Zinc Finger Protein Hzf1 Gene Function And Mechanisms

Human mature blood cells derive from Hematopoietic Stem Cells(HSCs). Commitment of stem cells to specific hematopoietic lineages and further differentiation and development mature of the committed cells are controlled at least partially through the combinatorial action of lineage-restricted and more widely expressed transcription factors. Some transcription factors containing C2H2 zinc finger motif have been found to play an important part in differentiation and development of blood cells.In 2000, we cloned a novel zinc finger cDNA HZF1 (GenBank accession No. AF244088) by screening a human bone marrow cDNA library. The HZF1 encodes 670 amino acid residues including 15 typical C2H2 and 2 C2RH zinc finger motifs. The HZF1 mRNA was expressed extensively and highly in brain, heart, skeletal muscle and fetal liver. The HZF1 mRNA was detected in various hematopoietic cell lines. The repression of intrinsic expression of HZF1 was also revealed in K562/pAVu6HZF1i transfectant cells and the hemin-induced erythroid differentiation of K562 cells was significantly blocked and the PMA-induced megakaryocytic differentiation was obviously reduced. All these results proved that the zinc finger protein HZF1 play an important role in the erythroid and megakaryocytic differentiation of K562 cells. Based on these results, my study focused on the research of the functions of HZF1 by yeast two hybrid, monitoring the ERK signaling pathway and the analysis of gene chip.To finding the proteins interacting with HZF1, yeast two hybrid was used by screening a human bone marrow cDNA library and using the fragment of HZF1 as the bait protein. 288 candidated positive clones which was identified from 1478 clones was retested phenotypes, then 59 false positive clones was excluded. 229 candidated positive clones was sorted and sequenced, then blasted on the NCBI website. The non-coding cDNA sequence and the cDNA sequence which could not the correct protein was excluded, finally 30 cDNA sequences which ORF were correct were found. These protein was involved in eukaryotic translation elongation, differatiation, cell cycle and immune etc. Two false positive clones that could autonomously the report gene was excluded by the bait and prey plasmid cotransform experiment. Three positive clones 8-23,15-83 and 11-20 was choosed to proceed the next study. 8-23 is the part of the ORF of inhibitor of CDK interacting with cyclin A1 (INCA1). 15-83 is the part of the ORF of four and a half LIM domains 1 (FHL1). 11-20 is the part of the ORF of zinc finger protein 7 (ZNF7). The full coding sequence of FHL1 was cloned from the cDNA. The interaction of HZF1 and INCA1, as well as the interaction of HZF1 and FHL1, were confirmed by CoIP and Western Blot experiments, but the interaction of HZF1 and ZNF7 wasn't found.The expression of INCA1 during the the hemin-induced erythroid differentiation of K562 cells was detected by real-time PCR. The result showed INCA1 mRNA expression increased following erythroid differentiation of K562 cells induced by hemin and reached its crest at 24h, which was consistent with the case of HZF1.To study the change of ERK signaling pathway during the hemin-induced erythroid differentiation or the PMA-induced megakaryocytic differentiation of K562 cell line when the intrinsic expression of HZF1 was repressed, phosphorylation of ERK and MEK in K562/pAVu6HZF1i (intrinsic expression of HZF1 repressed by RNAi) and the control K562/pAVu6 were detected by Western Blot experiment. Whether the induce agents were exist or not, the phosphorylation of ERK and MEK were higher than the control cells K562/pAVu6. During the hemin- or PMA-induced differentiation of K562/pAVu6HZF1i, the change of phosphorylation of ERK and MEK as time was going were the same as the K562/pAVu6 cells. These results suggested that the repression of HZF1 probably effected the expression of some genes upstream MEK gene, resulting in the increase of phosphorylation of ERK and MEK.In order to find more clues to study the function of HZF1, the differention of gene expression between K562/pAVu6HZF1i and K562/pAVu6 was detected by using gene chip. Then, the results were analysized using the The NetAffy^TM Analysis Center(www.affymetrix.com). The genes that their expression increased or decreased were found and sortsed according to their functions. A number of genes involved signal transduction, cell cycle, differentiation, development and transcript regulation etc.INCA1, the interaction partner of HZF1, was found and confirmed in our study. INCA1( inhibitor of CDK interacting with cyclin A1) is a inhibitor of cyclin A1-CDK complex. The results also showed when the differentiation was repressed, the phosphorylation of ERK and MEK increased. Combination with the results of gene chip analysis, these results suggested HZF1 probably promotes the differentiation of K562 cell line by effecting ERK signaling pathway and regulating cell cycle.