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FOXP2 promotes the nuclear translocation of POT1, but FOXP2(R553H), mutation related to speech-language disorder, partially prevents it
Biochemical and Biophysical Research Communications 410 (2011) 593–596
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
FOXP2 promotes the nuclear translocation of POT1, but FOXP2(R553H), mutation related to speech-language disorder, partially prevents it Yuko Tanabe a, Eriko Fujita a,b, Takashi Momoi a,c,⇑ a
Division of Development and Differentiation, National Institute of Neuroscience, NCNP, 4-1-1 Ogawahigasi, Kodaira 187-8511, Japan Department of Pediatrics, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan c Center for Medical Science, International University of Health and Welfare, 2600-1 Kitakanamaru, Otawara, Tochigi 324-8501, Japan b
a r t i c l e
i n f o
Article history: Received 5 May 2011 Available online 12 June 2011 Keywords: FOXP2 POT1 FOXP2(R553H) Telomerase Speech-language disorder Forkhead
a b s t r a c t FOXP2 is a forkhead box-containing transcription factor with several recognizable sequence motifs. However, little is known about the FOXP2-associated proteins except for C-terminal binding protein (CtBP). In the present study, we attempted to isolate the FOXP2-associated protein with a yeast two-hybrid system using the C-terminal region, including the forkhead domain, as a bait probe, and identified protection of telomeres 1 (POT1) as a FOXP2-associated protein. Immunoprecipitation assay confirmed the association with FOXP2 and POT1. POT1 alone localized in the cytoplasm but co-localized with FOXP2 and the forkhead domain of FOXP2 in nuclei. However, both FOXP2 with mutated nuclear localization signals and (R553H) mutated forkhead, which is associated with speech-language disorder, prevented the nuclear translocation of POT1. These results suggest that FOXP2 is a binding partner for the nuclear translocation of POT1. As loss of POT1 function induces the cell arrest, the impaired nuclear translocation of POT1 in the developing neuronal cells may be associated with the pathogenesis of speech-language disorder with FOXP2(R553H) mutation. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction FOXP2 is a forkhead box, approximately 110 amino acids in length with a winged-helix DNA binding domain, -containing transcriptional factor [1]. Nuclear localization signals (NLSs) are found at the C-terminal and N-terminal ends of the forkhead [2]. FOXP2 also has leucine zipper and zinc finger domains [1]. FOXP2 expresses in the various tissues including lung and brain [1]. This protein is believed to have diverse roles during development, such as morphogenesis, differentiation, and proliferation. In the brain, the expression of the human and mouse homologues is very similar during development and in adulthood [3,4]. The missense mutation (R553H) in the forkhead-containing domain in the FOXP2 gene has been found in patients of speechlanguage disorder, which exhibits autosomal dominant trait and severe abnormalities in speech and language [5]. The relationship between this FOXP2 mutation and the pathogenesis of speech-language disorder has been extensively studied. FOXP2(R553H)
demonstrates reduced DNA binding and a defect in nuclear localization [2,6]; some of the FOXP2(R553H) is translocated into the nucleus in the dimeric form, while the rest of the FOXP2(R553H) remains in the cytoplasm [2]. Therefore, in addition to a defect in FOXP2(R553H) DNA binding activity, the binding partners of FOXP2 may fail to function in the nucleus if they are associated with FOXP2(R553H) in the cytoplasm [6]. Little is known about the molecular mechanism of FOXP2mediated regulation, although FOXP2 negatively regulates gene expression by associating with the co-repressor C-terminal binding protein (CtBP) via binding motif located at N-terminal region [7]. In the present study, we attempted to isolate the FOXP2-associated protein with a yeast two-hybrid system using the C-terminal region, including the forkhead domain, as bait. We have identified protection of telomeres 1 (POT1) as the associated protein, and demonstrate that FOXP2(R553H) prevents the nuclear translocation of POT1.
2. Materials and methods Abbreviations: FOXP2, forkhead box P2; POT1, protection of telomeres 1; KI, knock-in; USV, ultrasonic vocalization; CtBP, C-terminal binding protein. ⇑ Corresponding author at: Center for Medical Science, International University of Health and Welfare, 2600-1 Kitakanamaru, Otawara, Tochigi 324-8501, Japan. Fax: +81 287 24 3001. E-mail address: [email protected] (T. Momoi). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.06.032
2.1. DNA construction POT1 cDNA was isolated from a human fetal cDNA library by yeast two-hybrid screening. The enzyme-digested PCR DNA
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fragments corresponding to full-length FOXP2, FOXP2(484–715), FOXP2(484–598) containing forkhead domain (FOXP2-forkhead), FOXP2(484–598) with R553H mutation (FOXP2-R553H-forkhead), FOXP2(576–715), FOXP2-mutated NLS (mNLS) [2], and full-length POT1 were subcloned into the following vectors: pGAD, pGBK, pEGFP-C1 (Clontech), and FLAG vector pcDEF3 [8]. 2.2. Yeast two-hybrid assay Yeast two-hybrid assays were performed by transforming AH109 or Y187 yeast cells with both pGBK- and pGAD-based constructs according to the manufacturer’s instructions (Clontech). AH109 transformants were selected on SD-Leu/-Trp/-His/-Ade plates for 2–3 days. After dilution, 2–3 ml of cells were spotted onto SD-Leu/-Trp/-His/-Ade plates and grown for 3–4 days. The respective b-galactosidase activities of Y187 yeast transformed with pGBK and pGAD constructs were assayed according to the manufacturer’s instructions. 2.3. Transfection COS cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2, using a-MEM medium supplemented with 10% fetal bovine serum. For immunoprecipitation and immunoblotting, cells were transfected with 16 lg of FLAG-POT1, alone or in combination with 4 lg of GFP-FOXP2s. Transfections were conducted using Lipofectamin 2000 (Invitrogen) according to manufacturer’s instructions. For immunostaining, cells were cotransfected with FLAG-POT1 and GFP-FOXP2s using the calciumphosphate method, as described previously [8]. 2.4. Immunoprecipitation and immunblot analysis Cells were lysed in lysis buffer [50 mM Tris–HCl pH 8.0, 150 mM NaCl, 10% glycerol, 0.5% IGEPAL CA630, and protease inhibitors;
Complete mini (Roche Diagnostics)] at 4 °C for 15 min, and sonicated. After insoluble materials were removed by centrifugation, cell lysates were subjected to immunoprecipitation as described previously [2] and the resulting precipitates were subjected to immunoblot analysis using anti-GFP (MBL) and antiFLAG (Sigma) antibodies. Immunoreactivity was visualized using alkaline phosphatase-conjugated anti-mouse or anti-rabbit IgG antibodies, respectively, with Nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-1-phosphate (Roche Diagnostics). 2.5. Immunostaining After transfected COS cells were cultured for 24 h and then fixed in 2% paraformaldehyde in phosphate buffered saline (PBS) at 4 °C for 30 min, they were then subjected to immunostaining using mouse anti-FLAG antibody in incubation buffer at 4 °C overnight, as described previously [2]. Mouse rhodamine-labeled IgG was prepared from Leinco Technologies. Nuclei were labeled with Hoechst 33342 (Molecular Probes). Immunoreactivity was viewed using a fluorescence microscope (Leica AF6000, Leica Microsystems).
3. Results 3.1. Isolation of POT1 via yeast two-hybrid screening We examined FOXP2-associated protein by yeast two-hybrid assay. We used the C-terminal region of FOXP2 including the forkhead domain, FOXP2(484–715), as bait (Fig. 1A) and isolated POT1 cDNA (from the eighth amino acid to the 30 -UTR). Yeast AH109 cotransformed with pGAD-POT1 and pGBK-FOXP2(484–715) or pGBK-FOXP2(484–598) containing forkhead domain grew on SDLeu/-Trp/-His/-Ade plates, while cells co-transformed with pGADPOT1 and pGBK-FOXP2(576–715) lacking forkhead domain did
Fig. 1. Yeast two-hybrid screening for FOXP2 binding protein. (A) A schematic representation on the relationship between functional domains of FOXP2 and bait probes used in yeast two-hybrid screening. FOXP2(484–715, including forkhead domain), FOXP2(484–598, including forkhead domain), FOXP2(576–715, lacking forkhead domain) were used as bait probes. PolyQ, poly-glutamine; ZF, zinc finger; LZ, leucine zipper; Forkhead, forkhead domain. POT1 was isolated by yeast two-hybrid screening using FOXP2(484–715) as a probe. The prey, pGAD-POT1, interacted with pGBK-FOXP2(484–598), but not with pGBK-FOXP2(576–715), in yeast. (B) The b-galactosidase activity of the transformed cells. The relative b-galactosidase activity of the transformed cells with FOXP2(484–598), FOXP2(484–598) and POT1, FOXP2-R553H(484–598), FOXP2R553H(484–598) and POT1, FOXP2(576–715), and FOXP2(576–715) and POT1 were 0.33 ± 0.04, 117.06 ± 8.26, 0.41 ± 0.04, 50.33 ± 12.09, 0.40 ± 0.05, and 0.50 ± 0.07, respectively.
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not (Fig. 1A). Thus, FOXP2-forkhead associated with POT1, a component of the telomerase ribonucleoprotein [9]. In contrast with FOXP2-forkhead located in the nucleus, the forkhead with (R553H), mutation related to speech-language disorder, partially localizes in both the cytoplasm and the nucleus [2]. To examine the interaction between POT1 and FOXP2(484– 598) or FOXP2(484–598) with R553H mutation {FOXP2-R553H(484–598)} quantitatively, b-galactosidase activity was measured in the yeast Y187 strain co-transformed with pGAD-POT1 and pGBK-FOXP2-forkhead or mutated one (Fig. 1B); The relative b-galactosidase activity of the co-transformed cells with pGADPOT1 and pGBK-FOXP2-R553H(484–598) was about 2-fold lower than that of the co-transformed cells with pGAD-POT1 and pGBK-FOXP2(484–598). Thus, pGBK-FOXP2-R553H(484–598) also associated with POT1. On the other hand, the co-transformed cells with pGAD-POT1 and pGBK-FOXP2(576–715) did not demonstrate the b-galactosidase activity. 3.2. The interaction between FOXP2 and POT1and nuclear translocation of POT1 We confirmed the interaction between GFP-FOXP2 and FLAG-POT1 by immunoprecipitation and immunoblot analysis (Fig. 2). When GFP-FOXP2 and FLAG-POT1 were co-transfected into COS cells, anti-FLAG detected both FLAG-POT1 and GFP-FOXP2 in the precipitates; GFP-FOXP2 was not detected in the precipitates in the absence of FLAG-POT1 (Fig. 2A). GFP-FOXP2 alone localized
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to the nucleus, while FLAG-POT1 alone localized in the cytoplasm. In the presence of GFP-FOXP2, FLAG-POT1 co-localized with GFPFOXP2 in the nucleus (Fig. 2B). NLSs are located at both the N-terminal and C-terminal sides of the forkhead domain [2]; GFP-FOXP2 with a mutated NLS (GFP-FOXP2-mNLS) localized in the cytoplasm. FLAG-POT1 associated with GFP-FOXP2-mNLS (Fig. 2A), but they co-localized in the cytoplasm (Fig. 2B). 3.3. Effect of mutated FOXP2(R553H) on the nuclear translocaton of POT1 As FOXP2-R553H-(484–598) (FOXP2-R553H-forkhead), interacted with POT1 (Fig. 1B), we also examined the influence of R553H mutation on the nuclear translocation of POT1. GFPFOXP2-forkhead localized in the nucleus, while FOXP2-R553Hforkhead was observed in both the cytoplasm and the nucleus [2]. FLAG-POT1 co-localized with GFP-FOXP2-forkhead in the nucleus (Fig. 3), but co-localized with FOXP2-R553H-forkhead in both the cytoplasm and the nucleus (Fig. 3). Therefore, the nuclear translocation of POT1 was affected by the localization of FOXP2 and its derivatives containing forkhead domain and NLS. 4. Discussion POT1 is a component of the telomerase ribonucleoprotein complex that is essential for the replication of chromosome termini [9].
Fig. 2. Interaction between POT1 and FOXP2 and their co-localization. (A) Immunoprecipitation and immunoblot analysis on the interaction between the GFP-FOXP2 or FOXP2-mutated NLS (mNLS) and POT1. (B) Localization of FOXP2 and POT1. GFP-FOXP2 localized in the nuclei. While FLAG-POT1 localized in the cytoplasm, it co-localized with GFP-FOXP2 and GFP-FOXP2-mNLS in the nuclei (arrowheads) and the cytoplasm (arrows), respectively. Scale bar, 15 lm.
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Fig. 3. Effect of FOXP2-R553H-forkhead on the nuclear localization of POT1. While FLAG-POT1 co-localized with GFP-FOXP2-forkhead in the nuclei (arrowheads), it colocalized with FOXP2-R553H-forkhead in both the cytoplasm (arrows) and the nucleus (arrowhead). Scale bar, 20 lm.
A six-protein complex (TRF1, TRF2, RAP1, TIN2, POT1, and TPP1), known as shelterin, protects the telomeres of human chromosomes [10]. POT1 directly binds the single-stranded 30 extension at the chromosome end [11,12], which is bridged with TIN2 through protein–protein interactions via TPP1 [10,13]. POT1-TPP1 switches from inhibiting to allowing telomerase access to the telomere [14]. Defects in POT1 activate the DNA damage response, leading to propagation of a DNA damage signal and inappropriate DNA repair reaction, and resulting in cell cycle arrest [15]. Because POT1 does not have putative NLS sites, a nuclear protein with an NLS is necessary as a binding partner for the nuclear localization of POT1. In the present study, we showed that both FOXP2 and FOXP2-mNLS associated with POT1 and their localization had the influence on the localization of POT1; POT1 alone was primarily located in the cytoplasm, but co-localized with FOXP2 in the nucleus, or co-localized with FOXP2-mNLS in the cytoplasm (Fig. 2), suggesting that FOXP2 is a binding partner for the nuclear localization of POT1. FOXP2 has diverse roles as a transcriptional factor during development, including during survival or proliferation, in specific regions including the brain. FOXP2 may regulate the cell cycles via associating with POT1. This will be one of the next issues in future. Homozygotic knock-in mice with the FOXP2(R552H), mutation related to speech-language disorder, exhibit poor development of Purkinje cells and die within 3 weeks after birth [16]. The interaction between mutated R553H-forkhead and POT1 was 2-fold lower than that between wild-type forkhead and POT1 but interaction still remains (Fig. 1B). In contrast with forkhead domain, POT1 partly co-localized with mutated (R553H)-forkhead in the cytoplasm (Fig. 3), suggesting that cells expressing FOXP2(R553H) may have a higher risk of being mistaken for having broken DNA ends and being subjected to DNA repair by loss of the protection of the telomere terminus. Therefore, the neuronal cells expressing FOXP2-R553H in the brain may be damaged during development due to POT1 dysfunction. Loss of POT1 function in the nuclei of neurons in the developing brain may be associated with the pathogenesis of speech-language disorder with FOXP2 mutation. This will be another major issue in the future study. Acknowledgments Authors thank Dr. Akifumi Mizutani for introduction of yeasttwo-hybrid assay in the initial time of this study. This work was
supported by Grants-in-Aid for Scientific Research (KAKENHI) of the Ministry of Education, Culture, Sports, Science and Technology, Japan (21200011, 21700377); Grants-in-Aid for Health Labour Scientific Research of the Ministry of the Health, Labour and Welfare, Japan; and a Grant-in-Aid for Intramural Research (20B-13) for Neurological and Psychiatric Disorders of National Center of Neurology and Psychiatry (NCNP), Japan. References [1] W. Shu, H. Yang, L. Zhang, et al., Characterization of a new subfamily of winged-helix/forkhead (Fox) genes that are expressed in the lung and act as transcriptional repressors, J. Biol. Chem. 276 (2001) 27488–27497. [2] A. Mizutani, A. Matsuzaki, M.Y. Momoi, et al., Intracellular distribution of a speech/language disorder associated FOXP2 mutant, Biochem. Biophys. Res. Commun. 353 (2007) 869–874. [3] R.J. Ferland, T.J. Cherry, P.O. Preware, et al., Characterization of Foxp2 and Foxp1 mRNA and protein in the developing and mature brain, J. Comp. Neurol. 460 (2003) 266–279. [4] C.S. Lai, D. Gerrelli, A.P. Monaco, et al., FOXP2 expression during brain development coincides with adult sites of pathology in a severe speech and language disorder, Brain 126 (2003) 2455–2462. [5] C.S. Lai, S.E. Fisher, J.A. Hurst, et al., A forkhead-domain gene is mutated in a severe speech and language disorder, Nature 413 (2001) 519–523. [6] S.C. Vernes, J. Nicod, F.M. Elahi, et al., Functional genetic analysis of mutations implicated in a human speech and language disorder, Hum. Mol. Genet. 31 (2006) 54–67. [7] S. Li, J. Weidenfeld, E.E. Morrisey, Transcriptional and DNA binding activity of the Foxp1/2/4 family is modulated by heterotypic and homotypic protein interactions, Mol. Cell. Biol. 24 (2004) 809–822. [8] E. Fujita, A. Soyama, T. Momoi, RA175, which is the mouse ortholog of TSLC1, a tumor suppressor gene in human lung cancer, is a cell adhesion molecule, Exp. Cell Res. 287 (2003) 57–66. [9] P. Baumann, T.R. Cech, Pot1, the putative telomere end-binding protein in fission yeast and humans, Science 292 (2001) 1171–1175. [10] T. de Lange, Shelterin: the protein complex that shapes and safeguards human telomeres, Genes Dev. 19 (2005) 2100–2110. [11] D. Loayza, H. Parsons, J. Donigian, et al., DNA binding features of human POT1: a nonamer 50 -TAGGGTTAG-30 minimal binding site, sequence specificity, and internal binding to multimeric sites, J. Biol. Chem. 279 (2004) 13241–13248. [12] M. Lei, E.R. Podell, T.R. Cech, Structure of human POT1 bound to telomeric single-stranded DNA provides a model for chromosome end-protection, Nat. Struct. Mol. Biol. 11 (2004) 1223–1229. [13] D. Loayza, T. De Lange, POT1 as a terminal transducer of TRF1 telomere length control, Nature 423 (2003) 1013–1018. [14] F. Wang, E.R. Podell, A.J. Zaug, et al., The POT1–TPP1 telomere complex is a telomerase processivity factor, Nature 445 (2007) 506–510. [15] D. Hockemeyer, A.J. Sfeir, J.W. Shay, et al., POT1 protects telomeres from a transient DNA damage response and determines how human chromosomes end, EMBO J. 24 (2005) 2667–2678. [16] E. Fujita, Y. Tanabe, A. Shiota, et al., Ultrasonic vocalization impairment of Foxp2 (R552H) knockin mice related to speech-language disorder and abnormality of Purkinje cells, Proc. Natl Acad. Sci. USA 105 (2008) 3117–3122.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
FOXP2 promotes the nuclear translocation of POT1, but FOXP2(R553H), mutation related to speech-language disorder, partially prevents it Yuko Tanabe a, Eriko Fujita a,b, Takashi Momoi a,c,⇑ a
Division of Development and Differentiation, National Institute of Neuroscience, NCNP, 4-1-1 Ogawahigasi, Kodaira 187-8511, Japan Department of Pediatrics, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan c Center for Medical Science, International University of Health and Welfare, 2600-1 Kitakanamaru, Otawara, Tochigi 324-8501, Japan b
a r t i c l e
i n f o
Article history: Received 5 May 2011 Available online 12 June 2011 Keywords: FOXP2 POT1 FOXP2(R553H) Telomerase Speech-language disorder Forkhead
a b s t r a c t FOXP2 is a forkhead box-containing transcription factor with several recognizable sequence motifs. However, little is known about the FOXP2-associated proteins except for C-terminal binding protein (CtBP). In the present study, we attempted to isolate the FOXP2-associated protein with a yeast two-hybrid system using the C-terminal region, including the forkhead domain, as a bait probe, and identified protection of telomeres 1 (POT1) as a FOXP2-associated protein. Immunoprecipitation assay confirmed the association with FOXP2 and POT1. POT1 alone localized in the cytoplasm but co-localized with FOXP2 and the forkhead domain of FOXP2 in nuclei. However, both FOXP2 with mutated nuclear localization signals and (R553H) mutated forkhead, which is associated with speech-language disorder, prevented the nuclear translocation of POT1. These results suggest that FOXP2 is a binding partner for the nuclear translocation of POT1. As loss of POT1 function induces the cell arrest, the impaired nuclear translocation of POT1 in the developing neuronal cells may be associated with the pathogenesis of speech-language disorder with FOXP2(R553H) mutation. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction FOXP2 is a forkhead box, approximately 110 amino acids in length with a winged-helix DNA binding domain, -containing transcriptional factor [1]. Nuclear localization signals (NLSs) are found at the C-terminal and N-terminal ends of the forkhead [2]. FOXP2 also has leucine zipper and zinc finger domains [1]. FOXP2 expresses in the various tissues including lung and brain [1]. This protein is believed to have diverse roles during development, such as morphogenesis, differentiation, and proliferation. In the brain, the expression of the human and mouse homologues is very similar during development and in adulthood [3,4]. The missense mutation (R553H) in the forkhead-containing domain in the FOXP2 gene has been found in patients of speechlanguage disorder, which exhibits autosomal dominant trait and severe abnormalities in speech and language [5]. The relationship between this FOXP2 mutation and the pathogenesis of speech-language disorder has been extensively studied. FOXP2(R553H)
demonstrates reduced DNA binding and a defect in nuclear localization [2,6]; some of the FOXP2(R553H) is translocated into the nucleus in the dimeric form, while the rest of the FOXP2(R553H) remains in the cytoplasm [2]. Therefore, in addition to a defect in FOXP2(R553H) DNA binding activity, the binding partners of FOXP2 may fail to function in the nucleus if they are associated with FOXP2(R553H) in the cytoplasm [6]. Little is known about the molecular mechanism of FOXP2mediated regulation, although FOXP2 negatively regulates gene expression by associating with the co-repressor C-terminal binding protein (CtBP) via binding motif located at N-terminal region [7]. In the present study, we attempted to isolate the FOXP2-associated protein with a yeast two-hybrid system using the C-terminal region, including the forkhead domain, as bait. We have identified protection of telomeres 1 (POT1) as the associated protein, and demonstrate that FOXP2(R553H) prevents the nuclear translocation of POT1.
2. Materials and methods Abbreviations: FOXP2, forkhead box P2; POT1, protection of telomeres 1; KI, knock-in; USV, ultrasonic vocalization; CtBP, C-terminal binding protein. ⇑ Corresponding author at: Center for Medical Science, International University of Health and Welfare, 2600-1 Kitakanamaru, Otawara, Tochigi 324-8501, Japan. Fax: +81 287 24 3001. E-mail address: [email protected] (T. Momoi). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.06.032
2.1. DNA construction POT1 cDNA was isolated from a human fetal cDNA library by yeast two-hybrid screening. The enzyme-digested PCR DNA
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fragments corresponding to full-length FOXP2, FOXP2(484–715), FOXP2(484–598) containing forkhead domain (FOXP2-forkhead), FOXP2(484–598) with R553H mutation (FOXP2-R553H-forkhead), FOXP2(576–715), FOXP2-mutated NLS (mNLS) [2], and full-length POT1 were subcloned into the following vectors: pGAD, pGBK, pEGFP-C1 (Clontech), and FLAG vector pcDEF3 [8]. 2.2. Yeast two-hybrid assay Yeast two-hybrid assays were performed by transforming AH109 or Y187 yeast cells with both pGBK- and pGAD-based constructs according to the manufacturer’s instructions (Clontech). AH109 transformants were selected on SD-Leu/-Trp/-His/-Ade plates for 2–3 days. After dilution, 2–3 ml of cells were spotted onto SD-Leu/-Trp/-His/-Ade plates and grown for 3–4 days. The respective b-galactosidase activities of Y187 yeast transformed with pGBK and pGAD constructs were assayed according to the manufacturer’s instructions. 2.3. Transfection COS cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2, using a-MEM medium supplemented with 10% fetal bovine serum. For immunoprecipitation and immunoblotting, cells were transfected with 16 lg of FLAG-POT1, alone or in combination with 4 lg of GFP-FOXP2s. Transfections were conducted using Lipofectamin 2000 (Invitrogen) according to manufacturer’s instructions. For immunostaining, cells were cotransfected with FLAG-POT1 and GFP-FOXP2s using the calciumphosphate method, as described previously [8]. 2.4. Immunoprecipitation and immunblot analysis Cells were lysed in lysis buffer [50 mM Tris–HCl pH 8.0, 150 mM NaCl, 10% glycerol, 0.5% IGEPAL CA630, and protease inhibitors;
Complete mini (Roche Diagnostics)] at 4 °C for 15 min, and sonicated. After insoluble materials were removed by centrifugation, cell lysates were subjected to immunoprecipitation as described previously [2] and the resulting precipitates were subjected to immunoblot analysis using anti-GFP (MBL) and antiFLAG (Sigma) antibodies. Immunoreactivity was visualized using alkaline phosphatase-conjugated anti-mouse or anti-rabbit IgG antibodies, respectively, with Nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-1-phosphate (Roche Diagnostics). 2.5. Immunostaining After transfected COS cells were cultured for 24 h and then fixed in 2% paraformaldehyde in phosphate buffered saline (PBS) at 4 °C for 30 min, they were then subjected to immunostaining using mouse anti-FLAG antibody in incubation buffer at 4 °C overnight, as described previously [2]. Mouse rhodamine-labeled IgG was prepared from Leinco Technologies. Nuclei were labeled with Hoechst 33342 (Molecular Probes). Immunoreactivity was viewed using a fluorescence microscope (Leica AF6000, Leica Microsystems).
3. Results 3.1. Isolation of POT1 via yeast two-hybrid screening We examined FOXP2-associated protein by yeast two-hybrid assay. We used the C-terminal region of FOXP2 including the forkhead domain, FOXP2(484–715), as bait (Fig. 1A) and isolated POT1 cDNA (from the eighth amino acid to the 30 -UTR). Yeast AH109 cotransformed with pGAD-POT1 and pGBK-FOXP2(484–715) or pGBK-FOXP2(484–598) containing forkhead domain grew on SDLeu/-Trp/-His/-Ade plates, while cells co-transformed with pGADPOT1 and pGBK-FOXP2(576–715) lacking forkhead domain did
Fig. 1. Yeast two-hybrid screening for FOXP2 binding protein. (A) A schematic representation on the relationship between functional domains of FOXP2 and bait probes used in yeast two-hybrid screening. FOXP2(484–715, including forkhead domain), FOXP2(484–598, including forkhead domain), FOXP2(576–715, lacking forkhead domain) were used as bait probes. PolyQ, poly-glutamine; ZF, zinc finger; LZ, leucine zipper; Forkhead, forkhead domain. POT1 was isolated by yeast two-hybrid screening using FOXP2(484–715) as a probe. The prey, pGAD-POT1, interacted with pGBK-FOXP2(484–598), but not with pGBK-FOXP2(576–715), in yeast. (B) The b-galactosidase activity of the transformed cells. The relative b-galactosidase activity of the transformed cells with FOXP2(484–598), FOXP2(484–598) and POT1, FOXP2-R553H(484–598), FOXP2R553H(484–598) and POT1, FOXP2(576–715), and FOXP2(576–715) and POT1 were 0.33 ± 0.04, 117.06 ± 8.26, 0.41 ± 0.04, 50.33 ± 12.09, 0.40 ± 0.05, and 0.50 ± 0.07, respectively.
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not (Fig. 1A). Thus, FOXP2-forkhead associated with POT1, a component of the telomerase ribonucleoprotein [9]. In contrast with FOXP2-forkhead located in the nucleus, the forkhead with (R553H), mutation related to speech-language disorder, partially localizes in both the cytoplasm and the nucleus [2]. To examine the interaction between POT1 and FOXP2(484– 598) or FOXP2(484–598) with R553H mutation {FOXP2-R553H(484–598)} quantitatively, b-galactosidase activity was measured in the yeast Y187 strain co-transformed with pGAD-POT1 and pGBK-FOXP2-forkhead or mutated one (Fig. 1B); The relative b-galactosidase activity of the co-transformed cells with pGADPOT1 and pGBK-FOXP2-R553H(484–598) was about 2-fold lower than that of the co-transformed cells with pGAD-POT1 and pGBK-FOXP2(484–598). Thus, pGBK-FOXP2-R553H(484–598) also associated with POT1. On the other hand, the co-transformed cells with pGAD-POT1 and pGBK-FOXP2(576–715) did not demonstrate the b-galactosidase activity. 3.2. The interaction between FOXP2 and POT1and nuclear translocation of POT1 We confirmed the interaction between GFP-FOXP2 and FLAG-POT1 by immunoprecipitation and immunoblot analysis (Fig. 2). When GFP-FOXP2 and FLAG-POT1 were co-transfected into COS cells, anti-FLAG detected both FLAG-POT1 and GFP-FOXP2 in the precipitates; GFP-FOXP2 was not detected in the precipitates in the absence of FLAG-POT1 (Fig. 2A). GFP-FOXP2 alone localized
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to the nucleus, while FLAG-POT1 alone localized in the cytoplasm. In the presence of GFP-FOXP2, FLAG-POT1 co-localized with GFPFOXP2 in the nucleus (Fig. 2B). NLSs are located at both the N-terminal and C-terminal sides of the forkhead domain [2]; GFP-FOXP2 with a mutated NLS (GFP-FOXP2-mNLS) localized in the cytoplasm. FLAG-POT1 associated with GFP-FOXP2-mNLS (Fig. 2A), but they co-localized in the cytoplasm (Fig. 2B). 3.3. Effect of mutated FOXP2(R553H) on the nuclear translocaton of POT1 As FOXP2-R553H-(484–598) (FOXP2-R553H-forkhead), interacted with POT1 (Fig. 1B), we also examined the influence of R553H mutation on the nuclear translocation of POT1. GFPFOXP2-forkhead localized in the nucleus, while FOXP2-R553Hforkhead was observed in both the cytoplasm and the nucleus [2]. FLAG-POT1 co-localized with GFP-FOXP2-forkhead in the nucleus (Fig. 3), but co-localized with FOXP2-R553H-forkhead in both the cytoplasm and the nucleus (Fig. 3). Therefore, the nuclear translocation of POT1 was affected by the localization of FOXP2 and its derivatives containing forkhead domain and NLS. 4. Discussion POT1 is a component of the telomerase ribonucleoprotein complex that is essential for the replication of chromosome termini [9].
Fig. 2. Interaction between POT1 and FOXP2 and their co-localization. (A) Immunoprecipitation and immunoblot analysis on the interaction between the GFP-FOXP2 or FOXP2-mutated NLS (mNLS) and POT1. (B) Localization of FOXP2 and POT1. GFP-FOXP2 localized in the nuclei. While FLAG-POT1 localized in the cytoplasm, it co-localized with GFP-FOXP2 and GFP-FOXP2-mNLS in the nuclei (arrowheads) and the cytoplasm (arrows), respectively. Scale bar, 15 lm.
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Fig. 3. Effect of FOXP2-R553H-forkhead on the nuclear localization of POT1. While FLAG-POT1 co-localized with GFP-FOXP2-forkhead in the nuclei (arrowheads), it colocalized with FOXP2-R553H-forkhead in both the cytoplasm (arrows) and the nucleus (arrowhead). Scale bar, 20 lm.
A six-protein complex (TRF1, TRF2, RAP1, TIN2, POT1, and TPP1), known as shelterin, protects the telomeres of human chromosomes [10]. POT1 directly binds the single-stranded 30 extension at the chromosome end [11,12], which is bridged with TIN2 through protein–protein interactions via TPP1 [10,13]. POT1-TPP1 switches from inhibiting to allowing telomerase access to the telomere [14]. Defects in POT1 activate the DNA damage response, leading to propagation of a DNA damage signal and inappropriate DNA repair reaction, and resulting in cell cycle arrest [15]. Because POT1 does not have putative NLS sites, a nuclear protein with an NLS is necessary as a binding partner for the nuclear localization of POT1. In the present study, we showed that both FOXP2 and FOXP2-mNLS associated with POT1 and their localization had the influence on the localization of POT1; POT1 alone was primarily located in the cytoplasm, but co-localized with FOXP2 in the nucleus, or co-localized with FOXP2-mNLS in the cytoplasm (Fig. 2), suggesting that FOXP2 is a binding partner for the nuclear localization of POT1. FOXP2 has diverse roles as a transcriptional factor during development, including during survival or proliferation, in specific regions including the brain. FOXP2 may regulate the cell cycles via associating with POT1. This will be one of the next issues in future. Homozygotic knock-in mice with the FOXP2(R552H), mutation related to speech-language disorder, exhibit poor development of Purkinje cells and die within 3 weeks after birth [16]. The interaction between mutated R553H-forkhead and POT1 was 2-fold lower than that between wild-type forkhead and POT1 but interaction still remains (Fig. 1B). In contrast with forkhead domain, POT1 partly co-localized with mutated (R553H)-forkhead in the cytoplasm (Fig. 3), suggesting that cells expressing FOXP2(R553H) may have a higher risk of being mistaken for having broken DNA ends and being subjected to DNA repair by loss of the protection of the telomere terminus. Therefore, the neuronal cells expressing FOXP2-R553H in the brain may be damaged during development due to POT1 dysfunction. Loss of POT1 function in the nuclei of neurons in the developing brain may be associated with the pathogenesis of speech-language disorder with FOXP2 mutation. This will be another major issue in the future study. Acknowledgments Authors thank Dr. Akifumi Mizutani for introduction of yeasttwo-hybrid assay in the initial time of this study. This work was
supported by Grants-in-Aid for Scientific Research (KAKENHI) of the Ministry of Education, Culture, Sports, Science and Technology, Japan (21200011, 21700377); Grants-in-Aid for Health Labour Scientific Research of the Ministry of the Health, Labour and Welfare, Japan; and a Grant-in-Aid for Intramural Research (20B-13) for Neurological and Psychiatric Disorders of National Center of Neurology and Psychiatry (NCNP), Japan. References [1] W. Shu, H. Yang, L. Zhang, et al., Characterization of a new subfamily of winged-helix/forkhead (Fox) genes that are expressed in the lung and act as transcriptional repressors, J. Biol. Chem. 276 (2001) 27488–27497. [2] A. Mizutani, A. Matsuzaki, M.Y. Momoi, et al., Intracellular distribution of a speech/language disorder associated FOXP2 mutant, Biochem. Biophys. Res. Commun. 353 (2007) 869–874. [3] R.J. Ferland, T.J. Cherry, P.O. Preware, et al., Characterization of Foxp2 and Foxp1 mRNA and protein in the developing and mature brain, J. Comp. Neurol. 460 (2003) 266–279. [4] C.S. Lai, D. Gerrelli, A.P. Monaco, et al., FOXP2 expression during brain development coincides with adult sites of pathology in a severe speech and language disorder, Brain 126 (2003) 2455–2462. [5] C.S. Lai, S.E. Fisher, J.A. Hurst, et al., A forkhead-domain gene is mutated in a severe speech and language disorder, Nature 413 (2001) 519–523. [6] S.C. Vernes, J. Nicod, F.M. Elahi, et al., Functional genetic analysis of mutations implicated in a human speech and language disorder, Hum. Mol. Genet. 31 (2006) 54–67. [7] S. Li, J. Weidenfeld, E.E. Morrisey, Transcriptional and DNA binding activity of the Foxp1/2/4 family is modulated by heterotypic and homotypic protein interactions, Mol. Cell. Biol. 24 (2004) 809–822. [8] E. Fujita, A. Soyama, T. Momoi, RA175, which is the mouse ortholog of TSLC1, a tumor suppressor gene in human lung cancer, is a cell adhesion molecule, Exp. Cell Res. 287 (2003) 57–66. [9] P. Baumann, T.R. Cech, Pot1, the putative telomere end-binding protein in fission yeast and humans, Science 292 (2001) 1171–1175. [10] T. de Lange, Shelterin: the protein complex that shapes and safeguards human telomeres, Genes Dev. 19 (2005) 2100–2110. [11] D. Loayza, H. Parsons, J. Donigian, et al., DNA binding features of human POT1: a nonamer 50 -TAGGGTTAG-30 minimal binding site, sequence specificity, and internal binding to multimeric sites, J. Biol. Chem. 279 (2004) 13241–13248. [12] M. Lei, E.R. Podell, T.R. Cech, Structure of human POT1 bound to telomeric single-stranded DNA provides a model for chromosome end-protection, Nat. Struct. Mol. Biol. 11 (2004) 1223–1229. [13] D. Loayza, T. De Lange, POT1 as a terminal transducer of TRF1 telomere length control, Nature 423 (2003) 1013–1018. [14] F. Wang, E.R. Podell, A.J. Zaug, et al., The POT1–TPP1 telomere complex is a telomerase processivity factor, Nature 445 (2007) 506–510. [15] D. Hockemeyer, A.J. Sfeir, J.W. Shay, et al., POT1 protects telomeres from a transient DNA damage response and determines how human chromosomes end, EMBO J. 24 (2005) 2667–2678. [16] E. Fujita, Y. Tanabe, A. Shiota, et al., Ultrasonic vocalization impairment of Foxp2 (R552H) knockin mice related to speech-language disorder and abnormality of Purkinje cells, Proc. Natl Acad. Sci. USA 105 (2008) 3117–3122.