The information about when and where each gene is to be expressed is mainly encoded in the DNA sequence of enhancers, sequence elements that comprise binding sites (motifs) for different transcription factors (TFs). Most of the research on enhancer sequences has been focused on TF motif presence, whereas the enhancer syntax, that is, the flexibility of important motif positions and how the sequence context modulates the activity of TF motifs, remains poorly understood. Here, we explore the rules of enhancer syntax by a two-pronged approach in Drosophila melanogaster S2 cells: we (1) replace important TF motifs by all possible 65,536 eight-nucleotide-long sequences and (2) paste eight important TF motif types into 763 positions within 496 enhancers. These complementary strategies reveal that enhancers display constrained sequence flexibility and the context-specific modulation of motif function. Important motifs can be functionally replaced by hundreds of sequences constituting several distinct motif types, but these are only a fraction of all possible sequences and motif types. Moreover, TF motifs contribute with different intrinsic strengths that are strongly modulated by the enhancer sequence context (the flanking sequence, the presence and diversity of other motif types, and the distance between motifs), such that not all motif types can work in all positions. The context-specific modulation of motif function is also a hallmark of human enhancers, as we demonstrate experimentally. Overall, these two general principles of enhancer sequences are important to understand and predict enhancer function during development, evolution, and in disease.

中文翻译：

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增强子表现出受限的序列灵活性和基序功能的上下文特异性调节

有关每个基因何时何地表达的信息主要编码在增强子的 DNA 序列中，增强子是包含不同转录因子 (TF) 的结合位点（基序）的序列元件。大多数关于增强子序列的研究都集中在 TF 基序的存在上，而增强子语法，即重要基序位置的灵活性以及序列上下文如何调节 TF 基序的活性，仍然知之甚少。在这里，我们通过双管齐下的方法在果蝇S2 细胞中探索增强子语法的规则：我们 (1) 用所有可能的 65,536 个八核苷酸长序列替换重要的 TF 基序，以及 (2) 将八种重要的 TF 基序类型粘贴到496 个增强子中有 763 个位置。这些互补策略揭示了增强子表现出受限的序列灵活性和基序功能的上下文特异性调节。重要的基序可以在功能上被构成几种不同基序类型的数百个序列所取代，但这些只是所有可能的序列和基序类型的一小部分。此外，TF 基序具有不同的内在强度，这些强度受到增强子序列上下文（侧翼序列、其他基序类型的存在和多样性以及基序之间的距离）的强烈调节，因此并非所有基序类型都可以在所有位置发挥作用。正如我们通过实验证明的那样，基序功能的上下文特异性调节也是人类增强子的标志。总体而言，增强子序列的这两个一般原则对于理解和预测发育、进化和疾病期间的增强子功能非常重要。