Mitotic chromatin and implications in erythroid transcriptional regulation

 描述(由申请人提供)：通过有丝分裂的进展对细胞转录程序的维持提出了挑战，因为它迫使RNA聚合酶II和大多数转录调节因子从染色质中被驱逐出去。最近的工作已经确定了一些转录因子和组蛋白翻译后修饰，它们仍然与有丝分裂染色质相关，因此可能为细胞提供其转录程序和细胞身份的“书签”。在这里，我们将结合重点机械实验和全球分析来研究有丝分裂书签的原理。我们发现，必需的红系转录因子GATA1在有丝分裂过程中稳定地保留在其间期结合位点的一个子集上。有丝分裂染色质与GATA1结合是有丝分裂后红系特异基因及时重新激活所必需的。在目标1中，我们将确定GATA1在有丝分裂过程中与其间期结合位点的子集结合的机制和功能。计算分析未能提供线索，说明是什么将仅限相间部位(I-部位)与保留GATA1的部位(IM-部位)分开。然而，初步数据表明，当基因组GATA1结合元件异位插入红系基因组时，I和IM结合特征被重述。这表明局部序列上下文决定了I与IM的结合，并为分析这些元件的特异性提供了一种方便的方法。我们将通过截断和点突变来定义GATA1在有丝分裂染色质上保留的必要条件和充分条件。我们将使用基因组编辑来直接测试在有丝分裂后内源性I-和IM-位点在基因重新激活中的作用。在确定了GATA1 IM特性所需的顺式元件(S)之后，我们将鉴定并功能研究GATA1介导的红系细胞书签中的相关反式作用因子(S)。在目标2中，我们将与Ben Garcia博士的实验室合作，通过使用高度纯化的有丝分裂红系细胞群体的定量质谱学，在全球范围内检验组蛋白修饰的动态。这将是有丝分裂期间全球组蛋白标记水平的第一次表征。选择活力和稳定的标记，以及放置它们的酶，将通过全基因组定位分析进行研究。然后，我们将研究结合IM位点富集组蛋白标记的蛋白质在促进GATA1有丝分裂染色质占有率和基因重新激活方面的影响。这些研究有望更好地理解基于染色质和基于转录因子的红系细胞有丝分裂书签机制，并可能对理解与细胞分化和谱系稳定相关的转录稳定性以及可塑性具有重要意义。

 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.