Transcriptional regulatory sequences within the first intron of the chicken apolipoproteinAI (apoAI) gene

Previous studies demonstrated that the -82 to +87 nucleotides (nt) 5'-upstream region of the chicken apolipoprotein (apoAI) gene are necessary for maximum reporter chloramphenicol acetyl transferase (cat) gene activation in chicken hepatocarcinoma (LMH) cells [Bhattacharyya, N., Chattapadhyay, R., Oddoux, C., Banerjee, D,, 1993. Characterisation of the chicken apolipoprotein A-I gene 5'-flanking region. DNA Cell Biol. 12, 597-604]. The -82 to +87 nt contain the 5'-untranslated nt, part of the first intron, and the upstream regulatory sequences. In this study, we examined the role of the first intron in the transcriptional regulation of the chicken apoAI gene. Six different reporter cat gene constructs with or without part of the first intron were prepared and transfected into LMH, normal rat kidney (NRK) and human hepatocarcinoma (HepG2) cells. Cell extracts were prepared from each transfected cell line, and CAT activities were measured. All three cell-lines readily expressed CAT, indicating that transcriptional regulatory sequences are present within the first intron region of the chicken apoAI gene. Tn an enhancer assay, the first intron containing cat, construct exhibited a 5.4-fold increase of reporter activity in NRK cells when compared to a SV 40 promoter containing cat plasmid, suggesting the presence of a moderate enhancer element within +29 to +87 nt of the first intron. DNase I protection assays, electrophoretic mobility shift assays and binding experiments with nuclear proteins isolated from different chicken tissues and LMH cells showed interaction with +29 to +87 nt. Nuclear proteins isolated from tissues like liver and intestine, that actively express apoAI gene, failed to interact with +29 to +87 nt, whereas nuclear proteins isolated from tissues that are less active in apoAI gene expression readily interacted with this region. To show the binding of the LMH-specific tl ans-acting factors to the +50 to +68 nt intron region, DNA-affinity chromatography step was performed by using H-3-labeled nuclear proteins. These studies demonstrate that the first intron region of the apoAI gene interacts with transacting proteins and plays an important role in transcriptional regulation of the apoAI gene. (C) 1999 Elsevier Science B.V. All rights reserved.