Regulation Of Erythroid Gene Expression

PU.1是一种与GATA-1 DNA结合相互作用的转录因子 这两种蛋白质之间存在相互抑制，这两种蛋白质决定了细胞在造血系中的命运。PU.1 反式激活(TAD)和DNA结合域被报道为 参与了互动，我们注意到PU.1 TAD 与p53的TAD有结构上的同源性。与 吉姆·奥米金斯基的实验室已经证明，P53TAD在体外和体内也与GATA-1DNA结合域相互作用。相互作用需要GATA-1的连接子和C-Finger。这些蛋白质相互抑制 红系前体细胞系6C2中彼此的反式激活活性。在红细胞形成之前的核浓缩和去核过程中，或在巨核细胞多倍化过程中，可能需要GATA-1来防止p53的诱导。在与Masi Yamamoto的合作中，我们正在测试GATA-1的突变体，这些突变体在GATA1.05细胞和小鼠的救援试验中不与P53相互作用，以确定这种相互作用在造血过程中的作用。 另一个项目涉及与EKLF转录因子相关的贫血。 EKLF DNA结合域的突变发生在贫血患者中。在CBA患者和NAN(新生儿贫血)小鼠中，EKLF第二个锌指上的一个氨基酸发生了突变。在与Jim Bieker的合作中，我们试图确定贫血表型是否基于一些EKLF靶基因与野生型NAN-EKLF的DNA结合亲和力不同。

PU.1 is a transcription factor that interacts with the GATA-1 DNA binding domain, and there is reciprocal inhibition between the two proteins that determines cell fate within the hematopoietic lineages. The PU.1 transactivation (TAD) and DNA binding domains are reported to be involved in the interaction and we have noted that the PU.1 TAD shares structural homology with the TAD of p53. 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 linker and C-finger of GATA-1 are required for the interaction. 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 or during the polyploidization of megakaryocytes. In collaboration with Masi Yamamoto we are testing mutants of GATA-1 that do not interact with p53 in rescue assays in GATA1.05 cells and mice to determine the role of this interaction during hematopoiesis. Another project involves anemias associated with the EKLF transcription factor. Mutations in the EKLF DNA binding domain occur in people with anemias. An amino acid in the second zinc finger of EKLF is mutated in patients with CBA and in the Nan (Neonatal anemia) mouse. In collaboration with Jim Bieker we are trying to determine if the anemic phenotype is based on different DNA binding affinities of Nan-EKLF relative to wild-type for some EKLF target genes.