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Fig. 7. HIF1
directly binds to the human LAMA3 promoter and stimulates its activity. (A) Luciferase reporter assays of the human LAMA3 promoter activity performed in HaCaT cells transfected with either an empty vector (pcDNA3) or constructs encoding either HIF1 or USF1. Results are expressed as the fold stimulation of the luciferase activity measured in cells transfected with the pcDNA3 empty vector. (B) Functional activity of the HIF1 construct was verified by cotransfecting HIF1 together with a reporter plasmid containing three HRE consensus sites cloned upstream a minimal thymidine kinase (TK) promoter and the luciferase reporter gene. (C) Partial DNA nucleotide sequence of the LAMA3 promoter. Nucleotide numbering is relative to the first nucleotide of the transcription initiation site (+1). AP1a, AP1b and AP1c indicate the position of the three reported AP1 sites. The HRE consensus sequence is represented by a box at position –108. Arrows indicate the transcription start site and the beginning of the DNA coding sequence. (D) Effect of HIF1 on the transcriptional activity of the human LAMA3 promoter mutated at the –108 HRE consensus site. The Mut hLAMA3 promoter driving the expression of the luciferase reporter gene was cotransfected in HaCaT cells with either an empty vector (pcDNA3) or a construct encoding HIF1. A control condition where the Mut hLAMA3-Luc promoter was cotransfected with an USF1-encoding plasmid was also performed. In A, B and D, the histograms show the mean ± s.e.m. of the results obtained in three independent experiments resulting from transfections performed using different plasmid preparations. (E) A ChIP assay was performed on extracts from 6 hour CoCl2-stimulated HaCaT keratinocytes. HIF1 immunoprecipitation was performed using a specific anti-HIF1 antibody. Primers spanning the LAMA3 (upper panel) or the VEGF (lower panel) promoter regions were used for PCR amplification. In (input) represents the control of the PCR amplification performed using HaCaT genomic DNA, which showed a 142 bp and a 104 bp band corresponding to the amplification of the LAMA3 and VEGF promoter regions, respectively. The IgG (M,R) and Pol2 lanes indicate the PCR amplification obtained when the extracts were immunoprecipitated with a non immune mouse immunoglobulin (M), a non relevant rabbit antibody (R) or an antibody raised against the RNA polymerase II (Pol2). The Pol2 antibody was included in the kit and represents a supplemental positive control, according to the manufacturer. PG, amplification after immunoprecipitation with protein-G-coupled beads only.
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