Molecular Biology of the Ribonuclease A Gene Superfamily

在本报告期间，我们研究了加塔-1和加塔-2在激活分泌性核糖核酸酶，嗜酸性粒细胞衍生的神经毒素（EDN/RNase 2）的转录中的作用。 在嗜酸性粒细胞中检测到加塔-1和加塔-2的增强表达，并且EDN/RNase 2基因的延伸的5'启动子中的两个共有GATA结合位点中的一个或两个的缺失或突变导致报告基因活性的显著降低。抗体增强的电泳迁移率变化和染色质免疫沉淀分析表明，加塔-1和加塔-2蛋白结合到EDN启动子中的两个功能性加塔共有序列。有趣的是，单独的加塔-1的RNA沉默对EDN/RNase 2表达没有影响;加塔-2的沉默导致EDN表达减少，并且在丁酸诱导的HL-60克隆15细胞和分化的源自CD 34（+）造血祖细胞的人嗜酸性粒细胞中也导致加塔-1表达减少。同样，过表达加塔-2导致EDN/RNase 2和加塔-1的转录增加。总之，我们的数据表明，加塔-2通过与EDN启动子的相互作用直接起作用，并且还通过其调节分化中的嗜酸性粒细胞中加塔-1表达的能力间接起作用（Qiu等人，J Biol Chem.2009）。 另一份出版物关注哺乳动物RNase A核糖核酸酶的多样性，并完成了Wei Zhao博士在我们小组担任客座教授期间的一些杰出工作。 这份手稿鉴定了啮齿动物RNase 1基因的一个新簇，也用于完成和纠正GenBank数据库中的几个错误条目（Siegel et al. Mamm Genome，2009，in press）。 我继续担任生物化学杂志的编辑委员会（自2008年以来），这主要归功于我在核糖核酸酶生物学和生物化学方面的专业知识。

During this reporting period, we examined the roles of GATA-1 and GATA-2 in activating transcription of the secretory ribonuclease, the eosinophil-derived neurotoxin (EDN/RNase 2). Augmented expression of both GATA-1 and GATA-2 was detected in eosinophils and deletion or mutation of one or both of the two consensus GATA-binding sites in the extended 5' promoter of the EDN/RNase2 gene resulted in profound reduction in reporter gene activity. Antibody-augmented electrophoretic mobility shift and chromatin immunoprecipitation analyses indicate that GATA-1 and GATA-2 proteins bind to both functional GATA consensus sequences in the EDN promoter. Interestingly, RNA silencing of GATA-1 alone had no impact on EDN/RNase2 expression; silencing of GATA-2 resulted in diminished expression of EDN, and also diminished expression of GATA-1 in both butyric acid-induced HL-60 clone 15 cells and in differentiating human eosinophils derived from CD34(+) hematopoietic progenitors. Likewise, overexpression of GATA-2 in resulted in augmented transcription of both EDN/RNase2 and GATA-1. Taken together, our data suggest that GATA-2 functions directly via interactions with the EDN promoter and also indirectly, via its ability to regulate the expression of GATA-1 in differentiating eosinophils (Qiu et al. J Biol Chem. 2009). Another publication focuses on the diversity of mammalian RNase A ribonucleases, and completes some outstanding work from Dr. Wei Zhao's visiting professorship with our group. This manuscript, which identifies a novel cluster of rodent RNase 1 genes, serves also to complete and to correct several erroneous entries in GenBank database (Siegel et al. Mamm Genome, 2009, in press) I continue to serve on the Editorial Board of Journal of Biological Chemistry (since 2008), a largely due to my expertise in Ribonuclease Biology and Biochemistry.