Mitotic chromatin and implications in erythroid transcriptional regulation

有丝分裂染色质及其对红细胞转录调控的影响

• 批准号: 9047724
• 负责人: Vivek Behera
• 金额: $ 4.8万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9047724
• 关键词: Affinity; Beryllium; Binding; Binding Sites; Biological Assay; Cell Cycle; Cell Line; Cell Maintenance; Cells; ChIP-seq; Characteristics; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Computer Analysis; DNA; DNA Polymerase II; DNA Sequence; Data; Data Set; Defect; Disease; Elements; Enzymes; Epigenetic Process; Erythroblasts; Erythroid; Erythroid Cells; Erythropoiesis; GATA1 gene; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genome; Genomics; Hematopoietic; Histones; Interphase; Knowledge; Laboratories; Location; Maintenance; Mass Spectrum Analysis; Mediating; Mitosis; Mitotic; Mus; Mutate; Nuclear Extract; Phenotype; Plant Roots; Play; Point Mutation; Population; Post-Translational Protein Processing; Protein Binding; Proteins; RNA; RNA Polymerase II; RNA Sequences; Reader; Reagent; Recruitment Activity; Risk; Role; Site; Specific qualifier value; Specificity; Surveys; System; Techniques; Testing; Trans-Activators; Transcription Regulatory Protein; Transcriptional Regulation; Work; base; daughter cell; erythroid differentiation; genome editing; genome-wide; genome-wide analysis; histone modification; human GATA1 protein; loss of function; programs; public health relevance; research study; small hairpin RNA; transcription factor; transcriptome sequencing

 DESCRIPTION (provided by applicant): Progression through mitosis presents a challenge to the maintenance of cellular transcriptional programs by forcing the eviction of RNA Polymerase II and of most transcriptional regulators from chromatin. Recent work has identified a number of transcription factors and histone post-translational modifications that remain associated with mitotic chromatin and may thus provide cells with a "bookmark" of their transcriptional programs and cellular identity. Here we will combine focused mechanistic experimentation with global analyses to study principles of mitotic bookmarking. We found that the essential erythroid transcription factor GATA1 is stably retained at a subset of its interphase binding sites during mitosis. Mitotic chromatin binding by GATA1 is necessary for the timely reactivation of select erythroid lineage specific genes following mitosis. In Aim 1 we will determine the mechanism and function of GATA1 binding to a subset of its interphase binding sites during mitosis. Computational analysis failed to provide clues as to what separates interphase only sites (I-sites) from sites at which GATA1 is retained (IM-sites). However, preliminary data indicate that I- and IM-binding characteristics are recapitulated when genomic GATA1 binding elements are ectopically inserted into the erythroid genome. This indicates that local sequence context determines I vs IM binding and provides a convenient assay to analyze the specificity of these elements. We will define the minimal sequence context necessary and sufficient for GATA1 retention on mitotic chromatin through truncations and point mutations. We will use genome editing to directly test in both gain- and loss-of-function studies the role of endogenous I- and IM-sites on gene reactivation following mitosis. Following the identification of cis-element(s) required for GATA1 IM characteristics, we will identify and functionally study the relevant trans acting factor(s) in GATA1 mediated bookmarking in erythroid cells. In Aim 2 in collaboration with the lab of Dr. Ben Garcia we will examine the dynamics of histone modifications on a global scale by quantitative mass spectrometry using highly purified mitotic erythroid cell populations. This will be the first characterization of global histone mark levels during mitosis. Select dynami and stable marks, as well as the enzymes placing them, will be studied by genome wide location analysis. We will then examine the impact of proteins that bind IM-site enriched histone marks in promoting GATA1 mitotic chromatin occupancy and in gene reactivation. These studies are expected to provide a better understanding of chromatin based and transcription factor based mechanisms of mitotic bookmarking in erythroid cells and might have implications for understanding the transcriptional stability as well as plasticity associated with cellular differentiation and lineage stability.

 描述（由申请人提供）：通过有丝分裂的进展通过迫使RNA聚合酶II和大多数转录调节因子从染色质中驱逐而对细胞转录程序的维持提出了挑战。最近的工作已经确定了一些转录因子和组蛋白的翻译后修饰，仍然与有丝分裂染色质，并因此可能提供细胞与他们的转录程序和细胞身份的“书签”。在这里，我们将结合联合收割机集中的机制实验与全球分析，研究有丝分裂书签的原则。我们发现，在有丝分裂过程中，红系细胞必需转录因子GATA 1稳定地保留在其间期结合位点的一个子集。有丝分裂染色质结合GATA 1是必要的选择红细胞谱系特异性基因的有丝分裂后及时重新激活。在目标1中，我们将确定GATA 1在有丝分裂过程中结合其间期结合位点的机制和功能。计算分析未能提供线索，以区分间期只有网站（I-网站）的网站，其中GATA 1被保留（IM网站）。然而，初步的数据表明，I-和IM-结合特性重演时，基因组GATA 1结合元件异位插入到红细胞基因组。这表明局部序列环境决定了I与IM结合，并提供了一种方便的测定法来分析这些元件的特异性。我们将定义通过截短和点突变使GATA 1保留在有丝分裂染色质上所必需和足够的最小序列背景。我们将使用基因组编辑直接测试获得和功能丧失研究中内源性I-和IM-位点对有丝分裂后基因再激活的作用。在鉴定GATA 1 IM特征所需的顺式元件之后，我们将鉴定并功能性研究红系细胞中GATA 1介导的书签化中的相关反式作用因子。在Aim 2中，我们将与Ben Garcia博士的实验室合作，使用高度纯化的有丝分裂红细胞群，通过定量质谱法在全球范围内研究组蛋白修饰的动态。这将是有丝分裂期间全球组蛋白标记水平的第一个表征。将通过全基因组定位分析研究选择的动态和稳定标记以及放置它们的酶。然后，我们将研究结合IM位点富集的组蛋白标记的蛋白质在促进GATA 1有丝分裂染色质占据和基因再激活中的影响。这些研究有望更好地理解基于染色质和转录因子的红系细胞有丝分裂书签机制，并可能对理解转录稳定性以及与细胞分化和谱系稳定性相关的可塑性产生影响。