Nuclear protein interactions with the human KDR/flk-1 promoter in vivo. Regulation of Sp1 binding is associated with cell type-specific expression.
The endothelial cell type-specific tyrosine kinase KDR/flk-1 is a receptor for vascular endothelial growth factor and a critical regulator of endothelial cell growth and development. To study mechanisms of endothelial cell differentiation and gene regulation, we have analyzed the topology of the proximal promoter of human KDR/flk-1. A protected sequence between base pairs -110 and -25 was defined by in vitro DNase I footprinting analysis in human umbilical vein endothelial cells (HUVECs). Purified Sp1 alone produced similar protection, and electrophoretic mobility shift assays demonstrated that Sp1 was indeed the major nuclear protein binding to this region. Despite the cell type specificity of KDR/flk-1 expression, no cell type differences were observed in DNA-protein interactions in vitro. In contrast, in vivo footprinting assays demonstrated marked differences in core promoter interactions between cell types. Protection of Sp1 binding sites was observed in HUVECs by in vivo DNase I footprinting, whereas in human fibroblasts and HeLa cells a pattern consistent with nucleosomal positioning was observed. In vivo dimethylsulfate footprinting confirmed that DNA-protein interactions occurred within Sp1 elements in HUVECs but not in nonendothelial cells. It is possible that distant elements coordinate Sp1 binding and chromatin structure to regulate cell type-specific expression of KDR/flk-1.
Patterson
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C
Division of Cardiology, University of Texas Medical Branch, Galveston, Texas 77555-1064, USA. camp@cardiology.utmb.edu
Wu
Y
Y
Lee
M E
ME
DeVault
J D
JD
Runge
M S
MS
Haber
E
E
D001483:Base Sequence; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D017978:Receptors, Growth Factor / Q000235:genetics* / Q000378:metabolism; D011975:Receptors, Mitogen / Q000235:genetics* / Q000378:metabolism; D040262:Receptors, Vascular Endothelial Growth Factor; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013329:Structure-Activity Relationship
D002843:Chromatin; D017978:Receptors, Growth Factor; D011975:Receptors, Mitogen; D016329:Sp1 Transcription Factor; D004247:DNA; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor