A Computational Biology Approach to Mapping Nucleosomes in Stem Cells

描述（由申请人提供）： 多水平的表观遗传调控对维持胚胎干细胞的多能性状态至关重要。组蛋白修饰和DNA甲基化已被证明控制ES细胞的干细胞性。尽管核小体定位在表观遗传调控中的重要性，但核小体是否以及如何调控干细胞功能仍然定义不清，部分原因是在高等生物中获得高分辨率核小体图谱的技术障碍。最近，我们通过结合一种新的化学作图方法和贝叶斯去卷积算法，获得了一个碱基对分辨率的酵母核小体图谱。目前的项目旨在扩展这种新方法，以绘制小鼠基因组的核小体。使用表达工程组蛋白H4的ES细胞，我们的初步结果证明了构建高等生物高分辨率核小体图谱的可行性。化学作图需要在组蛋白H4的47位引入独特的半胱氨酸，以共价连接巯基反应性铜螯合标记。由于小鼠基因组中存在多个组蛋白H4基因拷贝，该过程变得复杂。因此，我们的第一个目标是开发一种化学作图方案，并在培养的哺乳动物细胞中进行全基因组化学作图。为了基于化学数据确定核小体的中心位置，需要对来自局部重叠核小体的切割信号进行解卷积。我们以前为酵母开发的贝叶斯算法对小鼠数据的计算效率很低。因此，我们的第二个目标是开发一个更有效的计算算法和软件工具。在实现上述目标的基础上，我们将为多能ES细胞和分化的成纤维细胞生成核小体的化学图谱，并通过RNA-seq实验平行定量基因表达。我们将对小鼠基因组的核小体定位的全局特征进行高分辨率分析，并研究核小体如何与其他表观调节因子协调调节基因表达。最后，使用化学核小体图谱，我们的目标是确定抑制性染色质标记H3 K27 me 3对整个基因组核小体定位的影响。总而言之，拟议的工作将以前所未有的细节和准确性描绘胚胎干细胞的核小体景观，为多能细胞状态的表观遗传调控提供新的方面。

DESCRIPTION (provided by applicant): Multiple levels of epigenetic regulation are essential to maintain the pluripotent state of embryonic stem (ES) cells. Histone modification and DNA methylation have been shown to control the stemness of ES cells. Despite the importance of nucleosome positioning in epigenetic regulation, whether and how nucleosomes regulates stem cell functions remains poorly defined, in part due to technical obstacles to obtain high-resolution nucleosome maps in higher organisms. Recently we obtained a yeast nucleosome map at base-pair resolution by combining a novel chemical mapping approach and a Bayesian deconvolution algorithm. This current project seeks to extend this new approach to map nucleosomes for the mouse genome. Using ES cells expressing an engineered histone H4, our preliminary results have demonstrated the feasibility of constructing high-resolution nucleosome maps for higher organisms. The chemical mapping requires introducing a unique cysteine into histone H4 at position 47 to covalently attach a sulfhydryl-reactive copper-chelating label. This procedure is complicated by existence of multiple copies of histone H4 genes in the mouse genome. Thus our first aim is to develop a chemical mapping protocol, and carry out genome-wide chemical mapping in cultured mammalian cells. To define the center positions of nucleosomes based on chemical data requires deconvolution of cleavage signals from locally overlapping nucleosomes. The Bayesian algorithm we developed previously for yeast is computationally inefficient for the mouse data. Thus our second aim is to develop a more efficient computing algorithm and software tools. With above aims achieved, we will generate chemical maps of nucleosomes for both pluripotent ES cells and differentiated fibroblast cells, and quantify the gene expression in parallel by RNA-seq experiments. We will perform high-resolution analysis on global features of nucleosome positioning for the mouse genome, and investigate how nucleosomes regulate gene expression in coordination with other epi-regulators. Lastly, using chemical nucleosome maps we aim to determine the impact of the repressive chromatin mark H3K27me3 on nucleosome positioning throughout the genome. Taken together, the proposed work will delineate the nucleosome landscape of embryonic stem cells in unprecedented details and accuracy, providing insight into a new aspect of epigenetic regulation of the pluripotent cellular state.