Advancing an Innovative NGS Approach to Discover and Investigate Histone Tail Proteolysis

推进创新的 NGS 方法来发现和研究组蛋白尾部蛋白水解

• 批准号: 10575717
• 负责人: JUDD C RICE
• 金额: $ 24.75万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-17 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10575717
• 关键词: ATAC-seq; Adipocytes; Binding; Biological Process; Cell Line; Cells; Chromatin; Chromatin Structure; Communities; Computer Analysis; Data Set; Development; Disease; Epigenetic Process; Eukaryota; Event; Excision; Fatty acid glycerol esters; Future; Gene Activation; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomics; Goals; Health; Heterogeneity; Histone H3; Histones; Human; In Vitro; Maps; Methods; Modeling; Modification; Multipotent Stem Cells; Mus; Muscle Cells; Muscle Fibers; N-terminal; Nucleosome Core Particle; Nucleosomes; Osteogenesis; Outcome Study; Pathway interactions; Peptide Hydrolases; Post-Translational Protein Processing; Proteins; Proteolysis; Regulation; Reporting; Role; Series; Site; Specific qualifier value; Structure; Tail; Testing; Transcriptional Activation; United States National Institutes of Health; bone; cell type; chromatin remodeling; developmental disease; epigenetic regulation; epigenomics; genome-wide; histone modification; human disease; human tissue; innovation; insight; lipid biosynthesis; multipotent cell; myogenesis; next generation sequence data; novel; osteoblast differentiation; precursor cell; progenitor; programs; public health relevance; recruit; stem cells; transcription factor; transcriptome sequencing

ABSTRACT One of the epigenomic changes frequently observed during eukaryotic differentiation is the proteolysis of the histone H3 N-terminal tail (H3NT) within chromatin. Although H3NT proteolysis was first described over 60 years ago and is a common feature of diverse eukaryotic developmental pathways, the functional significance of this programmed epigenetic event remains largely undetermined. Progress in the field has been significantly hindered by the lack of methods to identify the specific loci targeted for H3NT proteolysis. To overcome this barrier we pioneered the first genome-wide method to discover and investigate H3NT-cleaved (H3cl) loci, called ChIPac-Seq. The goal of this proposal is to optimize the technical and computational aspects of ChIPac- Seq, which would support future efforts to adapt the ChIPac-Seq approach for high-throughput applications. To this end we will leverage ChIPac-Seq to discover the cell type-specific loci selectively targeted for H3NT proteolysis in differentiated osteoblasts, myocytes and adipocytes derived from the same multipotent progenitor cell in vitro. The expected low concordance of H3cl loci between cell types provides the unique opportunity to optimize the ChIPac-Seq computational analysis to identify bona fide H3cl loci with the highest degree of rigor and to simplify and streamline the pipeline for use by the broad scientific community. Furthermore, the direct functional effects of H3NT proteolysis in generating specific epigenetic changes at the H3cl loci in terms of histone modifications, chromatin structure and transcription factor binding will be examined by leveraging the wealth of publically available NGS datasets. The anticipated outcomes of this study will yield transformative insights into the sites and mechanistic functions of H3NT proteolysis as a novel epigenetic regulator and, importantly, produce an optimized ChIPac-Seq pipeline for use by the general scientific community.

抽象的 真核细胞分化过程中经常观察到的表观基因组变化之一是蛋白质水解 染色质内的组蛋白 H3 N 末端尾 (H3NT)。尽管 H3NT 蛋白水解作用首次被描述超过 60 多年前，这是多种真核发育途径的共同特征，其功能意义 这种程序化的表观遗传事件的发生在很大程度上仍然是未知的。该领域进展显着 由于缺乏识别 H3NT 蛋白水解的特定位点的方法而受到阻碍。为了克服这个 我们首创了第一个全基因组方法来发现和研究 H3NT 裂解 (H3cl) 位点， 称为 ChIPac-Seq。该提案的目标是优化 ChIPac 的技术和计算方面 Seq，这将支持未来将 ChIPac-Seq 方法应用于高通量应用的努力。到 为此，我们将利用 ChIPac-Seq 来发现选择性针对 H3NT 的细胞类型特异性位点 来自同一多能细胞的分化成骨细胞、肌细胞和脂肪细胞中的蛋白水解作用 体外祖细胞。细胞类型之间 H3cl 位点的预期低一致性提供了独特的 有机会优化 ChIPac-Seq 计算分析，以识别真实的 H3cl 位点 严格程度，并简化和精简管道以供广大科学界使用。 此外，H3NT 蛋白水解在产生特定表观遗传变化方面的直接功能作用 将检查组蛋白修饰、染色质结构和转录因子结合方面的 H3cl 位点 通过利用丰富的公开 NGS 数据集。这项研究的预期结果将产生 对 H3NT 蛋白水解作用位点和机制功能的变革性见解作为一种新型表观遗传学 调节器，重要的是，产生优化的 ChIPac-Seq 管道供一般科学界使用 社区。