Core transcription factors, Oct4, Sox2 and Nanog, individually form complexes with nucleophosmin (Npm1) to control embryonic stem (ES) cell fate determination.

Embryonic stem (ES) cells have therapeutic potential in regenerative medicine, although the molecular mechanism controlling their pluripotency is not completely understood. Depending on interaction partners most proteins can be involved in several different cellular mechanisms. We screened for novel protein-protein interactions using in situ proximity ligation assays together with specific antibodies directed against known important ES cell proteins. We found that all three core transcription factors, namely Oct4, Sox2 and Nanog, individually formed complexes with nucleophosmin (Npm1). We showed that the Npm1/Sox2 complex was sustained when cells were induced to differentiate by retinoic acid, while decreased in the other differentiation pathways. Moreover, Oct4 also formed individual complexes with translationally controlled tumor protein (Tpt1). Downregulation of Npm1 or Tpt1 increased mRNA levels for genes involved in mesoderm and ectoderm differentiation pathways, respectively, indicative of their involvement in ES cell maintenance. We have here described four novel protein-protein interactions in ES cell involving all three core transcription factors. Our findings improve the current knowledge about ES cell-specific protein networks and indicate the importance of Npm1 and Tpt1 to maintain the ES cell phenotype.


INTRODUCTION
still no further proteins have been assigned important for the iPS cell creation.
Nucleophosmin/nucleoplasmin family member 1 (Npm1, also known as B23, Numatrin or NO38) is expressed at high levels in mouse [8] and human [9] ES cells. It is a multifunctional phosphoprotein that has been implicated in cell proliferation [10] as well as regulation of transcription, where it appears to be able to both repress [11] or stimulate [12][13] transcription. We recently showed that Npm1 and translationally controlled tumor protein (Tpt1, also referred to as TCTP, Fortilin, Histamine-releasing factor HRF, or P23) form a complex in ES cells and that this complex is important for cell proliferation [14]. Tpt1 has previously been shown to improve reprogramming efficiency of somatic cell nuclear transfers [15], which is another method for dedifferentiation of somatic cells. As previously stated, cell proliferation has been shown to be a criteria for successful iPS cell creation and Npm1 has also previously been shown to interact with one of the four factors for iPS cell creation i.e. c-Myc [16]. In view of these findings, we here investigated if Npm1 and Tpt1 network with other factors identified as important in iPS cell creation and ES cell maintenance.

Oct4 interacts physically with Npm1 and Tpt1 in ES cells
Oct4 is needed for maintenance of ES cells and iPS cell creation, but its relation to Npm1 and Tpt1, two factors found to be important for ES cell proliferation, have not been addressed and remained elusive. In situ proximity ligation assay (PLA) [17] is a powerful tool to screen rather easily for protein-protein interactions. Confocal micrographs collected at 0.38 µm intervals and merged together, show high number of Npm1/Oct4 complexes in the nucleoplasm of interphase ES cells ( Figure 1A, each red dot represents one detected interaction). Interaction was also observed in mitotic cells using an antibody only recognizing Npm1 phosphorylated at residue T198 ( Figure 1B, red dots). Oct4 also formed individual complexes with Tpt1 and a considerable number of Oct4/Tpt1 complexes are seen in the nucleus of interphase ES cells ( Figure 1C, red dots).
In brief, both Npm1 and Tpt1 physically interact individually with Oct4 in ES cells, and the interactions are not cell cycle dependent.

Npm1 physically interacts with Sox2 in ES cells
In addition to Oct4, Sox2 is another of the three important core transcription factors identified in ES cells. Using in situ PLA the possible interaction of Sox2 with Npm1 and Tpt1 was investigated. Confocal micrographs collected at 0.38 µm intervals and merged together, showed a substantial number of Npm1/Sox2 complexes in the nucleus of interphase cells (Figure 2A, red dots). The same pattern was observed with another set of Npm1/Sox2 antibodies (anti-Sox2 [MAB2018, R&D Systems] and anti-Npm1 [ab15440, abcam]; data not shown).
To further verify these results, extract prepared from ES cells was subjected to co-immunoprecipitation with anti-Sox2 followed by Western blot. Npm1 was coimmunoprecipitated with anti-Sox2 ( Figure 2B, IP Sox2: 1 M NaCl and 0.1 M Citrate) but not with IgG control (data not shown).

Figure 1. Oct4 physically in teracts with Npm1 and Tpt1 in ES cells. Immunofluorescence confocal microscopy in combination
with in situ PLA, which detects protein-protein complexes, was used to explore interactions between Oct4 to Npm1 and Tpt1. Each detected complex is represented by a red dot. DNA was counterstained by Hoechst 33342 (blue). Scale bar represents 10 µm. (A) Complexes between endogenous Npm1 and Oct4 were found in the nucleoplasm of interphase cells. (B) Complexes between Npm1 and Oct4 during mitosis using an antibody specific to phosphorylated Npm1. (C) Complexes between endogenous Oct4 and Tpt1 in the nucleoplasm of interphase cells.

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No interaction was observed between Tpt1 and Sox2 and was therefore used as one of the negative controls for the in situ PLA method (Suppl. Figure 1). The PLA together with co-immunoprecipitation establishes that endogenous Npm1 physically interacts with endogenous Sox2 in ES cells.

Npm1/Sox2 interaction changes during differentiation
Both Sox2 and Npm1 protein levels are changing when ES cells start to differentiate. To investigate how the interaction is affected in the beginning of differentiation, ES cells were treated toward different differentiation pathways (retinoic acid, dimethyl sulfoxide and withdrawal of leukemia inhibitory factor) in combination with in situ PLA. Npm1/Sox2 complexes were shown to decrease when differentiation was induced by dimethyl sulfoxide or withdrawal of leukemia inhibitory factor ( Figure 3). Conversely, such complexes remained, or even increased in number when differentiation was induced by addition of retinoic acid. This analysis showed that the Npm1/Sox2 interaction is reduced during conditions known to induce differentiation of ES cells into mesoderm and endoderm, whereas differentiation by retinoic acid into ectodermal lineage is induced in the continuous presence of Npm1/Sox2 complexes.

Npm1 also interacts with the third core transcription factor, Nanog
As stated in the introduction, three core transcription factors, namely Oct4, Sox2 and Nanog, have been  www.impactaging.com proven essential for ES cell maintenance. We have now shown that Npm1 form protein-protein complexes with both Oct4 and Sox2, which prompted us to investigate how the interactions are between Nanog, Npm1 and Tpt1, using is situ PLA in combination with confocal microscopy.
Confocal micrographs collected at 0.38µm intervals and merged together, showed that Nanog interacts with Npm1 ( Figure 4A, red dots), whereas no interaction was seen between Nanog and Tpt1 ( Figure 4B).
These analyses conclude that endogenous Npm1 interacts with all three core transcription factors in ES cells since it also interacts with endogenous Nanog.

Tpt1 and Npm1 are involved in the differentiation of ES cells
ES cells have the capacity to differentiate into all three germ layers: endoderm, mesoderm and ectoderm. We studied whether shRNA mediated downregulation of Tpt1 or Npm1 affected the ES cell maintenance analyzed by qPCR. As shown in Figure 5, shRNA mediated downregulation of Tpt1 gave a minor increase in the of Oct4 levels, whereas levels of Sox2 and the ectodermal differentiation marker Nestin was increased (blue bars). Npm1 downregulation neither affected levels of Oct4, nor Sox2 notably, but increased the mesodermal marker Brachyury (black bars). qPCR data from shRNA mediated downregulation of Npm1 and Tpt1 respectively, indicates that both proteins seem to be involved in different differentiation pathways.

DISCUSSION
Before iPS stem cells can be used in regenerative medicine several obstacles have to be overcome. One of these is the poor efficiency of the process. While some cell types readily are dedifferentiated, other cells are resistant to the same viral treatment. Tpt1 have previously been implicated in regulation of Oct4 in somatic cell nuclear transfers [18], but somewhat contradictory, our shRNA knockdown of Tpt1 in ES cells did not notably affect Oct4 transcription, if any effect it was a slight increase in Oct4 levels. A possible explanation of this opposite effect could be that we only Immunofluorescence confocal microscopy in combination with in situ PLA, which detects protein-protein complexes, was used to explore interactions between Nanog to Npm1 and Tpt1. Each detected complex is represented by a red dot. (A) Complexes between endogenous Npm1 and Nanog were found in the nucleoplasm in some of the interphase ES cells. (B) No interaction was observed between Tpt1 and Nanog in ES cells. DNA was counterstained by Hoechst 33342 (blue). Scale bar represents 10 µm. www.impactaging.com had a 50% decrease of Tpt1 and that the remaining amount of Tpt1 was enough to keep Oct4 normal. We show that Tpt1 interacts with Oct4, while no interaction was found between Tpt1 and Sox2, or together with Nanog. The interaction between Tpt1 and Oct4 may be associated with the finding that Tpt1 increases Oct4 levels in somatic nuclear transfers [18], since Oct4 is known to self-regulate [19]. Interestingly, Sox2 and the ectodermal differentiation marker Nestin increased during downregulation of Tpt1. In support of our findings, Tpt1 protein levels decrease during neural cell differentiation [20]. Other reports also implicate a role for Tpt1 in embryonic development. Knockout mice deficient in both Tpt1 alleles are embryonic lethal and depending on if the entire gene [21] or part of the gene [22] is deleted, they die around E3.5 and E9.5, respectively. E3.5 is around the blastocyst stage from which ES cells are propagated from the inner cell mass of the developing embryo [23]. Altogether, these results imply that Tpt1 is required for ES cell maintenance as well as involved in ectoderm lineage formation.
Further, we found that Npm1 interacts with all three core transcription factors, namely Oct4, Sox2 and Nanog in ES cells. The Npm1/Oct4 interaction is ES cell specific due to the fact that Oct4 is an ES cell specific protein which becomes downregulated when the cells differentiate [24]. Available data regarding Npm1 and ES cells show that Npm1 is needed for ES cell proliferation, but neither affect levels of Oct4 or Nanog, nor differentiation [25]. This is in accordance with our shRNA downregulation of Npm1 which did not notably affect levels of neither Oct4 nor Sox2. Sox2 is crucial for ES cells. However, it is still expressed after differentiation and it has been shown to be an important protein for development of epiblast and extraembryonic ectoderm [26]. Therefore the Npm1/Sox2 interaction was investigated during different paths of differentiation. By withdrawal of leukemia inhibitory factor or addition of dimethyl sulfoxide the number of complexes and their intensity were decreased, while the number of complexes was stable or even slightly increased by addition of retinoic acid. This implies that Npm1/Sox2 has a function not only in ES cells but also functions in ectodermal cells, at least during the first stages of differentiation. This is supported by our finding that Npm1 depletion increased the expression of mesodermal marker Brachyury and did not affect the ectodermal marker Nestin. Suppression of Npm1 in neural stem cells inhibits cell proliferation, induces apoptosis through the p53 pathway but does not affect cell differentiation [27]. This may imply that the Npm1/Sox2 interaction has a role in pushing ES cells towards ectodermal differentiation, but that once they have started to differentiate into neural stem cells, they can differentiate further without Npm1 involvement. Knockout mice deficient in both Npm1 alleles are embryonic lethal. The mice show aberrant organogenesis and die around E11.5 owing to severe anemia resulting from defects in primitive haematopoiesis [28]. Thus, Npm1 seems to have several different important functions during the embryonic development, and further studies are needed to explore these roles.
Nanog is to some extent very different from the other two core transcription factors. Heterogeneous expression of Nanog is observed in ES cells [29] and overexpression of Nanog is enough to keep ES cell maintenance in the absense of LIF [30]. Nanog has also recently been implicated in G1 to S transition, where Nanog overexpression results in quicker cell cycle progression through accelerated S-phase entry by direct binding and regulation of two proteins important for this process [31]. We have previously shown that overexpression of Npm1 also results in higher cell proliferation rates [14], so our newly found interaction between Nanog and Npm1 might very well play a part in cell cycle regulation. Although, given that Npm1 shows individual interactions with all three core transcription factors, it argues also for a role in transcriptional regulation. Previously it has been shown that Npm1 functions as a histone chaperone that remodels local chromatin structures [32]. Therefore one logical explanation for these three interactions would be that Npm1 remodels the chromatin structure so that the different transcription factors can bind and activate the specific genes.
We have shown that both Npm1 and Tpt1 are involved in ES cell maintenance and they both form individual complexes with Oct4. Npm1 also forms complexes with Sox2 and Nanog and the Npm1/Sox2 interaction are sustained in the early parts of ectodermal differentiation. Since Npm1 interacts with several factors identified necessary for iPS cell creation, it may has a critical role for successful dedifferentiation. Especially since cell proliferation has been shown to be a crucial criterion for successful iPS cell creation and Npm1 is essential for cell proliferation in both ES [14,25], neural stem cells [27] and in other cell systems [10].
Cell extracts. Whole cell extracts were prepared by harvesting confluent cell cultures containing approximately 3×10 7 cells. Harvested cells were incubated in ice cold extraction buffer (10 mM Tris-HCl pH 7.4, 10 mM MgCl 2 , 10 mM KCl, 1.0 mM DTT) containing protease inhibitor cocktail tablet (Complete, Roche Diagnostics) for 10 min at 4°C. The addition of NP40 to 1% (v/v) was followed by incubation for 10 min, 4°C. Cell lysates were homogenized and NaCl was added to a final concentration of 420 mM followed by incubation for 1 h, 4°C. The extracts were cleared by centrifugation (19,000×g, 1 h, 4°C)  1% Triton X-100/PBS/1% fetal calf serum were added for 2 h. Duolink (Olink Biosciences) in situ proximity ligation assay (PLA) was performed according to the manufacturer's protocol. PLA probes were diluted in 0.1% Triton X-100/PBS/1% fetal calf serum and incubated in a pre-heated humidity chamber for 1 h at 37°C, followed by hybridization, ligation, amplification and detection. The distance between the two primary antibodies needs to be less than 40 nm to generate a signal in this assay, making the methodology highly specific for physically interacting protein-protein complexes. Slides were analyzed using an inverted Zeiss LSM 510 META confocal microscope equipped with a Zeiss image processing system. An 63×/1.4 NA oil objective and sequential scanning with narrow bandpass filters was used (420-480 nm for Hoechst 33342 and 560-615 nm for Alexa 613).
Co-immunoprecipitation. Co-immunoprecipitation was performed with Dynabeads Protein G (Invitrogen) according to the manufacturer's protocol by addition and crosslinking with dithiobispropionimidate-2HCl of Quantitative RT-PCR. Total RNA was isolated from shRNA transfected cells using RNeasy mini kit with the addition of RNase-Free DNase to eliminate contaminating genomic DNA (Qiagen). 1 µg of RNA was subjected to reverse transcription into cDNA using SuperScript III (Invitrogen), according to the manufacturer's protocol. Endogenous gene copy numbers were determined by qPCR analysis using the ABI PRISM 7900 system with SYBR Green mix reagents (Applied Biosystems). Briefly, the qPCR www.impactaging.com mixture contained 1 µl cDNA, 1×SYBR Green mix reagent and 50 nM of each primer in a total reaction volume of 20 µl. All samples were analyzed in triplicates using the primer pairs listed in Table 1. Each primer pair yielded a single product, confirmed by dissociation curve analysis, and gave no product in the no-template control. To analyze the obtained data, all samples were normalized to an internal reference gene (GAPDH) to eliminate sample variances and toward a sample transfected with a vector containing a nonsense shRNA construct to eliminate effects from transfection. Following primer pairs were used to measure endogenous mRNA levels of proteins of interest, regarding ES cell maintenance (Oct4, Sox2), ectodermal differentiation (Nestin, Sox2), mesodermal differentiation (Brachyury), endodermal differentiation (GATA4) and reference gene (GAPDH) for internal normalization, after shRNA mediated downregulation of Tpt1 or Npm1. www.impactaging.com