Systematic analysis of effects of naturally occurring insertions and deletions that alter transcription factor spacing identifies tolerant and sensitive transcription factor pairs [ChIP-seq]

Regulation of gene expression requires the combinatorial binding of sequence-specific transcription factors (TFs) at promoters and enhancers. Prior studies showed that alterations in the spacing between TF binding sites can influence promoter and enhancer activity. However, the relative importance of TF spacing alterations resulting from naturally occurring insertions and deletions (InDels) has not been systematically analyzed. To address this question, we first characterized the genome-wide spacing relationships of 75 TFs in K562 cells as determined by ChIP-sequencing. We found a dominant pattern of a relaxed range of spacing between collaborative factors, including forty-six factors exclusively exhibiting relaxed spacing with their binding partners. Next, we exploited millions of InDels provided by genetically diverse mouse strains and human individuals to investigate the effects of altered spacing on TF binding and local histone acetylation. Spacing alterations resulting from naturally occurring InDels are generally tolerated in comparison to genetic variants directly affecting TF binding sites. A remarkable range of tolerance was further established for PU.1 and C/EBPß, which exhibit relaxed spacing, by introducing synthetic spacing alterations ranging from 5-bp increase to >30-bp decrease using CRISPR/Cas9 mutagenesis. These findings provide implications for understanding mechanisms underlying enhancer selection and for interpretation of non-coding genetic variation. Overall design: ChIP-seq for wild-type ER-HoxB8 cells

基因表达调控依赖于序列特异性转录因子（transcription factors, TFs）在启动子与增强子区域的组合结合。既往研究表明，转录因子结合位点间的间距改变可影响启动子及增强子的活性。然而，由自然发生的插入缺失变异（insertions and deletions, InDels）所导致的转录因子间距改变的相对重要性，尚未得到系统性分析。
为解答这一科学问题，本研究首先通过染色质免疫共沉淀测序（ChIP-sequencing）技术，解析了K562细胞中75种转录因子的全基因组间距关联模式。我们发现协作因子间存在以宽松间距范围为特征的主导模式，其中46种转录因子与其结合伴侣仅表现出宽松间距特征。
随后，我们利用遗传多样性小鼠品系及人类个体携带的数百万个插入缺失变异，探究了间距改变对转录因子结合及局部组蛋白乙酰化的影响。相较于直接作用于转录因子结合位点的遗传变异，自然发生的插入缺失变异所导致的间距改变通常可被耐受。通过CRISPR/Cas9诱变技术引入从增加5bp到减少超过30bp的人工间距改变，进一步证实了表现出宽松间距特征的PU.1与C/EBPβ具有显著的耐受范围。
上述研究结果为理解增强子选择的潜在机制以及解读非编码遗传变异提供了重要参考依据。整体实验设计：野生型ER-HoxB8细胞的染色质免疫共沉淀测序