HISTONE H3 METHYLATION AND EPIGENOMICS IN ACUTE LEUKEMIA

急性白血病中的组蛋白 H3 甲基化和表观基因组学

• 批准号: 8581843
• 负责人: Lukas D Wartman
• 金额: $ 15.37万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8581843
• 关键词: 5' Untranslated Regions; Acute Myelocytic Leukemia; Acute Promyelocytic Leukemia; Acute leukemia; Address; Alleles; Animals; Antibodies; Biological Assay; Bone Marrow Cells; Cathepsin G; Cells; ChIP-seq; Complementary DNA; Control Animal; Data; Development; Engineering; Epigenetic Process; Event; Exons; Gene Expression; Genetic Recombination; Genomics; Goals; Health; Hematopoiesis; Hematopoietic; Histone H3; Histones; Human; Journals; Knock-out; Knockout Mice; Lead; Lentivirus Vector; Link; Malignant - descriptor; Malignant Neoplasms; Marrow; Mediating; Mentors; Methodology; Methylation; Modeling; Mus; Mutate; Mutation; Pancytopenia; Penetrance; Phenotype; Physicians; Population; Publishing; RARA gene; RNA; Research; Research Personnel; Role; Sampling; Scientist; Site; Somatic Mutation; Stem cells; Sum; Therapeutic; Training; Training Support; Transgenic Mice; X Chromosome; base; career; cell growth; epigenomics; exome sequencing; genome sequencing; in vivo; leukemia; leukemogenesis; male; mouse model; progenitor; protein expression; research study; self-renewal; skills; stem; transcriptome sequencing; tumor; tumor progression

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to provide the training and support for the applicant to become an independent physician-scientist with a career focused on studying the genomics and epigenomics of acute leukemia. The goals for the applicant include: obtaining practical and didactic training in scientific methodology, refining technical skills, and publishing the outlined research in top-tier scientific journals. The long-tem goal of the research is to define the role of KDM6A in normal and leukemic hematopoiesis. KDM6A encodes a H3K27 histone demethylase on the X chromosome that is mutated in a wide range of human cancers, including acute myeloid leukemia. Inactivating mutations of Kdm6a are the most common acquired progression events in our mouse model of acute promyelocytic leukemia (APL). To define the functional consequences of Kdm6a inactivation, we propose the following aims: Specific Aim 1: We will analyze a Kdm6a conditional knockout mouse to define the role of Kdm6a in normal and leukemic hematopoiesis. We will cross Kdm6a conditional knockout mice (with LoxP sites flanking the 3rd exon) with Vav-Cre transgenic mice to selectively delete Kdm6a in the hematopoietic lineage. We will characterize normal hematopoiesis using competitive repopulation assays, flow-based analysis of hematopoietic progenitors, and serial replating experiments. For these experiments, we will analyze 6-8 week old male Vav-Cre mice hemizygous for the floxed Kdm6a allele, along with the appropriate control mice. We will establish a tumor watch to determine if Kdm6a inactivation is associated with spontaneous leukemia development or bone marrow failure. We will intercross Kdm6a conditional knockout mice (with the Vav-Cre allele) with Ctsg-PML-RARA mice to define changes in cell growth, differentiation, and self renewal, and we will determine whether Kdm6a deficiency alters APL latency, penetrance, or phenotype in a tumor watch study. Specific Aim 2: We will define the epigenetic consequences of Kdm6a inactivation using RNA-Seq and ChIP-Seq. We will perform RNA-Seq on mouse APL tumors with and without spontaneous Kdm6a deletions or mutations, and on RNA derived from bone marrow cells from young, male Vav-Cre mice hemizygous for the Kdm6a conditional allele, along with the appropriate control animals. We will perform ChIP-Seq on mouse APL tumors with and without spontaneous Kdm6a deletions or mutations. We will use an anti-H3(tri-methyl K27) antibody to determine if changes in H3K27 methylation occur, and also with an anti-H3(tri-methyl K4) antibody, since Kdm6a inactivation may alter H3K4 methylation. Finally, we will perform RNA-Seq and ChIP-Seq on selected cell populations and leukemias (if they develop) in the male Vav-Cre mice hemizygous for the Kdm6a conditional allele, and APL tumors that arise in Ctsg-PML-RARA mice with and without the engineered deletion of Kdm6a. We will therefore comprehensively identify and analyze the epigenetic and transcriptional alterations associated with Kdm6a inactivation in wild type vs. PML-RARA expressing hematopoietic and leukemia cells.

描述（由申请人提供）：该提案的长期目标是为申请人提供培训和支持，使申请人成为独立的医师科学家，其职业专注于研究急性白血病的基因组学和表观基因组学。申请人的目标包括：在科学方法论，完善技术技能以及在顶级科学期刊上发表概述的研究。该研究的长期目标是定义KDM6A在正常和白血病造血中的作用。 KDM6A在X染色体上编码H3K27组蛋白脱甲基酶，该染色体在包括急性髓样白血病在内的广泛的人类癌症中突变。 KDM6A的灭活突变是我们急性前临床白血病的小鼠模型中最常见的获得进展事件（APL）。为了定义KDM6A灭活的功能后果，我们提出以下目的：特定目标1：我们将分析KDM6A条件敲除小鼠，以定义KDM6A在正常和白血病造血中的作用。我们将用VAV-CRE转基因小鼠在造血谱系中选择性地删除KDM6A，将有条件的敲除小鼠（带有LOXP位点在第三外显子两侧）穿越KDM6A。我们将使用竞争性重新群测定，造血祖细胞的基于流动的分析以及实验的串行反应来表征正常的造血作用。对于这些实验，我们将分析6-8周大的雄性VAV-CRE小鼠半合子的KDM6A等位基因，以及适当的对照小鼠。我们将建立一种肿瘤观察，以确定KDM6A失活是否与自发性白血病发育或骨髓衰竭有关。我们将与CTSG-PML-RARA小鼠（与VAV-CRE等位基因）进行跨KDM6A有条件的敲除小鼠，以确定细胞生长，分化和自我更新的变化，我们将确定KDM6A缺陷会改变肿瘤观察中的APL潜伏期，渗透率，渗透率，渗透率或表型。具体目标2：我们将使用RNA-Seq和ChIP-Seq定义KDM6A灭活的表观遗传后果。我们将对有或没有自发的KDM6A缺失或突变的小鼠APL肿瘤进行RNA-Seq，并在来自年轻的，雄性VAV-CRE小鼠的骨髓细胞中衍生自KDM6A有条件等位基因的RNA，以及适当的对照动物。我们将对具有和不具有自发KDM6A缺失或突变的小鼠APL肿瘤进行CHIP-SEQ。我们将使用抗H3（Tri-甲基K27）抗体来确定H3K27甲基化的变化是否发生，并且使用抗H3（Tri-甲基K4）抗体，因为KDM6A失活可能会改变H3K4甲基化。最后，我们将对选定的细胞群体和白血病（如果它们发育）在雄性Vav-Cre小鼠中进行RNA-SEQ和CHIP-SEQ，用于KDM6A有条件等位基因的Hemizygous，并且在带有KDM6A的工程删除的CTSG-PML-RARA小鼠中出现的APL肿瘤。因此，我们将全面识别和分析与野生型和PML-RARA表达造血和白血病细胞相关的与KDM6A失活有关的表观遗传和转录改变。