Crif1 is a novel transcriptional coactivator of STAT3
2008; Springer Nature; Volume: 27; Issue: 4 Linguagem: Inglês
10.1038/sj.emboj.7601986
ISSN1460-2075
AutoresMin Chul Kwon, Bon‐Kyoung Koo, Jin-Sook Moon, Yoon Young Kim, Ki Cheol Park, Nam-Shik Kim, Mi Yi Kwon, Myung-Phil Kong, Ki‐Jun Yoon, Sun-Kyoung Im, Jaewang Ghim, Yong-Mahn Han, Sung Key Jang, Minho Shong, Young–Yun Kong,
Tópico(s)Protein Tyrosine Phosphatases
ResumoArticle17 January 2008free access Crif1 is a novel transcriptional coactivator of STAT3 Min-chul Kwon Min-chul Kwon Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Bon-Kyoung Koo Bon-Kyoung Koo Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Jin-Sook Moon Jin-Sook Moon Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Yoon-Young Kim Yoon-Young Kim Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Ki Cheol Park Ki Cheol Park Department of Internal Medicine, Chungnam National University School of Medicine, Daejeon, South Korea Search for more papers by this author Nam-Shik Kim Nam-Shik Kim Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Mi Yi Kwon Mi Yi Kwon Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Myung-Phil Kong Myung-Phil Kong Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Ki-Jun Yoon Ki-Jun Yoon Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Sun-Kyoung Im Sun-Kyoung Im Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Jaewang Ghim Jaewang Ghim Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Yong-Mahn Han Yong-Mahn Han Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, South Korea Search for more papers by this author Sung Key Jang Sung Key Jang Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Minho Shong Minho Shong Department of Internal Medicine, Chungnam National University School of Medicine, Daejeon, South Korea Search for more papers by this author Young-Yun Kong Corresponding Author Young-Yun Kong Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Min-chul Kwon Min-chul Kwon Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Bon-Kyoung Koo Bon-Kyoung Koo Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Jin-Sook Moon Jin-Sook Moon Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Yoon-Young Kim Yoon-Young Kim Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Ki Cheol Park Ki Cheol Park Department of Internal Medicine, Chungnam National University School of Medicine, Daejeon, South Korea Search for more papers by this author Nam-Shik Kim Nam-Shik Kim Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Mi Yi Kwon Mi Yi Kwon Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Myung-Phil Kong Myung-Phil Kong Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Ki-Jun Yoon Ki-Jun Yoon Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Sun-Kyoung Im Sun-Kyoung Im Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Jaewang Ghim Jaewang Ghim Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Yong-Mahn Han Yong-Mahn Han Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, South Korea Search for more papers by this author Sung Key Jang Sung Key Jang Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Minho Shong Minho Shong Department of Internal Medicine, Chungnam National University School of Medicine, Daejeon, South Korea Search for more papers by this author Young-Yun Kong Corresponding Author Young-Yun Kong Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea Search for more papers by this author Author Information Min-chul Kwon1, Bon-Kyoung Koo1, Jin-Sook Moon1, Yoon-Young Kim1, Ki Cheol Park2, Nam-Shik Kim1, Mi Yi Kwon1, Myung-Phil Kong1, Ki-Jun Yoon1, Sun-Kyoung Im1, Jaewang Ghim1, Yong-Mahn Han3, Sung Key Jang1, Minho Shong2 and Young-Yun Kong 1 1Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, South Korea 2Department of Internal Medicine, Chungnam National University School of Medicine, Daejeon, South Korea 3Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, South Korea *Corresponding author. Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Kyungbuk, Pohang 790-784, South Korea. Tel.: +82 54 279 2287; Fax: +82 54 279 2199; E-mail: [email protected] The EMBO Journal (2008)27:642-653https://doi.org/10.1038/sj.emboj.7601986 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Signal transducer and activator of transcription 3 (STAT3) is a transcriptional factor that performs a broad spectrum of biological functions in response to various stimuli. However, no specific coactivator that regulates the transcriptional activity of STAT3 has been identified. Here we report that CR6-interacting factor 1 (Crif1) is a specific transcriptional coactivator of STAT3, but not of STAT1 or STAT5a. Crif1 interacts with STAT3 and positively regulates its transcriptional activity. Crif1−/− embryos were lethal around embryonic day 6.5, and manifested developmental arrest accompanied with defective proliferation and massive apoptosis. The expression of STAT3 target genes was markedly reduced in a Crif1−/− blastocyst culture and in Oncostatin M-stimulated Crif1-deficient MEFs. Importantly, the key activities of constitutively active STAT3-C, such as transcription, DNA binding, and cellular transformation, were abolished in the Crif1-null MEFs, suggesting the essential role of Crif1 in the transcriptional activity of STAT3. Our results reveal that Crif1 is a novel and essential transcriptional coactivator of STAT3 that modulates its DNA binding ability, and shed light on the regulation of oncogenic STAT3. Introduction Signal transducer and activator of transcription 3 (STAT3) is a latent cytoplasmic transcriptional factor that can be activated by cytokines and growth factors. Disruption of STAT3 by gene targeting generated early embryonic lethality (∼embryonic day 6.5) (Takeda et al, 1997), and tissue-specific removal of STAT3 revealed its broad functional spectrum, including cell proliferation, differentiation, and apoptosis (Levy and Darnell, 2002). STAT3 has also gained attention because it is persistently active in a high proportion of human cancers and is required for tumor cell survival (Bromberg, 2001). STAT3 activates the transcription of several genes involved in cell cycle progression, such as Myc, Pim1, and Fos, and also upregulates antiapoptotic genes such as Bcl-2 and Bcl-XL (Yu et al, 2007). Upon cytokine stimulation, STAT3 is activated through phosphorylation by Janus kinase (Jak) family members, dimerizes, and then translocates into the nucleus, where it binds specific promoter sequences of target genes and induces their transcription (Levy and Darnell, 2002; Levy and Lee, 2002). Significant progress has been made in the elucidation of the positive and negative regulation of STAT3 signalling (Levy and Darnell, 2002; Heinrich et al, 2003). Negative regulators, such as SOCS3, PIAS3, and GRIM-19, play important roles in cellular function by limiting cytokine signals (Chung et al, 1997; Lufei et al, 2003; Alexander and Hilton, 2004). In contrast, EZI and Gfi-1 have been suggested as positive regulators of STAT3 signalling. EZI, a nuclear zinc-finger protein, augments STAT3 activity by keeping it in the nucleus (Nakayama et al, 2002), and another zinc-finger protein, Gfi-1, also enhances STAT3 signalling by interacting with the STAT3 inhibitor PIAS3 (Rodel et al, 2000). Although the STAT3-signalling pathway from the plasma membrane to the nucleus has been delineated in detail, the molecular bases that govern gene transcription by STAT3 require further elucidation. Like many other transcription factors, STAT3 associates with the transcriptional coactivators cAMP response element binding protein-binding protein/p300 (CBP/p300) and steroid receptor coactivator 1 (NcoA/SRC1a). These interactions enhance the transcriptional activity of STAT3 (Nakashima et al, 1999; Giraud et al, 2002). In addition, other transcriptional activators, such as c-Jun and glucocorticoid receptor, function synergistically with STAT3 to activate gene expression (Shuai, 2000). However, these coactivators are not specific to STAT3, but are also implicated in the functions of other STAT family members as well as in oncoproteins (such as Myb, Jun, and Fos), transforming viral proteins (such as E1A, E6, and large T antigen) and tumor-suppressor proteins (such as p53, E2F, Rb, Smads, RUNX, and BRCA1) (Shuai, 2000; Litterst and Pfitzner, 2002; Iyer et al, 2004). Recently, MCM5 and CoaSt6 have been suggested as specific coactivators of STAT1 and STAT6, respectively, although their in vivo relevance needs to be determined (Snyder et al, 2005; Goenka and Boothby, 2006). These findings raise the possibility of the existence of an additional coactivator that binds specifically to STAT3 and modulates its transcriptional activity. Using yeast two-hybrid screening, we identified CR6-interacting factor 1 (Crif1) as a novel binding partner of STAT3, and found that overexpression of Crif1 enhances the transcriptional activity of STAT3. Crif1 interacts with Gadd45α,β,γ, and Nur77, and has been suggested as a potential regulator of cell cycle progression and cell growth (Chung et al, 2003; Park et al, 2005). To determine the role of Crif1 in vivo, we generated mice with a disruption in the gene encoding Crif1. Interestingly, Crif1-deficient embryos showed early embryonic lethality before the gastrulation stage, and Crif1-deficient blastocysts exhibited reduced expression of STAT3 target genes. Mouse embryonic fibroblasts (MEFs) with the Crif1 gene disrupted using Cre recombinase exhibited impaired STAT3 transcriptional activities, although the upstream signalling events were intact. Chromatin immunoprecipitation (ChIP) experiments and an electrophoretic mobility shift analysis (EMSA) showed that Crif1 is essential for the DNA-binding activity of STAT3. Furthermore, cellular transformation by the constitutively active form of STAT3 was completely abolished in the Crif1-null MEFs. Our findings revealed that Crif1 is an essential and specific transcriptional coactivator of STAT3. Results STAT3 interacts with Crif1 A yeast two-hybrid screen identified Crif1 as a novel binding partner of STAT3 (Supplementary Figure 1A). To confirm this interaction, Myc-tagged Crif1 was transfected into HEK 293 cells, and lysates were immunoprecipitated with an anti-Myc antibody and then immunoblotted with an anti-STAT3 antibody to detect endogenous STAT3 (Figure 1A). As a result, Crif1 indeed interacted with STAT3. To test whether Crif1 also interacts with other STAT proteins, we used MEFs overexpressing HA-tagged Crif1 (HA-Crif1). In contrast to the strong binding with STAT3, Crif1 did not interact with STAT1 and STAT5a, suggesting a specific functional link between Crif1 and STAT3 (Figure 1B and C). To identify the domain of Crif1 responsible for STAT3 binding, we generated three HA-Crif1 deletion mutants (Supplementary Figure 1B). These HA-Crif1 mutants were each expressed in HEK 293 cells and immunoprecipitated, which revealed that the C-terminal coiled-coil domain (CCD) of Crif1 interacted with STAT3 (Figure 1D). We used a yeast two-hybrid system to define the domain of STAT3 that interacts with Crif1. The analysis revealed that the CCD of STAT3 was sufficient for the interaction with Crif1 (Figure 1E). Taken together, these results suggest that Crif1 and STAT3 interact via their CCDs. Figure 1.Interaction of Crif1 with STAT3. (A) Interaction between Crif1 and STAT3. HEK 293T cells were transfected with Myc-Crif1 and immunoprecipitated (IP) with an anti-Myc Ab, followed by western blotting (IB) with an anti-STAT3 Ab. (B, C) Specific binding of Crif1 with STAT3. MEFs overexpressing mock or HA-Crif1 were mock-stimulated (B) or stimulated with IFNγ (20 ng/ml) for 30 min (C), and lysates were immunoprecipitated with an anti-HA Ab, followed by western blotting with anti-STAT3 (B), STAT5a (B), and STAT1 (C) Abs. (D) HA-tagged Crif1 deletion mutants were transfected into HEK 293T cells and then immunoprecipitated with an anti-HA Ab. Total cell extracts and immunoprecipitates were blotted with anti-HA and anti-STAT3 Abs. (E) Schematic representation of the STAT3 constructs and their interactions with full-length Crif1 in a yeast two-hybrid system. NTD, N-terminal domain; CCD, coiled-coil domain; DBD, DNA-binding domain; LK, linker domain; SH2, SH2 domain; TAD, transactivation domain. Download figure Download PowerPoint Oncostatin M stimulation enhances the interaction between Crif1 and STAT3 To examine the spatio-temporal interaction of Crif1-STAT3 upon stimulation with Oncostatin M (OSM), a member of the IL-6 cytokine subfamily, we generated NIH3T3 cell lines stably expressing HA-Crif1 (HA-Crif1 NIH3T3), and examined the localization of HA-Crif1 and STAT3 in the HA-Crif1 NIH3T3 cells before and after the OSM stimulation. Consistent with previous reports, Crif1 was localized predominantly in the nucleus (Gornemann et al, 2002; Chung et al, 2003). Without OSM stimulation, STAT3 was found in both the nucleus and cytoplasm, but OSM stimulation resulted in the accumulation of STAT3 in the nucleus, where Crif1 was located (Figure 2A). The merged pictures of Crif1 and STAT3 indicated the colocalization of both proteins in the nucleus after OSM stimulation. Furthermore, when the cell lysates were immunoblotted with an anti-phospho-STAT3 antibody or immunoprecipitated with an anti-HA antibody, followed by an anti-STAT3 antibody, STAT3 phosphorylation was detected at 5 min after treatment with OSM, and began to diminish at 60 min (Figure 2B). Interestingly, the amount of STAT3 bound to Crif1 apparently increased upon OSM stimulation, suggesting that the interaction between Crif1 and STAT3 is dependent on the OSM stimulation. Figure 2.Crif1 enhances the transcriptional activity of STAT3. (A) Colocalization of Crif1 and STAT3. HA-Crif1 expressing NIH3T3 cells were stimulated with OSM (10 ng/ml) and immunostained with anti-HA (in green) and anti-STAT3 (in red) Abs. (B) Enhanced interaction between Crif1 and STAT3 upon OSM stimulation. HA-Crif1-expressing NIH3T3 cells were stimulated with OSM (10 ng/ml), and lysates were immunoprecipitated with an anti-HA Ab, followed by western blotting with an anti-STAT3 Ab. (C–H) Enhanced transcriptional activity of STAT3 by Crif1. NIH 3T3 cells were cotransfected with the Crif1 expression vector and the STAT3 responsive m67-luciferase construct, and were cotransfected with the STAT3 expression vector (C) or were stimulated with LIF (D) and OSM (E) for 8 h. HCT116 (F), SNU387 (G) and MDA-MB 468 (H) human cancer cell lines were cotransfected with the Crif1 expression vector and the STAT3 responsive m67-luciferase construct. Luciferase activity was measured 36 h after transfection. The results are representative of three independent experiments (*P<0.05, **P<0.002, ***P<0.02). A full-colour version of this figure is available at The EMBO Journal Online. Download figure Download PowerPoint Since Crif1 resides and might function in the nucleus, we tested whether the interaction between Crif1 and STAT3 upon OSM stimulation results from the nuclear accumulation of activated STAT3 using STAT3Y705F, which has a mutation at a phosphorylation site for dimerization (Kaptein et al, 1996; Bhattacharya and Schindler, 2003). V5-tagged STAT3 and V5-tagged STAT3Y705F plasmids were each co-electroporated with HA-tagged Crif1 into the MEFs, which were then stimulated with OSM for 20 min. Anti-HA immunoprecipitates from the whole-cell extracts were subjected to a western blot analysis with an anti-V5 antibody. OSM stimulation enhanced the Crif1–STAT3 interaction, but not the Crif1–STAT3Y705F interaction (Supplementary Figure 1F). However, the basal level of interaction between Crif1 and STAT3Y705F might represent the basal import pathway (Bhattacharya and Schindler, 2003). In addition, Crif1 interacted with TAD domain-deleted STAT3, which lacks phosphorylation sites for dimerization (Supplementary Figure 1C and D). These results suggest that the nuclear enrichment of STAT3 by OSM stimulation enhances the interaction between Crif1 and STAT3. Crif1 enhances the transcriptional activity of STAT3 The stimulation-dependent interaction between Crif1 and STAT3 might affect the biological activity of STAT3, as a transcriptional factor. To evaluate whether Crif1 alters the transcriptional activity of STAT3, we performed reporter gene assays, using a luciferase reporter construct containing four STAT-binding sites (m67-luc) (Bromberg et al, 1999). Crif1 alone slightly enhanced the STAT3-mediated transcriptional activity in NIH3T3 cell lines. However, when Crif1 was coexpressed with full-length STAT3, Crif1 substantially increased the STAT3-mediated transcriptional activity (Figure 2C). Furthermore, Crif1 enhanced the STAT3-mediated transcriptional activity in the presence of LIF and OSM (Figure 2D and E). We also examined the effect of Crif1 using human cancer cell lines, HCT116 (human colon cancer cell line), SNU387 (human hepatic cancer cell line), and MDA-MB 468 (human breast cancer cell line), which have constitutively phosphorylated STAT3 (Yoshida et al, 1996; Garcia et al, 1997; Siddiquee et al, 2007). As expected, the transcriptional activity of STAT3 in these cell lines was dramatically increased, by over 20-fold, by the ectopic expression of Crif1 without any stimulation (Figure 1F–H). The relatively small induction in the NIH3T3 cell line by ectopic Crif1 expression might be due to different cellular contexts, such as the endogenous expression of Crif1. These results suggest that Crif1 positively regulates the STAT3-mediated transcriptional activity. Generation of Crif1−/− mice STAT3 is essential and sufficient to maintain the pluripotency of murine embryonic stem cells (Niwa et al, 1998), and its disruption causes embryonic lethality before gastrulation (Takeda et al, 1997). To elucidate the physiological relevance of the STAT3-Crif1 interaction, Crif1−/− mice were generated (Supplementary Figure 2). The Crif1+/− mice were healthy and fertile. However, when the heterozygous mice were intercrossed, no Crif1−/− mice were detected among 330 offspring, indicating that Crif1 deficiency results in postnatal or embryonic lethality. To determine when the Crif1−/− mice died, time-pregnant heterozygous females were killed at different gestational stages. Among 66 embryos analysed from E7.5 to E9.5, none were Crif1−/−. Among 30 embryos assayed from E6.5 embryos, only three were Crif1−/− (Supplementary Figure 2D). In contrast, Crif1−/− blastocysts were morphologically normal and appeared in the expected Mendelian ratio (Supplementary Figure 2D). These data indicate that Crif1−/− embryos die around E6.5. Crif1−/− embryos show defective proliferation and massive apoptosis At E6.5, all of the Crif1−/− embryos were smaller and developmentally retarded, as compared with their control littermates (Figure 3A). A histological analysis of 18 E6.5 decidua generated from heterozygous intercrosses revealed two distinct morphological classes. Fifteen embryos (83.3%) exhibited normal cellularity and cytoarchitecture in all embryonic and extra-embryonic structures (Figure 3B), and three (16.7%) were severely growth retarded and showed abnormal structures, such as the absence of a proamniotic cavity (Figure 3C). Therefore, we examined the rate of proliferation using BrdU incorporation and the extent of apoptosis by performing TUNEL assays in Crif1 mutant and control littermates at E6.5. BrdU labelling showed that the proliferation rate in the Crif1−/− embryos was significantly reduced relative to that of the control littermates (Figure 3D and G). Furthermore, few apoptotic cells were observed in the control embryos, but many TUNEL-positive cells were scattered throughout the Crif1−/− embryos at E6.5 (Figure 3E and H). These results suggest that the growth deficit of the Crif1−/− embryos resulted from both defective cellular proliferation and increased cell death. Figure 3.Defective proliferation and massive apoptosis in Crif1−/− embryos. (A) Gross appearance of PCR-verified E6.5 Crif1+/− (left) and Crif1−/− embryos (right). The genotypes of both embryos, determined by PCR analysis, are indicated at the bottom of the figure. (B–I) Sagittal sections of E6.5 Crif1+/− (B, D–F) and Crif1−/− (C, G–I) embryos were stained with haematoxylin and eosin (B, C), anti-BrdU Ab (D, G), TUNEL (E, H), and Hoechst (F, I). Download figure Download PowerPoint Defective outgrowth of the inner cell mass from Crif1−/− blastocysts To directly assess the growth capability of Crif1−/− embryos, we collected E3.5. blastocysts from heterozygote intercrosses and cultured them individually for 7 days. Two days after culture, all of the blastocysts had successfully attached to the bottom of culture dish and hatched from the zona pellucida. After 3 days in culture, all of the blastocysts produced apparently normal trophoblast giant cells, a process necessary to induce the decidual reaction during implantation. However, the inner cell mass (ICM), which forms the future embryonic tissues, did not exhibit outgrowth in the Crif1−/− blastocysts. Longer periods of blastocyst culture confirmed the inability of ICM outgrowth from 6 of the 9 Crif1−/− blastocysts (Figure 4A, case no. 1). Although the other Crif1−/− blastocysts showed marginal ICM outgrowth, these ICM-like cells did not grow further (Figure 4A, case no. 2). At 3 days of culture, we also examined whether the Crif1−/− blastocysts were undergoing apoptosis. Whereas the Crif1+/− blastocysts showed no apoptotic cells, the Crif1−/− blastocysts displayed many TUNEL-positive apoptotic cells (Figure 4B). Taken together, these results suggest that Crif1 is required for the normal ICM outgrowth of blastocysts. Figure 4.Defective outgrowth of ICM (A) and massive apoptosis (B) in Crif1−/− blastocysts. (A) Crif1+/+ (+/+), Crif1+/− (+/−), and Crif1−/− (−/−) blastocysts from Crif1+/− intercrosses were individually cultured in LIF-containing ES medium for 7 days. The outgrowths of ICM were inspected daily and photographed on the indicated day. (B) Crif1+/− (+/−), and Crif1−/− (−/−) blastocysts cultured for 3 days were subjected to a TUNEL assay. A DNase I-treated Crif1+/− blastocyst was used as a positive control for DNA fragmentation (Pos). Download figure Download PowerPoint Expression of Crif1 in the early developmental stages Crif1 mRNA was previously reported to be expressed ubiquitously, and at notably high levels in the thyroid gland, heart, lymph nodes, trachea, and adrenal tissues (Chung et al, 2003). In the blastocysts, Crif1 was expressed in the ICM, similarly to Oct4 and Nanog (Supplementary Figure 3A and B; data not shown). During early development, Crif1 was expressed in embryonic and extra-embryonic tissues, and the highest level of Crif1 expression was seen in the chorion, allantois, and amnion (Supplementary Figure 3C). Since Crif1 is critical for ICM outgrowth, we examined whether Crif1−/− blastocysts have an intact ICM. Sixty-nine blastocysts from seven heterozygote intercrosses were randomly divided into three groups and subjected to in situ hybridization and genotyping (Supplementary Figure 3E and F). One group (n=16) was genotyped; as expected, four blastocysts were Crif1−/−, consistent with the expected Mendelian ratio. The second group (n=26) was assayed for the presence of Oct4 expression: all were positive, indicating that all of the blastocysts had an apparently normal ICM (Supplementary Figure 3E). The third group (n=27) was assayed for Crif1 expression. Unexpectedly, it was detected in all of them, whereas 25% were expected to be homozygous mutants. This result suggests the possibility that an initial supply of Crif1 mRNA might be delivered maternally (Supplementary Figure 3F). Interestingly, the Crif1 mRNA in the Crif1−/− blastocysts disappeared after 3 days of culture (Supplementary Figure 4). Therefore, we examined the Crif1 mRNA expression patterns before the blastocyst stage, using an RT–PCR analysis. As expected, Crif1 mRNA was detected in unfertilized eggs and persisted until the blastocyst stage, although its expression level was decreased (Supplementary Figure 3D). These results suggest that the maternally derived Crif1 mRNA is not degraded until the blastocyst stage, and thus it might contribute to the initial outgrowth and survival of the ICM in Crif1−/− blastocysts. Decreased expression of STAT3 target genes in cultured Crif1−/− blastocysts Since Crif1 interacted with STAT3, and regulated its transcriptional activity, we predicted that the lethality would, at least partially, result from impaired STAT3 signalling. To investigate this possibility, we examined the expression levels of STAT3 target genes in cultured blastocysts, because STAT3 is responsible for the ICM outgrowth in this period (Takeda et al, 1997). Three days after the blastocyst culture in the presence of LIF, one-third of an individual colony was used for genotyping by genomic PCR, and the rest of the colony with the same genotype was pooled. RNA was extracted from each pooled colony lysate and analysed by semi-quantitative RT–PCR and real-time RT–PCR. Although the maternally derived Crif1 mRNA still remained in the Crif1−/− blastocysts before the culture (Supplementary Figure 3F), it was undetectable in the Crif1−/− colonies 3 days after the blastocyst culture, whereas the Crif1+/− colonies showed about half of the expression, as compared with that of the Crif1+/+ colonies (Supplementary Figure 4). Intriguingly, the expression levels of four known STAT3 target genes, Myc, Socs3, c-Fos, and JunB, were significantly decreased in the Crif1−/− colonies, as compared with the Crif1+/+ and Crif1+/− colonies (Supplementary Figure 4). In contrast, the expression levels of unrelated genes (Oct4, Id1, Id3, and Sox2) were not affected. Real-time RT–PCR also showed decreased expression levels of STAT3 target genes (Myc, Socs3, c-Fos, and JunB), whereas those of unrelated genes, Id1 and Id3, were not changed (Figure 5). These results indicate that Crif1 is critical for the expression of STAT3 target genes, and that it might work as a positive regulator of STAT3. Figure 5.Decreased expression of STAT3 target genes in colonies from Crif1−/− blastocysts. (A, B) Real-time RT–PCR analyses of STAT3 target genes (Myc, Socs3, c-Fos, and JunB) and unrelated genes (Id1 and Id3). Crif1+/+ (+/+), Crif1+/− (+/−), and Crif1−/− (−/−) blastocysts were individually cultured in LIF-containing ES medium for 3 days. One-third of an individual colony was used for genotyping by genomic PCR, and the rest of the colony with the same genotype was pooled. RNA was extracted from each pooled colony lysate and analysed by real-time RT–PCR. The error bars indicate the standard deviation. Oct4 was used for normalization. The results are representative of three independent experiments. Significant differences are *P<0.0001, **P<0.01, ***P<0.0005, and ****P<0.0001. Download fi
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