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

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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.