Function of Putative Determinant in Hematopoiesis

摘要 细胞限制性转录调控因子在基因选择过程中起着关键作用 在造血过程中的调节。我们一直在研究它的分子和生物学功能 红系Krüppel样因子(EKLF；KLF1)。EKLF是一种细胞限制性转录因子，是一种全球性的 对红系程序至关重要的基因的调节器。我们的建议是以意想不到的小说为基础的 在上一个资金周期中提出的意见，将这项长期研究扩展到创新 使用说明： 我们发现EKLF是通过单甲基化和双甲基化在精氨酸残基上修饰的。这个 目标1的实验将解决这些变化的背景及其功能，并确定 负责的酶。 我们发现小鼠Nan-EKLF和人CDA-KLF1突变型红系的代谢物 细胞发生改变；这包括一种参与组蛋白染色质相关变化的代谢物。 修改。AIM 2的实验将检验这些修改的全局性质 小鼠原代细胞和我们新开发的CDA患者来源的细胞系。 我们发现在EKLFKO和Nan/+细胞中染色质的可及性发生了变化。这个 目标3的实验将在减少和集合的上下文中检查这些变化 在这些遗传背景中异位表达的基因，这一分析将扩展到 包括CDA患者来源的细胞。 我们发现EKLF KO细胞的代谢体发生了改变，并导致了DNA的改变 表观遗传学。目标4的实验将在全球范围内分析这些变化 在没有EKLF的情况下，观察到RNA表达和染色质可及性改变。 这些研究将得到分子、代谢和生化分析的辅助， 遗传方法，以及使用原代或最少操纵的细胞，这些细胞是新的 已经成立了。阐明EKLF在调节现象中的作用将继续阐明 红系生物学和细胞限制性转录因子发挥作用的基本机制 对遗传表达、新陈代谢和表观遗传学的各种但高度可控的影响。

SUMMARY Cell-restricted transcriptional modulators play critical roles in the process of selective gene regulation during hematopoiesis. We have been investigating the molecular and biological function of Erythroid Krüppel-like Factor (EKLF; KLF1). EKLF is a cell-restricted transcription factor that is a global regulator of genes essential for the erythroid program. Our proposal builds on unanticipated novel observations made during the previous funding cycle that extend this long-term study towards innovative directions: We found that EKLF is modified at arginine residues by mono- and di-methylation. The experiments of Aim 1 will address the context for these alterations and their function, and identify the enzymes responsible. We found that the metabolomes of the mouse Nan-EKLF and human CDA-KLF1 mutant erythroid cells are altered; this includes a metabolite involved in chromatin-associated changes in histone modification. The experiments of Aim 2 will examine the global nature of these modifications in mouse primary cells and in our newly-developed CDA patient-derived cell lines. We find that chromatin accessibility is altered in EKLF KO and in the Nan/+ cell. The experiments of Aim 3 will examine these changes in the context of the sets of decreased and ectopically expressed genes in these genetic backgrounds, an analysis that will be expanded to include CDA patient-derived cells. We found that the metabolome of the EKLF KO cell is altered, and leads to changes in DNA epigenetics. The experiments of Aim 4 will globally analyze these changes in the context of the altered RNA expression and chromatin accessibility observed in the absence of EKLF. These studies will be aided by the use of molecular, metabolic, and biochemical analyses, genetic approaches, and use of primary or minimally manipulated cells that have been newly established. Elucidating EKLF’s role in regulatory phenomena will continue to illuminate novel aspects of erythroid biology and the essential mechanisms by which a cell-restricted transcription factor can exert varied yet highly controlled influences on genetic expression, metabolism, and epigenetics.