The Role of EHZF in Erythroid Differentiation of K562 Cells.

Early hematopoietic zinc finger protein (EHZF), the human homolog to mouse Evi3, is a transcription factor with 30 zinc fingers and a highly conserved N-terminal FOG repression motif. The EHZF mRNA expression is abundant in early hematopoietic progenitors and declines during differentiation. In most of the leukemic cells from acute myelogenous leukemia patients, significant levels of EHZF mRNA are detected. These findings suggest that EHZF play an important role in hematopoietic differentiation.

In the present study we investigated whether siRNA-mediated depletion of EHZF affected the erythroid differentiation of K562 cells. Three kind of EHZF siRNA were designed and their expression vectors were constructed. After transient expression of these siRNAs by electroporation, hemoglobinization of K562 cells were analyzed by Benzidine staining. In all of these siRNAs introduced cells, the levels of hemoglobinizations were abundant compared to control cells, which suggested that EHZF might influence the erythroid differentiation of K562 cells. To further analyze the effects of EHZF siRNA in erythroid differentiation, we established K562 clones in which EHZF was depleted by siRNA. EHZF siRNA expression vectors were introduced into K562 cells by electroporation and the expression of EHZF mRNA were examined by RT-PCR in G418 resistant clones. The depressions of EHZF mRNA in the clones were confirmed by Northern blotting. We isolated several EHZF depleted clones in each kind of EHZF siRNA introduced cells. Most of the EHZF depleted clones showed marked hemoglobinization by Benzidine staining compared to control clones, and surface expressions of glycophorin A were also increased in these clones. These results confirmed the relevant role of EHZF in erythroid differentiation of K562 cells. Comparison of mRNA expressions between an EHZF depleted clone and a control clone using DNA array showed increase expressions of hemoglobin alpha, beta, delta mRNA in EHZF depleted clone compared to control clone. Several transcription factors which are involved in erythroid differentiation also showed the difference mRNA levels between two clones.

Studies are underway to elucidate the mechanisms underlying the role of EHZF in erythroid differentiation, the relationship to other transcription factors (GATA-1, GFi-1B, FOG-1 etc) which involved in erythroid differentiation.