Phase Transitions in Chromatin Organization that cause Cancer Progression

项目摘要 尽管测序、成像和筛查技术取得了进展，但细胞核的形态变化 结构仍然被用作癌症的常见和可靠的诊断方法。健康的细胞总是有椭圆形， 细胞核形态光滑，染色质分布明显，而癌细胞的特点是不规则， 核形状参差不齐，染色质分布中断。与此相关的是，最常见的突变之一 人类癌症中的蛋白质是染色质重构体BAF复合体ARID1A的一种成分。这些 突变失去核小体滑动活性，导致转录组改变和癌症进展。作为一名 BAF复合体的结构支架，ARID1A中许多导致癌症的突变导致早期 截断会导致曝气生物滤池复合体的损失。然而，驱动程序突变也存在于相对 ARID1a的分子机制未知的未确定的内在无序区域。 对核组织的调查已经确定了本质上无序的区域的重要性 在决定整个核结构和组织以及形成核体方面的相分离 像核仁和异染色质一样的染色质隔间，但直到最近我们还不能 来控制这一组织，或最终将其与染色质隔间功能联系起来。布兰格温实验室有 开发了光遗传工具，允许控制凝析油的体内生物物理行为。 作为马克癌症研究基金会的一名生命科学研究员，我决定 染色质-染色质交联蛋白HP1a在决定核形状和力学中的作用 已发现的由相分离决定的染色质内含、排除和紧致规则 车厢。在这项提议中，我将扩大我的研究范围，以了解发生在 这些导致癌症的ARID1A突变上的核隔室，以及这是如何与它们的 疾病表型。在目标1中，我将询问决定缩合物如何与染色质相互作用的相互作用， 并利用光触发的相分离来调节活细胞中染色质的紧凑和转录。在……里面 目标2，我将确定染色质聚合物如何引导相分离，以及核刚性如何影响 核体体积和功能输出。在目标3中，在我的共同导师Cigall Kadoch博士的指导下，我 将利用在AIMS 1和2中获得的知识来构建BAF复合蛋白ARID1A的分子机制 和ARID1B在肿瘤发生中的作用，包括它们的IDR在靶向BAF复合体活性中的作用以及 与癌症相关的凝聚行为、序列靶向和转录输出的突变。 在我导师的支持下，以及普林斯顿大学和 在达纳·法伯癌症研究所，我将获得独特的工具，并将接受癌症生物学方面的培训 方法、生物物理学理论和下一代测序分析。这些目标加在一起，将提供一个新的 展望癌症中的核组织，可能导致新的治疗场所。

Project Summary Despite advances in sequencing, imaging and screening technologies, morphological changes in nuclear structure are still utilized as a common and reliable diagnosis of cancer. Healthy cells invariably have ellipsoid, smooth nuclear shape and distinctive chromatin distribution, while cancerous cells are characterized by irregular, jagged nuclear shape and disrupted chromatin distribution. Relatedly, one of the most commonly mutated proteins in human cancers is a component of the chromatin remodeler BAF complex known as ARID1A. These mutations lose nucleosome sliding activity, leading to altered transcriptome and cancer progression. As a structural scaffold of the BAF complex, many of the mutations in ARID1A that cause cancer result in early truncation and lead to loss of BAF complex assembly. However, driver mutations also exist in the relatively uncharacterized intrinsically disordered regions of ARID1A whose molecular mechanism is unknown. Investigations into nuclear organization have identified the importance of intrinsically disordered regions and phase separation in dictating overall nuclear structure and organization, as well as forming nuclear bodies like the nucleolus, and chromatin compartments like heterochromatin, but until recently we have not been able to control this organization, or link it conclusively to chromatin compartment function. The Brangwynne lab has developed optogenetic tools that allow for control of in vivo biophysical behavior of condensates. As a Life Science Research Fellow through the Mark Foundation for Cancer Research, I have determined the role of chromatin-chromatin crosslinking protein HP1a in determining nuclear shape and mechanics, as well as discovered rules for chromatin inclusion, exclusion, and compaction dictated by phase separated compartments. In this proposal I will expand my research to understand the biophysical changes that occur in these nuclear compartments upon ARID1A mutations that drive cancer, and how this is connected to their disease phenotype. In Aim 1, I will interrogate interactions that dictate how condensates interact with chromatin, and utilize light-triggered phase separation to modulate chromatin compaction and transcription in living cells. In Aim 2, I will determine how the chromatin polymer directs phase separation, and how nuclear stiffness influences nuclear body volume and functional output. In Aim 3, under the guidance of my co-mentor Dr. Cigall Kadoch, I will utilize knowledge gained in Aims 1 and 2 to build a molecular mechanism of BAF complex proteins ARID1A and ARID1B in oncogenesis, including the roles of their IDRs in targeting BAF complex activity and effects of cancer-associated mutations on condensation behavior, sequence targeting and transcriptional output. With the support of my mentors and the greater research environment at both Princeton University and the Dana Farber Cancer Institute, I will have access to unique tools, and will receive training in cancer biology methods, biophysical theory, and next-generation sequencing assays. Together, these aims will provide a new perspective on nuclear organization in cancer that may lead to novel venues of therapeutics.