Myocardin-dependent activation of the CArG box-rich smooth muscle gamma-actin gene: preferential utilization of a single CArG element through functional association with the NKX3.1 homeodomain protein

Abstract

Serum response factor (SRF) is a ubiquitously expressed transcription factor that binds a 10-bp element known as the CArG box, located in the proximal regulatory region of hundreds of target genes. SRF activates target genes in a cell- and context-dependent manner by assembling unique combinations of cofactors over CArG elements. One particularly strong SRF cofactor, myocardin (MYOCD), acts as a component of a molecular switch for smooth muscle cell (SMC) differentiation by activating cytoskeletal and contractile genes harboring SRF-binding CArG elements. Here we report that the human ACTG2 promoter, containing four conserved CArG elements, displays SMC-specific basal activity and is highly induced in the presence of MYOCD. Stable transfection of a non-SMC cell type with Myocd elicits elevations in endogenous Actg2 mRNA. Gel shift and luciferase assays reveal a strong bias for MYOCD-dependent transactivation through CArG2 of the human ACTG2 promoter. Substitution of CArG2 with other CArGs, including a consensus CArG element, fails to reconstitute full MYOCD-dependent ACTG2 promoter stimulation. Mutation of an adjacent binding site for NKX3.1 reduces MYOCD-dependent transactivation of the ACTG2 promoter. Co-immunoprecipitation, glutathione S-transferase pulldown, and luciferase assays show a physical and functional association between MYOCD and NKX3.1; no such functional relationship is evident with the related NKX2.5 transcription factor despite its interaction with MYOCD. These results demonstrate the ability of MYOCD to discriminate among several juxtaposed CArG elements, presumably through its novel partnership with NKX3.1, to optimally transactivate the human ACTG2 promoter.

FIGURE 1.

ACTG2 expression in cell lines and tissues. A, RT-PCR analysis showing mRNA expression of SMC γ-actin (Actg2) and other SMC markers and transcription factors in various tissues and cell culture models. Note the widespread expression of full-length (upper band) Srf but restricted expression of the δ5 splice variant (lower band) of Srf (47) to cultured cells and skeletal muscle and heart, but not aortic medial SMC. B, RT-PCR analysis of the indicated markers in various prostate cell lines treated with either 1 (Strom and Epi) or 10 nm (PC-3 and LNCaP) androgen (R1881). Abbreviations used are prostate stromal cells, Strom; and prostate epithelial cells, Epi. C, RT-PCR analysis of the indicated markers in prostate (Pr) and bladder (Bl). D, ACTG2 protein expression (brown) in coronary artery (d), aorta (e), and microvessels of rat perivascular tissue (arrows in f). Specificity of staining is indicated by the absence of signal when using an isotype-matched IgG control antibody (panels a–c). Magnifications are ×200.

FIGURE 2.

Basal ACTG2 promoter activity. A, VISTA plot of the 9-exon human (Hsa) ACTG2 gene (denoted at the top with the bent arrow indicating the transcription start site) versus orthologs in chimp (Ptr), dog (Cfa), mouse (Mmu), and chicken (Gga). Blue vertical peaks indicate exons, whereas pink peaks indicate at least 75% sequence homology over 100 bp of non-coding sequence. Note the progressive loss in non-coding sequence homology from chimp to chicken. B, sequence logos for the 5 major cis-acting elements in the immediate 5′ promoter region of ACTG2. These logos represent position weight matrices derived from human, chimp, dog, mouse, and chicken orthologous sequence elements (position of human elements relative to transcription start site indicated at the left of each logo). Note that CArG4 and CArG2 are 100% homologous across all species analyzed. Where sequences diverge in other elements, the height of the letters diminishes with inclusion of variant base sequences. C, firefly luciferase activities of various human ACTG2 promoter constructs in 3 distinct SMC lines (PAC1 pulmonary artery, human SKLMS uterine SMC, and A7r5 aortic SMC) versus two non-SMC types (COS7 and L6). Luciferase activity is presented here as the fold-activation of normalized (ratio of luciferase to Renilla control) luciferase to the minimal −55 promoter, set to 1. Each bar represents the average (and standard deviation) of 4 replicates in each of the indicated cells lines. These experiments were performed multiple times with similar trends in relative fold-activation. Note the y axis here and in Fig. 3 is based on a logarithmic scale.

FIGURE 3.

MYOCD-dependent stimulation of ACTG2 promoter and endogenous mRNA. COS7 (A) or PAC1 SMC (B) were co-transfected with the indicated human ACTG2 promoters linked to luciferase in the absence (open bars) or presence (closed bars) of MYOCD and resulting activities determined as described in the legend to Fig. 1. Similar results were found in two independent experiments. C, semi-quantitative RT-PCR analysis shows endogenous Actg2 mRNA induction in three independent L6 myoblast clones stably transfected with Myocd, but not in similarly grown L6 cells carrying an empty expression vector. A housekeeping gene (Gapd) serves as a loading control. D, knockdown of SRF in BC₃H1 cells reveals decreases in Actg2 mRNA expression. shEGFP, short hairpin enhanced green fluorescent protein (shEGFP).

FIGURE 4.

SRF-MYOCD ternary complex formation on the ACTG2 promoter. EMSA showing SRF/FLAG-MYOCD binding to a radiolabeled ACTG2 −205 probe containing CArG2, CArG1, and NKE (labeled A, B, and C, respectively, in the schematic at the bottom). The probe supports SRF binding (lane 2), which is supershifted with an antibody to SRF (lane 3), but not MYOCD binding alone (lane 4). A ternary complex is seen when SRF and MYOCD are combined (lane 5) and this complex is supershifted with antibodies to SRF (lane 6) or FLAG (lane 7). Ternary complex is competed with the cold −205 probe (lane 8) or CArG2 (lane 9), but not CArG1 (lane 10) or NKE (lane 11) oligonucleotides. Similar results are seen when using whole cell lysates of L6 myoblasts stably transfected with FLAG-MYOCD as the source of MYOCD (data not shown).

FIGURE 5.

ACTG2 CArG2 is the major determinant for SRF-MYOCD binding and activity. A, luciferase activities (normalized fold-change versus wild type ACTG2 −285 set to 1) in COS7 cells co-transfected with the indicated mutant promoters and MYOCD. Each bar represents the average (and S.D.) of 4 replicates and is representative of several independent experiments. B, EMSA of in vitro translated SRF and MYOCD with wild type and various mutants of the ACTG2 −285 promoter, used as radiolabeled probes. Presence or absence of nucleoprotein complexes with wild type (lanes 1 and 6), mutant CArG1–4 (lane 2), mutant CArG1, -3, and -4 (lane 3), mutant CArG2 (lane 4), or mutant NKE (lane 5) probes. Variable reductions in SRF-MYOCD nucleoprotein complexes with the −285 ACTG2 probe are seen with ×100 cold competitor oligonucleotides to CArG2 (lane 7), CArG1 (lane 8), CArG3 (lane 9), CArG4 (lane 10), and NKE (lane 11). SRF-MYOCD complexes with wild type −285 ACTG2 probe (lane 12) and the same probe with CArG substitutions as follows: CArG2 > CArG1 (lane 13), CArG2 > CArG3 (lane 14), or CArG2 > CArG4 (lane 15). C, luciferase activities (as in panel A) following MYOCD-dependent activation of the indicated ACTG2 −285 promoter constructs in COS7 cells. Single and double asterisks indicate statistical significance at 0.05 and 0.01, respectively.

FIGURE 6.

NKX3.1 potentiates MYOCD-dependent activation of the ACTG2 promoter. A, COS7 cells were co-transfected with the ACTG2 −285 promoter plus the indicated plasmids, and luciferase activity (per “Experimental Procedures”) was measured 24 h later. The amount of MYOCD input was 50 ng, whereas NK factors were used at 500 ng. Results are displayed as the mean ± S.D. and are representative of two independent studies. B, similar study as in panel A only the amount of NKX3.1 input was varied and balanced by the empty pcGN vector control. C, the wild type ACTG2 −285 promoter or the indicated mutants were co-transfected with 50 ng of MYOCD and 500 ng of NKX3.1 and luciferase activity measured (per “Experimental Procedures”). Results are displayed as those in panels A and B.

FIGURE 7.

Interaction of myocardin with NKX3.1 and NKX2.5. A and B, COS7 cells were transfected with cDNAs for FLAG-tagged full-length myocardin (WT) or its truncated mutant with deleted C-terminal transactivation domain (ΔC) together with HA-tagged NKX3.1 or NKX2.5. Cells were lysed and the cleared cell extracts were subjected to immunoprecipitation (IP) with FLAG antibodies followed by Western blotting (WB) of immune complexes (IP) or total cell lysates with FLAG or HA antibodies. C, in vitro binding of [³⁵S]MYOCD with GST-Nkx3.1.