Abstract
The apolipoprotein A-II (apoA-II) gene regulatory region -911 to +29 strongly promotes the transcription of the promotorless chloramphenicol acetyltransferase (CAT) gene in cells of hepatic (HepG2) and intestinal (CaCo2) origin but not in HeLa cells. Deletion of nucleotides -911 to -860 decreased the hepatic and intestinal transcription to 7% and 18% of control, respectively. Further progressive deletions extending to nucleotides -614, -440, -230, and -80 abolished both hepatic and intestinal transcription, indicating that the distal promoter region -911 to -614 contains regulatory elements that are essential for intestinal and hepatic transcription. An internal deletion of the -614 to -230 region decreased hepatic transcription 60% while it increased intestinal transcription 140% of control indicating that the elements which control hepatic and intestinal transcription of the apoA-II gene may be different within this regulatory region. DNase I footprinting analysis with rat liver nuclear extracts identified 14 protected regions: A, -40 to -33; B, -65 to -42; C, -126 to -110; D, -276 to -255; E, -377 to -364; F, -404 to -384; G, -468 to -455; H, -573 to -554; I, -706 to -680; J, -734 to -716; K, -760 to -743; L, -803 to -773; M, -853 to -829, and N, -903 to -879, as the DNA binding sites for nuclear factors. Five of the regions (B, C, G, H, and K) bind to heat-stable factors. DNA binding gel electrophoretic assays indicated that region N (-903 to -879), which is essential for efficient transcription, binds predominantly a nuclear activity designated AIIN3. This activity is present in cells of hepatic and intestinal origin but absent in HeLa cells. Similar analysis showed that region H (-573 to -554) binds to the liver-specific factor HNF1/LFB1. Deletion of this region decreased hepatic and intestinal transcription 80 and 64% of control, respectively, suggesting that HNF1/LFB1 or a related activity contributes to optimal transcription but is not essential for the tissue-specific expression of the human apoA-II gene.
Highlights
The apolipoprotein A-I1 gene regulatory [1].In plasma, apoA-I1 exists as a dimer of two 77-aminoregion -911 to +29 strongly promotes the transcription of the promotorless chloramphenicol acetyltransferase (CAT) gene in cells of hepatic (HepG2) and intestinal (CaCo2) origin but not in HeLa cells
Bind to the Regulatory Region N (-903 to -879) of the Human apoA-I1 Gene-As shown in Fig. 2A, the element N is an essential constituent of the apoA-I1 promoter since deletion of this element drastically reduced both hepatic and intestinal transcription
Thespecific interaction of hepatic nuclear factorswithdomain N was investigated using DNA binding gel electrophoretic assays. These assays performed with a double-stranded oligonucleotide corresponding to region N (-903 to -879) and rat liver nuclear extracts showed that thisregion binds predominantly a nuclear activity designated AIIN3and two minor activities
Summary
May contain elements that are contributing tooptimal transcription,theyarenotessentialforthe cell type-specific. For this analysis, apoA-I1 promoter regions tion. -911 to +29 promoter region has strong promoteractivity in gion -911 to +29 were end-labeled and used in DNaseI hepatic and intestinal cells which compares with the activity footprinting assays. This analysis showed the presence of 14 of the apoB -899/+24 and RSV promoters (Fig. 2 F ). The single activity which binds in region H is designated AIIH1
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