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

摘要（项目2） 项目2旨在阐明增强子功能障碍在调节细胞的时空组织中的作用。 染色质和癌症中的信息传递。多种遗传学和生物学证据表明， 急性髓性白血病（AML）和骨髓增生异常（MDS）病例的子集患有增强子， 功能障碍我们假设增强子功能障碍导致染色质的物理状态改变， 包括三维环的异常状态，导致转录、起始和 延长复合物并破坏细胞中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， 纳米细胞计数法和动物核心将用于阐明染色质的异常结构和功能。 最初的研究将检验这一假设，即减少染色质修饰剂、粘附素和MLL 3的表达， 编码一种特定的组蛋白甲基转移酶，导致细胞分化和 在骨髓细胞系和红细胞系中的增殖和甘氨酸引导的分化。这将是完成 通过使用功能丧失突变体来探测这些染色质调节剂在细胞增殖、细胞周期 调节、凋亡和DNA修复和基因表达。然后，该模型系统将用于关联 这些生物学结果通过测量它们对染色质构象变化的影响， 增强子/启动子成环、染色体区室化和折叠。RNA-Seq的大数据集， ChIP seq和“5C”染色体构象捕获数据将被整合，正常和非正常染色体的3D模型将被整合。 恶性染色质将被构建来解释这些染色质调节因子的单个等位基因的丢失是如何 可以重新编程细胞并将其推向过度恶性的途径。这些体外观察结果将 使用正常骨髓生成的体内模型、粘附素和MLL 3损失的动物模型进一步测试，以及 主要人类标本的分析，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构型是否会导致对表观遗传靶向的 毒品从这些染色质变化的功能后果的研究中获得的见解 在离子浓度影响的研究中， 在改变恶性细胞染色质结构中的作用（项目1）以及缩合蛋白介导的折叠在 染色质结构和功能在巨噬细胞和染色体规模（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).