Target gene-specific modulation of myocardin activity by GATA transcription factors

Abstract

Myocardin is a transcriptional coactivator that regulates cardiac and smooth muscle gene expression by associating with serum response factor. We show that GATA transcription factors can either stimulate or suppress the transcriptional activity of myocardin, depending on the target gene. Modulation of myocardin activity by GATA4 is mediated by the physical interaction of myocardin with the DNA binding domain of GATA4 but does not require binding of GATA4 to DNA. Paradoxically, the transcription activation domain of GATA4 is dispensable for the stimulatory effect of GATA4 on myocardin activity but is required for repression of myocardin activity. The ability of GATA transcription factors to modulate myocardin activity provides a potential mechanism for fine tuning the expression of serum response factor target genes in a gene-specific manner.

FIG. 1.

Myocardin and GATA4 synergistically activate the Nkx2.5 enhancer. (A) Primary neonatal cardiomyocytes were infected with adenoviruses encoding myocardin or lacZ (as a negative control). Four days later, expression of Nkx2.5 transcripts was measured by semiquantitative reverse transcription-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts were detected as a loading control. (B) Gel mobility shift assays were performed with a radiolabeled oligonucleotide corresponding to the CArG box in the Nkx2.5 enhancer and in vitro-translated SRF and myocardin proteins, as indicated. Myocardin formed a stable ternary complex with SRF on this sequence (lane 3). Anti-SRF antibody supershifted the SRF complex (lane 2), and anti-Flag antibody supershifted the ternary complex formed by SRF and Flag-myocardin (lane 4). (C) COS cells were transiently transfected with the luciferase reporter plasmids shown above each panel and the indicated amounts (in nanograms) of myocardin expression vector. Luciferase activity was assayed and is expressed as fold activation above the level of expression of the reporter gene alone. (D) COS cells were transiently transfected with the luciferase reporter plasmids shown above each panel and the indicated amounts (in nanograms) of myocardin expression vector with and without a GATA4 expression vector (100 ng), and luciferase activity was determined as in panel C.

FIG. 2.

Differential effects of GATA4 on myocardin-dependent transcription. COS cells were transiently transfected with luciferase reporter plasmids controlled by (A) the ANF promoter or a mutant promoter lacking the two GATA4 binding sites, (B) the SM22 promoter, or (C) four copies of the SM22 CArG box and the indicated amounts (in nanograms) of myocardin and GATA4 expression vectors. Luciferase activity was assayed and is expressed as fold activation above the level of expression of the reporter gene alone.

FIG. 3.

Mapping the region of myocardin that responds to GATA. (A) Schematic diagrams of myocardin mutants. The potential of each protein to be stimulated by GATA4 on the 4xSM22-CArG promoter or repressed by GATA4 on the SM22 promoter is indicated. (B and C) COS cells were transiently transfected with the luciferase reporter plasmids shown above each panel and expression vectors encoding GATA4 (100 ng) and myocardin or myocardin mutants (100 ng) shown in panel A, as indicated. Luciferase activity was assayed and is expressed as fold activation above the level of expression of the reporter gene alone. (D) Schematic diagrams of GAL4-myocardin fusion proteins. The potential of each protein to be stimulated by GATA4 is indicated. (E) COS cells were transiently transfected with an upstream activation sequence-luciferase reporter plasmid and expression vectors encoding GAL4-myocardin fusion proteins (100 ng) shown in panel D with and without a GATA4 expression vector (100 ng), as indicated. Luciferase activity was assayed and is expressed as fold activation above the level of expression of the reporter gene alone.

FIG.4.

Mapping the region of myocardin that interacts with GATA4. (A) A GST-GATA4 fusion protein encompassing amino acids 177 to 332 was incubated with [³⁵S]methionine-labeled myocardin proteins translated in vitro. Myocardin proteins associated with GST-GATA4 are shown at the top, and 10% of the input proteins are shown at the bottom. (B) Summary of the GST-GATA4 pulldown and coimmunoprecipitation assays (data not shown). (C) GST pulldown assays were performed with the indicated myocardin mutants and GST-GATA4 as described for panel A. (D) Summary of the GST-GATA4 pulldown assays in panel C.

FIG. 5.

Mapping the region of GATA4 that interacts with myocardin. (A) Schematic diagrams of GATA4 mutants and their activities. (B) A GST-myocardin fusion protein encompassing amino acids 129 to 510 was incubated with [³⁵S]methionine-labeled GATA proteins translated in vitro. GATA4 proteins associated with GST-myocardin are shown at the top, and 10% of the input proteins are shown at the bottom. (C) COS cells were transiently transfected with the luciferase reporter plasmids shown above each panel and expression vectors encoding myocardin (100 ng) and GATA4 or GATA4 mutants (100 ng) shown in panel A, as indicated. Luciferase activity was assayed and is expressed as fold activation above the level of expression of the reporter alone. (D) Point mutants in Cf of GATA4 and their activities. (E) A GST-myocardin fusion protein encompassing amino acids 129 to 510 was incubated with [³⁵S]methionine-labeled GATA proteins translated in vitro. GATA4 proteins associated with GST-myocardin or GST are shown at the top and middle, respectively, and 10% of the input proteins are shown at the bottom. (F) COS cells were transiently transfected with the luciferase reporter plasmids shown above each panel and expression vectors encoding myocardin (100 ng) and GATA4 or GATA4 mutants (100 ng) shown in panel D, as indicated. Luciferase activity was assayed and is expressed as fold activation above the level of expression of the reporter alone.

FIG. 6.

Schematic diagram of myocardin and a model. (A) The domains of myocardin are shown. (B) The regions of GATA4 that associate with myocardin and mediate activation and repression are shown.