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, 19 (4), 2577-84

Myogenic Basic Helix-Loop-Helix Proteins and Sp1 Interact as Components of a Multiprotein Transcriptional Complex Required for Activity of the Human Cardiac Alpha-Actin Promoter

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Myogenic Basic Helix-Loop-Helix Proteins and Sp1 Interact as Components of a Multiprotein Transcriptional Complex Required for Activity of the Human Cardiac Alpha-Actin Promoter

E Biesiada et al. Mol Cell Biol.

Abstract

Activation of the human cardiac alpha-actin (HCA) promoter in skeletal muscle cells requires the integrity of DNA binding sites for the serum response factor (SRF), Sp1, and the myogenic basic helix-loop-helix (bHLH) family. In this study we report that activation of the HCA correlates with formation of a muscle-specific multiprotein complex on the promoter. We provide evidence that proteins eluted from the multiprotein complex specifically react with antibodies directed against myogenin, Sp1, and SRF and that the complex can be assembled in vitro by using the HCA promoter and purified MyoD, E12, SRF, and Sp1. In vitro and in vivo assays revealed a direct association of Sp1 and myogenin-MyoD mediated by the DNA-binding domain of Sp1 and the HLH motif of myogenin. The results obtained in this study indicate that protein-protein interactions and the cooperative DNA binding of transcriptional activators are critical steps in the formation of a transcriptionally productive multiprotein complex on the HCA promoter and suggest that the same mechanisms might be utilized to regulate the transcription of muscle-specific and other genes.

Figures

FIG. 1
FIG. 1
Formation of a muscle-specific protein complex on HCA promoter. (A) EMSA analysis for binding of nuclear factors. Samples (10 μg) of C2C12 myotube nuclear extracts were employed with radiolabeled HCA promoter fragment in the absence of specific competitor (lane 1) and in the presence of a 50-fold molar excess of the unlabeled fragment (lane 2). (B) Competition analysis. Nuclear extracts from C2C12 myotubes were assayed in the absence of specific competitor (lane 1) and in the presence of a 100-fold molar excess of unlabeled synthetic double-stranded oligonucleotides containing the following sequences: the normal and mutated GC boxes (lanes 2 and 3), the normal and mutated CArG boxes (lanes 4 and 5), the normal and mutated E boxes (lanes 6 and 7). (C) The shifted protein complex is cell type specific and differentiation dependent. An EMSA of the radiolabeled HCA promoter incubated with equal amounts of nuclear extracts from HeLa (lane 1) and from C2C12 myotubes (lane 2) cells is shown. Equivalent amounts of nuclear extracts derived from either undifferentiated C2C12 myoblasts (MB, lane 3) or differentiated myotubes (MT, lane 4) were analyzed by EMSA with the radiolabeled HCA promoter.
FIG. 2
FIG. 2
Identification of the protein components of the shifted complex. (A) Proteins eluted from the shifted complex (Fig. 1, lane 1) were subjected to Western blotting with a polyclonal rabbit antiserum raised against Sp1 protein. Lanes: 1, in vitro-synthesized SRF protein; 2, recombinant Sp1 protein; 3, blank lane; and 4, proteins eluted from the shifted complex. (B) Complex proteins were subjected to Western blotting with a monoclonal antibody against myogenin (FD5). Lanes: 1, in vitro-synthesized myogenin; 2, recombinant Sp1 protein; 3, blank lane; and 4, proteins eluted from the shifted complex. (C) Complex proteins were subjected to Western blotting with polyclonal rabbit antiserum raised against SRF protein. Lanes: 1, in vitro-synthesized SRF protein; 2, recombinant Sp1 protein; 3, blank lane; and 4, proteins eluted from the complex.
FIG. 3
FIG. 3
In vitro assembly of a protein complex on the HCA promoter by purified MyoD, E12, SRF, and Sp1. (A) EMSA was performed with the radiolabeled HCA promoter with different combinations of GST-MyoD, E12, His-SRF, and Sp1 purified proteins. The arrow points to a shifted complex containing MyoD-E12 heterodimers. The asterisk indicates a low-mobility complex observed exclusively in the presence of MyoD, E12, SRF, and Sp1. (B) Competition analysis of the low-mobility complex described in panel A. Oligonucleotides containing the GC, CArG, or E box but not their respective mutated sequences compete for the formation of the low-mobility complex. (C) EMSA was performed with radiolabeled HCA promoter fragments derived from the HCA wild type and the HCA-μGC, HCA-CArGM, and HCA-E-sm constructs (see Materials and Methods) and purified MyoD, E12, SRF, and Sp1 proteins. The low-mobility complex described in panel A and indicated by the asterisk is observed when the HCA wild type (lane 1) but not the HCA mutants (lanes 2 to 4) are employed.
FIG. 4
FIG. 4
The presence of the protein complex correlates with the HCA promoter activity in muscle cells. (A) Schematic representation of the HCA promoter fragments used in transfection and EMSA assays. Mutations introduced in the GC, CArG, and E boxes are in lowercase letters. The relative promoter activity is expressed as a percentage and refers to the luciferase activities generated by the different HCA constructs when transiently transfected in C2C12 cells. The numbers in parenthesis represent standard deviations. (B) Gel retardation analysis for binding of nuclear factors from C2C12 cells with different HCA promoter fragments. Nuclear extract from C2 myotubes was employed with radiolabeled fragment of the wild type (wt) or HCA promoter containing mutated GC box (μGC), CarG box (CArG-M), or E box (E-sm).
FIG. 5
FIG. 5
Sp1 interact with two myogenic bHLH proteins, myogenin and MyoD, in vitro and in vivo. (A) The GST protein (lane 3) or the GST-myogenin (lane 4) were mixed with 10 μl of reticulocyte lysate programmed by Sp1-encoding RNA and supplemented with [35S]methionine and then processed and resolved on a 10% denaturing polyacrylamide gel. Lane 2 shows the input radiolabeled Sp1. (B) C3H10T1/2 cells were transiently transfected with the UASx4-tk-LUC indicator plasmid and the Gal-Sp1, Gal-E12, and VP16-MyoD activators. To correct for transfection efficiency, the CMV-lacZ plasmid was added to the transfection reaction. After 48 h, cells were processed and luciferase and β-galactosidase assays were performed on an automated microtiter plate luminometer (MLX; Dynex Technology). Bars indicate standard deviations. (C) Nuclear extracts derived from metabolically radiolabeled C2C12 cells were incubated with unblocked (lane 2) or blocked (lane 3) Sp1 antiserum in low-stringency conditions. Double immunoprecipitation with α-myogenin antibody, followed by unblocked (lane 5) or blocked (lane 6) Sp1 antiserum in high-stringency conditions, with radiolabeled C2C12 nuclear extracts reveals that the protein associated with myogenin is bona fide Sp1. Lane 4 shows radiolabeled in vitro-synthesized Sp1.
FIG. 6
FIG. 6
A region of Sp1 spanning the DNA-binding domain and the HLH domain of myogenin mediate protein interactions. (A) Wild-type and different truncated version of radiolabeled Sp1 were affinity purified on GST-myogenin-coated agarose beads. The right panel shows input proteins. (B) Schematic representation of the Sp1 polypeptides employed in (A). Regions rich in serine and threonine (S/T), glutamic acid (Q), and the zinc finger motif are indicated at the top. (C) The C-terminal 168 amino acids of Sp1 fused to GST were reacted with several versions of radiolabeled myogenin. The left panel indicates the input proteins. The myogenin ΔN and ΔC proteins were synthesized by using the myogenin DM4-79 and DM158-224 constructs described elsewhere (35). (D) Schematic representation of the myogenin deletions employed in the experiments reported in panel C.

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