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. 2013 May 10;288(19):13876-84.
doi: 10.1074/jbc.M113.457648. Epub 2013 Apr 8.

Repression of myoblast proliferation and fibroblast growth factor receptor 1 promoter activity by KLF10 protein

Rajini Parakati  1 , Joseph X DiMario
Affiliations

Repression of myoblast proliferation and fibroblast growth factor receptor 1 promoter activity by KLF10 protein

Rajini Parakati et al. J Biol Chem. .

Abstract

Background: FGFR1 gene expression regulates myoblast proliferation and differentiation, and its expression is controlled by Krüppel-like transcription factors.

Results: KLF10 interacts with the FGFR1 promoter, repressing its activity and cell proliferation.

Conclusion: KLF10 represses FGFR1 promoter activity and thereby myoblast proliferation.

Significance: A model of transcriptional control of chicken FGFR1 gene regulation during myogenesis is presented. Skeletal muscle development is controlled by regulation of myoblast proliferation and differentiation into muscle fibers. Growth factors such as fibroblast growth factors (FGFs) and their receptors (FGFRs) regulate cell proliferation and differentiation in numerous tissues, including skeletal muscle. Transcriptional regulation of FGFR1 gene expression is developmentally regulated by the Sp1 transcription factor, a member of the Krüppel-like factor (KLF) family of transcriptional regulators. Here, we show that another KLF transcription factor, KLF10, also regulates myoblast proliferation and FGFR1 promoter activity. Expression of KLF10 reduced myoblast proliferation by 86%. KLF10 expression also significantly reduced FGFR1 promoter activity in myoblasts and Sp1-mediated FGFR1 promoter activity in Drosophila SL2 cells. Southwestern blot, electromobility shift, and chromatin immunoprecipitation assays demonstrated that KLF10 bound to the proximal Sp factor binding site of the FGFR1 promoter and reduced Sp1 complex formation with the FGFR1 promoter at that site. These results indicate that KLF10 is an effective repressor of myoblast proliferation and represses FGFR1 promoter activity in these cells via an Sp1 binding site.

Keywords: Cell Proliferation; DNA Transcription; Fibroblast Growth Factor Receptor (FGFR); Gene Regulation; Myogenesis.

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Figures

FIGURE 1.
FIGURE 1.
Effect of KLF10 gene expression on myoblast proliferation. A, immunodetection of proliferating myoblasts and cells transfected with pCMVKLF10. Myoblasts were transfected with pCMVKLF10, and transfected cells were identified using a FLAG epitope tag antibody followed by Texas Red-conjugated secondary antibody. Proliferating cells were identified by immunodetection of PCNA using a fluorescein-conjugated anti-PCNA antibody. Nuclei were visualized by DAPI staining. Arrows indicate the nucleus of a transfected cell expressing the KLF10 gene but not PCNA. B, the percentage of PCNA-expressing cells with and without transfection of pCMVKLF10. Nontransfected and transfected cells were scored for expression of PCNA based on immunostaining with the PCNA antibody. Transfection of pCMVKLF10 significantly reduced the percentage of PCNA-positive cells (p < 0.01). Error bars indicate S.D.
FIGURE 2.
FIGURE 2.
Expression of KLF10 gene in myoblasts and myotubes. A, RNA was isolated from myogenic cultures at 0, 24, 48, and 72 h as well as 10 days of incubation. KLF10 mRNA was reverse-transcribed, and the DNA was amplified using KLF10-specific primers (see “Experimental Procedures”). The control reaction did not include reverse transcriptase (−RT). KLF10 RNA was detected in extracts from both myoblasts and myotubes. B, KLF10 protein was detected by Western blot analysis of nuclear extracts from myogenic cultures at 0, 24, 48, and 72 h as well as 10 days of incubation. Immunodetection of E47 protein was used as a control for amount of nuclear extract in each analyzed sample. KLF10 protein was detected at the indicated time points.
FIGURE 3.
FIGURE 3.
Repression of FGFR1 promoter activity by KLF10. A, ED13 myoblasts were transfected with 3284FGFR1CAT and increasing amounts of pCMVKLF10. FGFR1 promoter activity was significantly reduced by KLF10 expression (p < 0.02). Error bars indicate S.D. B, Drosophila SL2 cells were transfected with 3284FGFR1CAT, a constant amount of pPacSp1 to activate FGFR1 promoter activity, and increasing amounts of pCMVKLF10. Expression of KLF10 decreased FGFR1 promoter activity (p < 0.02). Error bars indicate S.D.
FIGURE 4.
FIGURE 4.
Southwestern blot analysis of KLF10 binding to the −23-bp Sp1 binding site. Proteins within nuclear protein extract from differentiated myotubes were resolved in a polyacrylamide gel and probed with double-stranded oligonucleotides containing the wild type (−23) or mutated (mut-23) sequence of the proximal (−23-bp) Sp1 binding site. A control lane that was not hybridized to an oligonucleotide probe is included. Also included is a Western blot of KLF10, detected with the FLAG epitope tag antibody. The protein band detected with the wild type oligonucleotide has the same relative mobility as KLF10.
FIGURE 5.
FIGURE 5.
Electromobility shift analysis of protein complex formation with the proximal Sp1 binding site. A, nuclear protein extracts were prepared from nontransfected, control myoblasts (Con Extract) and from myoblasts transfected with the pCMVKLF10 expression construct (KLF10 Extract). These extracts were incubated with double-stranded oligonucleotides containing the wild type −23-bp Sp1 binding site sequence (−23 Sp Site) and the consensus Sp1 binding site (Sp1 Con Site). Arrows indicate protein-DNA complexes formed by incubation of control extract with either the −23-bp Sp Site or the Sp1 consensus site. These complexes were reduced when control extract was replaced by KLF10 extract. The arrowhead indicates a protein-DNA complex that was enhanced in the presence of KLF10 extract, particularly with the −23-bp Sp Site. B, nuclear extract from differentiated myotubes was incubated with oligonucleotides containing either the wild type (−23 Sp Site) or the mutated binding site (m23 Sp Site). Extract was also incubated with the KLF10 antibody (KLF10 Ab) prior to incubation with the wild type Sp binding site oligonucleotide. Arrows indicate protein-DNA complexes with lower relative mobility due to the addition of the KLF10 antibody.
FIGURE 6.
FIGURE 6.
Chromatin immunoprecipitation assays of KLF10 interaction with the FGFR1 promoter. A, chromatin was isolated from myogenic cells at 0, 24, 48, and 72 h of cell culture and immunoprecipitated with Sp1 (Sp1 Ab) or KLF10 antibodies (KLF10 Ab). FGFR1 DNA was amplified using FGFR1 promoter-specific forward and reverse primers that flanked all three proximal Sp1 binding sites. Lane 1, input chromatin prior to immunoprecipitation. Lane 2, immunoprecipitation without antibody (No Ab). Lane 3, immunoprecipitation with a nonspecific antibody (N.S. Ab). Lane 4, immunoprecipitation with the Sp1 antibody. Lane 5, immunoprecipitation with the KLF10 antibody. Lane 6, amplification of plasmid DNA containing the FGFR1 promoter sequence (Control). PCR products (input, light gray bars; Sp1 antibody precipitated, gray bars; KLF10 antibody precipitated, black bars) from 0 to 72 h were quantitated. Error bars indicate S.D. B, endogenous FGFR1 chromatin was prepared from nontransfected myotubes and immunoprecipitated using the KLF10 antibody. Chromatin was also prepared from myotubes transfected with pCMVKLF10, which expresses KLF10 coupled to the FLAG epitope tag. Myotubes were also transfected with the wild type FGFR1 promoter coupled to the luciferase reporter gene (3284FGFR1Luc) or the FGFR1 promoter-luciferase construct containing the mutation of the −23-bp Sp binding site (m23FGFR1Luc). Chromatin from transfected cells was immunoprecipitated with the FLAG epitope tag antibody. Endogenous FGFR1 DNA was amplified using FGFR1 promoter-specific forward and reverse primers that flanked all three proximal Sp1 binding sites. DNA containing either the wild type or the mutated FGFR1-luciferase sequence was amplified using the same FGFR1 promoter-specific forward primer and a luciferase gene-specific reverse primer. Lane 1, input chromatin prior to immunoprecipitation. Lane 2, immunoprecipitation without antibody. Lane 3, immunoprecipitation with a nonspecific antibody. Lane 4, immunoprecipitation with the KLF10 or FLAG antibody. Lane 5, amplification of plasmid DNA containing either the wild type or the mutated FGFR1 promoter sequence. PCR products (input, light gray bars; KLF10 antibody precipitated, black bars) from endogenous (Endo.), 3284FGFR1Luc (3284), and m23FGFR1Luc (m23) DNAs were quantitated. Error bars indicate S.D.
FIGURE 7.
FIGURE 7.
Model of transcriptional regulation of FGFR1 promoter activity in proliferating myoblasts and differentiated myotubes.
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