[Expression of human HZF1 in E. coli and preparation of antibody against human HZF1 protein].

OBJECTIVE: To express human HZF1 fusion protein in E. coli and to obtain an anti-HZF1 antibody. METHODS: A DNA fragment encoding non-zinc finger region of HZF1 protein was inserted into pET30a vector to get the recombination expression plasmid pET30a-HZF1. E. coli was transformed with pET30a-HZF1 and the selected clones were cultured with isopropy-beta-D-thiogalactoside induction. The proteins were prepared from the culture and the fusion protein was purified by Ni column. Rabbits were immunized and reinforced three times with the purified fusion protein. The antiserum was collected and the titer and the specificity of the antibody were checked by ELISA and Western blot. RESULTS: Antibody against HZF1 was obtained and its titer was more than 1:100 000, as proven by ELISA. Western blot analysis showed specific reaction between this antibody and HZF1 fusion protein or the endogenetic HZF1 protein in hemin-induced K562 cells. CONCLUSIONS: The specific antibody against HZF1 is obtained. The antibody may have potential application in farther HZF1 function study and HZF1 determination in tissues and cells.

[Isolation of new Zinc finger genes through cDNA library screening combined with RACE].

Most of the important functionally proteins contain the corresponding function domains that consist of conserved amino acid sequences. The study provided a method to identify novel genes that encode proteins containing important functionally domains with conserved sequences. First, primers were designed according to the sequence of the cDNA library vector and the ESTs that have been obtained by reverse PCR and degenerate primers encoding Zinc finger domain. The cDNA library DNA was used as template for PCR amplification. The amplified fragment that contains nonhomologous sequences of the cDNA was inserted into pGEM-T easy vector. The fragment was recovered and used as a probe for screening the cDNA library. Several cDNAs with full length that encode proteins with Zinc finger domain and represent the original ESTs have been successfully cloned from a human bone marrow cDNA library. This strategy can also be used in screening genes that encode proteins containing differential function domains with conserved sequences.

Zinc finger protein HZF1 promotes K562 cell proliferation by interacting with and inhibiting INCA1.

Previously, we characterized a zinc finger protein gene HZF1 (ZNF16) and demonstrated that it played a significant role in the erythroid and megakaryocytic differentiation of K562 cells by knockdown of the gene. In this study, we examined the effect of HZF1 on the proliferation and apoptosis of K562 cells and identified the possible mechanism for this effect. By lentivirus-mediated gene transfer, we obtained stable K562 transductants with HZF1 overexpression (K562/WPXL-HZF1) and stable control transductants (K562/WPXL). Significantly rapid cell amplification was observed in K562/WPXL-HZF1 cells compared to K562/WPXL cells. The cell cycles of the two transductants were analyzed and the results demonstrated that HZF1 overexpression promoted the S to G2/M phase transition. Additionally, we found that the overexpression of HZF1 slightly inhibits the apoptosis of K562 cells induced by sodium arsenate. Furthermore, using a yeast two-hybrid (Y2H) system we identified the HZF1-interacting proteins and screened 29 potential binding partners of HZF1. Using a co-immunoprecipitation (Co-IP) assay, we confirmed the interaction between HZF1 and the inhibitor of cyclin-dependent kinase (CDK) interacting with cyclin A1 (INCA1), and proved that this interaction leads to the inhibition of INCA1 function, which rescued the activity of CDK2 inhibited by INCA1. In conclusion, our results identified novel functions of the HZF1 gene and revealed a possible mechanism through which HZF1 affects K562 cell proliferation.

Human microRNA clusters: genomic organization and expression profile in leukemia cell lines.

MicroRNAs (miRNAs) play an important role in diverse physiological and developmental processes by negatively regulating expression of target genes at the post-transcriptional level. Here, we globally analyzed the genomic organization of all registered 326 human miRNA genes in miRNA registry 7.1 and found that 148 human miRNA genes appeared in a total of 51 clusters. Alignment of the miRNA sequences in different clusters revealed a significant number of miRNA paralogs among the clusters, implying an evolution process targeting the potentially conserved roles of these molecules. Then we performed Northern blot analysis for expression profiling of all clustered miRNAs in several human leukemia cell lines. Consistent expression of the miRNAs in a single cluster was revealed in 39 clusters, while inconsistent expression of members in a single cluster was detected in the other 12 clusters. Meanwhile, we identified several hematopoietic lineage-specific or -enriched miRNA clusters (e.g., the mir-29c, mir-302, mir-98, mir-29a, and let-7a-1 clusters) and individual miRNAs (e.g., mir-181c, mir-181d, mir-191, and mir-136). These findings may suggest vital roles of these miRNA clusters or miRNAs in human hematopoiesis and oncogenesis, and provide clues for understanding the function and mechanism of miRNAs in various biological processes.

Differential expression changes in K562 cells during the hemin-induced erythroid differentiation and the phorbol myristate acetate (PMA)-induced megakaryocytic differentiation.

K562 cell line has been used as a model of common progenitor of erythroblasts and magakaryocytes and can be differentiated into erythroid and megakaryocytic lineages by hemin and phorbol myristate acetate (PMA) respectively. We analyzed mRNA expression in un-induced, hemin-induced and PMA-induced K562 cells by differential display reverse transcription polymerase chain reaction (DDRT-PCR) method. 314 differential expression sequence tags (ESTs) were obtained. Among them, 201 ESTs displayed up-regulation and 85 ESTs down-regulation after hemin induction, 186 ESTs showed up-regulation and 72 ESTs down-regulation after PMA induction. The differentially expressed genes included those encoding transcription factors, signaling factors, apoptosis-associated factors and others. 45 of these ESTs stand for genes whose open reading frames were found but whose functions remain unknown. 4 ESTs represent possibly new genes. Furthermore we compared differences of gene expression during hemin-induced erythroid differentiation and PMA-induced megakaryocytic differentiation and found that the expressional changes of some transcription factors and metabolism proteins are the common but the expressional changes of some signal pathways in these two differentiation processes are different. These results suggested that erythroid differentiation and megakaryocytic differentiation are associated in activation and repression of different signal pathways.

T to C substitution at -175 or -173 of the gamma-globin promoter affects GATA-1 and Oct-1 binding in vitro differently but can independently reproduce the hereditary persistence of fetal hemoglobin phenotype in transgenic mice.

The T to C substitution at position -175 of the gamma-globin gene has been identified in some individuals with non-deletion hereditary persistence of fetal hemoglobin (HPFH). In this study, the HPFH phenotype was reestablished in transgenic mice carrying the mu'LCRAgamma(-175)psibetadeltabeta construct, which contained a 3.1-kb mu'LCR cassette linked to a 29-kb fragment from the Agamma-to beta-globin gene with the natural chromosome arrangement but with the -175 mutation, which provided evidence for this single mutation as the cause of this form of HPFH. The HPFH phenotype was also reproduced in transgenic mice carrying the mu'LCRAgamma(-173)psibetadeltabeta construct, in which the -175 T to C Agamma gene was substituted with the -173 T to C Agamma gene. In vitro experiments proved that the -175 mutation significantly reduced binding of Oct-1 but not GATA-1, whereas the -173 mutation dramatically decreased binding of GATA-1 but not Oct-1. These results suggest that abrogation of either GATA-1 or Oct-1 binding to this promoter region may result in the HPFH phenotype. An in vivo footprinting assay revealed that either the -175 mutation or the -173 mutation significantly decreased overall protein binding to this promoter region in adult erythrocytes of transgenic mice. We hypothesize that a multiprotein complex containing GATA-1, Oct-1, and other protein factors may contribute to the formation of a repressive chromatin structure that silences gamma-globin gene expression in normal adult erythrocytes. Both the -173 and -175 T to C substitutions may disrupt the complex assembly and result in the reactivation of the gamma-globin gene in adult erythrocytes.