Expression of the human apolipoprotein A-I gene in rat myogenic L6E9 cells. DNA methylation and regulation of gene activity

• Published in: The Journal of biological chemistry 1988-02, Vol.263 (4), p.1739-1744
• Main Authors: Ruiz-Opazo, N, Zannis, V I
• Format: Article
• Language: English
• Subjects: Animals; Apolipoprotein A-I; Apolipoproteins A - genetics; Base Sequence; Biological and medical sciences; Biotechnology; Cell Line; DNA Restriction Enzymes - metabolism; Fundamental and applied biological sciences. Psychology; Gene expression; Gene Expression Regulation; Genetic engineering; Genetic technics; HeLa Cells - metabolism; Humans; Methods. Procedures. Technologies; Molecular and cellular biology; Molecular genetics; Muscles - metabolism; Protein Processing, Post-Translational; Rats; Transcription, Genetic; Transfection; Tumor Cells, Cultured - metabolism; Vectors (cloning, transfer, expression). Insertion sequences and transposons
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)77938-1

An 11-kilobase EcoRI fragment containing the human apoA-I gene was cloned in the pSV2gpt vector. The recombinant plasmid was then used to transfect a rat myogenic cell line (L6E9) and HeLa cells. Selection and analysis of cell clones resistant to hypoxanthine/aminopterin/thymidine and mycophenolic acid showed that the apoA-I gene is expressed in L6E9 but not in HeLa cells. The apoA-I mRNA transcribed by either the transfected L6E9 or control human liver cell line, HepG2, has a major transcription initiation site located 26 nucleotides downstream from the CATAAA box. The apoA-I synthesized by the L6E9 cells is secreted into the culture medium and is indistinguishable from the apoA-I secreted by HepG2 cells. These results demonstrate that the L6E9 cells contain the necessary factors required for the expression of the apoA-I gene. Further analysis showed that the endogenous rat apoA-I gene is not expressed in L6E9 cells and exists in a hypermethylated configuration. In contrast, the exogenous human apoA-I gene was found to be hypomethylated in the transfected L6E9 cells and in other apoA-I-producing cells. These findings are consistent with the hypothesis that the expression of the endogenous rat apoA-I gene in L6E9 cells is prevented by hypermethylation, suggesting that compared to other mechanisms which control initiation of transcription, DNA methylation may play a higher hierarchical role in the regulation of expression of eukaryotic genes.