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HGNC Approved Gene Symbol: ZNF224
Cytogenetic location: 19q13.31 Genomic coordinates (GRCh38) : 19:44,094,361-44,109,826 (from NCBI)
Zinc finger proteins (ZNFs) are DNA-binding transcriptionl regulators, and ZNFs containing Kruppel-associated box (KRAB) domains typically mediate transcriptional repression. The ZNF224 gene encodes 2 splice variants, ZNF224 and ZNF255 (also known as BMZF2), that are differentially expressed in adult and fetal tissues and encode proteins with different subcellular localizations. ZNF224 contains a KRAB domain, localizes to the nucleus, and functions as a transcriptional repressor of the aldolase A (ALDOA; 103850) gene. ZNF255 lacks the KRAB domain and localizes throughout the cell, including nucleus, cytosol, and nucleolus. Both ZNF224 and ZNF255 interact physically and functionally with specific isoforms of WT1 (607103) (Medugno et al., 2007; Florio et al., 2010).
Using PCR primers based on conserved sequences of Kruppel-like genes to screen a bone marrow cDNA library, followed by database searching and 3-prime and 5-prime RACE, Han et al. (1999) isolated cDNAs encoding several zinc finger proteins, including ZNF255. The deduced 623-amino acid ZNF255 protein contains 18 tandemly repeated C-terminal zinc finger motifs with interspersed conserved knuckle sequences, as well as an N-terminal Kruppel-related novel box (KRNB), which is also found in FDZF2 (ZNF230). RT-PCR analysis detected wide expression that was highest in hemopoietic tissues and cell lines.
By affinity chromatography to isolate HeLa cell nuclear proteins that interact with WT1, followed by peptide analysis and RT-PCR of HeLa cell mRNA, Lee et al. (2002) obtained a cDNA encoding ZNF255, which they called BMZF2. The deduced 622-amino acid protein has a calculated molecular mass of 72 kD. Northern blot analysis detected expression only in fetal tissues. Transcripts of about 5.0 and 4.0 were detected in fetal brain, lung, liver, and kidney, and a transcript of about 3.4 kb was also detected in fetal lung. Western blot analysis of cytosolic and nuclear fractions obtained from transfected cells indicated that ZNF255 is expressed in the nucleus.
Medugno et al. (2003) identified ZNF224 as a 97-kD protein that interacted with the ALDOA (103850) negative regulatory element (NRE). By database analysis, they identified a ZNF224 cDNA. The deduced 707-amino acid protein contains 19 tandemly repeated C2H2-type zinc finger motifs separated by 7-amino acid linkers.
Medugno et al. (2007) determined that ZNF224 and ZNF255 are splice variants of the same gene that differ at their 5-prime UTRs and 5-prime coding regions. The longer ZNF224 protein contains an N-terminal KRAB domain and 19 C-terminal zinc fingers. ZNF255 lacks the KRAB domain of ZNF224, but it has the 19 zinc fingers. Semiquantitative PCR detected variable ZNF224 expression in all adult and fetal tissues examined. Expression of ZNF255 was more restricted in adult tissues and showed generally lower expression in adult tissues compared with fetal tissues. Immunofluorescence analysis revealed that ZNF224 was expressed exclusively in the nucleus. In contrast, ZNF255 was expressed in both nucleus and cytoplasm, and it was also distributed in the nucleolus in 20 to 30% of cells, where it colocalized with fibrillarin (FBL; 134795).
Florio et al. (2010) noted that mouse does not contain an ortholog of the ZNF224 gene.
Han et al. (1999) showed that the KRNB box of ZNF255 has a slight but significant transactivation activity in yeast cells, but not in mammalian cells.
Lee et al. (2002) demonstrated that ZNF255 interacted with WT1 in vitro and in vivo. Mutation analysis showed that zinc fingers 6 to 10 of ZNF255 were required to interact with the zinc finger region of WT1. ZNF255 inhibited transcriptional activation by WT1, and the presence of a repressor domain within ZNF255 was confirmed in a reporter assay.
By gel shift assay, Medugno et al. (2003) confirmed that ZNF224 bound the negative cis element NRE in the ALDOA gene. Progressive deletion of ZNF224 C-terminal zinc fingers reduced the ALDOA NRE binding. Expression of ZNF224 in COS cells inhibited reporter activity from a promoter containing 2 ALDOA NRE core elements in a dose-dependent manner. Medugno et al. (2003) concluded that ZNF224 negatively regulates ALDOA gene expression during the cell cycle and differentiation.
Using chromatin immunoprecipitation and reporter gene assays with transfected HEK293 cells, Medugno et al. (2007) showed that ZNF255 was weaker than ZNF224 in binding the NRE in the ALDOA promoter region and in repressing expression of an ALDOA reporter plasmid.
By coimmunoprecipitation analysis of human cell lines, Florio et al. (2010) discovered that ZNF224 interacted specifically and exclusively with the -KTS isoform of WT1, whereas ZNF255 interacted with both the +KTS and -KTS isoforms of WT1. Using a reporter plasmid containing the promoter region of a WT1 target gene, VDR (601769), Florio et al. (2010) showed that cotransfection of ZNF224 caused a dose-dependent enhancement of WT1(-KTS)-mediated VDR expression, while ZNF255 had no effect. Chromatin immunoprecipitation analysis showed that ZNF224 was recruited with WT1 to the VDR promoter. Knockdown of ZNF224 reduced VDR mRNA and protein. In contrast, ZNF255, but not ZNF224, colocalized with WT1(+KTS) in the polysome fraction of HEK293 cells and copurified with poly(A) ribonuclear particles.
Medugno et al. (2007) determined that the ZNF224 gene contains 6 exons. The first 3 exons are noncoding. The 5-prime UTR of the ZNF255 transcript corresponds to intron 5, and the coding region of ZNF255 is completely contained within exon 6.
By analysis of somatic cell hybrids and by in situ hybridization, Rousseau-Merck et al. (1993) demonstrated that the KOX22 (ZNF224) gene and the KOX5 (ZNF45; 194554) genes mapped to 19q13.2-qter. Furthermore, by pulsed field gel electrophoresis experiments they showed that the pair of genes lie within a DNA segment less than 300 kb long.
Using FISH, Han et al. (1999) mapped the ZNF224 gene to chromosome 19q13, where the ZNF256 gene (606956) maps.
Florio, F., Cesaro, E., Montano, G., Izzo, P., Miles, C., Costanzo, P. Biochemical and functional interaction between ZNF224 and ZNF255, two members of the Kruppel-like zinc-finger protein family and WT1 protein isoforms. Hum. Molec. Genet. 19: 3544-3556, 2010. [PubMed: 20591825] [Full Text: https://doi.org/10.1093/hmg/ddq270]
Han, Z.-G., Zhang, Q.-H., Ye, M., Kan, L.-X., Gu, B.-W., He, K.-L., Shi, S.-L., Zhou, J., Fu, G., Mao, M., Chen, S.-J., Yu, L., Chen, Z. Molecular cloning of six novel Kruppel-like zinc finger genes from hematopoietic cells and identification of a novel transregulatory domain KRNB. J. Biol. Chem. 274: 35741-35748, 1999. [PubMed: 10585455] [Full Text: https://doi.org/10.1074/jbc.274.50.35741]
Lee, T. H., Lwu, S., Kim, J., Pelletier, J. Inhibition of Wilms tumor 1 transactivation by bone marrow zinc finger 2, a novel transcriptional repressor. J. Biol. Chem. 277: 44826-44837, 2002. [PubMed: 12239212] [Full Text: https://doi.org/10.1074/jbc.M205667200]
Medugno, L., Costanzo, P., Lupo, A., Monti, M., Florio, F., Pucci, P., Izzo, P. A novel zinc finger transcriptional repressor, ZNF224, interacts with the negative regulatory element (AldA-NRE) and inhibits gene expression. FEBS Lett. 534: 93-100, 2003. [PubMed: 12527367] [Full Text: https://doi.org/10.1016/s0014-5793(02)03783-3]
Medugno, L., Florio, F., Cesaro, E., Grosso, M., Lupo, A., Izzo, P., Costanzo, P. Differential expression and cellular localization of ZNF224 and ZNF255, two isoforms of the Kruppel-like zinc-finger protein family. Gene 403: 125-131, 2007. [PubMed: 17900823] [Full Text: https://doi.org/10.1016/j.gene.2007.07.036]
Rousseau-Merck, M.-F., Hillion, J., Jonveaux, P., Couillin, P., Seite, P., Thiesen, H.-J., Berger, R. Chromosomal localization of 9 KOX zinc finger genes: physical linkages suggest clustering of KOX genes on chromosomes 12, 16, and 19. Hum. Genet. 92: 583-587, 1993. [PubMed: 8262519] [Full Text: https://doi.org/10.1007/BF00420943]