Localization and analysis of the principal promoter for human tenascin-X

Tenascin-X is a large extracellular matrix protein expressed in connective tissues. Mutations in TNXB are a cause of Ehlers-Danlos syndrome. Comparison of 25 kb of human and mouse DNA near the TNXB untranslated exon identified eight regions of >80% identity. Of 17 cell types and lines screened, TNXB expression was abundant only in fibroblasts and HT1080 human skin fibrosarcoma cells. Expression of TNXB promoter/reporter constructs in HT1080 cells showed that region E, near the untranslated exon, had the greatest activity, and the two regions of greatest identity, 5.0 and 3.3 kb upstream, had no activity. Mobility shift assays identified six protein-binding regions. Regions I, II, and IV bound Sp1 and Sp3, but only I and IV were functional in HT1080 cells. Regions III and V bound unknown proteins and exerted strong enhancer-like activity. Mutation of regions III and V in promoter/reporter constructs decreased TNXB transcription and identified functionally important Sp1 and Sp3 sites. These experiments provide an essential foundation for understanding the regulation of this vital protein.