THE SEQUENCE OF REGULATORY EVENTS CONTROLLING THE EXPRESSION OF THE GAMMA-D-CRYSTALLIN GENE DURING FIBROBLAST GROWTH FACTOR-MEDIATED RAT LENS FIBER CELL-DIFFERENTIATION

The transcriptional activation of tissue-specific genes during termina l differentiation must be preceded by the priming of the chromatin and the appearance of the required transacting factors. We have timed the se events for the transcriptional activation of the rat gamma D-crysta llin gene, a lens fiber cell-specific gene that encodes a structural l ens protein, during the (basic fibroblast growth factor (bFGF)-induced ) in vitro differentiation of rat lens fiber cells. In vitro, in the p resence 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 measurin g the methylation state of the ThaI site at -16, occurs much sooner, w ithin 1 day. By genomic footprinting, the first protein interaction wi th the promoter region was visible at Day 8; full occupancy of the pro moter region could be detected only at Day 12. The genomic footprint i dentified four putative regulatory regions: -141/-131, -88/-71, -55/-4 5, 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 t ransfected gamma D/CAT reporter genes and also abolished interaction w ith 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 fi ber cell differentiation, as a tetramer of the -85/ -67 sequence silen ced a tk/CAT construct when transfected into fiber cells at a late sta ge, but not at an early stage, of in vitro differentiation. To time th e 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 c onstruct was measured during fiber cell differentiation. The -73/+45 c onstruct was active between Day 5 and Day 14, with a maximum at Day 12 . The additional sequence information present in the -1100/+45 constru ct 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 tr ansacting factors during lens fiber cell differentiation controls the timing of first the activation and then the shutdown of the gamma D-cr ystallin gene promoter. (C) 1996 Academic Press, Inc.