Initial Exploration Of ZNFD Biological Functions And ZBTB9 Cloning And Expression Of Mouse Zinc Finger Protein

The C2H2-type zinc finger proteins that function as transcription factors perform important roles in various cellular functions, including transcriptional regulation, cell proliferation, differentiation and apoptosis. We cloned two mouse zinc finger gene ZNFD (NM₀01004061)and ZBTB9(NM₀01005916) by RT-PCR analysis.SMART analysis showed that ZNFD protein contains a C2H2-type zinc finger in the C terminal; ZBTB9 protein contains a BTB domain in the N terminal and two C2H2-type zinc fingers in the C terminal, which suggests that they are both important members of the C2H2-type zinc finger protein family. There are rare studies about the function of ZNFD and ZBTB9 in mice, so we carry out preliminary exploration on these two genes. (1) Firstly, we cloned the ZNFD gene from mouse testis cDNA by RT-PCR, then identified by sequencing. Bioinformatics databases were used to analyze and predict its function. Results showed that ZNFD gene had a 1002bp full open reading frame (ORF), and it was localized on chromosome 18qD1. The ZNFD gene encoded a protein with 333 amino acids. The molecular weight was predicted to be 37.4 kDa. The expression pattern analysis of mouse ZNFD revealed that it was most probably a testis-specific expression gene and had a high expression level. The ZNFD could not be detected until 13days. Multiple alignment analysis of ZNFD in seven species showed that ZNFD shared many similarities with other species.To verify the subcelluar location of ZNFD protein, we contructed the plasmid pEGFP-ZNFD, which expressed the fused protein GFP-ZNFD. Results showed that ZNFD was a nuclear protein. To identify whether the N-terminal domain and the zinc finger domain of ZNFD have effect on its nuclear localization, we constructed a series of truncated fragments of ZNFD-pEGFP. Results suggested that the N-terminal amino acids(1-214) is required for its nuclear localization, and the ZNF domain have little function in localization of the ZNFD protein.Here we performed Dual-luciferase reporter assay system to investigate the potentially role of ZNFD on signaling pathway including AP1(PMA),HSE,GRE,NF-κB,AP1,CRE,SRE,p53. Results indicated that ZNFD functioned as a dose-dependent activator of the HSE reporter gene. Further study of the functional domain of ZNFD suggested that ZNFD needed to maintain its structural integrity in transcriptional regulation.The pHSE-luc plasmid is designed to monitor the activation of heat shock factor (HSF) and heat shock-mediated signal transduction pathways. HSE elements commonly exist in the promoter region of heat shock protein genes. After the transcription factors binds to HSE, the transcription of heat shock protein genes is induced. It is suggest that ZNFD may probably participate in the development of mice testis and spermatogenesis by regulating the HSE-mediated expression of HSPs.(2) In silico analysis of mouse ZBTB9 showed that it contains an ORF of 1380bp and encodes 459 amino acid protein.The translation start codon is ATG and the stop codon is TAG. It was localized to 17qA3.3. Subcellular localization analysis demonstrated that ZBTB9 was localized to the nucleus, forming dot-like structures. After expression and purification,we obtained the purified recombinant protein. In this study, the cloning and expression of ZBTB9 provided a reliable tool for the further study of its biological function.