RESUMO
We previously identified the ZNF85 (HPF4) KRAB zinc finger gene, a member of the human ZNF91 family. Here, we show that the ZNF85 gene is highly expressed in normal adult testis, in seminomas, and in the NT2/D1 teratocarcinoma cell line. Immunocytochemical localization of a panel of beta-Gal/ZNF85 fusion proteins revealed that ZNF85 contains at least one nuclear localization signal located in the spacer region connecting the KRAB domain with the zinc finger repeats. Bacterially expressed ZNF85 zinc finger domain bound strongly and exclusively to DNA in vitro in a zinc-dependent manner. The KRAB(A) domain of the ZNF85 protein and of several other members of the ZNF91 family exhibited repressing activity when tested in Gal4 fusion protein assays. The repression was significantly enhanced by the addition of the KRAB (B) domain, whereas further addition of other conserved regions had no effect. The ZNF85 KRAB(A) and (B) domains in vitro bound several nuclear proteins that might constitute critical cofactors for repression.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Proteínas Repressoras/fisiologia , Testículo/metabolismo , Dedos de Zinco/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Núcleo Celular/metabolismo , DNA/metabolismo , DNA Complementar , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Expressão Gênica , Humanos , Fatores de Transcrição Kruppel-Like , Masculino , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Zinco/metabolismo , Dedos de Zinco/genéticaRESUMO
Two novel KRAB (Krüppel associated box) type zinc finger protein encoding cDNAs, named Kzf1 and Kzf2 (Kzf for KRAB zinc finger), were identified by screening of a rat embryonic brain cDNA library with a human ZNF91 KRAB probe. Kzf1 and Kzf2 encode proteins with an amino-terminal KRAB domain and a carboxy-terminal zinc finger cluster containing 9 and 13 zinc finger units, respectively. While Kzf2 appears to be ubiquitously expressed, Kzf1 is preferentially expressed in the testis. Within the testis, Kzf1 mRNA is restricted to germ cells. The Kzf1 protein exhibits DNA binding activity and its KRAB domain can function as a repressor module in transcription. Using somatic cell hybrid analysis, the Kzf1 gene was mapped to chromosome 6.
Assuntos
Proteínas de Ligação a DNA/genética , Testículo/metabolismo , Dedos de Zinco , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Humanos , Masculino , Dados de Sequência Molecular , Ratos , Espermatogênese/fisiologiaRESUMO
Six Krüppel-type zinc finger (ZF) genes were cloned from a seminoma cDNA library. One, ZFS-1, showed high sequence homology to the ZNF91 KRAB (Krüppel-associated box) ZF gene family and also the same chromosomal assignment. Interestingly, Northern blot analyses using ZFS-1 and ZNF91 revealed that multiple ZF genes on chromosome 19 were predominantly expressed in seminomas. In addition, the testis and the seminoma showed specific expression of 2.3 kb transcript. Our results suggest that ZF genes on chromosome 19 may be implicated in the development and/or growth of seminomas.
Assuntos
Cromossomos Humanos Par 19 , Seminoma/genética , Dedos de Zinco/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Mapeamento Cromossômico , DNA Complementar/química , DNA Complementar/isolamento & purificação , Humanos , Dados de Sequência MolecularRESUMO
KRAB zinc-finger proteins (KRAB-ZFPs) constitute a large subfamily of ZFPs of the Krüppel C2H2 type. KRAB (Krüppel-associated box) is an evolutionarily conserved protein domain found N-terminally with respect to the finger repeats. We report here the characterization of a particular subgroup of highly related human KRAB-ZFPs. ZNF91 is one representative of this subgroup and contains 35 contiguous finger repeats at its C-terminus. Three mRNA isoforms with sequence identity to ZNF91 were isolated by the polymerase chain reaction. These encode proteins with a KRAB domain present, partially deleted or absent. Five genomic fragments were characterized, each encoding part of a gene: the ZNF91 gene or one of four distinct, related KRAB-ZFP genes. All exhibit a common exon/intron organization with the variant zinc finger repeats organized in a single exon and the KRAB domain encoded by two separate exons. This positioning of introns supports the hypothesis that the mRNA isoforms encoding polypeptides with variability in the KRAB domain could arise by alternative splicing. By in situ chromosomal mapping studies and by analysis of fragments from a human genomic yeast artificial chromosome library containing KRAB-ZFP genes, we show that these genes occur in clusters; in particular, a gene complex containing over 40 genes has been identified in chromosomal region 19p12-p13.1. These ZNF91-related genes probably arose late during evolution since no homologous genes are detected in the mouse and rat genomes. Although the transcription of members of this KRAB-ZFP gene subgroup is detectable in all human tissues, their expression is significantly higher in human T lymphoid cells.
Assuntos
Cromossomos Humanos Par 19 , Proteínas de Ligação a DNA/genética , Genes , Linfócitos T/metabolismo , Dedos de Zinco , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Mapeamento Cromossômico , Clonagem Molecular , DNA/genética , Expressão Gênica , Ligação Genética , Humanos , Fatores de Transcrição Kruppel-Like , Camundongos , Dados de Sequência Molecular , Família Multigênica , RNA Mensageiro/genética , Ratos , Mapeamento por Restrição , Alinhamento de SequênciaRESUMO
We have previously shown that the human genome includes hundreds of genes coding for putative factors related to the Krüppel zinc-finger protein, which regulates Drosophila segmentation. We report herein that about one-third of these genes code for proteins that share a very conserved region of about 75 amino acids in their N-terminal nonfinger portion. Homologous regions are found in a number of previously described finger proteins, including mouse Zfp-1 and Xenopus Xfin. We named this region the Krüppel-associated box (KRAB). This domain has the potential to form two amphipathic alpha-helices. Southern blot analysis of "zoo" blots suggests that the Krüppel-associated box is highly conserved during evolution. Northern blot analysis shows that these genes are expressed in most adult tissues and are down-regulated during in vitro terminal differentiation of human myeloid cells.
Assuntos
Proteínas de Ligação a DNA/química , Hormônios Juvenis/química , Proteínas Repressoras , Fatores de Transcrição , Dedos de Zinco , Sequência de Aminoácidos , Clonagem Molecular , DNA/genética , Proteínas de Ligação a DNA/genética , Biblioteca Gênica , Humanos , Hormônios Juvenis/genética , Fatores de Transcrição Kruppel-Like , Dados de Sequência Molecular , Células Tumorais CultivadasRESUMO
Our aim was to identify new human proteins with potential DNA binding activity, related to the Krüppel protein which regulates Drosophila segmentation. We screened a human placenta cDNA library and a human genomic DNA library with a synthetic oligonucleotide probe corresponding to the H/C link region that connects finger loops in the multifingered Krüppel protein. We found more than 100 different mRNAs encoding Krüppel multifingered proteins in the human placenta. In the whole human genome, the number of genes encoding such proteins reaches about 300. Sequence analysis of 14 cloned cDNAs indicated that they code for at least nine undescribed human finger proteins. The sequences of the 106 finger repeats present in these nine proteins are highly homologous. Most of the variability lies in a limited number of positions located in their postulated alpha-helical structure, and therefore could be implicated in their DNA-binding specificity.
