Development of mouse models to delineate the tumour suppressor function of INI1/hSNF5 gene
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Studies in our laboratory have demonstrated that re-introduction of INI1/hSNF5 gene back to INI1-/- cell lines derived from human MRT causes G0/G1 arrest and that this arrest is correlated with the ability of INI1/hSNF5 to directly mediate the HDAC-dependent transcriptional repression of the Cyclin D1 gene. This would provide a potential downstream target for the INI1/hSNF5 gene and de-repression of CyclinD1 gene due to INI1/hSNF5 loss could be a key mechanism of tumor formation.;To test the hypothesis that ablating the key downstream targets that are repressed by INI1/hSNF5 gene will result in the loss of tumor formation, we carried out the following experiments for this thesis project. We (i) generated mouse models for AT/RT carrying heterozygous deletions in Ini1 gene by using ES cell technology and targeted disruption through homologous recombination; (ii) crossed these mice to cyclinD1-/- mice to determine the effect of ablating cyclinD1 gene on the frequency and nature of the tumors induced due to Ini1 loss in Ini1+/- heterozygous mice; and (iii) have further characterized the mechanism of transcriptional repression by human INI1 by studying INI1-interacting proteins.;Our results indicate that while homozygous deletion of Ini1 gene results in embryonic lethality, heterozygous knockout mice develops a variety of brain and head tumors with characteristic rhabdoid cells, with a penetrance of about 25--40%. Results of our initial analyses indicate the presence of mutations in exons of ini1allele in some of these tumors and absence of INI1 protein in some other tumors. These results are consistent with the mechanism of tumor formation due to loss of heterozygocity in the ini1 gene.;To determine the mechanism of INI1 mediated repression, we have analyzed the INI1-interacting proteins. Recently, genetic and biochemical evidence in yeast suggested that components of Sin3-Rpd3 histone deacetylase complex (HDAC), which regulates transcriptional repression, are targets of regulation by Cyclophilin A. We have found that Cyclophilin A directly interacts with INI1. Our study indicates that IN1 mediated repression is inhibited in a dose dependent manner by the addition of cyclosporine A, an inhibitor of Cyclophilins.;In summary, we have analyzed the effect of downstream targets and interacting proteins on the tumor suppressor function and transcriptional activity of Ini1. Our analysis indicate that ablating cyclinD1 reduces the tumor formation by INI1 and suggests that cyclinD1 could be an effective drug target to treat AT/RT. (Abstract shortened by UMI.).
Source: Dissertation Abstracts International, Volume: 64-09, Section: B, page: 4181.;Advisors: Ganjam V. Kalpana.