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Deficiency of the dual ubiquitin/SUMO ligase Topors results in genetic instability and an increased rate of malignancy in mice

by
Henderson D. Marshall
BA, 2003
Rutgers, the State University

Thesis Advisor: Eric H. Rubin, MD
Graduate Program of Cellular and Molecular Pharmacology

Child Health Institute
Room 3101
New Brunswick

Tuesday, April 29, 2008
3:00 pm


Abstract

The nuclear protein Topors was first identified as a topoisomerase I- and p53-binding protein. It contains a conserved N-terminal RING domain and was shown to function as a RING-dependent ubiquitin E3 ligase for p53 and other proteins. Moreover, it was demonstrated that Topors sumolyates p53 in vitro and in cells, distinguishing it as the first example of a protein with dual ubiquitin and SUMO E3 ligase activity. A proteomic screen for Topors substrates yielded several proteins involved in chromatin modification, and a Drosophila Topors ortholog was shown to regulate a chromatin insulator. Expression studies of human Topors suggest that it may function as a tumor suppressor in colon, lung and brain malignancies. To investigate the role of Topors in mammals, I generated a knockout mouse model using a gene-trapped allele. The mutant allele expresses a fusion transcript containing exons 1 and 2 of Topors with exon 3 replaced by vector-derived beta-galactosidase sequence. The protein derived from the fusion transcript lacks regions implicated in both the ubiquitin and SUMO ligase activities of TOPORS. Breeding studies indicated that mice homozygous for the mutant allele were born at the expected Mendelian frequency. However, mice lacking Topors were smaller than their littermates and exhibited a higher rate of perinatal and adult mortality. In a cohort study of tumor formation, a single tumor (papillary adenoma of the Harderian gland) was detected among 40 Topors +/+ mice (2.5% incidence). By contrast, among 69 Topors+/- mice, 12 tumors (17% incidence) of various histologies were identified. The relative risk of tumor development in the Topors+-/- mice was 7.0 (95% confidence interval 0.94 to 51.5, p = 0.0167, Fisher`s Exact Test). Furthermore, Topors-/- primary murine embryonic fibroblasts (pMEFs) exhibited a capacity for anchorage-independent growth and an increased rate of foci formation in monolayer culture. Additional analyses indicated that Topors -/- pMEFs grew slower than wild-type cells but exhibited an increased rate of aneuploidy, indicating that Topors acts as a tumor suppressor by affecting genomic stability. Loss of Topors was associated with mislocalization of the heterochromatin protein HP1alpha to centromeres as well as derepression of transcription at major satellite repetitive DNA. However, Topors-deficient pMEFs did not exhibit increased sensitivity to DNA-damaging agents or to the microtubule-targeting drug vinblastine, suggesting that alterations in pericentric chromatin rather than DNA repair of a checkpoint defect underly the aneuploidy observed in Topors-/- cells. In addition, while loss of Topors did not affect total cellular levels of ubiquitin or SUMO-1 conjugates, a decrease in high molocular weight SUMO-2/3 conjugates was apparent in Topors-deficient embryonic fibroblasts. Using the Connectivity Map, Topors -/- cells were found to have a transcriptional state similar to that of cells exposed to histone deacetylase inhibitors (HDACi) and were resistant to the anti-proliferative effects of the histone deacetylase inhibitor trichostatin A, consistent with a role for Topors in the regulation of chromatin modifications. Collectively, these findings suggest that Topors functions as a tumor suppressor by maintenance of genomic stability via regulation of pericentric heterochromatin.


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