Early events in angiogenesis: cloning an alpha-prolyl 4-hydroxylase-like gene

To identify and early response factor by exposure to ionizing radiation, we cloned the cDNA of a novel Krüppel-type zinc finger (ZNF) gene, ZK1, from a cDNA library constructed from the human leukemia cell line, CMK86, using degenerate primers. This cDNA encoded a protein of 671 amino acids with an A box of Krüppel-associated box (KRAB) domain at the N-terminus, followed by 15 ZNF motifs. The expression level of the ZK1 mRNA in human leukemia cells lines, CMK86 and U937, was increased after exposure to ionizing radiation. Furthermore, murine myeloid precursor 32D cells that were stably transfected with ZK1 cDNA had higher sensitivity to ionizing radiation than the 32D parent cells. These data suggest that the ZK1 gene is one of early response genes by exposure to ionizing radiation, and may have some functions on radiation-induced apoptotic cell death on hematopoietic cells.     

Functional analysis of ZNF85 KRAB zinc finger protein, a member of the highly homologous ZNF91 family

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.     

HUB1, a novel Krüppel type zinc finger protein, represses the human T cell leukemia virus type I long terminal repeat-mediated expression

We have shown that human T-cell leukemia virus type I (HTLV-I) gene expression is negatively regulated by the U5 repressive element (U5RE) of its long terminal repeat (LTR). To isolate factors binding to U5RE, we screened a cDNA expression library by south-western blotting with a U5RE probe. Screening 2 x10(6) clones gave a positive clone with a 3.8 kb insert encoding a novel 671 residue polypeptide, named HTLV-I U5RE binding protein 1 (HUB1), with five zinc finger domains and a Krüppel-associated box like domain; HUB1 may be related to a repressor belonging to the Krüppel type zinc finger protein. A 4.0 kb mRNA for HUB1 is ubiquitously expressed among all human tissues tested. HUB1 recognizes the TCCACCCC sequence as a core motif and exerts a strong repressive effect on HTLV-I LTR-mediated expression. A new repressive domain, named HUB1 repressive (HUR) domain, was identified, rather than the Krüppel-associated box like domain. The N-terminal region upstream of HUR domain seemed to be also indispensable to the repression. Thus, we propose that HUB1 is a new type repressor and plays an important role in the HTLV-I U5-mediated repression.     

Complex beta-satellite repeat structures and the expansion of the zinc finger gene cluster in 19p12

We investigated the organization, architecture, and evolution of the largest cluster ( approximately 4 Mb) of Krüppel-associated box zinc finger (KRAB-ZNF) genes located in cytogenetic band interval 19p12. A highly integrated physical map ( approximately 700 kb) of overlapping cosmid and BAC clones was developed between genetic STS markers D19S454 and D19S269. Using ZNF91 exon-specific probes to interrogate a detailed EcoRI restriction map of the region, ZNF genes were found to be distributed in a head-to-tail fashion throughout the region with an average density of one ZNF duplicon every 150-180 kb of genomic distance. Sequence analysis of 208,967 bp of this region indicated the presence of two putative ZNF genes: one consisting of a novel member of this gene family (ZNF208) expressed ubiquitously in all tissues examined and the other representing a nonprocessed pseudogene (ZNF209), located 450 kb proximal to ZNF208. Large blocks of ( approximately 25-kb) inverted beta-satellite repeats with a remarkably symmetrical higher order repeat structure were found to bracket the functional ZNF gene. Hybridization analysis using the beta-satellite repeat as a probe indicates that beta-satellite interspersion between ZNF gene cassettes is a general property for 1.5 Mb of the ZNF gene cluster in 19p12. Both molecular clock data as well as a retroposon-mapping molecular fossil approach indicate that this ZNF cluster arose early during primate evolution (approximately 50 million years ago). We propose an evolutionary model in which heteromorphic pericentromeric repeat structures such as the beta satellites have been coopted to accommodate rapid expansion of a large gene family over a short period of evolutionary time. [The sequence data described in this paper have been submitted to GenBank under accession nos. AC003973 and AC004004.]     

Direct interaction of the KRAB/Cys2-His2 zinc finger protein ZNF74 with a hyperphosphorylated form of the RNA polymerase II largest subunit

We previously identified ZNF74 as a developmentally expressed gene commonly deleted in DiGeorge syndrome. ZNF74 encodes an RNA-binding protein tightly associated with the nuclear matrix and belongs to a large subfamily of Cys2-His2 zinc finger proteins containing a KRAB (Kruppel-associated box) repressor motif. We now report on the multifunctionality of the zinc finger domain of ZNF74. This nucleic acid binding domain is shown here to function as a nuclear matrix targeting sequence and to be involved in protein-protein interaction. By far-Western analysis and coimmunoprecipitation studies, we demonstrate that ZNF74 interacts, via its zinc finger domain, with the hyperphosphorylated largest subunit of RNA polymerase II (pol IIo) but not with the hypophosphorylated form. The importance of the phosphorylation in this interaction is supported by the observation that phosphatase treatment inhibits ZNF74 binding. Double immunofluorescence experiments indicate that ZNF74 colocalizes with the pol IIo and the SC35 splicing factor in irregularly shaped subnuclear domains. Thus, ZNF74 sublocalization in nuclear domains enriched in pre-mRNA maturating factors, its RNA binding activity, and its direct phosphodependent interaction with the pol IIo, a form of the RNA polymerase functionally associated with pre- mRNA processing, suggest a role for this member of the KRAB multifinger protein family in RNA processing.     

Interleukin-10 and beta-thalassaemia major: what role?

The gene for human Krüppel-related protein 4, (HKR4, gene symbol GLI4), a zinc finger protein of unknown function, has been localized by fluorescence in situ hybridization to chromosome 8q24.3, distal to c-MYC.