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

原癌基因Ets-1和非依赖生长因子-1(GFI-1)参与了多种类型细胞的生长和分化，其表达失控参与了肿瘤的形成。我们发现Ets-1和GFI-1通过相互作用影响基因表达。免疫共沉淀和谷胱甘肽-S-转移酶下拉实验表明，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。利用人工报告进行荧光素酶(Luc)检测表明，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协同减少了激活。对Bax启动子的EBS和GBS进行的定点突变表明，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…进一步证实，即使在EBS或GBS突变的情况下，Bax启动子上也形成了Ets-1-GFI-1复合体。导入针对Ets-1和/或GE-1的小干扰RNA可增强内源Bax基因的表达。我们的结果表明，Ets-1和GFI-1之间的相互作用促进了它们与Bax启动子上的特定位置的结合，并在体内抑制了Bax的表达。PU.1是髓系发育的物质调节因子，GFI-1B是红系/巨核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PU1与GFI-1B在体内相互作用。GST下拉分析表明，结合位点位于PU.1的Ets结构域和GFI-1B的锌指结构域。荧光素酶报告分析表明，PU1和GFI-1B以剂量依赖的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表明，Mac-1的表达减少，尽管这种减少并不太强，不足以完全抑制PU.1诱导的髓系分化。此外，在人脐血造血祖细胞中转导带有GFI1B的PU1显著抑制红系细胞的生长和集落形成，而单独转导GFI1b可显著促进红系细胞的生长和集落形成。GFI-1B与Ets结构域(PU.)N端和133/134区缺失突变PU.1共表达不与GATA-1结合但仍与GFI-1B结合的LANA133/34)也能抑制GFI-1B诱导的红系集落形成。我们的结果提示PU.1通过阻断GFI-1B功能和GATA-1功能来抑制红系细胞的生长和分化。较少

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