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

摘要 在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。 染色质内组蛋白H3 N-末端尾（H3 NT）。虽然H3 NT蛋白水解首次被描述超过60 年前，是一个共同的特点，不同的真核生物发育途径，功能意义 这一程序化的表观遗传事件仍在很大程度上尚未确定。该领域的进展显著 由于缺乏鉴定H3 NT蛋白水解靶向的特异性位点的方法而受阻。为了克服这个 我们开创了第一个全基因组方法来发现和研究H3 NT切割（H3cl）基因座， 叫做ChIPac-Seq该提案的目标是优化ChIPac的技术和计算方面， Seq，这将支持未来的努力，以适应高通量应用的ChIPac-Seq方法。到 为此，我们将利用ChIPac-Seq来发现选择性靶向H3 NT的细胞类型特异性基因座 在分化的成骨细胞、肌细胞和脂肪细胞中的蛋白质水解， 体外祖细胞。细胞类型之间H3cl基因座的预期低一致性提供了独特的 有机会优化ChIPac-Seq计算分析，以鉴定真正的H3cl基因座， 这是为了提高严谨程度，简化和精简管道，供广大科学界使用。 此外，H3 NT蛋白水解在产生特定的表观遗传变化中的直接功能作用在细胞中被证实。 H3cl基因座在组蛋白修饰，染色质结构和转录因子结合方面将进行检查 通过利用丰富的可用的NGS数据集。这项研究的预期结果将产生 对H3 NT蛋白水解作为一种新的表观遗传学机制的位点和机制功能的变革性见解 调节器，重要的是，产生一个优化的ChIPac-Seq管道，供一般科学 社区

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.