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 +87nt 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. In 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 +87nt 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 +87nt. Nuclear proteins isolated from tissues like liver and intestine, that actively express apoAI gene, failed to interact with +29 to +87nt, 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 trans-acting factors to the +50 to +68nt intron region, DNA-affinity chromatography step was performed by using 3H-labeled nuclear proteins. These studies demonstrate that the first intron region of the apoAI gene interacts with trans-acting proteins and plays an important role in transcriptional regulation of the apoAI gene.
Bhattacharyya
N
N
Department of Membrane Biochemistry, Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10021, USA. nisanb@box-n.nih.gov
Banerjee
D
D
D000818:Animals; D016632:Apolipoprotein A-I / Q000235:genetics*; D001483:Base Sequence; D002461:Cell Line, Transformed; D002645:Chickens; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D006801:Humans; D007438:Introns / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D016632:Apolipoprotein A-I; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I