Regulation Of Erythroid Gene Expression

GATA-1的DNA结合域包含许多赖氨酸残基，这些残基可以被乙酰化、泛素化和磷酸化修饰，这些修饰可能对确定GATA-1的功能很重要。我们正在研究这些残基在GATA-1活性和因子识别中的作用。在与Masyuki Yamamoto实验室的合作中，我们已经证明了GATA-1 DNA结合域中的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自结合而起作用。这些残基位于被CBP/ p300乙酰化的赖氨酸中，但乙酰化似乎与所观察到的表型的产生无关。其中两个残基在n连接体中一个在c指中。这些残基不是DNA结合所必需的。在这些残基中突变的GATA-1不能使GATA-1.05基因敲低的小鼠免于胚胎死亡，因为它不能支持明确的红细胞生成。这些残基（K到A）的突变降低了GATA-1自结合的能力。这些小鼠对GATA-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GATA-1自我关联仅对其控制的一部分基因重要的观点是一致的。本研究发现了一个新的GATA-1靶基因转铁蛋白受体。

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.