MAE-seq refines regulatory elements across the genome.
MAE-seq refines regulatory elements across the genome.
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DOI：
10.1093/nar/gkad1129
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发表时间：
2024-01-25
影响因子：
14.9
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中科院分区：
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正确的细胞命运决定依赖于调控元件（REs）与其靶基因之间精确的时空全基因组合作。然而，使用不同方法定义的REs长度各不相同，这表明存在序列冗余，并且基因组的背景可能难以理解。我们开发了一种名为MAE-seq（大规模活性增强子测序）的方法来实验鉴定25 bp范围内的功能性REs。本研究利用MAE-seq分别在小鼠胚胎干细胞（mESCs）、C2C12和HEK 293T中鉴定了626879、541617和554826个25-bp增强子。利用约1.6万亿个25 bp DNA片段和筛选120亿个细胞，我们在mESCs中鉴定出626879为活性增强子。对比分析显示，大多数组蛋白修饰数据集都有MAE-Seq位点注释。此外，33.85%（212195）的增强子为新生增强子，未发生表观遗传修饰。有趣的是，在控制新的和已知的增强子时，不同的染色质状态决定了对不同辅因子的需求。验证结果表明，这些25bp序列可以作为一个功能单元，其表达模式与先前定义的较大元件相同或相似。增强的分辨率有助于识别许多细胞特异性增强子并将其准确注释为超级增强子。此外，我们还发现了能够增强基因活性的新元件。通过整合高分辨率的Hi-C数据，55.64%以上的新元件可能与不同的靶基因有远端关联。例如，我们发现Cdh1基因与mESCs中一个新的和两个已知的REs相互作用。利用CRISPR-Cas9研究了这些相互作用的生物学效应，揭示了它们在协调Cdh1基因表达和mESC增殖中的作用。我们的研究提出了一种实验方法，将REs细化到25bp分辨率，提高了基因组注释的精度，并揭示了潜在的基因组背景。这种新颖的方法不仅促进了我们对基因调控的理解，而且为全面探索基因组景观开辟了道路。
Proper cell fate determination relies on precise spatial and temporal genome-wide cooperation between regulatory elements (REs) and their targeted genes. However, the lengths of REs defined using different methods vary, which indicates that there is sequence redundancy and that the context of the genome may be unintelligible. We developed a method called MAE-seq (Massive Active Enhancers by Sequencing) to experimentally identify functional REs at a 25-bp scale. In this study, MAE-seq was used to identify 626879, 541617 and 554826 25-bp enhancers in mouse embryonic stem cells (mESCs), C2C12 and HEK 293T, respectively. Using ∼1.6 trillion 25 bp DNA fragments and screening 12 billion cells, we identified 626879 as active enhancers in mESCs as an example. Comparative analysis revealed that most of the histone modification datasets were annotated by MAE-Seq loci. Furthermore, 33.85% (212195) of the identified enhancers were identified as de novo ones with no epigenetic modification. Intriguingly, distinct chromatin states dictate the requirement for dissimilar cofactors in governing novel and known enhancers. Validation results show that these 25-bp sequences could act as a functional unit, which shows identical or similar expression patterns as the previously defined larger elements, Enhanced resolution facilitated the identification of numerous cell-specific enhancers and their accurate annotation as super enhancers. Moreover, we characterized novel elements capable of augmenting gene activity. By integrating with high-resolution Hi-C data, over 55.64% of novel elements may have a distal association with different targeted genes. For example, we found that the Cdh1 gene interacts with one novel and two known REs in mESCs. The biological effects of these interactions were investigated using CRISPR-Cas9, revealing their role in coordinating Cdh1 gene expression and mESC proliferation. Our study presents an experimental approach to refine the REs at 25-bp resolution, advancing the precision of genome annotation and unveiling the underlying genome context. This novel approach not only advances our understanding of gene regulation but also opens avenues for comprehensive exploration of the genomic landscape.
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