The Sequence of Regulatory Events Controlling the Expression of the γD-crystallin Gene during Fibroblast Growth Factor-Mediated Rat Lens Fiber Cell Differentiation

AbstractThe 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 γD-crystallin gene, a lens fiber cell-specific gene that encodes a structural lens protein, during the (basic fibroblast growth factor (bFGF)-induced)in vitrodifferentiation of rat lens fiber cells.In vitro,in the presence of bFGF only, the endogenous γD mRNA accumulates between Day 10 and Day 15. When insulin is added as well, the differentiation process is accelerated and γD mRNA starts to accumulate at Day 8. Demethylation of the γD promoter region, as assessed by measuring the methylation state of theThaI 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 γD/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 γD 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, ofin vitrodifferentiation. To time the appearance of regulatory factors, the activity of a −73/+45 γD/CAT (containing the activator region) and of a −1100/+45 γD/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 γD 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 γD-crystallin gene promoter