Reconstructing dynamic epigenetic genome partitioning in single stem cells

项目摘要/摘要 对于多细胞生物体来说，要保持独特和专门的细胞类型，给定谱系的基因组具有 被分割，使不想要的发展计划自然地被压抑。此分区是 这是由一种叫做异染色质的核超微结构完成的。我们现在知道，在干细胞中，基因组 分割是高度动态的，因为先前存在的小的异染色质区域逐渐扩大(“扩散”) 在差异化方面。这一动态行为与我们先前的理解形成了鲜明对比，这表明 异染色质区域由编码“开始”和“停止”位置的硬连线DNA序列分隔。然而，这 动态的异染色质扩散是正常分化所绝对需要的，而我们不需要 理解它是如何机械地完成或由细胞调节的。在这里，我们建议揭开 胚胎干细胞分化中异染色质动态扩散的生化和遗传学基础。 动态扩散反应仍然是不透明的，因为它没有在适当的长度被寻址 刻度和时间刻度。异染色质的扩散发生在染色体的延伸上，100秒到1000秒 核小体，并超过一定时间窗后的S相。以前的研究主要集中在记录 全基因组效应，在静态快照中获得。为了在正确的长度上捕捉过程的动态- 和时间尺度，我们将部署两个尖端的、专门建造的试验台：1)我们将跟踪异染色质 随着分化程序的诱导，在单个干细胞中实时传播。这将被启用 通过我们最近验证的基于多重荧光报告的异染色质扩散传感器。动能 到目前为止还没有被视觉化。2)我们将在生物化学上重建 使用模块化条码染色质试剂的异染色质铺展工艺。我们将确定 组蛋白甲基化的传播动力学、扩散信号和边界的生化活性 停止这一进程的要素。到目前为止，扩散反应还没有成功地重建。 使用这些试验床，我们可以提出几个关于动态的、异染色质驱动的基因组的关键问题 分割：细胞如何调节动态传播？一种假设是扩散反应 它本身就是调谐的。另一种假说是，通过移除“块”，即通过拆除，来促进传播 扩展边界，或重新排列染色体结构区域。我们的试验台将允许我们 区分这些假设。此外，我们将开始解决这样一个问题：传播 一个分化的驱动力，或它的结果，利用我们的单细胞系统的精致分辨率。 最后，我们打算使用动态异染色质扩散作为识别和补救低效的一种手段 从诱导的多能细胞中分化出谱系。我们将利用我们的试验床组合来设计 人工调节随意传播反应的工具，以实现遗传稳定的世系决定 并为再生医学提供高度可预测的分化。

Project Summary/Abstract For multicellular organisms to maintain distinct and specialized cell types, the genomes of a given lineage have to be partitioned such that unwanted developmental programs become heritably repressed. This partitioning is carried out by a nuclear ultrastructure called heterochromatin. We now understand that in stem cells, genome partitioning is highly dynamic, in that pre-existing small heterochromatin regions progressively expand (“spread”) in differentiation. This dynamic behavior contrasts with our prior understanding, which indicated that heterochromatin regions are delimited by hard-wired, DNA sequence encoded “start” and “stop” sites. Yet, this dynamic heterochromatin spreading is absolutely required for normal differentiation, and we do not understand how it is mechanistically accomplished or regulated by the cell. Here, we propose to uncover the biochemical and genetic basis of dynamic heterochromatin spreading in embryonic stem cell differentiation. The dynamic spreading reaction remains opaque because it has not been addressed at the appropriate length scale and timescale. Heterochromatin spreading occurs over stretches of the chromosome, 100s to 1000s of nucleosomes, and over a certain time window after S-phase. Previous studies have focused on documenting genome-wide effects, obtained in static snapshots. To capture the dynamics of the process at the correct length- and timescale, we will deploy two cutting edge, purpose built testbeds: 1) We will follow heterochromatin spreading in real time in single stem cells, following the induction of differentiation programs. This will be enabled by a multiple fluorescent reporter based heterochromatin spreading sensor we recently validated. The dynamics of long-range spreading have to date not been visualized. 2) We will biochemically reconstitute the heterochromatin spreading process using modular, barcoded chromatin reagents. We will determine the propagation kinetics of histone methylation, the spreading signal, and the biochemical activity of boundary elements in stopping this process. The spreading reaction has to date not been successfully reconstituted. Using these testbeds, we can ask several critical questions about dynamic, heterochromatin-driven genome partitioning: How does the cell regulate dynamic spreading? One hypothesis would be that the spreading reaction itself is tuned. An alternative hypothesis is that spreading is promoted by removal of a “block”, i.e. via dismantling of spreading boundaries, or rearranging chromosome structural territories. Our testbeds will allow us to distinguish between these hypotheses. Additionally, we will begin to address the question whether spreading is a driver of differentiation, or a consequence of it, exploiting the exquisite resolution of our single cell system. Finally, we intend to use dynamic heterochromatin spreading as a means to identify and remedy inefficiencies in differentiating lineages from induced pluripotent cells. We will use the combination of our testbeds to devise tools to artificially tune the spreading reaction at will, in order to achieve genetically stable lineage decisions and provide highly predictable differentiation for regenerative medicine.