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 the lens structural protein epsilon-crystallin are encoded by the same single copy gene. Transcription of the gene is initiated from two closely spaced start sites, at -28 and +1. The usage of the downstream site is greatly enhanced in lens. Deletion mapping of the promoter shows that the region -70/+18 specifies the enhanced promoter activity in the lens. A critical role is played by the consensus Sp1 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 transcription initiation site downstream. By band shift experiments, supershift mobility assays and methylation interference assays, Sp1 was shown to bind to the Sp1 consensus binding site at -50 using either heart or lens nuclear extracts. Co-expression of Sp1 or Sp1-like factors inhibited the activity of an LDH-B/epsilon-crystallin promoter construct by approximately 60% in lens and by 40% in heart cells. Co-expression of Pax-6, a transcription factor shown to be involved in the lens-enhanced expression of a number of other crystallin genes, did not influence the promoter activity of the -130/+650 LDH-B/epsilon-crystallin promoter construct. In contrast to other crystallin promoters, the LDH-B/epsilon-crystallin promoter does not appear to contain a lens-specific element, rather our data lead to a model in which a factor transmitting the effect of Sp1, bound at -50, to the transcription initiation complex is responsible for the lens-preferred expression of the LDH-B/epsilon-crystallin promoter.
Brunekreef
G A
GA
Department of Molecular Biology and Cell Biology, University of Nijmegen, The Netherlands.
Kraft
H J
HJ
Schoenmakers
J G
JG
Lubsen
N H
NH
D000818:Animals; D002478:Cells, Cultured; D003459:Crystallins / Q000235:genetics*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000502:physiology*; D004372:Ducks / Q000235:genetics*; D005136:Eye Proteins; D018507:Gene Expression Regulation, Developmental; D018398:Homeodomain Proteins; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase / Q000235:genetics*; D007908:Lens, Crystalline / Q000502:physiology*; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins; D016329:Sp1 Transcription Factor / Q000502:physiology*; D014158:Transcription, Genetic
D003459:Crystallins; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D018398:Homeodomain Proteins; D007527:Isoenzymes; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; D016329:Sp1 Transcription Factor; D007770:L-Lactate Dehydrogenase