Patterns of transcriptional activity are encoded in our genome through regulatory elements such as promoters or enhancers that, paradoxically, contain similar assortments of sequence-specific transcription factor (TF) binding sites^1,2,3. Knowledge of how these sequence motifs encode multiple, often overlapping, gene expression programs is central to understanding gene regulation and how mutations in non-coding DNA manifest in disease^4,5. Here, by studying gene regulation from the perspective of individual transcription start sites (TSSs), using natural genetic variation, perturbation of endogenous TF protein levels and massively parallel analysis of natural and synthetic regulatory elements, we show that the effect of TF binding on transcription initiation is position dependent. Analysing TF-binding-site occurrences relative to the TSS, we identified several motifs with highly preferential positioning. We show that these patterns are a combination of a TF’s distinct functional profiles—many TFs, including canonical activators such as NRF1, NFY and Sp1, activate or repress transcription initiation depending on their precise position relative to the TSS. As such, TFs and their spacing collectively guide the site and frequency of transcription initiation. More broadly, these findings reveal how similar assortments of TF binding sites can generate distinct gene regulatory outcomes depending on their spatial configuration and how DNA sequence polymorphisms may contribute to transcription variation and disease and underscore a critical role for TSS data in decoding the regulatory information of our genome.

转录活性模式通过启动子或增强子等调节元件在我们的基因组中编码，矛盾的是，这些元件包含类似种类的序列特异性转录因子 （TF） 结合位点^1,2,3。了解这些序列基序如何编码多个通常重叠的基因表达程序，对于理解基因调控以及非编码 DNA 中的突变如何在疾病中表现出来至关重要^4,5。在这里，通过从单个转录起始位点 （TSS） 的角度研究基因调控，使用自然遗传变异、内源性 TF 蛋白水平的扰动以及天然和合成调节元件的大规模平行分析，我们表明 TF 结合对转录起始的影响是位置依赖性的。分析相对于 TSS 的 TF 结合位点出现，我们确定了几个具有高度优先定位的基序。我们表明这些模式是 TF 不同功能谱的组合——许多 TF，包括 NRF1、NFY 和 Sp1 等经典激活剂，根据它们相对于 TSS 的精确位置激活或抑制转录起始。因此，TF 及其间距共同指导转录起始的位点和频率。更广泛地说，这些发现揭示了相似的 TF 结合位点如何根据其空间配置产生不同的基因调控结果，以及 DNA 序列多态性如何导致转录变异和疾病，并强调了 TSS 数据在解码我们基因组的调控信息中的关键作用。