Mapping H4K20me3 onto the chromatin landscape of senescent cells indicates a function in control of cell senescence and tumor suppression through preservation of genetic and epigenetic stability

Background: Histone modification H4K20me3 and its methyltransferase SUV420H2 have been implicated in suppression of tumorigenesis. The underlying mechanism is unclear, although H4K20me3 abundance increases during cellular senescence, a stable proliferation arrest and tumor suppressor process, triggered by diverse molecular cues, including activated oncogenes. Here, we investigate the function of H4K20me3 in senescence and tumor suppression. Results: Using immunofluorescence and ChIP-seq we determine the distribution of H4K20me3 in proliferating and senescent human cells. Altered H4K20me3 in senescence is coupled to H4K16ac and DNA methylation changes in senescence. In senescent cells, H4K20me3 is especially enriched at DNA sequences contained within specialized domains of senescence-associated heterochromatin foci (SAHF), as well as specific families of non-genic and genic repeats. Altered H4K20me3 does not correlate strongly with changes in gene expression between proliferating and senescent cells; however, in senescent cells, but not proliferating cells, H4K20me3 enrichment at gene bodies correlates inversely with gene expression, reflecting de novo accumulation of H4K20me3 at repressed genes in senescent cells, including at genes also repressed in proliferating cells. Although elevated SUV420H2 upregulates H4K20me3, this does not accelerate senescence of primary human cells. However, elevated SUV420H2/H4K20me3 reinforces oncogene-induced senescence-associated proliferation arrest and slows tumorigenesis in vivo. Conclusions: These results corroborate a role for chromatin in underpinning the senescence phenotype but do not support a major role for H4K20me3 in initiation of senescence. Rather, we speculate that H4K20me3 plays a role in heterochromatinization and stabilization of the epigenome and genome of pre-malignant, oncogene-expressing senescent cells, thereby suppressing epigenetic and genetic instability and contributing to long-term senescence-mediated tumor suppression

Chemical carcinogens transform BHK cells by inducing a recessive mutation.

Treatment of BHK cells with mutagenic carcinogens induced neoplastic transformation in a single step. This transformation displayed the characteristics expected for a recessive mutation. Increasing doses of carcinogens induced transformants with kinetics similar to the kinetics with which they induced 6-thioguanine-resistant or ouabain-resistant mutants in the same population of cells. Transformants with temperature-restricted phenotypes were easily induced by carcinogens which cause mutations by base changes, but when ICR frameshift mutagens were used, the proportion of temperature-limited transformants was inversely related to the frequency with which a particular mutagen induced frameshift mutations. In hybrids between pseudodiploid isogenic strains of normal and transformed BHK cells, transformation was expressed as a dominant trait when the transformed parent was induced by a papovavirus, but was suppressed as a recessive trait when the transformed parent arose spontaneously or was chemically induced. Segregation of transformation was observed upon growth of suppressed normal hybrids, and the transformed phenotype which was reexpressed was in most cases characteristics of the original transformed parent

Radioisotopic labeling of human papovavirus (BK) by iodination and reductive alkylation

Purified virions of the GS strain of the BK group of human papovaviruses were labeled with 125I using chloramine T or lactoperoxidase or with tritium using sodium borohydride. All viral polypeptides were labeled. Tryptic digests of iodinated VP1 were analyzed.</jats:p

Virion polypeptide composition of the human papovavirus BK: comparison with simian virus 40 and polyoma virus

The polypeptide composition of labeled BK virus was compared with that of simian virus 40 (SV40) and polyoma virus by co-electrophoresis of disrupted virions in polyacrylamide gels containing approximately 73% of the capsid protein and had a molecular weight of 39,000. It was smaller than VP1 of SV40 and polyoma virus. The other polypeptides of BK virus were similar in molecular weight to those of SV40. A comparison of the proteins of BK virus and SV40 iodinated with chloramine T before and after disruption in alkaline buffer at pH 10.5 revealed differences between the two viruses in the number and distribution of tyrosines available for iodination. The tryptic peptides of VP1, VP3, VP4, and VP5 combined of SV40 were compared with those of the same polypeptides of BK virus. Among the 19 peptides of VP1 resolved, only two were common to both viruses. The analyses of VP4 and VP5, the histone-like proteins, however, showed more similarity between the viruses, with 6 of 15 resolved peptides in common. The tryptic digests of VP3 were completely different.</jats:p

Separation of lytic and transforming functions of the simian virus 40 A region: two mutants which are temperature sensitive for lytic functions have opposite effects on transformation

Thirty-six of 40 rat cell clones transformed to anchorage independence at low multiplicity of infection by simian virus 40 tsA58 were heat sensitive for continued expression of the transformed phenotype. tsA1499 is an 81-base-pair deletion at 21 map units which is like tsA58 in that it is also heat sensitive for lytic growth, belongs to the A complementation group, and produces rat cell transformants which contain a thermolabile T antigen. Unlike tsA58, however, tsA1499 generated rat cell transformants efficiently at the temperature at which it was lytically defective, and 10 of 17 clones transformed by tsA1499 were cold rather than heat sensitive for the continued maintenance of the transformed phenotype. The lytic and transforming activities of the A region thus appeared to function independently in mutant tsA1499.</jats:p