Analysis of mechanisms of the differentiation inhibition in erythroid cells induced by Ets transcription factor PU. I

寻找与PU诱导的小鼠红白血病细胞分化抑制有关的基因(S)。1、筛选了PU过表达后MEL细胞中表达上调或下调的基因。1采用mRNA差异显示(DD)策略。在筛选结果的基础上，我们将研究扩展到以下几个方面：(1)由于DD已经证明了一些骨髓单核细胞特异性基因的表达上调，我们扩大了分析并证明了多种骨髓单核细胞特异性基因的表达上调。此外，随着PU的过度表达。1、MEL细胞贴壁、吞噬。另一方面，突变的PU不能诱导骨髓单核细胞特异性基因的表达。1，表达了同样抑制MEL细胞红系分化的部分激活结构域。这些结果表明，PU。1诱导MEL细胞向粒单核细胞的谱系转换，提示谱系转换的途径与抑制细胞红系分化的途径不同。(2)PU过表达后在MEL细胞中表达上调的新基因之一。1在T细胞和胚胎癌细胞中正常表达。我们已经克隆了这个基因。由于发现了一个与人钙调素依赖性蛋白依赖性激酶I-样激酶(CKLiK)基因同源的开放阅读框，该新基因被认为是人CKLiK基因的小鼠同源基因。然而，开放阅读框的3个部分的核苷酸序列与人CKLiK基因的核苷酸序列存在差异，存在两种转录本，它们的3端存在差异，这可能是由于选择性剪接所致。我们正计划研究新基因在PU中的功能作用。1介导的MEL细胞分化抑制及PU1参与该基因的转录调控。

To identify a gene(s) involved in the differentiation inhibition of murine erythroleukemia (MEL) cells induced by PU. 1, we had screened the genes whose expression is up- or downregulated in MEL cells after overexpression of PU. 1 by using the mRNA differential display (DD) strategy. Based on the results of the screening, we have extended the study as follows.(1) Because DD had shown that the expression of some of myelomonocyte-specific genes is up-regulated, we expanded analysis and demonstrated that expression of a variety of myelomonocyte-specific genes is up-regulated. Furthermore, following overexpression of PU. 1, MEL cells became adherent and phagocytic. On the other hand, expression of myelomonocyte-specific genes was not induced when a mutant PU. 1, with part of the activation domain deleted, which also inhibits erythroid differentiation of MEL cells was expressed. These results indicate that PU. 1 induces lineage switch in MEL cells toward myelomonocytic cells and suggest that the pathway of lineage switch is distinct from that of inhibition of the erythroid differentiation of the cells.(2) One of the novel genes whose expression is up-regulated in MEL cells after overexpression of PU. 1 was found to be expressed normally in T cells and embryonal carcinoma cells. We have cloned this gene. As an open reading frame was identified with homology to human calcium-calmodelin-dependent kinase I-like kinase (CKLiK) gene, the novel gene was thought to be the mouse homologue of human CKLiK gene. However, the nucleotide sequence of 3 portion of the open reading frame was diverged from that of human CKLiK gene Two kinds of transcripts showmg difference in their 3 ends, which is presumably due to alternative splicing, were present We are now planning to investigate the functional role of the novel gene in the PU. 1-mediated differentiation inhibition of MEL cells and involvement of PU.1 in transcriptional regulation of the gene.