Gene expression in mature neutrophils

说明(申请人提供)：中性粒细胞是宿主抵御微生物感染的第一道防线，在炎症和组织损伤中发挥重要作用。以前对中性粒细胞中RNA表达的研究表明，对各种刺激的激活有非常强烈的转录反应。我们在髓系细胞系终末分化模型中的研究也表明，在细胞成熟过程中，大量转录因子的mRNA水平会发生变化。我们现在建议对发育中和成熟的中性粒细胞中转录活性的转录区域和调节因子进行协调和全面的研究。我们将调查： 1.《新奇》成绩单。我们将使用平铺阵列来确定在维甲酸诱导的终末分化过程中，在静息和激活的外周血中性粒细胞和NB4早幼粒细胞中表达的新转录物(即与已知基因不对应)的细胞特异性和链结构。 2.转录因子。我们将使用染色质免疫沉淀以及启动子和基因组阵列来鉴定在静息和激活的中性粒细胞中表达的序列特异性转录因子结合的启动子。我们还将研究在NB4细胞分化过程中哪些基因启动子与转录因子结合，以确定新的因子是否影响重要的下游靶点。 3.染色质结构和重塑蛋白：我们将使用寡核苷酸拼接阵列来测试中性粒细胞是否通过DNA与染色质重塑蛋白或特定修饰的组蛋白结合的改变来调节激活反应。我们还将在NB4细胞的终末分化过程中确定特定启动子序列与染色质修饰蛋白和修饰的组蛋白的关联。 4.DNA甲基化：利用互补系统确定CpG岛内外胞嘧啶残基的甲基化状态和位置，我们将研究激活的中性粒细胞和NB4细胞髓系成熟过程中胞嘧啶甲基化位点的变化。发现新的中性粒细胞特异性基因和调控网络可以为增强宿主防御、减轻炎症和治疗骨髓生成障碍提供新的靶点。

DESCRIPTION (provided by applicant): Neutrophils provide the first line of host defense against microbial infections and play a major role in inflammation and tissue damage. Previous studies of RNA expression in neutrophils have revealed a remarkably vigorous transcriptional response to activation by various stimuli. Our studies in a myeloid cell line model of terminal differentiation have also indicated that mRNA levels for a large number of transcription factors change during cell maturation. We now propose a coordinated and comprehensive investigation of the transcribed regions and the regulators of transcriptional activity in developing and mature neutrophils. We will investigate: 1. "Novel" transcripts. We will use tiling arrays to determine the cell specificity and strandedness of novel transcripts (i.e. not corresponding to known genes) expressed in resting and activated peripheral blood neutrophils and in NB4 promyelocytic leukemia cells during retinoid-induced terminal differentiation. 2. Transcription factors. We will use chromatin immunoprecipitation and both promoter and genomic arrays to identify promoters bound by sequence-specific transcription factors expressed in resting and activated neutrophils. We will also investigate which gene promoters are bound by transcription factors during NB4 cell differentiation to determine whether the novel factors affect important downstream targets. 3. Chromatin structure and remodeling proteins: We will use oligonucleotide tiling arrays to test whether neutrophils regulate activation responses by alterations in the association of DNA with chromatin remodeling proteins or specifically modified histones. We will also determine the association of specific promoter sequences with chromatin modifying proteins and modified histones during terminal differentiation of NB4 cells. 4. DNA methylation: Using complementary systems to determine the methylation status and location of cytosine residues, both within and outside CpG islands, we will investigate changes in the sites of cytosine methylation in activated neutrophils and during myeloid maturation in NB4 cells. Identification of novel neutrophil-specific genes and regulatory networks could provide new targets for augmentation of host defense, attenuation of inflammation, and treatments of disorders of myelopoiesis.