We have previously shown that the human genome includes hundreds of genes coding for putative factors related to the Krfippel zinc-ringer protein, which regulates

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.

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.

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.

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