Crotta S, Nicolis S, Ronchi A, Ottolenghi S, Ruzzi L, Shimada Y, Migliaccio A R, Migliaccio G
Dipartimento di Genetica e di Biologia dei Microrganismi, Università di Milano, Italy.
Nucleic Acids Res. 1990 Dec 11;18(23):6863-9. doi: 10.1093/nar/18.23.6863.
The transcriptional binding protein NFE-1 (also called GF-1 and Ery-f1) is thought to play a necessary, but not sufficient, role in the regulation of differentiation-related gene expression in a subset of hematopoietic lineages (erythroid, megakaryocytic, and basophil-mast cell). In order to clarify the mechanism which underlies the lineage-specificity of the NFE-1 expression, as well as the relationship between the expression of this factor and growth factor responsiveness, we have evaluated the capacity of erythropoietin (Epo)-, granulomonocytic (GM)-colony stimulating factor (CSF)-, and granulocyte (G)-CSF-dependent subclones derived from the interleukin 3 (IL-3)-dependent cell line 32D, to express 1) NFE-1 mRNA, 2) NFE-1-related nuclear proteins, and 3) chloramphenicol acetyl transferase (CAT) activity when transfected with a CAT gene under the control of NFE-1 cognate sequences. NFE-1 mRNA was found to be expressed not only in cells with mast cell (IL-3-dependent 32D) and erythroid (Epo-dependent 32D Epo1) phenotypes, but also in cells with predominantly granulocyte/macrophage properties, such as the GM-CSF- (early myelomonocytic) and G-CSF- (myelocytic) dependent subclones of 32D. However, a gradient of expression, correlating with the lineage, the stage of differentiation, and the growth factor responsiveness of the cell lines, was found among the different subclones: Epo greater than or equal to IL-3 greater than GM-CSF greater than G-CSF. Binding experiments demonstrated NFE-1 activity in all cell lines except the G-CSF-dependent line. Function of the NFE-1 protein was assessed by the expression of the CAT gene linked to the SV40 promoter and a mutant (-175 T----C) HPFH gamma-globin promoter. High level CAT expression was seen only in the Epo1 cells although low level expression was also seen in the parent 32D. These results demonstrate that the specificity of the expression of NFE-1 for the erythroid--megakaryocytic--mast cell lineages is obtained by progressive inactivation of its expression in alternative lineages.
转录结合蛋白NFE-1（也称为GF-1和Ery-f1）被认为在造血谱系（红细胞系、巨核细胞系和嗜碱性粒细胞-肥大细胞系）的一个亚群中，对分化相关基因表达的调控起着必要但不充分的作用。为了阐明NFE-1表达的谱系特异性背后的机制，以及该因子的表达与生长因子反应性之间的关系，我们评估了源自白细胞介素3（IL-3）依赖细胞系32D的促红细胞生成素（Epo）、粒细胞-单核细胞（GM）集落刺激因子（CSF）和粒细胞（G）-CSF依赖的亚克隆表达以下内容的能力：1）NFE-1 mRNA，2）NFE-1相关核蛋白，以及3）当用NFE-1同源序列控制下的氯霉素乙酰转移酶（CAT）基因转染时的CAT活性。发现NFE-1 mRNA不仅在具有肥大细胞（IL-3依赖的32D）和红细胞系（Epo依赖的32D Epo1）表型的细胞中表达，而且在主要具有粒细胞/巨噬细胞特性的细胞中表达，例如32D的GM-CSF（早期骨髓单核细胞）和G-CSF（骨髓细胞）依赖的亚克隆。然而，在不同亚克隆中发现了与细胞系的谱系、分化阶段和生长因子反应性相关的表达梯度：Epo≥IL-3＞GM-CSF＞G-CSF。结合实验证明除了G-CSF依赖的细胞系外，所有细胞系中都有NFE-1活性。通过与SV40启动子和突变型（-175 T→C）HPFHγ-珠蛋白启动子相连的CAT基因的表达来评估NFE-1蛋白的功能。虽然在亲本32D中也观察到低水平表达，但仅在Epo1细胞中观察到高水平的CAT表达。这些结果表明，NFE-1在红细胞系-巨核细胞系-肥大细胞谱系中的表达特异性是通过其在替代谱系中的表达逐渐失活而获得的。
