Epigenetic control of developmental gene regulation

项目摘要 虽然多种表观遗传过程有助于发育基因调控，但其机制 它们在谱系特化过程中重新连接基因调控网络通常是不清楚的。表观遗传基因缺陷 调节导致发育障碍和疾病。核RNA，包括多种类型的 非编码RNA和编码基因的新生转录物有助于调节表观遗传景观， 发育基因调控我们已经集中在功能的R-环-RNA/DNA杂合体，大多数 当新生的转录本与它们的DNA模板杂交时，在表观遗传基因的控制下， 在小鼠胚胎干细胞（mESC）的调控。我们先前发现R环调节结合 Tip 60-p400染色质重塑复合物的缺失和mESC中R环的部分缺失损害了 区分的精确度。然而，R环在分化中的确切作用尚不清楚。我们最近 采取系统和公正的方法来确定R环如何影响mESC的表观基因组， 揭示R环的缺失如何破坏分化。此外，我们发现了R环结合蛋白， 可能会调节它们的一些调节功能。我们进一步开发了新的单细胞基因组工具 有必要揭示R环和其他类别的核RNA如何在不同的细胞中发挥作用， 血统在这里，我们提出了一种新的R环结合蛋白的功能特点， 基因调控，是细胞增殖所必需的。此外，我们还将研究R环在 转录延伸、保真度和RNA加工。最后，我们将利用我们开发的新工具， 同时分析表观遗传特征，以确定R环和表观遗传标记的位置， 原肠胚形成，其中三个初级胚层由多能祖细胞建立。根据这一 在体内图谱中，我们将使用可诱导RNaseH 1 mESC系来破坏ESC分化模型中的R环， 揭示了它们控制发育基因的细胞类型特异性靶点和机制 表情总之，这些研究将揭示R环依赖基因的关键介导因子的功能 调节，R环调节对转录保真度至关重要的表观遗传标记的机制，以及 R环的发育目标这些研究将大大扩展我们对RNA的理解- 介导的基因调控及其在建立哺乳动物身体计划中的作用。

PROJECT SUMMARY Although multiple epigenetic processes contribute to developmental gene regulation, the mechanisms by which they rewire gene regulatory networks during lineage specification are often unclear. Defects in epigenetic gene regulation contribute to developmental disorders and disease. Nuclear RNAs, including multiple classes of non-coding RNAs and nascent transcripts of coding genes help regulate the epigenetic landscape and facilitate developmental gene regulation. We have focused on the functions of R-loops—RNA/DNA hybrids that most often arise when nascent transcripts hybridize to their DNA templates—in the control of epigenetic gene regulation in mouse embryonic stem cells (mESCs). We previously discovered that R-loops modulate binding of the Tip60-p400 chromatin remodeling complex and partial depletion of R-loops from mESCs impairs the fidelity of differentiation. However, the precise roles of R-loops in differentiation are unclear. We have recently taken systematic and unbiased approaches to identify how R-loops impact the epigenome of mESCs and uncover how depletion of R-loops disrupts differentiation. In addition, we uncovered R-loop binding proteins that likely mediate some of their regulatory functions. We further developed new, single-cell genomic tools necessary to uncover how R-loops and other classes of nuclear RNAs meditate their effects in different lineages. Here, we propose to characterize the effects of one novel R-loop binding protein that functions in gene regulation and is essential for cell proliferation. In addition, we will examine the roles of R-loops in transcription elongation, fidelity, and RNA processing. Finally, we will leverage novel tools we developed for simultaneous profiling of epigenetic features to identify the locations of R-loops and epigenetic marks during gastrulation, where the three primary germ layers are established from pluripotent progenitors. Drawing on this in vivo atlas, we will use an inducible RNaseH1 mESC line to disrupt R-loops in an ESC differentiation model, uncovering their cell type-specific targets and mechanisms by which they control developmental gene expression. Together, these studies will uncover the functions of a key mediator of R-loop dependent gene regulation, the mechanisms by which R-loops modulate epigenetic marks critical for transcriptional fidelity, and the developmental targets of R-loops. These studies will significantly expand our understanding of RNA- mediated gene regulation and its roles in establishment of the mammalian body plan.