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• W2142853902 abstract "Article15 March 2007free access Sox15 and Fhl3 transcriptionally coactivate Foxk1 and regulate myogenic progenitor cells Annette P Meeson Annette P Meeson Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Xiaozhong Shi Xiaozhong Shi Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Matthew S Alexander Matthew S Alexander Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author R S Williams R S Williams Department of Medicine, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Ronald E Allen Ronald E Allen Department of Animal Sciences, University of Arizona, Tucson, AZ, USA Search for more papers by this author Nan Jiang Nan Jiang Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Ibrahim M Adham Ibrahim M Adham Institute of Human Genetics, University of Göttingen, Göttingen, Germany Search for more papers by this author Sean C Goetsch Sean C Goetsch Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Robert E Hammer Robert E Hammer Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Daniel J Garry Corresponding Author Daniel J Garry Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA Donald W Reynolds Cardiovascular Clinical Research Center at UT Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Annette P Meeson Annette P Meeson Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Xiaozhong Shi Xiaozhong Shi Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Matthew S Alexander Matthew S Alexander Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author R S Williams R S Williams Department of Medicine, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Ronald E Allen Ronald E Allen Department of Animal Sciences, University of Arizona, Tucson, AZ, USA Search for more papers by this author Nan Jiang Nan Jiang Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Ibrahim M Adham Ibrahim M Adham Institute of Human Genetics, University of Göttingen, Göttingen, Germany Search for more papers by this author Sean C Goetsch Sean C Goetsch Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Robert E Hammer Robert E Hammer Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Daniel J Garry Corresponding Author Daniel J Garry Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA Donald W Reynolds Cardiovascular Clinical Research Center at UT Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Author Information Annette P Meeson1,‡, Xiaozhong Shi1,‡, Matthew S Alexander1,‡, R S Williams2, Ronald E Allen3, Nan Jiang1, Ibrahim M Adham4, Sean C Goetsch1, Robert E Hammer5 and Daniel J Garry 1,6,7 1Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA 2Department of Medicine, Duke University Medical Center, Durham, NC, USA 3Department of Animal Sciences, University of Arizona, Tucson, AZ, USA 4Institute of Human Genetics, University of Göttingen, Göttingen, Germany 5Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA 6Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA 7Donald W Reynolds Cardiovascular Clinical Research Center at UT Southwestern Medical Center, Dallas, TX, USA ‡These authors contributed equally to this work *Corresponding author. Internal Medicine-Cardiology, NB11.118A, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-8573, USA. Tel.: +1 214 648 1654; Fax: +1 214 648 1450; E-mail: [email protected] The EMBO Journal (2007)26:1902-1912https://doi.org/10.1038/sj.emboj.7601635 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The regulation of myogenic progenitor cells during muscle regeneration is not clearly understood. We have previously shown that the Foxk1 gene, a member of the forkhead/winged helix family of transcription factors, is expressed in myogenic progenitor cells in adult skeletal muscle. In the present study, we utilize transgenic technology and demonstrate that the 4.6 kb upstream fragment of the Foxk1 gene directs β-galactosidase expression to the myogenic progenitor cell population. We further establish that Sox15 directs Foxk1 expression to the myogenic progenitor cell population, as it binds to an evolutionarily conserved site and recruits Fhl3 to transcriptionally coactivate Foxk1 gene expression. Knockdown of endogenous Sox15 results in perturbed cell cycle kinetics and decreased Foxk1 expression. Furthermore, Sox15 mutant mice display perturbed skeletal muscle regeneration, due in part to decreased numbers of satellite cells and decreased Foxk1 expression. These studies demonstrate that Sox15, Fhl3 and Foxk1 function to coordinately regulate the myogenic progenitor cell population and skeletal muscle regeneration. Introduction Skeletal muscle has the capacity for self-repair. Resident within adult skeletal muscle is a pool of undifferentiated myogenic progenitor cells (MPCs) that includes satellite cells (Miller et al, 1999; Mourkioti and Rosenthal, 2005). Satellite cells are small mononuclear cells that share a common basal lamina with the larger multinucleated myocytes, and are located in a peripheral (or satellite) position with respect to the multinucleated myofiber (Mauro, 1961). Following muscle injury, the MPCs are activated, they proliferate and withdraw from the cell cycle to form multinucleated myofibers in a manner that recapitulates the fundamental events of muscle development in the fetus (Sheehan and Allen, 1999; Seale and Rudnicki, 2000; Snider and Tapscott, 2003; Shi and Garry, 2006). The MPCs are self-renewing and replenish a pool of quiescent progenitors; however, this capacity for self-renewal is finite and repeated injuries ultimately lead to loss of muscle mass and myopathies (Conboy and Rando, 2002; Olguin and Olwin, 2004; Collins et al, 2005). MPCs are arrested at an early stage of the myogenic program such that they do not express any of the bHLH proteins of the MyoD family (Shi and Garry, 2006). Recent studies have begun to identify factors that are expressed in the satellite cell population, including Foxk1, Pax7, C-met, syndecan3/4 and Pax3, although the molecular regulation of this cell population remains ill-defined (Garry et al, 1997; Borycki et al. 1999; Seale et al. 2000; Cornelison et al, 2001; Cornelison et al, 2004; Deasy et al, 2005; Montarras et al, 2005). Members of the forkhead/winged helix (Fox) transcription factor family, which has more than 100 constituents, have been identified in a number of vertebrate cell lineages and are known to exert important regulatory functions during development in the control of cell fate, patterning, proliferation, differentiation and tissue morphogenesis (Lai et al, 1993; Ang and Rossant 1994). With respect to stem cells and/or tissue repair, a mammalian forkhead/winged helix protein termed Foxd3 (Genesis) is expressed selectively in embryonic stem cells and Foxa2 has been identified in regenerating hepatocytes (Sutton et al, 1996; Ye et al, 1997). We have previously shown that Foxk1 is expressed in the satellite cell population that is resident in adult skeletal muscle (Garry et al, 1997). Using a gene disruption strategy, we have observed that mice that lack Foxk1 have impaired muscle regeneration, dysregulation of the cyclin-dependent kinase inhibitor, p21 (Cdkn1a), and perturbed cell cycle progression of the MPC population (Garry et al, 2000; Hawke et al, 2003a). Sox transcription factors are found in all metazoan species and, like forkhead transcription factors, play key roles in embryonic development. Satellite cells also express Sox transcription factors (Béranger et al, 2000; Schmidt et al, 2003). Members of the Sox transcription factor family have relative homology of the high-mobility group (HMG) DNA binding domain (Wilson and Koopman, 2002). For example, Sox2 is an essential regulator of ES cells, whereas Sox4 and Sox5 are key regulators of cardiac development (Koopman et al, 2004). Sox transcription factors interact with protein partners and function to either activate or repress downsteam target gene expression (Koopman et al, 2004). Sox15, a member of this family, is expressed in satellite cells, and mice-lacking Sox15 are viable but appear to have impaired skeletal muscle regeneration (Lee et al, 2004). Overall, the functional role of Sox15 in the MPC population is unclear. Transcriptional regulators of gene expression interact with adaptor proteins to form a regulatory complex of nuclear proteins. The Four and a half LIM domain family of proteins are regulators of growth and discrete stages of development and cellular differentiation (Chu et al, 2000; Müller et al, 2002). The LIM domain is characterized by a double zinc-finger motif that mediates protein–protein interactions. Fhl3 has previously been described to be expressed in skeletal muscle and is localized to the cytoplasmic and the nuclear compartments, although the functional role of this factor is unclear (Morgan and Madgwick, 1998). In the present study, we pursued a strategy to decipher the regulatory mechanisms that govern cell cycle re-entry of quiescent satellite cells. We demonstrate that Sox15 recruits Fhl3 to form a complex that coactivates the Foxk1 gene. We further establish that Sox15 directs Foxk1 expression to the MPC population. These studies support the notion that Foxk1 is a direct downstream target of Sox15. This Sox15–Fhl3 interaction and subsequent regulation of Foxk1 provides a mechanism whereby quiescent satellite cells re-enter the cell cycle and regenerate injured skeletal muscle. These studies enhance our understanding of the molecular regulation of the MPC population and will provide a platform for therapeutic applications for the treatment of debilitating myopathies. Results We have previously reported that the forkhead/winged helix transcription factor, Foxk1, is expressed in MPCs that are resident in adult skeletal muscle using light microscopic and ultrastructural immunohistochemical techniques (Garry et al, 1997). We have further defined Foxk1 as an important cell cycle regulator of the MPC population (Garry et al, 2000; Hawke et al, 2003a). To examine the regulation of the Foxk1 gene, we characterized a 4.6 kb Foxk1 promoter fragment that contained evolutionarily conserved regions between mouse and human. We generated transgenic mice using this 4.6 kb Foxk1 promoter fragment to drive the lacZ reporter gene (Figure 1A). Expression was observed in three transgenic lines and in all three lines β-galactosidase staining was specific to the muscle precursor cells of the developing limbs (E11.5, E13.5 and E14.5) (Figure 1B–E). These results indicate that the 4.6 kb promoter of Foxk1 contains regulatory elements that control expression in a cell lineage that are destined to become skeletal muscle (the MPC population) in the developing limbs (Figures 1E–K). During midgestational stages of embryogenesis (E13.5), expression of the 4.6 kb Foxk1 promoter was colocalized with Foxk1 in developing muscle using immunohistochemical techniques, a Foxk1 antiserum and a β-galactosidase antiserum (Figure 1G and H). Whereas the 4.6 kb Foxk1-lacZ expression largely recapitulated endogenous Foxk1 expression in the developing limbs, trunk muscles (low level) and heart (low level), β-galactosidase expression was not observed in the developing somites (Supplementary Figure 1). Figure 1.Transgenic embryonic expression of the Foxk1 promoter fused to lacZ. (A) Schematic outlining the transgenic construct that consists of a 4.6 kb upstream fragment of the Foxk1 gene fused to the lacZ reporter. (B) Whole-mount β-galactosidase expression in limbs, diaphragm and branchial arch derivatives in E11.5 (B), E13.5 (C, E) and E14.5 (D) 4.6 kb Foxk1–lacZ transgenic embryos. (F) Transverse section of the E13.5 embryo reveals β-galactosidase expression in muscle precursor cells of the limb. (G, H) Coexpression of β-galactosidase (α-bgal) and Foxk1 in intercostal muscles of 4.6 kb Foxk1–lacZ transgenic embryos. Low magnification (I) and high magnification (J) of an E13.5 parasaggital section of the Foxk1 promoter-lacZ expression (blue) and desmin immunolocalization (brown). Desmin is an intermediate filament protein expressed early during myogenesis. Desmin staining is observed in the heart and is coexpressed in a small subpopulation of the Foxk1 promoter-lacZ-positive mesenchymal percursor cells of the limb. h, heart. Download figure Download PowerPoint Following birth, expression of the 4.6 kb Foxk1 promoter fragment is limited to the quiescent MPC population. In response to cardiotoxin-induced injury to hindlimb skeletal muscle, we have shown that the MPC population undergoes activation (D1 following injury) and a tremendous proliferative expansion (D2–D5) with the formation of newly regenerated myotubes (D5–D10) following cardiotoxin injury (Hawke et al, 2003a; Goetsch et al, 2003). In the present study, we observed that the expression of the 4.6 kb Foxk1 promoter recapitulates Foxk1 expression during muscle regeneration and identifies both quiescent satellite cells and proliferating myogenic progenitor cells, but is not expressed in differentiated myotubes using whole-mount (Figure 2A) and histological sections of 5 day injured (Figure 2B), 10 day injured (Figure 2C), 4.6 kb Foxk1:mdx (Figure 2D) and uninjured skeletal muscle (Figure 2E and F). The mdx mouse lacks dystrophin and is characterized by cycles of degeneration and regeneration of skeletal muscle. Figure 2.Foxk1 transgene expression is observed in MPCs of cardiotoxin-injured and uninjured adult skeletal muscle. (A) β-Galactosidase expression in 4.6 kb Foxk1 promoter-lacZ transgenic whole-mount stained gastrocnemius muscles at defined time periods following cardiotoxin-induced muscle injury. Note robust β-galactosidase expression in transgenic gastrocnemius muscles 2 days following injury (corresponding to satellite cell activation) and 5 days following injury (associated with satellite cell proliferation), with decreased expression within 10 days following injury (newly regenerated myofibers). (B) Transverse histological sections of transgenic (4.6 kb Foxk1 promoter-lacZ) hindlimb skeletal muscle 5 days following injury. Note robust expression in activated satellite cells (blue) but no expression in newly regenerated centronucleated myofibers (arrowhead). (C) The 4.6 kb Foxk1 promoter-lacZ expression (blue) is expressed in MPCs in skeletal muscle 10 days following injury (arrowhead). (D) Transverse section of the 4.6 kb Foxk1-lacZ transgenic:mdx tibialis anterior skeletal muscle reveals β-galactosidase expression in activated satellite cells (black arrowheads) and not newly regenerated centronucleated myofibers (white arrowheads). Scale bar, 40 μm. (E, F) Transverse sections of the 4.6 kb Foxk1 promoter-lacZ skeletal muscle examined for β-galasctosidase (blue) and laminin (brown) expression in uninjured skeletal muscle (arrowhead). Note that the β-galactosidase-expressing cells are sublaminar, consistent with expression in satellite cells (arrowheads). Scale bars, 20 μm. Download figure Download PowerPoint A detailed analysis of the Foxk1 upstream fragment has been undertaken using a transgenic strategy. We generated mice harboring either 1.6 kb Foxk1 promoter-lacZ or 0.6 kb Foxk1 promoter-LacZ transgenes and we observed that a distal 3 kb fragment contains an enhancer(s) that directs expression in the MPC population (Supplementary Table 1). Sox15 binds to the Foxk1 promoter Database analysis of this 3 kb upstream fragment of the Foxk1 gene revealed the presence of a Sox binding element (SBE) that was evolutionarily conserved between mouse and human (Figure 3A). Using electrophoretic mobility shift assays (EMSA) and a 23-oligonucleotide probe of the Foxk1 promoter that contains the SBE, we examined the capacity of a myc-Sox15 fusion protein to bind to this DNA probe. As outlined in Figure 3B (mouse) and Supplementary Figure 2 (human), we demonstrated that Sox15 binds to this site (i.e. SBE) in the Foxk1 promoter, and that the myc-Sox15 fusion protein–oligonucleotide complex could be competed with cold competitor. We further demonstrated that mutagenesis of three nucleotides within the SBE of the radiolabeled oligonucleotide probe precluded formation of the protein–DNA complex (Figure 3B). Additionally, the incubation of the wild-type oligonucleotide probe that contains the SBE with the myc-Sox15 fusion protein and anti-myc serum resulted in the supershift of the protein–DNA complex (Figure 3B). Figure 3.Sox15 binds to a conserved region of the Foxk1 promoter. (A) Alignment of the human, mouse and rat Foxk1 promoter sequences revealing the evolutionary conservation of the Sox binding element. (B) EMSA of the 32P-labeled SBE from the mouse Foxk1 promoter with in vitro-synthesized myc-Sox15 protein. Competition studies were performed with 0–350 ng of unlabeled Sox15 binding site DNA (lanes 2–5). Using the same probe that has a mutated Sox binding site, no protein–DNA complex is formed (lane 6; mut). Using a myc antibody that recognizes the myc-Sox15 fusion protein, the protein–DNA complex is supershifted (lane 7; SS). (C) ChIP assay was undertaken following the overexpression of the myc-Sox15 fusion protein in C2C12 myoblasts, crosslinking and IP of the myc-Sox15 fusion protein–DNA complexes. Co-IP of the targeted promoter DNA was analyzed by PCR amplification. DNA was also isolated without IP and used as input. IP of the myc-Sox15 fusion protein pulled down the Foxk1 promoter. Note that no DNA was immunoprecipitated with the control IgG. (D) Forced overexpression of Sox15 in C2C12 cells compared with overexpression of a GFP expression plasmid (control) reveals a two-fold increase in endogenous Foxk1 transcript expression (n=4 for each sample; *P<0.001). (E) Sox15 specifically transactivates the Foxk1 promoter. Schematic of the 4.6 kb Foxk1 promoter-luc plasmid used in these assays containing the Sox binding motif is shown. Overexpression of increasing amounts of Sox8 or Sox15 demonstrates a dose-dependent transcriptional activation of the Foxk1 promoter by Sox15 (n=9 for each sample; *P<0.001). In contrast, Sox8 does not activate the Foxk1 promoter. Western blot analysis of extracts isolated from C2C12 myoblasts following overexpression of myc-Sox15 reveals overexpression of the fusion protein in a dose-dependent manner in C2C12 myoblasts (α-tubulin is used as a loading control). (F) Transfection of Sox15 expression plasmids with wild-type (WT) or mutant (mut) Sox binding site reveals a 90% decrease in luciferase activity following transfection with the mutated Foxk1 vector (n=3 for each sample; *P<0.01). (G) Mutagenesis of the SBE in the 4.6 kb Foxk1 promoter-lacZ transgene extinguishes β-galactosidase expression in the limbs of E13.5 embryos compared with age-matched controls (a total of 15 mutant transgenics were evaluated and all had significantly decreased or no expression in the developing limbs). Download figure Download PowerPoint To further investigate whether Sox15 interacts and binds to the Foxk1 promoter, we performed chromatin immunoprecipitation (ChIP) assays. Chromatin fragments from lysates of C2C12 cells transfected with a plasmid expressing myc-Sox15 were precipitated with a myc antibody or control IgG (Figure 3C). DNA from the immunoprecipitates was subjected to PCR analysis using primers specific to the Foxk1 promoter harboring the SBE. As shown in Figure 3C, chromatin fragments containing the Sox15 binding site in the Foxk1 gene were specifically immunoprecipitated by the myc antibody in cells expressing the myc-Sox15 fusion protein. No chromatin fragments were precipitated by control IgG that further established the specificity of Sox15 binding to the Foxk1 promoter (Figure 3C). Sox15 transcriptionally activates Foxk1 gene expression We performed transcriptional assays to assess the specificity of Sox transcription factors as regulators of Foxk1 gene expression. We fused the 4.6 kb Foxk1 promoter to the luciferase reporter (Figure 3E). We transfected C2C12 myoblasts (a cell line generated from satellite cells) with the Foxk1 promoter–reporter construct and Sox factors that have been reported to be expressed in the MPC population (i.e. Sox8 and Sox15) (Schmidt et al, 2003; Lee et al, 2004). We observed that Sox15, in a dose-dependent manner, was a potent transcriptional regulator of Foxk1 gene expression as it resulted in more than a 40-fold activation (Figure 3E). In contrast, Sox8 did not transcriptionally activate the Foxk1 gene (Figure 3E). To further examine the specificity of Sox15 transcriptional activity, we evaluated whether Sox15 was capable of activation of the myoglobin gene (a well-characterized muscle-specific promoter in our laboratory) (Garry et al, 1996, 1998; Grange et al, 2002). We observed that Sox15 is not a transcriptional activator of the 2.0 kb myoglobin promoter-luc construct (Supplementary Figure 3). To further evaluate the capacity of Sox15 to transcriptionally activate Foxk1, we overexpressed Sox15 (pcDNA-GFP-Sox15) in C2C12 myoblasts and assayed for endogenous Foxk1 transcript expression using QRT–PCR analysis. We observed that 24 h following overexpression of Sox15, Foxk1 mRNA was significantly increased more than two-fold compared with the control (n=4 for each sample; *P<0.001) (Figure 3D). The induction of endogenous Foxk1 expression is consistent with the hypothesis that Sox15 is an upstream regulator of the Foxk1 gene. Mutagenesis of the Sox binding site attenuates Foxk1 gene expression We undertook in vitro and in vivo studies to further examine whether Sox15 is an upstream regulator of the Foxk1 gene. We mutagenized three nucleotides within the SBE located in the 4.6 kb Foxk1 promoter and performed transcriptional and transgenic analyses. Mutagenesis of the SBE in the 4.6 kb Foxk1-luc plasmid and cotransfection with a Sox15 expression plasmid resulted in a 90% reduction of luciferase activity in C2C12 cells compared with the wild-type control (n=3 for each group; *P<0.01) (Figure 3F). We further examined the endogenous expression of the 4.6 kb (ΔSBE)Foxk1-lacZ construct using transgenic technologies. Mutant transgenic embryos were harvested in parallel with controls, fixed, stained with X-gal and examined for β-galactosidase expression. Expression was observed in 15 ΔSBE mutant-Foxk1 transgenic embryos at E13.5 and all had a severe reduction of β-galactosidase expression in the fore and hindlimbs (Figure 3G and Supplementary Figure 4). These results further establish that Sox15 is a direct upstream transcriptional regulator of Foxk1 expression. Sox15 physically interacts with Fhl3 Having established that Sox15 is an essential upstream regulator of Foxk1, we undertook a yeast two-hybrid assay to define the proteins that interact with Sox15 in the regulation of Foxk1 and the MPC population. We utilized the longest Sox15 construct (1–181) that lacked autoactivation as bait and screened a commercially available adult skeletal muscle library (Figure 4A). We identified the LIM-only protein, Fhl3, as a Sox15 interacting factor, which was confirmed using full-length Fhl3 in the yeast two-hybrid assay (Figure 4B). Fhl3 did not interact with the GAL4 DNA binding domain alone and there was no interaction between the bait and the GAL4 activation domain (Figure 4B). This interaction was further confirmed in mammalian cells using co-immunoprecipitation (co-IP) assays after myc-tagged Sox15 and HA-tagged Fhl3 or HA-tagged Sox15 and myc-tagged Fhl3 were coexpressed in C2C12 cells (Figure 4C). Figure 4.Sox15 physically interacts with Fhl3 and synergistically transctivates Foxk1 gene expression. (A) Selected Sox15 constructs were tested for autoactivation. The Sox (1–181) construct lacked autoactivation and was used as bait in the yeast two-hybrid assay. (B) Two-hybrid results, following the screening of a skeletal muscle library, confirmed the interaction of full-length Sox15 and Fhl3. (C) Co-IP assay following overexpression of myc-Sox15 and HA-Fhl3 or HA-Sox15 and myc-Fhl3 confirmed the interaction. (D) Overexpression of the respective tagged proteins reveals colocalization (yellow nuclei in the merge panel) in the nuclear compartment of C2C12 cells (arrowhead). (E) Cotransfection of Fhl3, Sox15 and 4.6 kb Foxk1-luc in C2C12 cells reveals that Fhl3 by itself is unable to transactivate Foxk1 expression, but in tandem with Sox15 there is a synergistic coactivation of Foxk1 gene expression (n=6; *P<0.001). Download figure Download PowerPoint To further investigate the interaction between Fhl3 and Sox15, we transfected C2C12 myoblasts with HA-Fhl3 and myc-Sox15 and examined the localization of these fusion proteins using immunohistochemical and confocal microscopic techniques. Sox15 was restricted to the nuclear compartment and colocalized with Fhl3 (Figure 4D). In addition, Fhl3 was abundantly expressed in myoblasts in the cytoplasmic and nuclear compartment and decreased in expression following differentiation using immunohistochemical and Western blot analyses (Figure 4D and Supplementary Figure 5). Future studies may further examine the interacting partners for Sox15 using an array of in vitro and in vivo (i.e. FRET) techniques. Having established that Fhl3 interacts with Sox15 and is coexpressed in C2C12 myoblasts, we examined the role of Fhl3 as a transcriptional regulator of Foxk1. We found that Fhl3 alone had no transcriptional activity, but in combination with Sox15 there was a dose-dependent transcriptional synergy in the activation of Foxk1 gene expression (Figure 4E). These studies support the hypothesis that Sox15 interacts with a complex that includes Fhl3 to regulate Foxk1 gene expression and the MPC population. Identification of the Sox15–Fhl3 interacting domains To enhance our understanding regarding the molecular basis of the Sox15–Fhl3 interaction, we generated deletional constructs to identify the interaction domains for each protein. We mapped the interaction domains using yeast two-hybrid and GST pull-down assays. The Fhl3 binding domain of Sox15 was mapped using a series of Sox15 deletion mutants. Sox15 deletional mutants (1–181 and 136–181) strongly interacted with Fhl3 using a yeast two-hybrid assay (Figure 5A). These results were confirmed using GST pull-down assays, as full length (1–231) and the deletion mutant (136–181) interacted with Fhl3 (Figure 5B). In contrast, deletion constructs that contained the N-terminal (1–46) or the HMG domain (47–135) did not interact with Fhl3 (Figure 5A and B). We conclude that residues between 136 and 181 of Sox15 are sufficient to bind Fhl3. We further emphasize that unlike other Sox factors, where the HMG domain serves a dual role as a DNA binding domain and an interaction domain to partner with other nuclear factors, the Fhl3 interacting domain is a non-HMG domain (136–181). Figure 5.Interaction of Sox15 and Fhl3 requires specific domains. (A) Schematic of Sox15 and the deletion constructs used in the yeast two-hybrid assay and GST pull-down assays. Identification of the Sox15 binding domain that interacts with Fhl3 as determined by the yeast two-hybrid assay. (B) In vitro-translated Sox15 deletion constructs were used for GST pull-down assays. As shown in (B), the Sox15 construct (136–181) harbors the Fhl3 binding domain. This is further established, as full-length Sox15 lacking the 136–181 domain (Δ136–181) is unable to bind to Fhl3. (C) Schematic representation of Fhl3 full-length and deletional constructs used in the yeast two-hybrid and GST pull-down assays. The ability of the respective constructs to bind Sox15 using yeast two-hybrid assays is noted. (D) In vitro-translated Fhl3 deletion constructs were used for GST pull-down assays. Although all the LIM domains are able to interact with Sox15, LIM1 domain demonstrates the strongest affinity. These results support the conclusion that Fhl3 has multiple interacting domains with Sox15. (E) Schematic of full-length and deletional Sox15 constructs containing or lacking the Fhl3 binding site used in the transcriptional assays presented in (F). (F) Cotransfection of the respective Sox15 constructs and the 4.6 kb Foxk1-luc plasmid reveals that the Fhl3 bindin" @default.
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• W2142853902 date "2007-03-15" @default.
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• W2142853902 title "Sox15 and Fhl3 transcriptionally coactivate Foxk1 and regulate myogenic progenitor cells" @default.
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