Initial Exploration Of The Function Of ZNFD-a C₂H₂-type Zinc Finger Protein

C2H2 zinc finger (C2H2-ZNF) genes are one of the largest and most complex gene super-families in metazoan genomes, with hundreds of members in the human and mouse genome. We got a novel human zinc finger gene ZNFD by bioinformatics analyse and NCBI database screening. The gene ID is NM₁82761. The bioinformatics character suggested that ZNFD is a member of C2H2 type zinc finger protein, and this gene has not been investigated. The main complishments are as follows.Firstly, we cloned the ZNFD gene from testis cDNA by RT-PCR, then identified by sequencing. Bioinformatics databases were used to analyze and predict its function. We confirmed that ZNFD gene has a 990bp full open reading frame (ORF), which was localized on chromosome 5q23.1. It was composed of five exons and four introns. The ZNFD gene encoded a protein with 329 amino acids. The molecular weight was predicted to be 37kDa. RT–PCR was used to determine the expression pattern of ZNFD in human multiple tissue. The tissue distribution pattern of ZNFD mRNA showed that the band was detected in the prostate, testis, brain, spleen, pancreas, and uterus. ZNFD gene is strongly expressed in adult testis and brain.Multiple alignment analysis was performed by ClustalW software in 7 ZNFD orthologs. ZNFD was highly conservative in 17 to 260aa in different species. To verify the subcelluar location of ZNFD protein, we contruct the plasmid pEGFP-C1-ZNFD which express the fused protein EGFP-ZNFD, then transfected it into Hela cells. The result indicated that ZNFD is a nuclear protein. To verify the subcelluar location and the function of N-terminal residues and ZNF motif in location of our interested protein, pEGFP-C1-ZNFD, pEGFP-C1-D-ZNF, and pEGFP-C1-ZNF were transfected into Hela cells. These results indicate that ZNFD is a nuclear protein and the ZNF domain is not required for nuclear localization of the ZNFD protein in Hela cells.Many papers showed that many ZNF proteins function as transcription factors. Accordingly, it was reasonable to postulate that ZNFD possibly possesses transcription function. Here we performed Dual-luciferase reporter assay system to investigate the potentially role of ZNFD on signaling pathway including AP1(PMA), GRE, SRE, p53, CRE, HSE, NF-κB and AP1. The data indicate that ZNFD protein induces the activation of AP1(PMA) transcription, and ZNFD induced a dose- dependent activation of the AP1(PMA) reporter gene. The pAP1(PMA)-TA-luc plasmid contains the AP1(Activator of protein 1) elements, which are designed for monitoring the induction of the PKC signal transduction pathway. So it suggests that the ZNFD may act as a transcriptional activator in PKC signal pathway to mediate cellular functions. Addition, tandem affinity purification (TAP) is a generic two-step affinity purification protocol that enables the isolation of protein complexes under close-to-physiological conditions. So we construct the plasmid which expresses the fusion TAP-ZNFD protein to analysis of protein-protein interaction.