Abstract
Cell shrinkage, formation of apoptotic bodies and chromatin fragmentation through cleavage of nucleosomal DNA into different fragment sizes catalysed by the DNase CAD (DFF40) are hallmarks of apoptosis in eukaryotic cells. Moreover, post-translational histone modifications such as acetylation, methylation and phosphorylation greatly influence the formation of overall eukaryotic chromatin structures with defined functional consequence. The overall issue of this work is to analyse cause and effects of apoptosis-related changes in histone modifications and their influence on chromatin condensation and fragmentation. The question whether particular histone modifications determine preferential DNA cleavage sites is given special interest.Firstly, this work analyses overall time-dependent changes of histone modifications after treatment with topoisomerase inhibitors. The results show a hyperphosphorylation of H2AX histones at serine 139 10 min post-induction and a deubiquitylation of H2A histones 6 hours post-induction. Secondly, analyses of a potential apoptosis-related release of specifically modified nucleosomes/histones into the nucleoplasm and cytoplasm show a gradual increase of all four core histones (H2A, H2B, H3, H4) and their specific modifications (H3 di-/trimethyl K4, H3 dimethyl K9, H2AX phosphate S139) in both nuclear and cytoplasmic fractions. However, H3 acetyl K9 histones and H3 trimethyl K9 histones do not increase in either of the two fractions. Thirdly, analyses of fragmented versus non-fragmented chromatin point to an accumulation of H3 dimethyl K9 histones and H2AX phosphate S139 histones in fragmented chromatin and a depletion of H3 trimethyl K9 histones and H3 acetyl K9 histones in the same fraction. All these results support the concept that specifically modified histones play a crucial role in the subtly regulated process of apoptosis-related chromatin condensation and cleavage of nucleosomal DNA.
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