Journal: The Journal of Biological Chemistry
Article Title: Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *
Figure Lengend Snippet: Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).
Article Snippet: The analysis of the 3′ deletion mutants of ME.2 revealed that the small 19–141 fragment did not bind STAT1 ( lane 19–141 ), whereas the larger 19–298 fragment ( lane 19–298 ) as well as the full-length ME.2 ( lane 19–619 ) bound STAT1 efficiently, suggesting that the binding site of STAT1 is located in the 141/298 region of ME.2 ( C ).
Techniques: Transfection, Expressing, Plasmid Preparation, Over Expression, Activity Assay, Binding Assay, Sequencing, Negative Control, Positive Control, Western Blot