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.
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.
Sun
Qiang
Q
Aab Cardiovascular Research Institute of the University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Taurin
Sebastien
S
Sethakorn
Nan
N
Long
Xiaochun
X
Imamura
Masaaki
M
Wang
Da-Zhi
DZ
Zimmer
Warren E
WE
Dulin
Nickolai O
NO
Miano
Joseph M
JM
D000199:Actins / Q000235:genetics*; D000818:Animals; D002454:Cell Differentiation; D005786:Gene Expression Regulation; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000737:chemistry*; D006801:Humans; D051379:Mice; D008954:Models, Biological; D009119:Muscle Contraction; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000737:chemistry* / Q000502:physiology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D015534:Trans-Activators / Q000737:chemistry* / Q000502:physiology; D014157:Transcription Factors / Q000737:chemistry*; D000099251:Myocardin
D000199:Actins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D000099251:Myocardin; C491178:NKX3-1 protein, human; C102837:Nkx3-1 protein, mouse