Single-cell and single-molecule profiling of protein-DNA interactions by MACHA

通过 MACHA 对蛋白质-DNA 相互作用进行单细胞和单分子分析

• 批准号: 10593507
• 负责人: Kohta Ikegami
• 金额: $ 21.86万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-16 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10593507
• 关键词: Adopted; Adult; Antibodies; Bacteria; Binding; Binding Proteins; Binding Sites; CCCTC-binding factor; Cell Nucleus; Cell Separation; Cells; ChIP-seq; Chimeric Proteins; Chromatin; Chromosome Structures; Complex; Cytosine; DNA; DNA Binding; DNA Repair; DNA sequencing; DNA-Protein Interaction; Data; Detection; Development; Dinucleoside Phosphates; Enhancers; Event; Frequencies; Future; Gene Expression Profile; Gene Expression Profiling; Genetic Transcription; Heart; Heterochromatin; Human Cell Line; Individual; JUN gene; Lamin Type A; Lead; Length; Libraries; Life; Maps; Measurement; Measures; Methods; Methylation; Methyltransferase; Preparation; Procedures; Process; Proteins; Protocols documentation; Reaction; Recombinants; Recovery; Repression; Resolution; S-Adenosylmethionine; Sampling; Site; Specificity; Tissues; Transcription Coactivator; base; bisulfite; bisulfite sequencing; cell type; chromatin protein; computational pipelines; detection sensitivity; experimental study; genome-wide; histone modification; in vitro activity; induced pluripotent stem cell derived cardiomyocytes; interest; nanopore; novel; protein profiling; single molecule; transcription factor

Summary Protein-DNA interactions are fundamental to DNA-dependent processes such as transcription, replication, DNA repair, and chromosome organization in all domains of cellular life. Enrichment-based methods to detect protein-DNA interactions are widely used to obtain genome-wide high-resolution maps of transcription factor binding and chromatin states. However, these methods have severe shortcomings when applied to single cells. In single-cell applications, enrichment-based methods cannot distinguish between unbound DNA and unsuccessful recovery of protein-bound DNA since both lead to absent DNA fragments and the sparse data makes it difficult or impossible to obtain cell-type-resolved binding profiles of transcription factors starting from complex tissues. This limitation significantly reduces the information content of single-cell data. MACHA will detect protein-DNA interactions in cells as cytosine methylation catalyzed by an exogenous GpC methyltransferase (GpCMTase) that is directed to protein-bound DNA by a primary antibody recognizing the protein of interest.

总结 蛋白质-DNA相互作用是DNA依赖性过程的基础，例如转录，复制， DNA修复和染色体组织在细胞生命的所有领域。基于富集的检测方法 蛋白质-DNA相互作用被广泛用于获得转录因子的全基因组高分辨率图谱 结合和染色质状态。然而，这些方法在应用于单细胞时具有严重的缺点。 在单细胞应用中，基于富集的方法不能区分未结合的DNA和未结合的DNA。 蛋白质结合DNA的不成功回收，因为两者都导致DNA片段缺失和数据稀疏 这使得难以或不可能获得起始于转录因子的细胞类型分辨的结合谱， 复杂的组织这种限制大大减少了单细胞数据的信息内容。MACHA将 检测细胞中蛋白质-DNA相互作用，如由外源GpC催化的胞嘧啶甲基化 甲基转移酶（GpCMTase），其通过识别甲基转移酶（GpCMTase）的第一抗体导向蛋白质结合的DNA。 感兴趣的蛋白质