Project 2: Molecular Modulation of Chromatin and Nuclear Structure in Cancer

摘要(项目2) 项目2试图阐明增强子功能障碍在调节脑血管紧张素转换酶时空组织中的作用。 染色质与癌症中的信息传递。多条遗传和生物学证据表明， 部分急性髓系白血病(AML)和骨髓发育不良(MDS)患者患有增强子 功能障碍。我们假设增强子功能障碍会导致染色质的物理状态改变， 包括3-D循环的异常状态，导致转录、启动和 拉长复合体，破坏细胞内基因表达的正常调节。的后遗症 这些事件是MDS和AML前体的无效造血和克隆性扩增。新兴 CRISPR技术，复杂的染色质确认捕获和全基因组染色质调查 纳米细胞计数和动物核心将被用来阐明染色质的异常结构和功能。 初步研究将检验这一假设，即染色质修饰物、粘附素和MLL3的表达减少 编码特定的组蛋白甲基转移酶，导致细胞分化和 髓系和红系细胞系的增殖和细胞因子引导的分化。这将会实现的 通过使用功能缺失突变体来探索这些染色质调节剂在细胞增殖、细胞周期中的作用 调控、细胞凋亡、DNA修复和基因表达。然后，该模型系统将用于关联 这些生物结果通过测量它们对染色质构象变化的影响 增强子/启动子环，染色体区划和折叠。RNA-Seq的大数据集， 芯片序列和“5C”染色体构象捕获数据将被整合，并建立正常和 恶性染色质将被构建来解释这些染色质调节器的单个等位基因的丢失是如何 可以对细胞进行重新编程，并将其推上恶性程度更高的道路。这些体外观察将是 进一步使用正常骨髓生成的体内模型、粘附素和MLI3丢失的动物模型进行测试，以及 分析原生人类标本，后者与PDX肿瘤模型核心合作。最后我们 将确定异常的染色质配置是否会导致对表观遗传靶向性的易感性增强 毒品。从这些研究中获得的关于染色质变化的功能后果的见解 千基尺度的结构将与通过研究离子浓度的影响而产生的结构相关联 在改变恶性细胞染色质结构中的作用(项目1)和凝聚素介导的折叠在肿瘤中的作用 大数据库和染色体尺度上的染色质结构和功能(项目3)。

ABSTRACT (PROJECT 2) Project 2 seeks to elucidate the role of enhancer dysfunction in modulating the spatio-temporal organization of chromatin and information transfer in cancer. Multiple lines of genetic and biological evidence suggest that a subset of cases of acute myeloid leukemia (AML) and myelodysplasia (MDS) suffers from enhancer dysfunction. We hypothesize that enhancer dysfunction leads to an altered physical state of chromatin, including an abnormal state of 3-D looping, resulting in aberrant formation of transcriptional, initiating and elongating complexes and destroying the normal regulation of gene expression in the cell. The sequelae of these events are ineffective hematopoiesis and clonal expansion of MDS and AML precursors. Emerging CRISPR technology, sophisticated chromatin confirmation capture and genome wide chromatin surveys and the nanocytometry and animal cores will be used to elucidate the aberrant structure and function of chromatin. Initial studies will test the hypothesis that reduced expression of chromatin modifiers, cohesin and MLL3, which encodes a specific histone methyl transferase, leads to an imbalance between cell differentiation and proliferation and cytokine-guided differentiation in myeloid and erythroid cell lines. This will be accomplished by using loss-of-function mutants to probe the role of these chromatin modulators in cell proliferation, cell cycle regulation, apoptosis, and DNA repair and gene expression. This model system will then be used to relate these biological outcomes to changes in chromatin conformation by measuring their impact on enhancer/promoter looping, chromosomal compartmentalization and folding. Large data sets of RNA-Seq, ChIP seq and “5C” chromosome conformation capture data will be integrated and 3D models of the normal and malignant chromatin will be constructed to explain how the loss of a single allele of these chromatin regulators can reprogram the cell and push it on the pathway to over malignancy. These in vitro observations will be further tested using an in vivo model of normal myelopoiesis, an animal model of cohesin and MLL3 loss, and analysis of primary human specimens, the latter in collaboration with the PDX Tumor Model Core. Finally we will determine if aberrant chromatin configurations lead to enhanced susceptibility to epigenetically targeted drugs. The insights gained from these studies of the functional consequences of changes in chromatin structure at the kilobase-scale will interlock with those generated by studying the impact of ion concentrations in changing chromatin structure in malignant cells (Project 1) and the role of condensin-mediated folding in chromatin structure and function at the megabase and chromosomal scale (Project 3).