Non-linear transcriptional responses to gradual modulation of transcription factor dosage

Genomic loci associated with common traits and diseases are typically non-coding and likely impact gene expression, sometimes coinciding with rare loss-of-function variants in the target gene. However, our understanding of how gradual changes in gene dosage affect molecular, cellular, and organismal traits is currently limited. To address this gap, we induced gradual changes in gene expression of four genes using CRISPR activation and inactivation. Downstream transcriptional consequences of dosage modulation of three master trans-regulators associated with blood cell traits (GFI1B, NFE2, and MYB) was examined using targeted single-cell multimodal sequencing. We showed that guide tiling around the TSS is the most effective way to modulate cis gene expression across a wide range of fold-changes, with further effects from chromatin accessibility and histone marks that differ between the inhibition and activation systems. Our single-cell data allowed us to precisely detect subtle to large gene expression changes in dozens of trans genes, revealing that many responses to dosage changes of these three TFs are non-linear, including non-monotonic behaviours, even when constraining the fold-changes of the master regulators to a copy number gain or loss. We found that the dosage properties are linked to gene constraint and that some of these non-linear responses are enriched for disease and GWAS genes. Overall, our study provides a straightforward and scalable method to precisely modulate gene expression and gain insights into its downstream consequences at high resolution. Overall design: ECCITE-seq experiments obtaining three different modalities from a single cell: Protein Hashes (HTO) to identify CRISPRi or CRISPRa cell line of origin, cDNA as measurement of RNA levels and sgRNAs (GDO) to ienditfy the unique loci perturbation

与常见性状及疾病相关的基因组位点（genomic loci）通常为非编码序列，且大概率会影响基因表达，其区域有时会与靶基因内的罕见功能丧失变异（loss-of-function variant）重合。然而，目前学界对于基因剂量的渐进变化如何影响分子、细胞及机体性状的机制仍知之甚少。为填补这一研究空白，我们利用CRISPR激活（CRISPR activation）与CRISPR失活（CRISPR inactivation）技术，对4个基因的表达量进行渐进式调控。我们针对3个与血细胞性状相关的核心反式调控因子（trans-regulator）——GFI1B、NFE2及MYB，通过靶向单细胞多模态测序（targeted single-cell multimodal sequencing），分析了其剂量调控带来的下游转录变化。研究结果表明，在转录起始位点（transcription start site, TSS）周围设计向导RNA平铺文库，是在宽幅倍数变化范围内调控顺式基因表达的最有效手段；且染色质开放性（chromatin accessibility）与组蛋白修饰（histone marks）会进一步对调控效果产生影响，且这种影响在激活与抑制系统中存在差异。我们的单细胞数据能够精准检测数十个反式调控靶基因中从细微到显著的基因表达变化，结果显示，即使将核心转录因子（transcription factor, TFs）的剂量变化限制为拷贝数增加或缺失，这3个转录因子的剂量响应大多呈现非线性特征，包括非单调的行为模式。我们还发现，基因的剂量特性与基因约束性（gene constraint）相关，且部分此类非线性响应在疾病相关基因及全基因组关联分析（Genome-Wide Association Study, GWAS）基因中存在富集。总体而言，本研究提供了一种简便且可扩展的方法，能够精准调控基因表达，并在高分辨率下解析其下游调控效应。实验设计：采用ECCITE-seq技术从单个细胞中获取三种不同模态的数据：蛋白标签（Protein Hashes, HTO）用于识别CRISPR干扰（CRISPRi）或CRISPR激活（CRISPRa）细胞系的来源；互补脱氧核糖核酸（complementary DNA, cDNA）用于检测RNA水平；单引导RNA（single-guide RNAs, sgRNAs，原文标注为GDO）用于识别唯一的位点扰动信息。