Functional Analysis of ZNF85 KRAB Zinc Finger Protein, a Member of the Highly Homologous ZNF91 Family

Abstract

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.

Identification of Glis1, a Novel Gli-related, Krüppel-like Zinc Finger Protein Containing Transactivation and Repressor Functions

“…Plasmids-The reporter plasmid pFR-LUC, containing five copies of the Gal4 upstream activating sequence (UAS) upstream and referred to as (UAS) 5 -LUC, was obtained from Stratagene. The vectors pM and VP16, and the ␤-galactosidase reporter plasmid pCMV␤ were purchased from CLONTECH.…”

“…with 0.25 g of (UAS) 5 -LUC, 0 -1.0 g of pM-Glis1 plasmid containing various Glis1 deletions as indicated, 1.0 -0 g of pBSK, and 0.25 g of pCMV␤, which served as an internal control to monitor transfection efficiency. Cells were transfected in Opti-MEM (Invitrogen) and 3-6 l of FuGENE 6 transfection reagent (Roche Molecular Biochemicals).…”

“…subfamilies based on the number of zinc finger motifs, sequence homology between the zinc-fingers, and the presence of specific repressor and activation domains (3)(4)(5)(6).…”

“…To generate pQE32-GLI1(ZFD) encoding (His) 5 ), the GLI1(ZFD) region was amplified by PCR and inserted into the BamHI or HindIII sites of pQE32 (Qiagen). All constructs were verified by restriction analysis and DNA sequencing.…”

Gene structure and promoter function of a teleost ribosomal protein: a tilapia (Oreochromis mossambicus) L18 gene

“…To obtain tiL18-0.5LacZ, this fragment was introduced into the expression vector pGCV-LacZ [20], where the SV40 promoter had previously been deleted [18]. The tiL18-0.2LacZ expression vector was obtained from the tiL18-0.5LacZ by digestion with NcoI and XhoI followed by religation.…”