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

原癌基因Ets-1和生长因子非依赖性-1（Gfi-1）与各种类型细胞的细胞生长和分化有关，其表达失调与恶性形成有关。我们发现Ets-1和Gfi-1相互作用，并通过它们的串扰影响基因表达。免疫共沉淀分析和谷胱甘肽-S-转移酶（GST）pull-down分析表明，Ets-1通过其Ets结构域与Gfi-1直接结合，Gfi-1通过其锌指结构域与Ets-1结合。使用人工报告的荧光素酶（Luc）测定显示，Gfi-1抑制Ets-1介导的转录激活，Ets-1抑制Gfi-1介导的转录激活。然而，在bax启动子的Ets-和Gfr结合位点（EBS和GBS）是相邻的，Ets-1和Gfi-1合作减少激活。定点突变的EBS和GBS的bax启动子表明，这两个结合位点是必要的充分镇压。染色质免疫沉淀分析 关于我们 证实了Ets-1-Gfi-1复合物在bax启动子上形成，即使EBS或GBS突变。针对Ets-1和/或GE-1的小干扰RNA的引入增强内源性bax基因表达。我们的研究结果表明，Gfi-1和Gfi-1之间的相互作用，促进其结合到bax启动子上的特定位点，并在体内抑制bax的表达。PU.1是骨髓发育的物质调节因子，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。我们通过免疫沉淀实验发现PU.1与Gfi-1B在体内相互作用。GST pull-down分析表明，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以剂量依赖性方式拮抗彼此的转录活性。Gfi-1B在小鼠早期髓系祖细胞中的转导显示Mac-1的表达降低，尽管这种降低不太强而不能完全抑制PU.1诱导的髓系分化。此外，在人脐带血造血祖细胞中用Gfi 1B转导PU. 1显著抑制红系细胞的生长和集落形成，这通过单独转导Gfi-lb而强烈增强。Gfi-1B与在N-末端和Ets结构域的133/134区域缺失的突变体PU. 1的共表达（PU. lANA 133/34），其不与加塔-1结合但仍与Gfi-1B结合，也抑制GFi-1B诱导的红系集落形成。我们的结果表明，PU.1通过阻断GFi-1B功能以及通过阻断加塔-1功能来抑制红系细胞的生长和分化。少

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