Dirks R P, Klok E J, van Genesen S T, Schoenmakers J G, Lubsen N H
Department of Molecular Biology, University of Nijmegen, The Netherlands.
Dev Biol. 1996 Jan 10;173(1):14-25. 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.
在终末分化过程中，组织特异性基因的转录激活必须先于染色质的启动和所需反式作用因子的出现。我们已确定了大鼠γD-晶状体蛋白基因转录激活过程中这些事件的时间，该基因是一种晶状体纤维细胞特异性基因，编码一种晶状体结构蛋白，在大鼠晶状体纤维细胞（碱性成纤维细胞生长因子（bFGF）诱导的）体外分化过程中。在体外，仅在存在bFGF的情况下，内源性γD mRNA在第10天至第15天之间积累。当同时添加胰岛素时，分化过程加速，γD mRNA在第8天开始积累。通过测量-16处ThaI位点的甲基化状态评估，γD启动子区域的去甲基化发生得要早得多，在1天内。通过基因组足迹分析，与启动子区域的首次蛋白质相互作用在第8天可见；仅在第12天才能检测到启动子区域被完全占据。基因组足迹鉴定出四个推定的调控区域：-141/-131、-88/-71、-55/-45和-15/-4。对-55和-46处的G残基进行定点诱变导致转染的γD/CAT报告基因的启动子活性降低三到五倍，并且也消除了与核提取物因子的相互作用。-43处的G→T突变没有影响。因此，-55/-45足迹来自近端激活剂。-88/-71足迹鉴定出γD启动子在晚期纤维细胞分化中的一个沉默子，因为-85/-67序列的四聚体在体外分化的晚期而非早期转染到纤维细胞中时使tk/CAT构建体沉默。为了确定调控因子出现的时间，在纤维细胞分化过程中测量了-73/+45γD/CAT（包含激活区域）和-1100/+45γD/CAT构建体的活性。-73/+45构建体在第5天至第14天之间有活性，在第12天达到最大值。-1100/+45构建体中存在的额外序列信息将γD启动子活性限制在第8天至第13天之间，在第10天达到最大值。我们得出结论，在晶状体纤维细胞分化过程中反式作用因子的阶段性出现控制了γD-晶状体蛋白基因启动子首先激活然后关闭的时间。
