Stable expression and cell-specific chromatin structure of human α1-antitrypsin cosmid transgenes in rat hepatoma cells

The human gene encoding alpha 1-antitrypsin (alpha 1AT, gene symbol PI) resides in a cluster of serine protease inhibitor (serpin) genes on chromosome 14q32.1. alpha 1AT is highly expressed in the liver and in cultured hepatoma cells. We recently reported the chromatin structure of a >100 kb region around the gene, as defined by DNase I-hypersensitive sites (DHSs) and matrix-attachment regions, in expressing and non-expressing cells. Transfer of human chromosome 14 by microcell fusion from non-expressing fibroblasts to rat hepatoma cells resulted in activation of alpha 1AT transcription and chromatin reorganization of the entire region. In the present study, we stably introduced cosmids containing alpha 1AT with various amounts of flanking sequence and a linked neo selectable marker into rat hepatoma cells. All single-copy transfectants with >14 kb of 5' flanking sequence expressed wild-type levels of alpha 1AT mRNA in a position-independent manner. In contrast, expression of transgenes containing only similar to 1.5-4 kb of flanking sequence was highly variable. Long-term culture of transfectant clones in the absence of selection resulted in gradual loss of neo expression, but expression of the linked alpha 1AT gene remained constant. DHS mapping of cosmid transgenes integrated at ectopic sites revealed a hepatoma-specific chromatin structure in each transfectant clone. The implications of these findings are discussed.