplified from the cDNA of Drosophila embryos or To assess the transition to primed state, the mESCs were cultured in Drosophila genomic DNA. Dulbecco’s modified Eagle medium/Ham’s F-12 (Wako Pure Chemical, 048-29785), supplemented with 15% KSR, 1× sodium pyruvate, 1× Generation of inducible KO mESCs − nonessential amino acids, 10 4 M 2-mercaptoethanol, 20 ng/ml activin A EB5 mESCs (107) were electroporated with 100 µg linearized KO vector (human/mouse/rat recombinant, R&D Systems, 338-AC-010), 12 ng/ml DNA at 800 V and 3 µF in a 0.4-cm cuvette using a Gene Pulser (Bio-Rad), FGF2 (human recombinant, Wako Pure Chemical, 067-04031) and 2 µM followed by culture with 1.5 µg/ml puromycin (for Klf2 and Klf4 KO pac IWP2 (AdoQ Bioscience, A12707-5) on mitomycin C-treated mouse vector), 200 µg/ml Hygromycin B (for Klf4 KO Hytk vector) or 1.5 µg/ml of embryonic fibroblast cells (Sugimoto et al., 2015). puromycin and 200 µg/ml of G418 (for Klf5 KO vector) for 8 days. The resulting stem cell colonies were picked up, expanded and genotyped by Plasmid construction PCR using the primer pairs KO-PCR1 and KO-IRES. ForgenerationoftheKlf2 knockout (KO) vector, genomic DNA fragments For the removal of the Frt cassette, the correctly targeted clones were for 5′ and 3′ homology arms (Chr:8, 72317939-72319206 and 72321033- seeded in a well of a 48-well plate at 104 cells per well, and transfected with 72324220 in GRCm38), as well as the floxed region containing exons 2 and 3 1 µg circular pCAG-FLPe-IP plasmid using Lipofectoamine 2000 (Thermo (Chr:8, 72319207-72321032 in GRCm38), were amplified from EB5 Fisher Scientific, 11668027), followed by culture for 3 days. Then, these genomic DNA using the primers Klf2-5′,Klf2-3′ and Klf2-flox, transfected cells were re-plated and cultured for 8 days at clonal density (for respectively, shown in Table S1. The PCR fragments of 5′ and 3′ Klf2 KO and Klf4 KO pac) or normal density with 1 µM gancyclovir (for homology arms were assembled in the ClaI-NotIsitesofpBR-blue II, Klf4 KO Hytk and Klf5 KO) for 8 days. The resulting stem cell colonies were resulting in pBR-Klf2 5′+3′. The floxed region was subcloned in the NheIsite picked up, expanded and genotyped by PCR. of ploxP-BNA. Then, the SpeI fragment carrying the floxed genome was In the case of Klf2 KO and Klf4 KO, the selection of the gene conversion introduced into the SpeIsitebetweenthe5′ and 3′ homology arms of pBR- event was applied to isolate homozygous KO clones (Nakatake et al., 2013). Klf2 5′+3′, resulting in pBR-Klf2 5′+flox+3′.Finally,theXhoIfragmentof Heterozygous mESCs (106) carrying the Frt-IRES-pac-pA-Frt cassette were the Frt-IRES-pac-pA-Frt cassette was introduced into the BamHI site flanking seeded on a 90-mm dish with medium containing 6 µg/ml puromycin and the 5′ end of the 3′ loxP sequence of pBR-Klf2 5′+flox+3′ using the partial cultured for 3 days. Then, the medium was changed to reduce the concentration fill-in method, resulting in pBR-Klf2 floxKO. of puromycin to 1.5 µg/ml and the cells were cultured for 8 days. The resulting For generation of the Klf4 KO vector, genomic DNA fragments for 5′ and stem cell colonies were picked up, expanded and genotyped by PCR. 3′ homology arms (Chr:4, 55532490-55531410 and 55528124-55524067 To generate the inducible mESC lines, the clones in which the loxP sites in GRCm38), as well as the floxed region containing exons 3, 4 and 5 were correctly introduced in both alleles and the Frt cassette were removed, (Chr:4, 55531419-55528125 in GRCm38), were amplified from EB5 seeded in a well of a 48-well plate at 104 cells per well, and transfected with genomic DNA using the primers Klf4-5′, Klf4-3′ and Klf4-flox, 0.25 µg circular pPB-CAG-MerCreMer-IH, 0.25 µg circular pPB-EGFP-IZ respectively, shown in Table S1. These PCR fragments of 5′ and 3′ and 0.5 µg circular pCAG-PiggyBac transposase (PBase) plasmids using homology arms were assembled in the ClaI-NotI sites of pBR-blue II, Lipofectoamine 2000, followed by culture for 2 days. Then, these resulting in pBR-Klf4 5′+3′. The floxed region was subcloned in the NheI transfected cells were re-plated and cultured with 200 µg/ml Hygromycin site of ploxPP2272. Then, the SpeI fragment carrying the floxed genome B and 20 µg/ml Zeocin for 8 days. The resulting stem cell colonies were was introduced into the SpeI site between the 5′ and 3′ homology arms of picked up, expanded and assessed for the expression of Egfp by fluorescent pBR-Klf4 5′+3′, resulting in pBR-Klf4 5′+flox+3′. Finally, the XhoI microscopic analysis, and the function of MerCreMer was assessed by PCR fragment of the Frt-IRES-pac-pA-Frt cassette and Frt-IRES-Hytk [fusion of genotyping of the cells cultured with 1 µg/ml 4-hydroxy tamoxifen (Tx). hph and HSV-tk (Lupton et al., 1991)]-pA-Frt was introduced into the AgeI For the establishment of the KO clones, the inducible KO mESCs were site flanking the 3′ end of the 3′ loxP sequence of pBR-Klf4 5′+flox+3′ cultured with 1 µg/ml Tx for 3 days. Then, these cells were dissociated and using adaptor DNA, resulting in pBR-Klf4 floxKO pac and pBR-Klf4 floxKO seeded at clonal density (1000 cells per 90-mm dish), followed by culture for Hytk, respectively. 8 days. The resulting stem cell colonies were picked up, expanded and For generation of the Klf5 KO vector, genomic DNA fragments for 5′ and genotyped by PCR. A PCR reaction with three primers was applied to 3′ homology arms (Chr:14, 99299127-99300223 and 99303877-99307091 ensure a proper deletion event. in GRCm38), as well as the floxed region containing exons 2 and 3 (Chr:14, 99300224-99303885 in GRCm38), were amplified from EB5 genomic Production of chimeric embryos DNA using the primers Klf5-5′, Klf5-3′ and Klf5-flox, respectively, shown A dissociated single mESC was injected into a C57BL6 blastocyst by in Table S1. These PCR fragments were assembled in the SacII-XhoI sites of microinjection. The blastocyst was then transferred into the uterus of a pBR-blue II, resulting in pBR-Klf5 5′+3′. The floxed region was subcloned pseudo-pregnant female ICR mouse. Embryos were collected at 13.5 days in the BamHI site of ploxP-BNA. Then, the SpeI fragment carrying the postcoitum to evaluate the chimera contribution ability of mESCs by floxed genome was introduced into the AgeI site between the 5′ and analyzing with fluorescence microscopy. The efficiency of chimera 3′ homology arms of pBR-Klf5 5′+3′ using adaptor DNA, resulting in pBR- production with wild-type mESCs was shown in our previous reports Klf5 5′+flox+3′. Finally, the XhoI fragment of Frt-SA-IRES-neo-pA:PGK- (Ohtsuka et al., 2012; Ohtsuka and Niwa, 2015). All animal experiments pacΔtk [fusion of pac and HSV tk (Chen and Bradley, 2000)]-pA-Frt conformed to Institute Guidelines for the Care and Use of Laboratory cassette was introduced into the NheI site flanking the 3′ end of the 5′ loxP Animals and were approved by the Institutional Committee for Laboratory sequence of pBR-Klf5 5′+flox+3′ using the adaptor DNA, resulting in pBR- Animal Experimentation (RIKEN Kobe Institute). Klf5 floxKO. For generation of the Rex1-mCherry knock-in vector, mCherry-IRES- Immunostaining and western blotting pac-pA cassette was introduced into the SpeI-EcoRI sites of Rex1 5′+3′ Monoclonal antibody against Klf2 was obtained by immunization of a homology arms as described previously (Toyooka et al., 2008). mouse with purified fusion protein of GST and full-length Klf2, followed by The PiggyBac vectors pPBCAG-MerCreMer-IH, pPBCAG-EGFP-IZ, the standard method. Cells were fixed with 4% paraformaldehyde in PBS for pBRPBCAG-cHA-IN and pBRPBCAG-Ty1linker-cHA-IN were constructed 30 min at 4°C, followed by permeabilization with 0.2% Triton X-100 in based on pGG131 (Guo et al., 2009). Ty1 linker consists of 3× Ty1 PBS for 10 min at room temperature. These cells were incubated with the (EVHTNQDPLD) and glycine linker (GGSGGGGSGGGSSSS). For following primary antibodies overnight at 4°C: mouse monoclonal anti- construction of the expression vectors of the Klf family members, the Oct3/4 (Santa Cruz Biotechnology, sc-5279), 1:1000; rabbit polyclonal entire coding regions were amplified from the complementary DNA anti-Sox2 (antisera), 1:1000; rat monoclonal anti-Nanog (e-Bioscience, (cDNA) of mESCs using the primers shown in Table S1, and the PCR MLC-51), 1:1000; mouse monoclonal anti-Klf2 (home-made), 1:1000; goat products were digested and inserted into the XhoI-NotI site of either polyclonal anti-Klf4 (R&D Systems, AF3640), 1:1000; rabbit polyclonal DEVELOPMENT

10 STEM CELLS AND REGENERATION Development (2018) 145, dev162404. doi:10.1242/dev.162404 anti-Klf5 (Abcam, ab24331); and rabbit polyclonal anti-Tbx3 (antisera, Data availability Toyooka et al., 2008). After washing, cells were incubated with appropriate RNA-seq data are available at Gene Expression Omnibus (GEO), under accession Alexa Fluor 488-conjugated secondary antibodies for 1 h at room number GSE76143. temperature with Hoechst 33258, and fluorescent images were taken on an Olympus OX-71 equipped with a CCD camera. Western blotting was Supplementary information performed with the antibodies for immunostaining as well as mouse Supplementary information available online at http://dev.biologists.org/lookup/doi/10.1242/dev.162404.supplemental monoclonal anti-Ty1 (Diagnode, MAb-054-050, 1:10,000) and rabbit polyclonal anti-Cdk2 (Santa Cruz Biotechnology, sc-163, 1:1000) for 8 µg References of total cell lysates prepared in TDL buffer [150 mM NaCl, 50 mM Tris- Blakeley, P., Fogarty, N. M. E., del Valle, I., Wamaitha, S. E., Hu, T. X., Elder, K., HCl (pH 8.0), 0.5% deoxycholic acid, 1% NP-40, 0.1% SDS]. The signal Snell, P., Christie, L., Robson, P. and Niakan, K. K. (2015). Defining the three was detected by SuperSignal West Femto Maximum Sensitivity Substrate cell lineages of the human blastocyst by single-cell RNA-seq. Development 142, (Thermo Fisher Scientific) using the CCD camera system LAS 4000 Mini 3151-3165. (Fuji Film). Chen, Y.-T. and Bradley, A. (2000). A new positive/negative selectable marker, puDeltatk, for use in embryonic stem cells. Genesis 28, 31-35. Rescue assay in triple-KO mESCs Ema, M., Mori, D., Niwa, H., Hasegawa, Y., Yamanaka, Y., Hitoshi, S., Mimura, J., Kawabe, Y., Hosoya, T., Morita, M. et al. (2008). Krüppel-like factor 5 is essential The inducible triple-KO mESCs (Klf2fl/fl:Klf4fl/fl:Klf5fl/fl: CAG-MCM-IH: 4 for blastocyst development and the normal self-renewal of mouse ESCs. Cell CAG-Egfp-IZ) were seeded in a well of a 48-well plate at 10 cells per well, Stem Cell 3, 555-567. and transfected with 0.5 µg circular expression vector (derived from Factor, D. C., Corradin, O., Zentner, G. E., Saiakhova, A., Song, L., Chenoweth, pBRPBCAG-cHA-IN or pBRPBCAG-Ty1linker-cHA-IN) and 0.5 µg circular J. G., McKay, R. D., Crawford, G. E., Scacheri, P. C. and Tesar, P. J. (2014). pCAG-PiggyBac transposase (PBase) plasmid using Lipofectoamine 2000, Epigenomic comparison reveals activation of “seed” enhancers during transition followed by culture for 2 days with or without Tx. Then, the transfected cells from naive to primed pluripotency. Cell Stem Cell 14, 854-863. were re-plated and cultured with 200 µg/ml G418 with or without Tx for Fujii, S., Nishikawa-Torikai, S., Futatsugi, Y., Toyooka, Y., Yamane, M., 8 days. For evaluation of the rescue ability, the colonies were stained with Ohtsuka, S. and Niwa, H. (2015). Nr0b1 is a negative regulator of Zscan4c in mouse embryonic stem cells. Sci. Rep. 5, 9146. Leishman staining solution, the numbers of the colonies with compact Geiman, D. E., Ton-That, H., Johnson, J. M. and Yang, V. W. (2000). morphology were counted under a microscope, and the ratio between the Transactivation and growth suppression by the gut-enriched Kruppel-like factor presence and absence of Tx was calculated. For assessing the rescue event, the (Kruppel-like factor 4) are dependent on acidic amino acid residues and protein- pool of colonies was cultured for preparation of RNA for quantitative PCR protein interaction. Nucleic Acids Res. 28, 1106-1113. and protein lysate for western blotting. For the combinatorial transfection of Guo, G., Yang, J., Nichols, J., Hall, J. S., Eyres, I., Mansfield, W. and Smith, A. five transcription factors (Nanog, Tbx3, Esrrb, Gbx2 and Nr5a2), these (2009). Klf4 reverts developmentally programmed restriction of ground state cDNAs were subcloned into pBRPBCAG-cHA-IN. Then, 0.15 µg of each of pluripotency. Development 136, 1063-1069. Hall, J., Guo, G., Wray, J., Eyres, I., Nichols, J., Grotewold, L., Morfopoulou, S., the expression vectors and 0.25 µg of circular PBase vector were transfected. Humphreys, P., Mansfield, W., Walker, R. et al. (2009). Oct4 and LIF/Stat3 For the combination of fewer than five factors, the total amount of the additively induce Krüppel factors to sustain embryonic stem cell self-renewal. Cell expression vector was adjusted with pBRPBCAG-cHA-IN. Stem Cell 5, 597-609. Hashimoto, H., Wang, D., Steves, A. N., Jin, P., Blumenthal, R. M., Zhang, X. and Phylogenetic analysis of Klf family members Cheng, X. (2016). Distinctive Klf4 mutants determine preference for DNA The open reading frame sequences for the members of mouse Klf family methylation status. Nucleic Acids Res. 44, 10177-10185. (Klf1-17), mouse Sp1 and Sp5, and Drosophila luna were collected from Jeon, H., Waku, T., Azami, T., Khoa, L. T. P., Yanagisawa, J., Takahashi, S. and ̈ Ensembl genome database. The amino acid sequences of the zinc-finger Ema, M. (2016). Comprehensive identification of Kruppel-like factor family members contributing to the self-renewal of mouse embryonic stem cells and domains consist of the three tandem zinc fingers shown in Fig. S4, and were cellular reprogramming. PLoS ONE 11, e0150715. analyzed with the UPGMA program of GENETYX-MAC to obtain the Jia, J., Zheng, X., Hu, G., Cui, K., Zhang, J., Zhang, A., Jiang, H., Lu, B., Yates, J., evolutionary tree. III, Liu, C. et al. (2012). Regulation of pluripotency and self- renewal of ESCs through epigenetic-threshold modulation and mRNA pruning. Cell 151, 576-589. FACS analysis Jiang, J., Chan, Y.-S., Loh, Y.-H., Cai, J., Tong, G.-Q., Lim, C.-A., Robson, P., For apoptotic assay, Annexin V-APC (BD Biosciences) staining was Zhong, S. and Ng, H.-H. (2008). A core Klf circuitry regulates self-renewal of performed according to the manufacturer’s protocol using inducible triple- embryonic stem cells. Nat. Cell Biol. 10, 353-360. Kalkan, T., Olova, N., Roode, M., Mulas, C., Lee, H. J., Nett, I., Marks, H., Walker, KO mESCs and wild-type mESCs. R., Stunnenberg, H. G., Lilley, K. S. et al. (2017). Tracking the embryonic stem cell transition from ground state pluripotency. Development 144, 1221-1234. Real-time PCR analysis Katz, J. P., Perreault, N., Goldstein, B. G., Lee, C. S., Labosky, P. A., Yang, V. W. First-strand DNAwas synthesized from 500 ng of the total RNA prepared by a and Kaestner, K. H. (2002). The zinc-finger transcription factor Klf4 is required for QuickGene RNA cultured cell HC kit (Kurabo) in 20 µl of the reaction terminal differentiation of goblet cells in the colon. Development 129, 2619-2628. mixture containing oligo-dT primers using a ReverTra Ace first strand Kim, M. O., Kim, S.-H., Cho, Y.-Y., Nadas, J., Jeong, C.-H., Yao, K., Kim, D. J., Yu, synthesis kit (Toyobo). Real-time PCR was performed with D.-H., Keum, Y.-S., Lee, K.-Y. et al. (2012). ERK1 and ERK2 regulate embryonic THUNDERBIRD SYBR qPCR Mix (Toyobo) using a CFX384 Real-Time stem cell self-renewal through phosphorylation of Klf4. Nat. Struct. Mol. Biol. 19, System (Bio-Rad). Primer sequences are listed in Table S1. 283-290. Krishnakumar, R., Chen, A. F., Pantovich, M. G., Danial, M., Parchem, R. J., Labosky, P. A. and Blelloch, R. (2016). FOXD3 regulates pluripotent stem cell Acknowledgements potential by simultaneously initiating and repressing enhancer activity. Cell Stem We thank Dr Futatsugi-Nakai (RIKEN CDB) for critical editing of the manuscript. Cell 18, 104-117. Kuo, C. T., Veselits, M. L., Barton, K. P., Lu, M. M., Clendenin, C. and Leiden, Competing interests J. M. (1997). The LKLF transcription factor is required for normal tunica media The authors declare no competing or financial interests. formation and blood vessel stabilization during murine embryogenesis. Genes Dev. 11, 2996-3006. Author contributions Lin, S.-C. J., Wani, M. A., Whitsett, J. A. and Wells, J. M. (2010). Klf5 regulates Conceptualization: A.N., H.N.; Methodology: M.Y., S.O., K.M., H.N.; Validation: lineage formation in the pre-implantation mouse embryo. Development 137, H.N.; Formal analysis: H.N.; Investigation: M.Y., S.O., K.M., H.N.; Resources: A.N., 3953-3963. H.N.; Writing - original draft: H.N.; Writing - review & editing: H.N.; Visualization: Lupton, S. D., Brunton, L. L., Kalberg, V. A. and Overell, R. W. (1991). Dominant H.N.; Supervision: H.N.; Project administration: H.N.; Funding acquisition: H.N. positive and negative selection using a hygromycin phosphotransferase- thymidine kinase fusion gene. Mol. Cell. Biol. 11, 3374-3378. Funding Martello, G., Sugimoto, T., Diamanti, E., Joshi, A., Hannah, R., Ohtsuka, S., This work was supported by RIKEN and the Japan Science and Technology Agency Göttgens, B., Niwa, H. and Smith, A. (2012). Esrrb is a pivotal target of the Gsk3/

(CREST program to H.N.). Tcf3 axis regulating embryonic stem cell self-renewal. Cell Stem Cell 11, 491-504. DEVELOPMENT

11 STEM CELLS AND REGENERATION Development (2018) 145, dev162404. doi:10.1242/dev.162404

Martello, G., Bertone, P. and Smith, A. (2013). Identification of the missing Sakaki-Yumoto, M., Kobayashi, C., Sato, A., Fujimura, S., Matsumoto, Y., pluripotency mediator downstream of leukaemia inhibitory factor. EMBO J. 32, Takasato, M., Kodama, T., Aburatani, H., Asashima, M., Yoshida, N. et al. 2561-2574. (2006). The murine homolog of SALL4, a causative gene in Okihiro syndrome, is Masui, S., Nakatake, Y., Toyooka, Y., Shimosato, D., Yagi, R., Takahashi, K., essential for embryonic stem cell proliferation, and cooperates with Sall1 in Okochi, H., Okuda, A., Matoba, R., Sharov, A. A. et al. (2007). Pluripotency anorectal, heart, brain and kidney development. Development 133, 3005-3013. governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem Schuetz, A., Nana, D., Rose, C., Zocher, G., Milanovic, M., Koenigsmann, J., cells. Nat. Cell Biol. 9, 625-635. Blasig, R., Heinemann, U. and Carstanjen, D. (2011). The structure of the Klf4 McConnell, B. B. and Yang, V. W. (2010). Mammalian Krüppel-like factors in health DNA-binding domain links to self-renewal and macrophage differentiation. Cell. and diseases. Physiol. Rev. 90, 1337-1381. Mol. Life Sci. 68, 3121-3131. Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K., Segre, J. A., Bauer, C. and Fuchs, E. (1999). Klf4 is a transcription factor required Maruyama, M., Maeda, M. and Yamanaka, S. (2003). The homeoprotein Nanog for establishing the barrier function of the skin. Nat. Genet. 22, 356-360. is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell Smith, A. (2017). Formative pluripotency: the executive phase in a developmental 113, 631-642. Miyazaki, T., Miyazaki, S., Ashida, M., Tanaka, T., Tashiro, F. and Miyazaki, J.-I. continuum. Development 144, 365-373. (2013). Functional analysis of Tcl1 using Tcl1-deficient mouse embryonic stem Snow, M. H. L. (1977). Gastrulation in the mouse: growth and regionalization of the cells. PLoS ONE 8, e71645. epiblast. J. Embryo. Exp. Morph. 42, 293-303. Nakagawa, M., Koyanagi, M., Tanabe, K., Takahashi, K., Ichisaka, T., Aoi, T., Sugimoto, M., Kondo, M., Koga, Y., Shiura, H., Ikeda, R., Hirose, M., Ogura, A., Okita, K., Mochiduki, Y., Takizawa, N. and Yamanaka, S. (2008). Generation of Murakami, A., Yoshiki, A., Chuva de Sousa Lopes, S. M. et al. (2015). A simple induced pluripotent stem cells without Myc from mouse and human fibroblasts. and robust method for establishing homogeneous mouse epiblast stem cell lines Nat. Biotechnol. 26, 101-106. by wnt inhibition. Stem Cell Rep. 4, 744-757. Nakatake, Y., Fujii, S., Masui, S., Sugimoto, T., Torikai-Nishikawa, S., Adachi, K. Tai, C.-I. and Ying, Q.-L. (2013). Gbx2, a LIF/Stat3 target, promotes reprogramming and Niwa, H. (2013). Kinetics of drug selection systems in mouse embryonic stem to and retention of the pluripotent ground state. J. Cell Sci. 126, 1093-1098. cells. BMC Biotechnol. 13, 64. Takahashi, K. and Yamanaka, S. (2006). Induction of pluripotent stem cells from Niwa, H. (2018). The principles that govern transcription factor network functions in mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, stem cells. Development 145, dev157420. 663-676. Niwa, H., Miyazaki, J.-I. and Smith, A. G. (2000). Quantitative expression of Oct-3/ Takashima, Y., Guo, G., Loos, R., Nichols, J., Ficz, G., Krueger, F., Oxley, D., 4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. Santos, F., Clarke, J., Mansfield, W. et al. (2014). Resetting transcription factor 24, 372-376. control circuitry toward ground-state pluripotency in human. Cell 158, 1254-1269. Niwa, H., Ogawa, K., Shimosato, D. and Adachi, K. (2009). A parallel circuit of LIF Tesar, P. J., Chenoweth, J. G., Brook, F. A., Davies, T. J., Evans, E. P., Mack, signalling pathways maintains pluripotency of mouse ES cells. Nature 460, D. L., Gardner, R. L. and McKay, R. D. G. (2007). New cell lines from mouse 118-122. epiblast share defining features with human embryonic stem cells. Nature 448, Niwa, H., Nakamura, A., Urata, M., Shirae-Kurabayashi, M., Kuraku, S., Russell, 196-199. S. and Ohtsuka, S. (2016). The evolutionally-conserved function of group B1 Sox Toyooka, Y., Shimosato, D., Murakami, K., Takahashi, K. and Niwa, H. (2008). family members confers the unique role of Sox2 in mouse ES cells. BMC Evol. Identification and characterization of subpopulations in undifferentiated ES cell Biol. 16, 173. culture. Development 135, 909-918. Ohtsuka, S. and Niwa, H. (2015). The differential activation of intracellular signaling Weinberger, L., Ayyash, M., Novershtern, N. and Hanna, J. H. (2016). Dynamic pathways confers the permissiveness of embryonic stem cell derivation from different mouse strains. Development 142, 431-437. stem cell states: naive to primed pluripotency in rodents and humans. Nat. Rev. Ohtsuka, S., Nishikawa-Torikai, S. and Niwa, H. (2012). E-cadherin promotes Mol. Cell Biol. 17, 155-169. incorporation of mouse epiblast stem cells into normal development. PLoS ONE 7, Yamane, M., Fujii, S., Ohtsuka, S. and Niwa, H. (2015). Zscan10 is dispensable for e45220. maintenance of pluripotency in mouse embryonic stem cells. Biochem. Biophys. Respuela, P., Nikolić, M., Tan, M., Frommolt, P., Zhao, Y., Wysocka, J. and Res. Commun. 468, 826-831. Rada-Iglesias, A. (2016). Foxd3 promotes exit from naive pluripotency through Yeo, J.-C., Jiang, J., Tan, Z.-Y., Yim, G.-R., Ng, J.-H., Göke, J., Kraus, P., Liang, enhancer decommissioning and inhibits germline specification. Cell Stem Cell 18, H., Gonzales, K. A. U., Chong, H.-C. et al. (2014). Klf2 is an essential factor that 118-133. sustains ground state pluripotency. Cell Stem Cell 14, 864-872. DEVELOPMENT