Enhancers are DNA sequences composed of transcription factor binding sites that drive complex patterns of gene expression in space and time. Until recently, studying enhancers in their genomic context was technically challenging. Therefore, minimal enhancers, the shortest pieces of DNA that can drive an expression pattern that resembles a gene’s endogenous pattern, are often used to study features of enhancer function. However, evidence suggests that some enhancers require sequences outside the minimal enhancer to maintain function under environmental perturbations. We hypothesized that these additional sequences also prevent misexpression caused by a transcription factor binding site mutation within a minimal enhancer. Using the Drosophila melanogaster even-skipped stripe 2 enhancer as a case study, we tested the effect of a Giant binding site mutation (gt-2) on the expression patterns driven by minimal and extended enhancer reporter constructs. We found that, in contrast to the misexpression caused by the gt-2 binding site deletion in the minimal enhancer, the same gt-2 binding site deletion in the extended enhancer did not have an effect on expression. The buffering of expression levels, but not expression pattern, is partially explained by an additional Giant binding site outside the minimal enhancer. Deleting the gt-2 binding site in the endogenous locus had no significant effect on stripe 2 expression. Our results indicate that rules derived from mutating enhancer reporter constructs may not represent what occurs in the endogenous context.

增强子是由转录因子结合位点组成的 DNA 序列，这些位点在空间和时间上驱动复杂的基因表达模式。直到最近，在基因组背景下研究增强子在技术上仍具有挑战性。因此，最小增强子，即可以驱动类似于基因内源模式的表达模式的最短 DNA 片段，通常用于研究增强子功能的特征。然而，有证据表明，一些增强子需要最小增强子之外的序列才能在环境扰动下维持功能。我们假设这些额外的序列也可以防止由最小增强子内的转录因子结合位点突变引起的错误表达。使用Drosophila melanogaster even n-skipped以 stripe 2 增强子为例，我们测试了巨型结合位点突变 (gt-2) 对最小和扩展增强子报告基因构建体驱动的表达模式的影响。我们发现，与最小增强子中 gt-2 结合位点缺失引起的错误表达相反，扩展增强子中相同的 gt-2 结合位点缺失对表达没有影响。表达水平的缓冲，而不是表达模式，部分由最小增强子之外的额外巨人结合位点解释。删除内源基因座中的 gt-2 结合位点对 stripe 2 表达没有显着影响。我们的结果表明，从突变增强子报告器构造中派生的规则可能不代表内源性上下文中发生的情况。