Dynamic Transcriptional Regulatory Complexes, Including E2F4, p107, p130, and Sp1, Control Fibroblast Growth Factor Receptor 1 Gene Expression during Myogenesis

Abstract

Developmentally controlled transcriptional regulation of myogenic cell proliferation and differentiation via expression of the fibroblast growth factor receptor 1 (FGFR1) gene is positively regulated by Sp1 and negatively regulated by E2F4-based transcriptional complexes. We report that p107 and p130 formed transcriptional complexes with E2F4 on the FGFR1 promoter and repressed FGFR1 gene transcription in myogenic cells. However, in Drosophila melanogaster SL2 cells, only p107 was able to repress Sp1-mediated transactivation of the FGFR1 promoter. Gel shift assays using transfected myoblast nuclear extracts showed that ectopic p107 reduced Sp1 occupancy of the proximal Sp binding site of the FGFR1 promoter, and coimmunoprecipitation studies indicated that Sp1 interacts with p107 but not with p130. Gel shift assays also demonstrated that Sp1 interacted with p107 in E2F4-p107 transcriptional complexes in myoblasts. The nature of the repressor transcriptional complex was altered in differentiated muscle fibers by the relative loss of the E2F4-p107-Sp1 transcription complex and replacement by the repressor E2F4-p130 complex. These findings demonstrate that activation and repression of FGFR1 gene transcription is governed by interplay between Sp1, p107, p130, and E2F4 in distinct transcriptional complexes during skeletal muscle development. Fibroblast growth factor receptors (FGFRs)1 have diverse functional roles in mitogenesis, angiogenesis, cell migration, differentiation, mesoderm induction, bone growth and limb development (1). In skeletal muscle, FGFR1 mediates the mitogenic activity initiated by FGF1 and FGF2. During skeletal myogenesis, FGFR1 gene expression is positively regulated in proliferating myoblasts and negatively regulated in differentiated muscle fibers. The functional significance of regulated expression of the FGFR1 gene during myogenesis is demonstrated by overexpression in vivo. Chick embryos overexpressing wild-type FGFR1 displayed delayed myoblast differentiation and muscle fiber formation. On the contrary, chick embryos overexpressing a dominant-negative form of FGFR1 displayed premature muscle fiber formation with decreased muscle mass (2, 3).Although regulation of FGFR1 gene expression is important for normal growth and development of skeletal muscles, the molecular mechanism governing its transcription is poorly understood. Positive regulation of FGFR1 gene expression in proliferating myoblasts is governed by the Sp1 transcription factor. The chicken FGFR1 promoter contains two functional, distal Sp1 binding sites, and the proximal region contains three Sp1 binding sites, all of which are essential for full promoter activity in proliferating myoblasts (4, 5). Negative transcriptional regulators control FGFR1 promoter activity as FGFR1 gene expression declines during myogenic differentiation. We recently identified E2F4 as a negative regulator of FGFR1 gene expression in skeletal muscle cells (6). Its repressor activity was mediated by E2F4 binding to a proximal cis-element at Ϫ65 bp. Howev...

Repression of Myoblast Proliferation and Fibroblast Growth Factor Receptor 1 Promoter Activity by KLF10 Protein

“…For analyses of FGFR1 promoter activities, ED13 chicken myoblasts were transfected with 3284FGFR1CAT (1.5 g/35-mm culture dish) and increasing amounts (0 -6 g/35-mm culture dish) of pCMVKLF10. For transfection of Drosophila SL2 cells, 3284FGFR1CAT (2.5 g/35-mm culture dish) and pPacSp1 (750 ng) along with increasing amounts of pCMVKLF10 (0 -6 g) were co-transfected using Cellfectin (Invitrogen) as described previously (29). For all transfections related to promoter activities, pRSV␤GAL (1 g) was co-transfected to normalize for variations in transfection efficiencies.…”

“…Southwestern Analysis and Electromobility Shift AssaysSouthwestern blot analysis was performed as described previously (29). Nuclear extracts (75 g) from control, nontransfected myoblasts and from myoblasts transfected with pCMVKLF10 as above were electrophoresed in a 7.5% SDS-polyacrylamide gel and electroblotted to nitrocellulose membranes.…”

“…In addition, myoblasts were allowed to differentiate in vitro for 10 days, and the resulting myotubes were maintained in cell culture medium supplemented with AraC as described previously (9). Chromatin immunoprecipitation was then conducted as described previously (29). Chromatin was immunoprecipitated with 4 g of Sp1 or KLF10 antibody (Abcam).…”

“…Also, pBSKS plasmid DNA (Stratagene) was used to maintain constant DNA amounts in transfections. Promoter activity assays were performed as described previously (29).…”

Rb family proteins as modulators of gene expression and new aspects regarding the interaction with chromatin remodeling enzymes

“…Each of the Rb family proteins binds to distinct members of the E2F transcription factors, which regulate the expression of genes whose protein products are necessary for cell proliferation and to drive cell-cycle progression (Muller et al, 2001;Young et al, 2003;Attwooll et al, 2004). More recently a broad range of studies are showing that pRb-family proteins associate with a wide variety of transcription factors and chromatin remodeling enzymes to control gene expression (La Sala et al, 2003;Macaluso et al, 2003Macaluso et al, , 2005Macaluso et al, , 2006Gunawardena et al, 2004;Parakati and DiMario, 2005).…”