Cloning, Characterization And Functional Analysis Of A Novel Human Zinc Finger Gene HKid3

The C₂H₂ zinc finger protein family is one of the largest families of transcription factors. It has been estimated that the human genome contains up to 706 C₂H₂ zinc finger genes. The C₂H₂ zinc finger motif consists of 21-23 amino acid CX_2-4CX₃FX₅LX₂HX_3-4H, with highly conserved H/C linker TGEKPYX between the last histidine of the preceding finger motif and the first cysteine of the next finger. Diversity within the C₂H₂ zinc finger gene family is found in the conserved modular domains that lie outside of the zinc finger region. These N-terminal domains include the KRAB, BTB-POZ, SCAN domain, etc. The primary role of the identified C₂H₂ zinc finger proteins is to bind specific DNA sequence via their zinc fingers, resulting in the activation or repression of gene expression during diverse biological processes such as cell growth, differentiation, tumorigenesis and embryogenesis.In order to isolate the C₂H₂ zinc finger genes involved in early human development more efficiently, we constructed a human embryo C2H2-ZNF enriched cDNA library. A biotin-labeled antisense capture-probe was designed based on the highly conserved H/C linker of C₂H₂ zinc finger proteins. The probe was incubated with total RNA from 4-6 week human embryo, and then streptavidin-coated magnetic beads was used to enrich the C₂H₂-specific mRNA. The C2H2-ZNF enriched humanembryonic cDNA library was then constructed according to the manufacture's protocol. Sequence analysis of randomly picked clones form the library indicated that most of the inserts were derived from C₂H₂ zinc finger genes, of which a 2070-bp fragment of a novel gene, hKid3, was found to be human orthologue of mouse Kid3 gene. 5' RACE was performed and yielded a 393 bp fragment corresponding to the 5' end of the hKid3 cDNA. The patchwork cDNA sequence data reported here is available in GenBank with accession No. AF525463.The complete sequence of the cloned hKid3 cDN A was 2397 bp and had an open reading frame of 1662 bp. The predicted hKID3 protein contains a N-terminal KRAB domain and 11 tandemly repeated C₂H₂ zinc finger motifs at the C-terminus. The hKid3 gene is assigned to human chromosome 5q35 according to the mapping information in NCBI. It is composed of five exons, with spacer and all zinc finger motifs in exon 5, and the KRAB A and B box encoded by exon 3 and exon 4 respectively.Comparison of hKid3 amino acid sequence with the most related ZNF proteins Kidl and Kid2 revealed that it belonged to the Kid gene subfamily. A conserved degenerate zinc finger, which was linker of about 28 amino acids with mutation on the amino acid crucial for zinc coordination, was found between the fourth and the fifth zinc finger of Kidl or Kid2, while this degenerate zinc finger is absent in Kid3 proteins from human, mouse and rat. The phylogenetic tree analysis argues that Kidl and Kid2 belong to a single group which resulted from a more recent duplication, and that this group and Kid3 came from a relative ancient duplication and might now serve different cellular functions.Northern blot analysis revealed two transcripts of 6 kb and 8.5 kb mainly expressed in human fetal brain and kidney. The hKid3 gene had little expression in the fetal lung and liver. The 6 kb transcript also expressed in human adult skeletal muscle and very weakly in other adult tissues such as brain and heart. Of note, the 8.5 kb transcript could not be detected in adult tissues, suggesting that it might be an product of embryo-specific post-transcriptional processing, thus may be important for human embryonic development of brain and kidney.The sequence analysis above suggests that hKid3 is a putative transcription factor, and it should be localized to nucleus. To investigate this possibility, we expressed EGFP fusion proteins of hKid3 in COS7 cells by transient transfection. Although cells transfected with the pEGFP-Cl vector or pEGFP-KS(KRAB+spacer) showed GFP fluorescence both in the cytoplasm and nucleus, cells transfected with either pEGFP-FL(full length) or pEGFP-ZF(zinc finger domain) showed fluorescence only in nucleus, demonstrating that hKID3 protein is a nuclear protein and the ZF domain was both necessary and sufficient for nuclear localization of hKID3.The KRAB domain of hKid3 exhibits transcription repressor activity when tested in CAT reporter assay. To further investigate the possibility of hKid3 functioning as a sequence-specific transcription factor like other ZNFs, we generated an oligonucleotide library containing 25-bp random DNA sequences. Recombinant GST fusion protein of hKID3 ZF domain(GST-ZF) was purified and immobilized on beads to capture putative target DNA from the library. At last a consensus DNA-binding sequence of 5'-CCAC(c/g)-3' was identified for hKID3. Mutational analysis of the consensus binding sequence by competitive elctrophoretic mobility shift assay(EMSA) indicated that each base of the "CCAC" core sequence was crucial for hKID3 specific DNA-binding activity.These results indicate that hKid3 may function as a nuclear transcription repressor , through binding to "CCAC-containing DNA element and the KRAB-mediated transcription repress mechanism with regulated expression pattern during human development of brain and kidney. The expression profile of suggesting that it may be modulated by embryo-specific alternative transcript expression, and thus be important for human embryonic development of brain and kidney.