Targeted Disruption of the Mouse Ing1 Locus Results in Reduced Body Size, Hypersensitivity to Radiation and Elevated Incidence of Lymphomas

Oncogene (2006) 25, 857–866 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE Targeted disruption of the mouse ing1 locus results in reduced body size, hypersensitivity to radiation and elevated incidence of lymphomas JV Kichina1,2,6, M Zeremski2,6,7, L Aris2,6, KV Gurova1,3, E Walker4, RFranks 2, AY Nikitin5, H Kiyokawa2 and AV Gudkov1,3 1Department of Molecular Genetics, Lerner Research Institute, Cleveland, OH, USA; 2Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL, USA; 3Cleveland BioLabs, Inc., Cleveland, OH, USA; 4Department of Quantitative Health Sciences, The Cleveland Clinic Foundation, Cleveland, OH, USA and 5Department of Biomedical Sciences, Cornell University, Ithaca, NY, USA Ing1 belongs to the family of evolutionary conserved genes neoplastic transformation of mouse mammary epithelial encoding nuclear PHD finger-containing proteins impli- cells: the GSE that encoded an antisense RNA against a cated in a variety of processes, including tumorigenesis, previously unknown gene promoted transformation of replicative senescence, excision repair and response to fibroblasts and epithelia cells (Garkavtsev et al., 1996). genotoxic stress. We have generated mice deficient in all Since overexpression of cDNA for the identified gene the isoforms of Ing1 by targeted disruption of the exon resulted in growth repression of human fibroblasts, it that is common for all ing1 transcripts. Embryonic was named Ing1 (abbreviation from ‘INhibitor of fibroblasts from ing1-knockout mice were similar to the Growth’; Garkavtsev et al. (1996)). Decreased expres- wild-type cells in their growth characteristics, replicative sion of ING1 was found in several breast carcinomas lifespan in culture, p53 induction and sensitivity to various and the Ing1 gene was found rearranged in one cytotoxic treatments with minor alterations in cell cycle neuroblastoma cell line pointing at its putative tumor- distribution in response to genotoxic stress. ing1-deficient suppressor function (Garkavtsev et al., 1996). It was animals are characterized by reduced size with no obvious later shown that ING1 expression is increased in morphological, physiological or behavioral abnormalities, senescent human diploid fibroblasts and that expression indicating that ing1 function is dispensable for the viability of Ing1-derived antisense RNA prolongs the lifespan of of mice under normal physiological conditions. Loss of these cells in culture (Garkavtsev and Riabowol, 1997). ing1 was associated with earlier onset and higher incidence In addition, inhibition of Ing1 was associated with a of lymphomas. Consistent with the possible involvement of reduced apoptotic response in some cellular systems Ing1 in DNA repair, ing1-deficient mice were more (Helbing et al., 1997). sensitive to total body gamma radiation. Our observations Ing1 belongs to a family of evolutionally conserved are well in line with the suggested role of ing1 as a genes found in animals, plants and fungi that encode candidate tumor suppressor gene involved in control of nuclear PHD finger proteins. The Ing1 gene family in DNA damage response. humans includes five paralogs named Ing1, Ing2, Ing3, Oncogene (2006) 25, 857–866. doi:10.1038/sj.onc.1209118; Ing4 and Ing5 (He et al., 2005). Both mouse and human published online 19 September 2005 Ing1 genes encode several protein isoforms translated from different transcripts that are initiated from Keywords: ING1; gene knockout; lymphoma; mouse; alternative promoters and consist of different 50-and radiation sensitivity common 30- exons. The three alternative transcripts of mouse ing1 encode two proteins: p37 (Ing1b) and p31 (Ing1a and Ing1c), one of which is a truncated version of Introduction the other (Zeremski et al., 1999). The human Ing1 gene has similar general structure and expression with one ING1a The Ing1 gene was originally identified in the search for more isoform identified, p47 , which has not so far genetic suppressor elements (GSEs) that promoted been found in mice (Gunduz et al., 2000; Saito et al., 2000). ING1 is expressed in established cell lines and in all tissues of adult mice, with higher levels found in Correspondence: Dr AV Gudkov, Department of Molecular Genetics, thymus and testis (Zeremski et al., 1999). NC20, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA. ING1 was found to be functionally and physically E-mail: [email protected] linked to p53 tumor suppressor (Garkavtsev et al., 1998; 6These authors contributed equally to this work. Zeremski et al., 1999; Leung et al., 2002), further 7Current address: Center for the Study of Hepatitis C, Weill Medical supporting the putative tumor suppressor function of College of Cornell University, 525 E.68th Street, New York, NY 10021, USA. ING1. There is experimental evidence indicating the Received 19 May 2005; revised 28 July 2005; accepted 4 August 2005; involvement of ING1 in repair of UV-induced DNA published online 19 September 2005 damage (Cheung, Jr et al., 2001). Consistently, the Phenotype of ing1 knockout mice JV Kichina et al 858 deficiency in Ing1 homologues in yeast interferes with be functionally distinct may complicate interpretation of normal cell cycle control and drug sensitivity (Loewith previously published clinical studies since none of the et al., 2000). In keratinocytes (Cheung, Jr et al., 2000) latter distinguished among the different ING1 protein and melanocytes (Cheung, Jr et al., 2001), the levels of forms. ING1 protein increase upon UV irradiation and its Thus, despite accumulation of a considerable body of accumulation in nucleoli confirms ING1 involvement in evidence concerning structure, evolution, expression of genotoxic stress response (Scott et al., 2001a). Ing1 gene and biochemical characteristics of its pro- While functional analyses suggested that ING family ducts, little is understood about physiological signifi- members play a role in cell response to genotoxic stress cance of ING1 at the organismal level. Evolutionary and determination of sensitivity to DNA damage, conservation and the association of ING family proteins biochemical analysis of ING proteins revealed their with important regulators of gene expression and DNA involvement in the enzymatic complexes determining repair implied the existence of basic physiological protein acetylation and deacetylation (Skowyra et al., functions for these proteins which could be revealed 2001; Kuzmichev et al., 2002; Vieyra et al., 2002). by translation into severe phenotypic defects resulting Moreover, another mammalian member of the ING from deficiency in Ing genes. To directly address this family, ING2, was found to take part in acetylation of intriguing possibility, we generated mice with targeted p53 (Nagashima et al., 2001). Interestingly, p33ING1b was disruption of the ing1 locus. The resulting animals reported to affect the degree of physical association appeared viable and fertile, although smaller than the between proliferating cell nuclear antigen (PCNA) and wild-type (wt) ones, and were characterized by an p300 (Scott et al., 2001b). Importantly, cooperation of increased sensitivity to ionizing radiation and elevated ING1 with p53 and its involvement in histone acetyla- incidence of lymphomas, observations consistent with tion/deacetylation complexes are different for different tumor-suppressor function of ING1 and its proposed ING1 isoforms (Skowyra et al., 2001; Vieyra et al., role in DNA repair. 2002) pointing to a functional interplay among the products of Ing1 locus. In summary, although many mechanistic details are still missing, the accumulated data suggest that ING1 proteins are likely to function as Results a link between DNA repair, apoptosis, and chromatin remodeling via HAT/ING1/PCNA protein complexes Ing1À/À-deficient mice have reduced body weight, but no (Campos et al., 2004a; He et al., 2005). gross abnormalities Although the Ing1 gene was mapped to the near- The mouse ing1 gene is composed of four exons. The telomeric region of the long arm of human chromosome first three exons (1a, 1b and 1c) form the 50 ends of three 13 (Garkavtsev et al., 1997; Zeremski et al., 1997), which alternative ing1 transcripts initiated from different is frequently rearranged in squamous cell carcinomas of promoters. The fourth exon is common for all tran- head and neck (Maestro et al., 1996), no cancer-specific scripts and contains most of the ing1 protein-coding rearrangements of Ing1 sequences were found in these sequences including the conserved PHD finger domain. types of tumors (Sanchez-Cespedes et al., 2000) or in Three mRNAs give rise to two Ing1 proteins p31ing1 and colorectal cancers (Sarela et al., 1999), myeloid leuke- p37ing1 (Zeremski et al., 1999). To investigate the mias (Ito et al., 2002) and lymphoid malignancies biological function of ing1, this gene was disrupted by (Ohmori et al., 1999). Rare somatic mutations in Ing1 homologous recombination. A 3 kb genomic fragment were reported in esophageal squamous cell carcinomas containing the entire common ing1 exon was replaced (Gunduz et al., 2000; Chen et al., 2001), breast tumors with a neomycin resistance (neo) gene abolishing (Toyama et al., 1999; Tokunaga et al., 2000) and production of both p31ing1 and p37ing1 protein isoforms gastrointestinal carcinoma (Oki et al., 1999); Ing1 (Figure 1a) in embryonic stem (ES) cells in vitro using a mutations are more common in melanoma, occurring standard gene

Load more