FUNCTIONAL-ANALYSIS OF THE PROMOTER AND FIRST INTRON OF THE HUMAN LYSYL OXIDASE GENE

Alterations in the synthesis and activity of lysyl oxidase occur conco mitant with developmental changes in collagen and elastin deposition a nd with the pathogenesis of several acquired and heritable connective tissue disorders. To begin to unravel the mechanisms that control lysy loxidase gene expression, we have previously reported the complete exo n-intron structure of the human lysyl oxidase gene. We have now sequen ced this entire gene, including all six introns and 4 kb of DNA 5' of exon 1. Analysis of over 13 kb of intervening sequence and 5' flanking sequence revealed a concentration of conserved consensus sequence ele ments within the first intron and 1 kb immediately 5' of exon 1. Analy sis of intron 1 and the 5' Banking domain, using recombinant plasmids containing the chloramphenicol acetyl transferase (CAT) reporter gene, identified functional DNA sequence elements within these non-coding d omains responsible for inhibition and up-regulation of CAT activity in primary cultures of human skin fibroblasts, in smooth muscle cells, r evertant cells derived from an osteosarcoma cell Line and malignant c- Ha-ras-transformed osteosarcoma cells. DNA sequence elements within in tron I, in particular, resulted in a marked increase in CAT reporter a ctivity in cultured fibroblasts, smooth muscle cells and osteosarcoma cells. In c-Ha-ras-transform osteosarcoma cells, however, no such enha ncer activity of intron 1 sequence was observed. Ras-transformed osteo sarcoma cells exhibited reduced steady-state levels of lysyl oxidase m RNA that was primarily controlled through reduced transcription of the lysyl oxidase gene. The lack of any up-regulation of CAT activity in these ras-transformed cells by sequence elements within intron 1 sugge sts a complex interaction between cis-acting domains and trans-acting transcriptional factors in the 5'promoter domain and the first intron of the lysyl oxidase gene.