Cell type-specific regulation of von Willebrand factor expression by the E4BP4 transcriptional repressor.
Mechanisms of tissue-restricted patterns of von Willebrand factor (VWF) expression involve activators and repressors that limit expression to endothelial cells and megakaryocytes. The relative transcriptional activity of the proximal VWF promoter was assessed in VWF-producing and -nonproducing cells, and promoter activity was highest in endothelial cells followed by megakaryocytes. Only basal VWF promoter activity was seen in nonendothelial cells. Here we identify a negative response element located at nucleotides (nts) +96/+105 and demonstrate, using chromatin immunoprecipitation (ChIP) analysis, that in vivo this sequence interacts with the E4BP4 transcriptional repressor. Differences in size and relative abundance of nuclear E4BP4 were observed. In HepG2 cells, low levels of larger forms of E4BP4 are present that directly interact with the negative response element. In VWF-expressing cells, high levels of smaller forms predominate with no evidence of direct DNA binding. However, in endothelial cells, mutation of the VWF E4BP4 binding motif not only restores but also further elevates VWF promoter activity, suggesting that E4BP4 may be part of a coordinated binding complex. These observations implicate this binding motif in repressing both activated and basal levels of VWF transcription by different cell type-specific mechanisms, and support the hypothesis that E4BP4 sequesters negative regulators of transcription, thereby enhancing activated gene expression.
Hough
Christine
C
The Department of Pathology and Molecular Medicine, Richardson Laboratories, Queen's University, Kingston, ON, Canada, K7L 3N6.
Cuthbert
Carla D
CD
Notley
Colleen
C
Brown
Christine
C
Hegadorn
Carol
C
Berber
Ergul
E
Lillicrap
David
D
D000818:Animals; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D045744:Cell Line, Tumor; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism*; D005091:Exons / Q000235:genetics; D051790:G-Box Binding Factors; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000032:analysis / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014162:Transfection; D014841:von Willebrand Factor / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051790:G-Box Binding Factors; C497440:NFIL3 protein, human; C497441:Nfil3 protein, mouse; D009687:Nuclear Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D014841:von Willebrand Factor