Regulation Of Erythroid Gene Expression

红系基因表达的调控

• 批准号: 7733939
• 负责人: Gary Felsenfeld
• 金额: $ 33.92万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7733939
• 关键词: 1-Phosphatidylinositol 3-Kinase; Acetylation; Affect; Affinity; Amino Acids; Anemia; Binding; Binding Sites; Biochemical; Biological Assay; C-terminal; Cell Line; Cells; Child; Chimeric Proteins; Chromatin; Chromatin Structure; Collaborations; Complex; DNA; DNA Binding; DNA Binding Domain; Development; Disease; EP300 gene; Embryo; Erythrocytes; Erythroid; Erythroid Progenitor Cells; Erythropoiesis; Family; Fingers; Gene Expression; Gene Targeting; Genes; Genetic Polymorphism; Genotype; Hematopoietic; Human; In Vitro; Insulin; Insulin Resistance; Laboratories; Lead; Lymphocyte; Lysine; Mammals; Megakaryocytes; Messenger RNA; Modification; Mus; Mutate; Mutation; N-terminal; Nuclear; Numbers; Obesity; Patients; Peptides; Phenotype; Phosphoinositide-3-Kinase, Catalytic, Gamma Polypeptide; Phosphorylation; Phosphotransferases; Physical condensation; Point Mutation; Positioning Attribute; Property; Proteins; Range; Regulation; Role; Site; Specific qualifier value; Specificity; Structure; TP53 gene; Tissues; Transactivation; Transcription Repressor/Corepressor; Transferrin Receptor; Ubiquitination; Upper arm; Zinc Fingers; aminoglutethimide/danazol/hydrocortisone/tamoxifen; base; cofactor; cohort; cytarabine/daunorubicin/thioguanine; eosinophil; erythroid Kruppel-like factor; human GATA1 protein; in vivo; interest; lipid biosynthesis; mast cell; member; non-diabetic; preference; prevent; promoter; research study

The DNA binding domain of GATA-1 contains a number of lysine residues that are modified by acetylation sumolation, ubiquitination and phosphorylation, and these modifications may be important in specifying GATA-1 function. We are investigating the role of these residues in GATA-1 activity and in factor recognition. In collaboration with the laboratory of Masyuki Yamamoto, we have shown that 3 lysine residues in the GATA-1 DNA binding domain are critical to the function of GATA-1, and appear to contribute by allowing GATA-1 to self-associate. These residues are among the lysines that are acetylated by CBP/ p300, but acetylation does not seem to be involved in generating the observed phenotype. Two of these residues are in the N-linker and one in the C-finger. The residues are not required for DNA binding. GATA-1 mutated in these residues is unable to rescue GATA-1.05 knockdown mice from embryonic lethality because it cannot support definite erythropoiesis. Mutation of these residues (K to A) decreases the ability of GATA-1 to self-associate. These mice show both positive and negative affects on GATA-1 target genes, while the levels of other targets remain unchanged. This is consistent with the idea that GATA-1 self-association is important for only a subset of the genes it controls. A new GATA-1 target gene, the transferrin receptor, was identified through this study. In collaboration with the Bougnres lab, we have identified a complex GATA site in the promoter of the p110 subunit of the P13 kinase gene that may be involved in regulating insulin resistance. The C genotype of a previously identified T/C polymorphism was found to correlate with increased sensitivity to insulin in two cohorts of obese non-diabetic children. This polymorphism creates a strong GATA binding site between two weaker sites in the p110 gene promoter. Lymphocytes from multiple cohorts of obese children homozygous for the C polymorphism have 1.5 fold higher p110 mRNA levels, and 1.7 fold higher p110 protein levels than cohort members with the T genotype. The levels of the p85, the other subunit of the PI3 kinase, are the same throughout the cohorts. These increases most likely occur through enhanced activation of the p110 subunit gene by GATA-3. The C promoter is more active than its T counterpart in transient assays in GATA-3 containing cells. The C promoter has a higher affinity than the T for GATA-2 and -3, both of which are involved in adipogenesis. While lymphoctes, which are not physiologically relevant to insulin resistance, were used in these studies, insulin responsive tissues could not be collected from this group of children. All other known SNPs in the vicinity of the PI3K gene (N=12) have been analyzed and do not contribute to this phenotype. The number of patients currently totals 2500 with analysis completed on 2000 of these. In addition to DNA binding, the zinc fingers are also responsible for GATA-1 interactions with many other factors. The N-finger interacts with the critical GATA-1 partner FOG, while the C-finger interacts with PU.1. Regions of both fingers interact with Sp1, EKLF and CBP/p300. N-finger mutations that disrupt FOG binding are associated with severe macrothrombocytopenias and anemias. There are currently six members of the GATA family in mammals, and at least two more of these are also critical to hematopoietic development. All GATA factors have highly related DNA binding domains and can interact with many of the same cofactors. PU.1 interacts with the GATA-1 DNA binding domain through its transactivation(TAD) and DNA binding domains, and inhibits GATA-1 activity. There is reciprocal inhibition between the two proteins and the interaction with the PU.1 TAD is mainly responsible for inhibiting GATA-1. This TAD has homology to the TAD of p53, and in collaboration with the laboratory of Jim Omichinski we have shown that the p53 TAD also interacts in vitro and in vivo with the GATA-1 DNA binding domain. The proteins reciprocally inhibit the transactivation activity of one another in an erythroid precursor cell line, 6C2. GATA-1 may be required to prevent p53 induction during the nuclear condensation and enucleation that precedes erythrocyte formation.

GATA-1的DNA结合结构域包含许多赖氨酸残基，这些残基是通过乙酰化sumolation，泛素化和磷酸化来修饰的，并且这些修饰对于指定GATA-1功能可能很重要。我们正在研究这些残基在GATA-1活性和因子识别中的作用。与Yamamoto Masyuki实验室合作，我们已经表明，GATA-1 DNA结合结构域中的3个赖氨酸残基对GATA-1的功能至关重要，并且似乎通过允许GATA-1自相同作出贡献。这些残基是由CBP/ p300乙酰化的赖氨酸，但乙酰化似乎与产生观察到的表型无关。这些残基中的两个在N链接中，一个在C指定中。 DNA结合不需要残基。这些残基中突变的GATA-1无法从胚胎致死性中挽救GATA-1.05敲低小鼠，因为它不能支持明确的红细胞生成。这些残基的突变（k至a）降低了GATA-1自相关的能力。这些小鼠对GATA-1靶基因表现出正面和负面影响，而其他靶标的水平保持不变。这与GATA-1自我关联仅对于它控制的基因的子集很重要的想法是一致的。通过这项研究确定了一种新的GATA-1靶基因，即转铁蛋白受体。 与Bougnres实验室合作，我们在P110亚基的启动子中确定了一个复杂的GATA位点，该基因可能参与调节胰岛素耐药性。发现先前鉴定的T/C多态性的C基因型与在两个肥胖非糖尿病儿童中对胰岛素的敏感性提高相关。这种多态性在p110基因启动子中的两个弱位点之间产生了强大的GATA结合位点。来自C多态性的多个肥胖儿童来自多个肥胖儿童的淋巴细胞的P110 mRNA水平高1.5倍，而P110蛋白质水平比具有T基因型的同类成员高1.7倍。 p85的水平是PI3激酶的另一个亚基，在整个队列中都是相同的。这些增加很可能是通过GATA-3增强P110亚基基因的激活而发生的。 C启动子在包含细胞的GATA-3中的瞬态测定中比其t对应物更活跃。 C启动子的亲和力高于t对于GATA -2和-3的T，这两者都参与脂肪形成。尽管在这些研究中使用了与胰岛素耐药无关的淋巴结，但在这些研究中使用了胰岛素反应性组织，无法从这组儿童中收集。已经分析了PI3K基因（n = 12）附近的所有其他已知SNP，并且对这种表型没有贡献。目前在2000年完成分析的患者数量为2500。 除了DNA结合外，锌的手指还负责与许多其他因素的GATA-1相互作用。 N手指与临界GATA-1伴侣雾相互作用，而C指定与PU.1相互作用。两个手指的区域与SP1，EKLF和CBP/P300相互作用。 破坏雾结合的N手指突变与严重的巨骨细胞减少症和贫血有关。目前，哺乳动物中有六名GATA家族成员，其中至少有两个对于造血发展至关重要。所有GATA因子都具有高度相关的DNA结合域，并且可以与许多相同的辅助因子相互作用。 PU.1通过其反式激活（TAD）和DNA结合结构域与GATA-1 DNA结合结构域相互作用，并抑制GATA-1活性。 两种蛋白质之间存在相互抑制作用，与​​PU.1 TAD的相互作用主要负责抑制GATA-1。该TAD与p53的TAD具有同源性，并与Jim Omichinski的实验室合作，我们表明p53 TAD在体外也与GATA-1 DNA结合域相互作用。蛋白质在红斑前体细胞系6C2中相互抑制彼此的反式激活活性。可能需要GATA-1来防止在形成红细胞的核凝结和摘除过程中p53诱导。

项目成果

Transgenic rescue of GATA-1-deficient mice with GATA-1 lacking a FOG-1 association site phenocopies patients with X-linked thrombocytopenia.

用缺乏 FOG-1 关联位点的 GATA-1 转基因拯救 GATA-1 缺陷小鼠，可复制 X 连锁血小板减少症患者的表型。

• DOI: 10.1182/blood-2003-07-2514
• 发表时间: 2004
• 期刊: Blood
• 影响因子: 20.3
• 作者: Shimizu,Ritsuko;Ohneda,Kinuko;Engel,JamesDouglas;Trainor,CeceliaD;Yamamoto,Masayuki
• 通讯作者: Yamamoto,Masayuki