Analysis of biological and biochemical parameters for chromatin and nuclear matrix association of SV40 large T antigen in transformed cells

Abstract

We analysed biological and biochemical parameters for the association of the simian virus 40 (SV40) large tumor antigen (large T) with the cellular chromatin and the nuclear matrix in SV40-transformed cells. Nuclear subclasses of large T were isolated by in situ cell fractionation (Staufenbiel & Deppert, 1983) and first analysed for possible biological functions in the maintenance of cellular transformation. Like large T in SV40 wild-type transformants, large T in SV40 tsA mutant (tsA58)-transformed cells, expressing a temperature-dependent phenotype, was present in all nuclear subfractions (nucleoplasm, chromatin and nuclear matrix), when cells were kept at the growth temperature permissive for the expression of the transformed phenotype (32 degrees C). When tsA mutant-transformed cells were shifted to the non-permissive growth temperature (39 degrees C), they reverted to the normal phenotype. Concomitantly, large T lost its ability to associate with the cellular chromatin and the nuclear matrix, indicating that an association of large T with these subcellular structures may be important for the maintenance of cellular transformation. We next analysed the DNA-binding properties (sequence-specific binding to the SV40 origin of replication, ORI) of the nuclear subclasses of SV40 wild-type and of SV40 mutant large T defective in SV40 ORI binding in order to determine the influence of sequence-specific DNA binding on the association of large T with the chromatin and the nuclear matrix. Our detailed analyses show distinct differences in the ability of the various nuclear subclasses of large T to bind to the SV40 ORI, but suggest that the association of large T with the chromatin and the nuclear matrix is mediated by protein-protein interactions rather than by sequence-specific DNA binding.