Gérard Goubin

The pag gene product, a physiological inhibitor of c-abl tyrosine kinase, is overexpressed in cells entering S phase and by contact with agents inducing oxidative stress

The human pag gene product is an inhibitor of the c‐abl tyrosine kinase and belongs to a new family of proteins. We show here that higher levels of pag gene expression are observed following induction of proliferation and contact with compounds inducing oxidative stress such as diethyl maleate and sodium arsenate. A weaker overexpression is seen in a macrophage cell line using hydrogen peroxide or menadione as inducers. Pag gene expression increases in synchronized cells entering the S phase. This raises the possibility that elevated levels of pag counteract the cytostatic activity of abl. Treatment of growth arrested cells with diethyl maleate and sodium arsenate induces pag gene overexpression, independently of cell proliferation. Thus, enhanced pag gene expression occurs in two cellular events: proliferation and response to oxidative stress.

Amplification and over-expression of OZF, a gene encoding a zinc finger protein, in human pancreatic carcinomas

The OZF gene encodes a protein consisting of 10 zinc finger motifs and is located on chromosome 19q13.1. We report here the amplification and over‐expression of the OZF gene in pancreatic carcinomas. Increased gene copy number was detected in 3 of 12 tumour cell lines and 2 of 12 primary pancreatic carcinomas. Expression was detected in all cell lines, and the gene was over‐expressed in cell lines with OZF gene amplification. Five of 8 tumours, including 2 primary tumours with OZF gene amplification, displayed high levels of OZF protein, whereas normal pancreas expressed low levels. Immuno‐histochemical analysis showed that expression was restricted to tumour cells. Thus, high‐level expression of OZF is frequent in pancreatic carcinomas and may contribute to the development or progression of this tumour. Int. J. Cancer 80:369–372, 1999.

The zinc finger protein OZF (ZNF146) is overexpressed in colorectal cancer

Overexpression of the OZF gene has previously been demonstrated in the majority of pancreatic cancers. However, because the stages of tumour progression in this disease are poorly defined, no conclusion could be drawn concerning the relationship between OZF overexpression and the course of tumour progression. In contrast, initiation and progression steps are well defined in colorectal cancer. Most colon cancers are believed to arise from polypoid adenomas as a result of the gradual accumulation of genetic alterations, allowing the study of genetic events in the early stages of neoplasia. Accordingly, we wanted to assess the frequency of OZF overexpression in this tumour type and the relationship between overexpression and disease stage. Twenty‐five colon carcinoma specimens from different sites and at various stages were analysed by immunoblotting using a highly specific mouse monoclonal antibody. Each sample was compared with adjacent normal colonic mucosa. Complementary immunohistochemical analysis was also carried out on a commercially available tissue array to identify cells expressing OZF. Of the 25 tumours analysed by immunoblotting, 20 expressed higher levels of OZF protein than their adjacent normal mucosa. Immunohistochemistry revealed OZF expression in tumour cells of 56/59 carcinomas and occasionally in infiltrating lymphocytes but at lower levels. Little or no staining was observed in cells taken from normal or inflammatory colon specimens. In both immunoblot and immunohistochemistry experiments, no correlation was found between OZF expression and clinical parameters such as TNM classification, stage, localization and age. Immunostaining was observed in low‐grade adenomas, indicating that OZF expression occurs very early in the course of tumour progression. OZF expression, infrequent or absent in normal colonic mucosa, is present in more than 80% of colorectal cancers. Dysregulation of the OZF gene is an early event that may be implicated in the genesis of colonic carcinoma and may therefore provide a potential therapeutic target. Copyright

Cloning, characterization, and chromosome assignment of Zfp146 the mouse ortholog of human ZNF146, a gene amplified and overexpressed in pancreatic cancer, and Zfp260 a closely related gene

The human OZF gene (ZNF146), located in chromosome band 19q13.1, is amplified and overexpressed in pancreatic carcinomas. It encodes a protein consisting solely of ten Krüppel zinc finger motifs. We report here the isolation and the characterization of the murine OZF cDNA (Zfp146). Comparison of the deduced amino acid sequences between murine, human and bovine cDNAs revealed a strong identity (95%). A closely related gene, Zfp260, was also isolated and characterized. It encodes a putative protein consisting of three vestigial zinc finger motifs followed by ten Krüppel zinc fingers sharing 79% identity and no gap insertion with the Zfp146 zinc fingers. In vitro transcription/translation of both genes led to synthesis of proteins of the predicted size. Co-expression was observed at the mRNA level in eight adult mouse tissues. Two-color FISH revealed co-localization of both genes on mouse chromosome 7 (band B1–B3). The co-expression and co-localization of Zfp146 and Zfp260 together with the close similarity of their zinc finger domains, suggests that both participate in the same regulatory pathway.

A Kruppel zinc finger of ZNF 146 interacts with the SUMO-1 conjugating enzyme UBC9 and is sumoylated in vivo.

The OZF (ZNF146) protein is a 33 kDa Kruppel protein, composed solely of 10 zinc finger motifs. It is overexpressed in the majority of pancreatic cancers and in more than 80% of colorectal cancers. We have identified OZF interacting factors with a yeast two-hybrid screen. Half of the positive clones characterized encoded UBC9, the E2 enzyme involved in the covalent conjugation of the small ubiquitin-like modifier 1 (SUMO-1). SUMO-1 is a 17 kDa migrating protein that is conjugated to several proteins and has been reported to exhibit multiple effects, including modulation of protein stability, subcellular localization, and gene expression. In HeLa cells transfected with OZF and SUMO-1 expression vectors, immunoblot revealed a major band migrating at 50 kDa and a minor band at 67 kDa, corresponding to the attachment to OZF of one and two SUMO-1 proteins, respectively. The relative amount of the sumoylated proteins increased following transfection with a UBC9 expression vector. The presence of the sumoylated form in HeLa cells solely transfected by OZF indicates the physiological activity of the endogenous SUMO-1 conjugation pathway. Using deletion mutants, we showed that two SUMO-1 modification sites are located on the sixth zinc finger. Mutation of two lysine residues greatly reduced the amount of the sumoylated form of OZF though their surrounding sequences differ from the consensus sequence reported for most proteins modified by SUMO-1 conjugation. Despite the presence of the sixth zinc finger, an OZF mutant containing zinc fingers 1–6 was not modified by SUMO-1 and failed to interact with UBC9. Addition of zinc finger 7 restored SUMO-1 modification and UBC9 interaction and provides evidence that a region downstream of the target lysines is required for interaction with UBC9, in order to achieve SUMO-1 modification. This is the first report of in vivo conjugation of a SUMO-1 protein to a Kruppel zinc finger motif. (Mol Cell Biochem 271: 215–223, 2005)

Zinc finger protein overexpressed in colon carcinoma interacts with the telomeric protein hRap1

The OZF (ZNF146) protein is a 33 kDa Kruppel protein, composed solely of 10 zinc finger motifs. It is overexpressed in the majority of pancreatic cancers and in more than 80% of colorectal cancers. We found an interaction between OZF and the telomeric hRap1 protein with a yeast two‐hybrid screen. hRap1 (TERF2IP) is an ortholog of the yeast telomeric protein, scRap1 originally identified as a regulator of telomere length. In HeLa cells, it interacts with TRF2, a telomere repeat binding factor whose inactivation causes a dysregulation of telomere length and structure. Immunoprecipitation with anti‐hRap1 antibodies in conditions that allow the purification of proteins associated with hRap1, demonstrated that OZF binds to hRap1 in HeLa cells. Using deletion mutants, we mapped the interacting domain of each protein. The three zinc fingers at the C‐terminus of OZF interact with a region of hRap1 located downstream of the coil domain. It involves a stretch of at least 25 amino acids at the C‐terminus of hRap1 that interact with TRF2. This suggests that OZF overexpression in tumours may alter the balance between hRap1 and other telomeric proteins and therefore that OZF function may be linked to telomere regulation.

FISH mapping of bovine zinc finger protein 164 (U23) and X81804 (U9) genes to river buffalo chromosomes 17 and 18 respectively

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Targeted Expression of the only Zinc Finger Gene in Transgenic Mice is Associated with Impaired Mammary Development

The only zinc finger (OZF) gene encodes a protein consisting mainly of 10 zinc finger motifs of the Krüppel type of yet unknown function. To potentially assess its in vivo role, mammary targeted deregulation of the expression of the murine gene was performed in transgenic mice using a goat β-casein-based transgene. Mammary expression of the transgene was observed in the 11 lines obtained. In three expressing lines, this expression was tissue-specific and developmentally regulated. Further analysis of mice from two expressing lines revealed that transgene-homozygous females could not sustain full growth of their pups. This phenotype was associated with an impaired mammary gland development noticeable only after mid-gestation. It was characterised by an increase of the adipocyte to acini ratio and low or absence of fat globules within these acini compared to non-transgenic control animals. These transgenic observations strongly suggest that OZF is active in the mammary gland, interfering with the lactation process and thus that the described transgenic mice could be useful models to search for the cellular partner(s) of this protein.

Partial nucleotide sequence and chromosomal localization of a bovine zinc finger gene ZNF164

A clone carrying an open reading frame coding for a novel zinc finger protein of the Krüppel family was isolated from a bovine genomic library and designated ZNF 164 (zinc finger protein 164). Partial sequencing revealed that it contained at least 13 zinc finger motifs preceded by a lysine-rich region of 60 amino acids. The ZNF164 protein shared approximately 60% similarity with several zinc finger proteins but did not appear to be orthologous with a previously identified gene. Using fluorescence in situ hybridization, the ZNF164 gene was mapped to bovine chromosome band 17q24.

Genomic organization and promoter identification of ZNF146, a gene encoding a protein consisting solely of zinc finger domains

The ZNF146 gene (alias OZF) encodes a protein consisting solely of ten zinc finger motifs. It is amplified and overexpressed in pancreatic carcinomas. To better understand the mechanisms controlling its expression, we have isolated the human ZNF146 gene and performed an initial assessment of its promoter activity. ZNF146 encompasses 25 kb of sequence and consists of four non-coding exons located upstream of a single coding exon. The sequence of proximal 1.4 kb of ZNF146 promoter has a high GC content, is devoid of a TATA box and contains several potential transcriptional elements. This region directs high-level expression of a transfected reporter construct in human cell lines. Analysis of a series of 5′-deletion constructs indicated that the first 80 bp upstream of the potential start site of transcription carry minimal promoter activity whereas the first 550 bp are required for maximal promoter activity.