Regulation Of Erythroid Gene Expression

加塔-1的DNA结合结构域含有许多赖氨酸残基，这些赖氨酸残基通过乙酰化、泛素化和磷酸化修饰，并且这些修饰在指定加塔-1功能中可能是重要的。我们正在研究这些残基在加塔-1活性和因子识别中的作用。与Masyuki Yamamoto的实验室合作，我们已经证明加塔-1 DNA结合结构域中的3个赖氨酸残基对加塔-1的功能至关重要，并且似乎通过允许加塔-1自缔合而起作用。这些残基是由CBP/ p300乙酰化的赖氨酸，但乙酰化似乎不参与产生观察到的表型。这些残基中的两个在N-接头中，一个在C-指中。DNA结合不需要这些残基。在这些残基中突变的加塔-1不能拯救加塔-1.05敲除小鼠免于胚胎致死，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）降低了加塔-1自缔合的能力。这些小鼠对加塔-1靶基因表现出积极和消极的影响，而其他靶基因的水平保持不变。这与加塔-1自相关仅对它所控制的基因的子集重要的观点一致。通过本研究鉴定了一个新的加塔-1靶基因-转铁蛋白受体。 在与Bougnres实验室的合作中，我们已经确定了一个复杂的加塔网站的P13激酶基因的p110亚基的启动子，可能参与调节胰岛素抵抗。在两组肥胖非糖尿病儿童中，发现先前鉴定的T/C多态性的C基因型与胰岛素敏感性增加相关。这种多态性在p110基因启动子的两个较弱位点之间产生了一个强的加塔结合位点。来自多个队列的肥胖儿童的淋巴细胞中，C多态性纯合子的p110 mRNA水平是T基因型队列成员的1.5倍，p110蛋白水平是T基因型队列成员的1.7倍。PI 3激酶的另一个亚基p85的水平在整个队列中是相同的。这些增加很可能是通过加塔-3增强p110亚基基因的激活而发生的。 在含有加塔-3的细胞中的瞬时测定中，C启动子比其T对应物更有活性。C启动子对加塔-2和GATA-3的亲和力高于T启动子，这两个基因都参与脂肪形成。虽然在这些研究中使用了与胰岛素抵抗无生理相关性的淋巴细胞，但无法从该组儿童中收集胰岛素反应组织。已经分析了PI 3 K基因附近的所有其他已知SNP（N=12），这些SNP对该表型没有贡献。目前患者总数为2500例，其中2000例完成了分析。 除了DNA结合，锌指还负责加塔-1与许多其他因子的相互作用。N指与关键的加塔-1伙伴FOG相互作用，而C指与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FOG结合的N指突变与严重的巨噬细胞减少症和贫血有关。目前哺乳动物中有六个加塔家族成员，其中至少还有两个对造血发育也至关重要。所有的加塔因子都具有高度相关的DNA结合结构域，并且可以与许多相同的辅因子相互作用。PU.1通过其反式激活（transactivation，ERK）和DNA结合结构域与加塔-1 DNA结合结构域相互作用，并抑制加塔-1活性。 这两种蛋白之间存在相互抑制作用，并且与PU.1的相互作用主要负责抑制加塔-1。该酶与p53的酶具有同源性，并且与Jim Omichinski的实验室合作，我们已经表明p53酶也在体外和体内与加塔-1 DNA结合结构域相互作用。这些蛋白质在红系前体细胞系6C 2中抑制彼此的反式激活活性。可能需要加塔-1来防止在红细胞形成之前的核浓缩和去核过程中的p53诱导。

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.