Synthetic STARR-seq reveals how DNA shape and sequence modulate transcriptional output and noise

The binding of transcription factors to short recognition sequences plays a pivotal role in controlling the expression of genes. The sequence and shape characteristics of binding sites influence DNA binding specificity and have also been implicated in modulating the activity of transcription factors downstream of binding. To quantitatively assess the transcriptional activity of tens of thousands of designed synthetic sites in parallel, we developed a synthetic version of STARR-seq (synSTARR-seq). We used the approach to systematically analyze how variations in the recognition sequence of the glucocorticoid receptor (GR) affect transcriptional regulation. Our approach resulted in the identification of a novel highly active functional GR binding sequence and revealed that sequence variation both within and flanking GR’s core binding site can modulate GR activity without apparent changes in DNA binding affinity. Notably, we found that the sequence composition of variants with similar activity profiles was highly diverse. In contrast, groups of variants with similar activity profiles showed specific DNA shape characteristics indicating that DNA shape may be a better predictor of activity than DNA sequence. Finally, using single cell experiments with individual enhancer variants, we obtained clues indicating that the architecture of the response element can independently tune expression mean and cell-to cell variability in gene expression (noise). Together, our studies establish synSTARR as a powerful method to systematically study how DNA sequence and shape modulate transcriptional output and noise.

转录因子与短识别序列的结合，在调控基因表达过程中发挥关键作用。结合位点的序列与形状特征，既影响DNA结合特异性，也被证实可调控结合位点下游转录因子的活性。为实现对数万个人工设计的合成位点的转录活性进行平行定量评估，我们开发了STARR-seq的合成版本（synSTARR-seq）。我们利用该方法系统分析了糖皮质激素受体(GR)识别序列的变异如何影响转录调控。本研究不仅鉴定出一条全新的高活性功能性GR结合序列，还发现GR核心结合位点内部及其侧翼区域的序列变异，可在不显著改变DNA结合亲和力的前提下调控GR的活性。值得注意的是，我们发现活性谱相似的变体，其序列组成具有高度多样性；与之相反，活性谱相似的变体群则展现出特定的DNA形状特征，这表明DNA形状或许比DNA序列更能有效预测转录活性。最后，我们通过针对单个增强子变体的单细胞实验，获得了关键线索：应答元件的结构可独立调控基因表达的均值以及细胞间表达变异（即表达噪声）。综上，本研究确立了synSTARR-seq作为一种强大的研究工具，可系统探究DNA序列与形状如何调控转录输出及表达噪声。