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Cross-talks between ETS and GFI family of transcription factors in hematopoietic cell differentiation

ETS 和 GFI 转录因子家族在造血细胞分化中的串扰

基本信息

批准号：
17591019
负责人：
OIKAWA Tsuneyuki
金额：
$ 2.18万
依托单位：
Health Sciences University of Hokkaido (2006)Sasaki Institute (2005)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591019/
关键词：
transcription factors ETS Ets-1 PU.1 Gfi-1 GfilB cross-talk apoptosis hematopoiesis Gfi-1 bax アポトーシス血球分化

项目摘要

The protooncogene Ets-1 and growth factor independent-1 (Gfi-1) are implicated in cell growth and differentiation in various types of cells, and their deregulated expression is involved in malignant formation. We found that Ets-1 and Gfi-1 interacted and affected gene expression through their cross-talk. Co-immunoprecipitation analysis and glutathione-S-transferase (GST) pull-down assay revealed that Ets-1 bound directly to Gfi-1 via its Ets domain, and Gfi-1 bound to Ets-1 via its zinc-finger domain. Luciferase (Luc) assays using artificial reports showed that Gfi-1 repressed Ets-l-mediated transcription activation and Ets-1 repressed Gfi-l-mediated transcriptional activation. However, in the bax promoter where the Ets-and Gfrbinding sites (EBS and GBS) are adjacent, Ets-1 and Gfi-1 cooperatively reduced activation. Site-directed mutagenesis on the EBS and GBS of the bax promoter showed that both binding sites were necessary for full repression. Chromatin immunoprecipitation analyses … More confirmed that an Ets-1-Gfi-1 complex formed on the bax promoter even when either EBS or GBS was mutated. Introduction of small interfering RNA against Ets-1 and/or GE-1 enhanced endogenous bax gene expression. Our results suggest that the interaction between ETs-1 and Gfi-1 facilitates their binding to specific sites on the bax promoter and repress bax expression in vivo. PU.1 is a mater regulator for myeloid development and Gfi-1B is a hematopoietic transcription factor essential for growth and differentiation of the erythroid/megakaryocytic lineages. We found that PU.1 interacted with Gfi-1B in vivo by immunoprecipitation assay. GST pull-down assay showed that the binding sites were in the Ets domain of PU.1 and the zinc finger domain of Gfi-1B. Luciferase reporter assays revealed that PU.1 and Gfi-1B antagonized each other's transcriptional activity in a dose dependent manner. Transduction of Gfi-1B in mouse early myeloid progenitor cells showed that expression of Mac-1 was reduced, although this reduction was not too strong to inhibit PU.1-induced myeloid differentiation completely. Furthermore, transduction of PU.1 with Gfi1B in human cord blood hematopoietic progenitors significantly inhibited growth and colony formation of erythroid cells which were strongly enhanced by transduction of Gfi-lb alone. Co-expression of Gfi-1B with a mutant PU.1 deleted at the N-terminus and the 133/134 region of the Ets domain (PU. lANA133/34), which did not bind to GATA-1 but still bound to Gfi-1B, also inhibited GFi-1B-induced erythroid colony formation. Our results suggest that PU.1 inhibit growth and differentiation of erythroid cells by blocking GFi-1B function as well as by blocking GATA-1 function. Less

原癌基因Ets-1和生长因子独立-1 （Gfi-1）参与各种类型细胞的生长和分化，其不受调控的表达参与恶性肿瘤的形成。我们发现Ets-1和Gfi-1通过它们的串扰相互作用并影响基因表达。免疫共沉淀分析和谷胱甘肽- s转移酶（GST）下拉实验表明，Ets-1通过其Ets结构域直接与Gfi-1结合，而Gfi-1通过其锌指结构域与Ets-1结合。人工报告的荧光素酶（Luc）检测显示，Gfi-1抑制ets -l介导的转录激活，Ets-1抑制gfi -l介导的转录激活。然而，在ets和gfr结合位点（EBS和GBS）相邻的bax启动子中，Ets-1和Gfi-1协同降低了激活。对bax启动子的EBS和GBS进行定点诱变表明，这两个结合位点都是完全抑制所必需的。染色质免疫沉淀分析…More证实，即使EBS或GBS发生突变，在bax启动子上也会形成Ets-1-Gfi-1复合物。引入针对Ets-1和/或GE-1的小干扰RNA可增强内源性bax基因的表达。我们的研究结果表明，ETs-1和Gfi-1之间的相互作用促进了它们与bax启动子上的特定位点的结合，并抑制了体内bax的表达。PU.1是髓系发育的重要调节因子，Gfi-1B是红细胞/巨核细胞谱系生长和分化所必需的造血转录因子。通过免疫沉淀实验，我们发现PU.1在体内与Gfi-1B相互作用。GST下拉实验显示，结合位点位于PU.1的Ets结构域和Gfi-1B的锌指结构域。荧光素酶报告基因分析显示，PU.1和Gfi-1B以剂量依赖的方式相互拮抗对方的转录活性。Gfi-1B在小鼠早期髓系祖细胞中的转导表明，Mac-1的表达减少，但这种减少并不强烈，不足以完全抑制pu .1诱导的髓系分化。此外，Gfi1B在人脐带血造血祖细胞中的转导能显著抑制红系细胞的生长和集落形成，而Gfi-lb的单独转导能显著增强红系细胞的生长和集落形成。Gfi-1B与n端和Ets结构域133/134区缺失的突变体PU.1共表达。lANA133/34)不与GATA-1结合，但仍与Gfi-1B结合，也能抑制Gfi-1B诱导的红系集落形成。我们的研究结果表明，PU.1通过阻断GFi-1B和GATA-1的功能抑制红系细胞的生长和分化。少