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The sequence of regulatory events controlling the expression of the gamma D-crystallin gene during fibroblast growth factor-mediated rat lens fiber cell differentiation.

Authors: Dirks, RP  Klok, EJ  Van Genesen, ST  Schoenmakers, JG  Lubsen, NH 
Citation: Dirks RP, etal., Dev Biol. 1996 Jan 10;173(1):14-25.
Pubmed: (View Article at PubMed) PMID:8575616
DOI: Full-text: DOI:10.1006/dbio.1996.0003

The transcriptional activation of tissue-specific genes during terminal differentiation must be preceded by the priming of the chromatin and the appearance of the required transacting factors. We have timed these events for the transcriptional activation of the rat gamma D-crystallin gene, a lens fiber cell-specific gene that encodes a structural lens protein, during the (basic fibroblast growth factor (bFGF)-induced) in vitro differentiation of rat lens fiber cells. In vitro, in the presence of bFGF only, the endogenous gamma D mRNA accumulates between Day 10 and Day 15. When insulin is added as well, the differentiation process is accelerated and gamma D mRNA starts to accumulate at Day 8. Demethylation of the gamma D promoter region, as assessed by measuring the methylation state of the ThaI site at -16, occurs much sooner, within 1 day. By genomic footprinting, the first protein interaction with the promoter region was visible at Day 8; full occupancy of the promoter region could be detected only at Day 12. The genomic footprint identified four putative regulatory regions: -141/-131, -88/-71, -55/-45, and -15/-4. Site-directed mutagenesis of the G residues at -55 and -46 resulted in a three- to fivefold decrease in promoter activity of transfected gamma D/CAT reporter genes and also abolished interaction with nuclear extract factor(s). A G-->T mutation at -43 had no effect. The -55/-45 footprint thus derives from a proximal activator. The -88/-71 footprint identifies a silencer of the gamma D promoter in late fiber cell differentiation, as a tetramer of the -85/-67 sequence silenced a tk/CAT construct when transfected into fiber cells at a late stage, but not at an early stage, of in vitro differentiation. To time the appearance of regulatory factors, the activity of a -73/+45 gamma D/CAT (containing the activator region) and of a -1100/+45 gamma D/CAT construct was measured during fiber cell differentiation. The -73/+45 construct was active between Day 5 and Day 14, with a maximum at Day 12. The additional sequence information present in the -1100/+45 construct constrained gamma D promoter activity to between Day 8 and Day 13, with a maximum at Day 10. We conclude that the phased appearance of transacting factors during lens fiber cell differentiation controls the timing of first the activation and then the shutdown of the gamma D-crystallin gene promoter.

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CRRD Object Information
CRRD ID: 1601018
Created: 2007-04-03
Species: All species
Last Modified: 2007-04-03
Status: ACTIVE



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RGD is funded by grant HL64541 from the National Heart, Lung, and Blood Institute on behalf of the NIH.