Spatial Epigenomic Profiling of Immune Cell Signatures at Subcellular Resolution in Health and Disease

亚细胞免疫细胞特征的空间表观组学研究 关于健康和疾病的决议 超过10%的儿童癌症仍然无法治愈，需要新的治疗方法。表观遗传治疗 由于其特异性和低毒性，值得特别关注。在这里，我计划探索表观遗传学的概况 在Garry的指导下正常发育和血癌患者的免疫和癌细胞 诺兰，单细胞蛋白质组学技术开发，与Howard Chang合作 实施表观基因组学方法，如染色体可及性分析，并与Kara Davis一起 急性淋巴细胞白血病(ALL)耐药B细胞亚型的表观遗传学研究表观遗传 测量仅限于整体水平测序和连接分析或有限数量的成像 记号笔。为了解决这些局限性，我将使用一种新兴的三维蛋白质组成像 这种技术被称为3D多重离子束成像(MIBI)或3D MIBI。表观遗传学 3D MIBI的研究得益于高度多路传输(多达100个标记)和超分辨率成像 能力(20 nm x-y；5 nm z分辨率)，提供了研究基因组位置的令人兴奋的机会，甲基化 DNA、蛋白质因素和染色体可获得性，所有这些都在单一免疫和 异常(白血病)细胞。为了系统地确定表观遗传状态，我计划利用克隆B细胞系来 通过一种方法破译包括染色质状态、蛋白质因子和修饰因子在内的表观遗传成分的可变性 50标记3D MIBI面板(目标1)。这些实验将显示表观遗传因素的分布(线性或 它们的表达水平和染色质状态的空间变异(全局或局部)。那我会的 用从6种不同正常人骨髓抽提物中分离的原代B细胞进行实验 受试者(目标2)。我将把每个B细胞亚型的表观遗传特征与相应的发育联系起来 状态(祖先、前、后或成熟)。然后，我将对OP9进行原代B细胞的体外共培养 培养1-6周的基质细胞，随后将通过3D MIBI进行固定和轮廓。这些 扰动实验将显示来自邻近细胞的信号事件如何驱动必要的表观遗传 达到B细胞亚群所需的条件。最后，我将转向分离的初级B细胞 来自20名新诊断的ALL患者(目标3)。我将解剖分化和空间表观基因组 骨髓抽吸物中反应性B细胞亚群和耐药B细胞亚型的重塑 使用OP9联合培养。这些将显示治疗耐药性是如何由单一的表观遗传状态或 多个不同的表观遗传特征。然后我将筛查组蛋白脱乙酰酶抑制剂(HDACi)在同一 急性淋巴细胞白血病和基质细胞B细胞亚群的共培养。通过改变抑制浓度和持续时间 条件下，我将剖析表观遗传药物在空间染色质重塑中的作用 所有的表观遗传疗法。总之，这些实验将阐明表观遗传编程的作用 用于正常受试者和血癌患者免疫细胞信号的癌症治疗应用。

SPATIAL EPIGENOMIC PROFILING OF IMMUNE CELL SIGNATURES AT SUBCELLULAR RESOLUTION IN HEALTH AND DISEASE More than ten percent of childhood cancers are still incurable and need novel therapies. Epigenetic treatments deserve special attention with their specificity and reduced toxicity. Here I plan to explore epigenetic profiles of immune and cancer cells in normal development and blood cancer patients under the mentorship of Garry Nolan for single cell proteomics technology development, in collaboration with Howard Chang for implementation of epigenomic methods such as chromosome accessibility assays, and with Kara Davis for epigenetics studies of treatment resistant B cell subtypes in acute lymphoblastic leukemia (ALL). Epigenetic measurements have been limited to bulk level sequencing and ligation assays or limited number of imaging markers. To address these limitations, I will use an emerging three dimensional (3D) proteomic imaging technology in individual cells, termed as 3D Multiplexed ion beam imaging (MIBI) or 3D MIBI. Epigenetics research by 3D MIBI benefits from high degree multiplexing (up to 100 markers) and super resolution imaging capability (20 nm x-y; 5 nm z resolution), providing exciting opportunities to study genomic sites, methylated DNA, protein factors, and chromosome accessibility, all within the same experiments in single immune and aberrant (leukemic) cells. To systematically determine epigenetic states, I plan to utilize clonal B cell lines to decipher variability of epigenetic components including chromatin states, protein factors and modifiers by a fifty-marker 3D MIBI panel (Aim 1). These experiments will show distribution of epigenetic factors (linear or log-scale) in their expression levels and spatial variations (global or local) in the chromatin states. I will then perform experiments with primary B cells isolated from six different bone marrow aspirates of normal human subjects (Aim 2). I will correlate epigenetic signatures of each B cell subtype to corresponding development state (progenitor, pre, post, or mature). I will then perform an ex vivo co-culture of primary B cells on OP9 stromal cells over 1-6 weeks of culturing, which will be followed by fixation and profiling by 3D MIBI. These perturbation experiments will show how signaling events from neighboring cells drive necessary epigenetic conditions that are required for reaching a B cell subset. Finally, I will turn to primary B cells that are isolated from twenty newly diagnosed ALL patients (Aim 3). I will dissect differentiation and spatial epigenomic remodeling of responder B cell subsets and treatment resistant B cell subtypes from bone marrow aspirates using the OP9 co-culture. These will show how treatment resistance arises from a single epigenetic state or multiple distinct epigenetic signatures. I will then screen Histone deacetylase inhibitors (HDACi) on the same co-culture of B cell subtypes from ALL and stromal cells. By varying concentration and duration of inhibition conditions, I will dissect the role of epigenetic drugs in spatial chromatin remodeling toward development of epigenetic therapies in ALL. Together, these experiments will shed light on the role of epigenetic programming for cancer treatment applications from immune cell signatures in normal subjects and blood cancer patients.