Collaborative Research: URoL: Epigenetics 2: Phase separated genome compartments as drivers of epigenetic phenotypes

在特定的动物中，不同的细胞类型，如心脏细胞和脑细胞，表现出不同的行为，因为它们表达不同的基因集。然而，它们都有基本上相同序列的DNA，因此也就有相同的一组基因。同样的DNA是如何被用来产生不同类型的细胞的？哪些基因是开启的，哪些基因是关闭的，这取决于它们的基本DNA序列是如何包装的。DNA是通过包裹在称为组蛋白的特定蛋白质周围来包装的，以产生称为核小体的珠状结构。然后，一串串的核小体被进一步折叠，以浓缩下面的DNA，使其更难获得。被称为异染色质的结构被认为在压缩核小体串和关闭潜在基因方面特别有效。几年前，人们发现了一种名为HP1蛋白的蛋白质，它是异染色质的核心成分，可以将DNA隔离成液滴，这些液滴在不同的阶段从周围的溶液中分离出来。这一发现为思考DNA包装提供了一种新的方法，同时也提出了新的基本问题，如：这些基于液滴的DNA间隔是如何受细胞信号调节的，以及液滴介导的DNA组织的变化如何在整个动物水平上影响生物学？为了解决这些问题，PIs组建了一个多学科团队，汇集了老鼠生物学、尖端成像技术和先进生物物理方法方面的专业知识。该项目的另一个关键目标是为来自代表性不足社区的中学生提供实践经验，以进行包装DNA的实验。这项研究将：(I)阐明小分子集合如何在整个动物水平上驱动可遗传的变化；(Ii)向中学生介绍科学发现的奇迹。可遗传基因调控的一种主要形式是由异染色质驱动的，它使特定的基因亚群沉默，对细胞分化、环境适应和生物生理学是必不可少的。异染色质可以通过基于相分离的机制形成，这一发现导致了一种想象基因组组织的新范式，其中相分离使基因组隔离成为可能。鉴于这些发现的新颖性，许多基本问题仍然没有得到回答，例如：(I)异染色质相分离背后的物理化学规则是什么；(Ii)相分离赋予了哪些类型的新特性；(Iii)生理后果是什么？解决这些问题需要在多个学科的交叉点上工作。因此，这个项目是在Lomvardas博士研究的小鼠嗅觉受体调节的生理背景下组织的，并使用了由Larabell博士开创的新成像技术和由Narlikar博士开发的生物物理工具。该项目整合了多个尺度的实验询问，从对HP1行为的原子水平研究到对整个小鼠表型的评估。具体地说，PI将研究小鼠嗅觉受体(OR)的表达，这是由特殊的异染室(ORH)控制的。ORH在空间上不同于在着丝粒(PH)附近形成的异染色质，强烈表明有两种不同的相分离状态。不同类群HP1的ORH和PH分别为HP1和HP1。结合使用小鼠遗传学、软X射线断层扫描和定量相分离方法，PI将调查(I)ORH和PH是否具有阻止混合并实现不同生理功能的不同物理化学性质，以及(Ii)这些不同性质是否可能源于HP1和HP1相中形成的亚结构的不同。PI预计，这些研究将阐明HP1和HP1之间的原子尺度差异如何导致液滴结构的中观尺度差异，进而产生明确的生理影响。该项目的一个关键目标也是通过一年一度的暑期工作坊为中学生提供基于异染色质的相分离实验的动手体验。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Different cell types in a given animal, such as heart cells and brain cells display different behaviors because they express different sets of genes. Yet, they all have DNA with essentially the same sequence and thus the same set of genes. How is it that the same DNA is used to generate different cell types? Which genes are on and which genes are off is controlled by how their underlying DNA sequences are packaged. DNA is packaged by wrapping it around specific proteins called histones to generate bead-like structures called nucleosomes. Strings of nucleosomes are then further folded to condense the underlying DNA and make it less accessible. Structures called heterochromatin are thought to be particularly effective at compacting strings of nucleosomes and turning off the underlying genes. A few years ago it was discovered that proteins named HP1 proteins, which are core components of heterochromatin, can sequester DNA into droplets that are separated from the surrounding solution in a different phase. This discovery provides a novel way to think about DNA packaging while also raising new fundamental questions such as: how are these droplet-based DNA compartments regulated by cellular signals and; how do changes in droplet mediated DNA organization impact biology at the level of a whole animal? To address these questions the PIs have assembled a multi-disciplinary team that brings together expertise in mouse biology, cutting-edge imaging technology and advanced biophysical methods. Another key goal of the project is to provide middle school students from underrepresented communities hands-on experience in carrying out experiments with packaged DNA. The research will: (i) shed light on how small collections of molecules can drive heritable changes at the level of a whole animal and; (ii) introduce middle-school students to the wonders of scientific discovery. A major form of heritable gene regulation is driven by heterochromatin, which silences specific subsets of genes and is essential for cellular differentiation, environmental adaptation and organismal physiology. The discovery that heterochromatin can form by phase-separation based mechanisms have led to a new paradigm for imagining genome organization, in which phase-separation enables genome sequestration. Given the novelty of the findings many fundamental questions remain unanswered such as: (i) what are the physico-chemical rules underlying phase-separation by heterochromatin; (ii) what types of emergent properties are conferred by phase-separation and; (iii) what are the physiological consequences? Addressing these questions requires working at the intersection of multiple disciplines. Therefore this project is organized within the physiological context of mouse olfactory receptor regulation as studied by Dr. Lomvardas and uses new imaging technologies pioneered by Dr. Larabell and biophysical tools developed by Dr. Narlikar. The project integrates experimental enquiry across multiple scales, from atomic-level studies of HP1 behavior to assessment of whole mouse phenotypes. Specifically the PIs will study olfactory receptor (OR) expression in mice, which is controlled by specialized heterochromatic compartments (ORH). ORH is spatially distinct from heterochromatin formed near centromeres (PH), strongly indicative of two different phase-separated states. ORH and PH are enriched for different HP1 paralogs, HP1 and HP1, respectively. Using a combination of mouse genetics, soft-Xray tomography and quantitative phase-separation methods the PIs will investigate (i) whether ORH and PH have different physico-chemical properties that prevent mixing and enable distinct physiological functions and, (ii) whether these different properties may arise from differences in the substructures formed within HP1 vs. HP1 phases. The PIs anticipate these studies will illuminate how atomic scale differences in sequence between HP1; and HP1; result in meso-scale differences in droplet structure, which in turn have a defined physiological impact. A key goal of the project is also to provide middle school students a hands-on experience in heterochromatin based phase-separation experiments through an annual summer workshop.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.