THE MECHANISM OF RECRUITMENT OF THE LACTATE DEHYDROGENASE-B EPSILON-CRYSTALLIN GENE BY THE DUCK LENS/

In duck, the housekeeping enzyme lactate dehydrogenase B (LDH-B) and t he lens structural protein E-crystallin are encoded by the same single copy gene. Transcription of the gene is initiated from two closely sp aced start sites, at -28 and +1. The usage of the downstream site is g reatly enhanced in lens. Deletion mapping of the promoter shows that t he region -70/+18 specifies the enhanced promoter activity in the lens . A critical role is played by the consensus Spl binding site at -50; mutation of this site abolishes lens-preferred expression. Deletion of the +1 transcription initiation site also leads to a decrease in lens -preferred expression, which can be restored by moving the -28 transcr iption initiation site downstream. By band shift experiments, supershi ft mobility assays and methylation interference assays, Spl was shown to bind to the Spl consensus binding site at -50 using either heart or lens nuclear extracts. Go-expression of Spl or Spl-like factors inhib ited the activity oi an LDH-B/epsilon-crystallin promoter construct by approximately 60% in lens and by 40% in heart cells. Go-expression of Pax-6, a transcription factor shown to be involved in the lens-enhanc ed expression of a number of other crystallin genes, did not influence the promoter activity of the -130/+650 LDH-B/epsilon-crystallin promo ter construct. In contrast to other crystallin promoters, the LDH-B/ep silon-crystallin promoter does not appear to contain a lens-specific e lement, rather our data lead to a model in which a factor transmitting the effect of Spl, bound at -50, to the transcription initiation comp lex is responsible for the lens-preferred expression of the LDH-B/epsi lon-crystallin promoter. (C) 1996 Academic Press Limited