Re-evaluating enhancer modularity and boundaries with quantitative approaches in Drosophila

转录增强子控制在哪个细胞、在什么时间、以及从核心启动子转录多少给定基因。例如，这一过程的精确性对于所有多细胞生物的正常胚胎发育是必要的。传统上，增强子是在报告基因试验中发现和定义的，在细胞培养或转基因生物中测试DNA片段的调控活性。这种方法描绘了一幅教科书式的画面，其中增强子是短的（100- 1000bp）， DNA序列的离散延伸，作为独立的模块工作。利用一种不同的定量方法来绘制增强子，我们最近发现，在果蝇的翅膀中，驱动色素沉着基因黄色的增强子比文献预测的要宽得多（~4000 bp），并且纠缠在一起。初步证据表明，该结果可推广到蝇体中另一种黄色驱动增强剂。这种情况破坏了增强子模块化的概念，这是理解发育和进化生物学的关键概念。有了这个建议，我们想首先彻底描述监管信息在这些不同的增强剂之间混杂的程度。然后，我们希望了解增强器通过哪些机制实现其功能模块化。我们怀疑，通过调节DNA的可及性，调控信息可能在不同的组织中选择性地可用。我们将通过研究身体和翅膀增强子的顺式和反式增强子可及性的决定因素来具体验证这一假设。我们将结合转基因果蝇的报告基因分析、计算和定量图像分析以及基因组方法来实现这些目标。我们希望我们的研究结果能够重新定义转录增强子，同时也揭示了来自不同的、重叠的增强子的调控信息是如何以一种选择性的方式被动员起来的。

Transcriptional enhancers control in which cells, at what time, and how much a given gene is transcribed from a core promoter. The precision of this process is for instanceb necessary for normal embryonic development of all multicellular organisms. Classically, enhancers have been revealed and defined in reporter assays, where the regulatory activity of a DNA segment is tested in cell culture, or in transgenic organisms. This approach has painted a textbook picture where enhancers are short (100-1000 bp), discrete stretches of DNA sequence, working as independent modules. Using a different and quantitative approach to map enhancers, we have recently found that enhancers of the pigmentation gene yellow, driving activity in the wings of flies (Drosophila) appear much broader (~4000 bp) and entangled than the literature predicts. Preliminary evidence indicates that the results extend to another enhancer of yellow driving in the fly body. This situation undermines the notion of enhancer modularity, a key concept to understand Developmental and Evolutionary Biology. With this proposal, we would like to first thoroughly describe the degree to which regulatory information is intermingled among these different enhancers. We then want to understand through which mechanisms enhancers achieve their functional modularity. We suspect that regulatory information may be selectively available in different tissues by means of regulated accessibility of the DNA. We will specifically test this hypothesis by investingating the determinants of enhancer accessibility, in cis and in trans, for the body and wing enhancers. We will combine reporter assays in transgenic Drosophila, computational and quantitative image analysis, and genomic methods to achieve these goals. We expect our results to seed the redefinition of transcriptional enhancers, but also to reveal how regulatory information from different, overlapping enhancers is mobilized in a selective manner.