Physics of liquid-drop compartment interaction in gene function

基因组测序已经发展到了可以作为常规工具使用的地步。挑战已经转移到解释我们的基因组--找出它是如何工作的。要回答这个问题，光知道DNA碱基的线性序列是不够的：毕竟，我们体内的不同细胞(如皮肤、心脏、肝脏)包含相同的DNA信息。相比之下，基因组有错综复杂的3D空间组织，对生物环境敏感，并与功能密切相关。毫不奇怪，理解基因行为的一大开放挑战是破译基因组在空间中是如何组织的，以及这种组织如何影响其功能。在哺乳动物细胞内，基因组以染色质的形式存在，染色质是一种由称为核小体的积木组成的聚合物。我们对细胞核内染色质组织的了解目前受到缺乏近距离观察和分子水平的机制信息的限制，这些信息不知道核小体相互作用是如何受到许多高度耦合因素的调控的。去年，突破性实验(Gibson等人，2019，Cell 179,470-484)发现了一个影响染色质结构的关键但尚未探索的参数：由许多短染色质聚合物组成的系统具有进行液-液相分离并形成无膜室的基本能力。我的目标是开发一个染色质的最小粗粒度模型，以阐明染色质的内在属性(如连接体DNA长度、核小体DNA解缠、异质性)的变化如何影响其形成相分离液滴的能力。我的项目将解决的关键开放问题是：相分离结构域中的核小体的行为是什么？我们能解释染色质固有属性的变化导致不同相分离的染色质结构域的出现吗？我们能从核小体的不同行为中预测相分离染色质结构域的中尺度物理性质吗？不同相分离结构域之间不同的核小体行为如何影响不同结构域的相互作用和结合？这项研究将通过以下具体目标和新的方法来实现：建立一种新的低分辨率粗粒染色质模型，研究其液-液分离能力。这个模型将具有每个核小体一珠的分辨率，并将固定在Collepardo小组的高分辨率化学精确染色质模型(每个氨基酸和DNA碱基一个小珠)上，并根据已发表的实验数据(Gibson等人，2019，Cell 179,470-484)进行验证，以纳入一个关键但尚未解释的特征：自发的DNA核小体展开。应用该模型研究了染色质的液-液相分离通过调节固有性质的变化。我将应用上述模型通过直接共存模拟方法计算染色质的全相图。这将使我能够研究连接物DNA长度、离子环境和核小体DNA解缠的变化如何影响相分离的临界温度。将该模型扩展到解释相分离结构蛋白的结合，并用它来理解功能染色质结构域中的核小体组织。这将使我了解结构蛋白与染色质结构中固有特征的结合如何影响染色质的相行为。我们的目标是使用与哺乳动物基因组中活性、非活性和抑制结构域的形成相关的蛋白质来比较我们的结果。所获得的理解将是提出解释不同染色质相分离区域内核小体组织调节的分子机制的核心。

Genome sequencing has advanced to the point that it can now be used as a routine tool. The challenge has moved to interpreting our genome - finding out how it functions. To answer this, knowledge of the linear sequences of DNA bases is not enough: after all, the different cells in our body (e.g. skin, heart, liver) contain the same DNA information. In contrast, genomes have intricate 3D spatial organizations that are sensitive to the biological context and are intimately linked to function. Not surprisingly, one of the big open challenges to understand gene behaviour is deciphering how the genome is organized in space and how this organization influences its function. Inside mammalian cells, the genome is found in the form of chromatin - a polymer made of building blocks called nucleosomes. Our understanding of chromatin organization inside the cell nucleus is currently limited by the lack of 'close up views' and molecular-level mechanistic information of how nucleosome interactions are regulated by many highly coupled factors. Last year, breakthrough experiments (Gibson et al., 2019, Cell 179, 470-484), discovered a crucial, yet unexplored, parameter affecting chromatin structure: a system made of many short chromatin polymers has the fundamental ability to undergo liquid-liquid phase separation and form membraneless compartments. My aim is to develop a minimal coarse-grained model of chromatin to elucidate how changes in intrinsic properties of chromatin (e.g. linker DNA length, nucleosome DNA unwrapping, heterogeneity) module its ability to form phase-separated liquid droplets. Key open questions that my project will address are: What is the behaviour of nucleosomes inside phase separated domains? Can we explain the emergence of different phase-separated chromatin domains from changes in the intrinsic properties of chromatin? Can we predict the mesoscale physical properties of phase-separated chromatin domains from the differential behaviour of nucleosomes? How does differential nucleosome behaviour among phase-separated domains impact the interaction and coalescence of distinct domains? This research will be achieved through the following specific objectives and novel methodology:O1. To develop a novel low-resolution coarse-grained model of chromatin to investigate its liquid-liquid phase separating ability. This model will have a resolution of one bead per nucleosome and will be anchoredto the high-resolution chemically-accurate chromatin model of the Collepardo group (1 bead per amino acid and DNA basepair) and validated against published experimental data (Gibson et al., 2019, Cell 179, 470- 484), to incorporate a crucial, yet unaccounted for, feature: spontaneous DNA nucleosome unwrapping.O2. To apply the model to investigate changes in chromatin liquid-liquid phase separation by modulation of intrinsic properties. I will apply the model described above to compute the full phase diagrams of chromatin via the Direct Coexistence Simulation method. This will allow me to investigate how changes in linker DNA length, ionic environment, and nucleosome DNA unwrapping influences the critical temperature for phase separation.O3. To extend the model to account for binding of phase-separating architectural proteins and use it to understand nucleosome organization inside functional chromatin domains. This will allow me to understand how binding of architectural proteins coupled to intrinsic characteristics in chromatin structure influence the phase behaviour of chromatin. We will aim to compare our results using proteins that are correlated with the formation of active, inactive, and repressed domains in mammalian genomes. The understanding gained will be central to propose molecular mechanisms that explain the regulation of nucleosome organization inside structurally different chromatin phase-separated domains.