Physics of liquid-drop compartment interaction in gene function

基因组测序已经发展到现在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-找出它如何运作。要回答这个问题，仅仅知道DNA碱基的线性序列是不够的：毕竟，我们身体中的不同细胞（例如皮肤，心脏，肝脏）包含相同的DNA信息。相比之下，基因组具有复杂的3D空间组织，对生物背景敏感，并与功能密切相关。毫不奇怪，理解基因行为的一个巨大挑战是破译基因组如何在空间中组织以及这种组织如何影响其功能。在哺乳动物细胞内，基因组是以染色质的形式存在的，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理解目前受到缺乏“近距离观察”和核小体相互作用如何受许多高度耦合因子调控的分子水平机制信息的限制。去年，突破性的实验（吉布森等人，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区室的基本能力。我的目标是开发一个最小的粗粒度模型的染色质，以阐明如何改变染色质的内在属性（如连接DNA长度，核小体DNA解包裹，异质性）模块的能力，形成相分离的液滴。我的项目将解决的关键开放问题是：核小体在相分离域中的行为是什么？我们能否从染色质内在性质的变化中解释不同相分离的染色质结构域的出现？我们能否从核小体的差异行为预测相分离的染色质结构域的介观物理性质？相分离结构域之间的核小体差异行为如何影响不同结构域的相互作用和合并？这项研究将通过以下具体目标和新的方法来实现：O 1。建立一种新的低分辨率粗粒染色质模型，研究其液-液相分离能力。该模型将具有每个核小体一个珠的分辨率，并将被复制到Collepardo组的高分辨率化学精确染色质模型（每个氨基酸和DNA碱基对一个珠），并针对已发表的实验数据进行验证（吉布森等人，2019，Cell 179，470- 484），以结合一个关键的但未解释的功能：自发DNA核小体解包。应用该模型研究染色质液-液相分离的内在性质的改变。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。这将使我能够研究如何改变连接DNA的长度，离子环境，和核小体DNA解包影响相分离的临界温度。扩展该模型以解释相分离结构蛋白的结合，并使用它来理解功能染色质结构域内的核小体组织。这将使我了解如何结合的建筑蛋白质耦合到染色质结构的内在特征影响染色质的相行为。我们的目标是比较我们的结果，使用蛋白质，与形成的活性，非活性和抑制结构域在哺乳动物基因组。所获得的理解将是中央提出的分子机制，解释结构不同的染色质相分离的结构域内的核小体组织的调节。

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.