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Neutrophil elastase gene expression in health and disease Wong, Edmond Tan-Loon


Neutrophil elastase (NE) is a major protease carried by neutrophils and synthesized in the promyelocyte. A role of NE in the pathogenesis of emphysema was hypothesized over 25 years ago. Our understanding of the pathogenesis of this disease has remained obscure however, due to the lack of an accurate model for examining the causative role of proteases such as NE in the disease. The hypothesis is presented that over-expression of NE increases susceptibility to emphysema. Two transgenic models were proposed to 1) develop a lung-specific NE transgene to examine the effects of NE over-expression in the lung, and 2) determine the ability of the proximal human NE gene promoter region to drive expression of the gene in mice. The lung-specific promoter-driven NE transgene appeared to be toxic to the developing embryo, and the proximal promoter region of the human NE gene was insufficient to drive transgene expression, suggesting that additional regulatory elements are required to drive NE gene transcription in vivo. To better understand NE gene regulation in vivo, the chromatin structure of the NE locus was examined. Seventeen DNase I hypersensitive sites (DHS) were discovered in an early myeloid cell line. Examination of non-expressing cell lines revealed that the chromatin organization at the locus is unique to NE-transcribing cells. To determine whether DHS flanking the NE gene are important in mediating NE transcription, reporter constructs carrying these sites were introduced transiently into an early myeloid cell line expressing NE. These studies confirmed a minimal functional promoter. The other DHS had no additional effect on transcription in this assay. To determine if these sites play a role in the presence of chromatin, stable transfections were performed. In this case, the minimal promoter was transcriptionally silent. Addition of a distal upstream hypersensitive site, DHS-9, increased expression by three-orders of magnitude. Since this effect was not observed in the transient studies, it can be concluded that DHS-9 is a chromatin-dependent enhancer of transcription. The requirement of DHS-9 in mediating high levels of transcription in the presence of chromatin thus identifies an important role of chromatin organization in NE gene expression in vivo.

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