ChIP-chip from ex-vivo differentiated erythroblasts derived from human primary CD34+ cells. Homo sapiens

The molecular mechanisms underlying erythroid-specific gene regulation remain incompletely understood. Closely spaced binding sites for GATA, NF-E2/maf and CACCC interacting transcription factors play functionally important roles in globin and other erythroid-specific gene expression. We and others recently identified the CACCC-binding transcription factor ZBP-89 as a novel GATA-1 and NF-E2/mafK interacting partner. Here, we examined the role of ZBP-89 in human globin gene regulation and erythroid maturation using a primary CD34+ cell ex vivo differentiation system. We show that ZBP-89 protein levels rise dramatically during human erythroid differentiation, and that ZBP-89 occupies key cis-regulatory elements within the globin and other erythroid gene loci. ZBP-89 binding correlates strongly with RNA Pol II occupancy, active histone marks, and high-level gene expression. ZBP-89 physically associates with the histone acetyltransferases (HATs) p300 and Gcn5/Trrap, and occupies common sites with Gcn5 within the human globin loci. Lentiviral shRNA knockdown of ZBP-89 results in reduced Gcn5 occupancy, decreased acetylated histone 3 levels, lower globin and erythroid-specific gene expression, and impaired erythroid maturation. Addition of the HDAC inhibitor valproic acid partially reverses the reduced globin gene expression. These findings reveal an activating role for ZBP-89 in human globin gene regulation and erythroid differentiation. Keywords: Antibody ChIP-chip, Human Primary Erythroblasts Overall design: Genomic targets of Transcription factor ZBP-89 and Histone 3 acetylation signature were identified by ChIP-chip in human primary cells.

红细胞特异性基因调控的分子机制目前尚未完全阐明。GATA、NF-E2/maf与CACCC结合型转录因子的紧密间隔结合位点，在珠蛋白及其他红细胞特异性基因的表达调控中发挥关键功能作用。本课题组及其他研究团队近期证实，CACCC结合型转录因子ZBP-89是GATA-1与NF-E2/mafK的新型互作搭档。本研究采用原代CD34+细胞体外分化体系，探究了ZBP-89在人类珠蛋白基因调控及红细胞成熟过程中的功能。研究结果显示，ZBP-89的蛋白水平在人类红细胞分化过程中显著上调，且ZBP-89可结合于珠蛋白及其他红细胞基因位点内的关键顺式调控元件（cis-regulatory elements）。ZBP-89的结合与RNA聚合酶II（RNA Pol II）的结合占有率、活性组蛋白修饰标记以及高水平基因表达呈显著正相关。ZBP-89可与组蛋白乙酰转移酶（histone acetyltransferases, HATs）p300及Gcn5/Trrap发生物理性互作，且在人类珠蛋白基因位点内与Gcn5共享结合位点。通过慢病毒介导的短发夹RNA（short hairpin RNA, shRNA）敲低ZBP-89的表达，会导致Gcn5的结合占有率降低、组蛋白H3乙酰化水平下降、珠蛋白及红细胞特异性基因表达量降低，同时损害红细胞成熟进程。添加组蛋白去乙酰化酶（histone deacetylase, HDAC）抑制剂丙戊酸，可部分逆转珠蛋白基因表达的下调现象。上述研究结果揭示了ZBP-89在人类珠蛋白基因调控及红细胞分化过程中的激活性功能。关键词：抗体染色质免疫沉淀芯片（ChIP-chip）、人类原代成红细胞。实验设计：通过染色质免疫沉淀芯片（ChIP-chip）在人类原代细胞中鉴定转录因子ZBP-89的基因组靶位点以及组蛋白H3乙酰化特征谱。