nuclei of DLD-1 and MCF-7 cells.
An explanation why chromatin condensation in HFbs occurs already at a Chaetocin concentration of only 0,01µM could be a redox-uptake mechanism that has recently been described for a ETP-toxin (Bernardo et al., 2003). The concentration of the substance within a cell can be several orders of magnitude greater (up to 1500-fold) than the applied concentration. This accumulation appears to enhance the toxicity of ETPs.
It is much more difficult to uncover a mechanism in the context of regulating histone lysine methylation than histone acetylation (Turner, 2000). This is because the latter mainly depends on repulsion of charges whereas lysine methylation effector functions depend on the recruitment and interaction of many proteins (Bernstein et al., 2007; Li et al., 2007). Furthermore epigenetic “cross-talk” can occur, making research and interpretations even more complicated (Fischle et al., 2003b; Kouzarides, 2007). This means that all possible interaction partners of H3K9me3, HP1-alpha and SUV39H1 have to be taken into account when it comes to a discussion about the underlying mechanisms of these changes observed after Chaetocin treatment.
Another question that has to be addressed is whether the enzyme activity after Chaetocin application is as high as in control cells. To investigate enzymatic activity directly several types of enzyme assays have been developed over the decades (Lottspeich, 1998). Enzyme assays can be split into two groups according to their sampling method: continuous assays, where the assay gives a continuous reading of activity, and discontinuous assays, where samples are taken, the reaction stopped and then the concentration of substrates/products determined. To test the hypothesis if the changes of H3K9me3 after drug application are due to altered enzyme activity an enzyme activity assay against the HMT SUV39H1 should be executed in subsequent experiments as was applied for HATs (Brownell et al., 1999).
Since cancer cells obviously have mechanisms to escape the effects of drug treatment at least at low concentrations it seems necessary to investigate the effect of Chaetocin application in other “normal” cell-strains.
HP1-alpha, which, binds to H3K9me3 (Bannister et al., 2001; Lachner et al., 2001), can still be found co-localizing after Chaetocin treatment. In all three cell types HP1-alpha was found to be overlapping with H3K9me3 to some extent in untreated control cells and in Chaetocin treated cells, indicating that H3K9me3 as a binding site for HP1-alpha is not affected by the drug. In MCF-7 and DLD-1 cells the overlapping volume of HP1-alpha appears similar before and after drug treatment; only in untreated HFbs different overlapping volume compared to Chaetocin treated cells was observed. The observation that HP1-alpha remains overlapping
at least partly with H3K9me3 in HFbs raises again the question whether the HP1/H3K9me3- system involved in heterochromatin formation is affected at all or if other pathways are altered by Chaetocin treatment.
An important finding was that a rearrangement of chromatin was not observed after an incubation time of 8h. Together with the observation that changes in chromatin occur after 24h this finding argues for a cell cycle dependence of the described effect because the time window where changes can be detected correspond approximately to the duration of one cell cycle in HFbs (diploma thesis A. Engelhard, 2001). The described changes are more likely due to a process where the passing of S-phase is required.
In a rescue assay where Chaetocin was “washed out” two days after application no re- establishment of chromatin occured. Cells were not capable to rearrange their chromatin to the original state as observed in control cells. This would on the one hand argue for cytotoxicity or on the other hand confirm that the process initiated by Chaetocin application is irreversible, thereby suggesting a long term epigenetic alteration (Lachner et al., 2004; Reinberg et al., 2004).
The performed experiments to uncover the role of this drug on the level of nuclear architecture were a necessary completion to the exclusively biochemical experiments made when Chaetocin was found and proofed to be a specific inhibitor of SU(VAR)3-9.
5.3 Analysis of lysine sites with regard to chromatin segments
In this chapter the spatial relationship of histone modifications H3K4me3, H3K9me3 and H3K27me3 with whole chromosome territories (CT), chromosomal subdomains and selected gene loci is discussed.The gene poor chromosome 18 and the gene rich chromosome 19 differ in their enrichment for H3K4me3 staining but not for H3K9me3 and H3K27me3. Co-localization analysis for H3K9me3 and the CTs of chromosomes 18 and 19 respectively yielded almost identical values despite their different “compaction” state, gene content and overall transcriptional activity. The bulk of H3K9me3 can be assigned to blocs of pericentromeric heterochromatin containing tandem repeats (Peters et al., 2003; Rice et al., 2003; Zinner et al., 2006). Since chromosome specific painting probes do not represent these large heterochromatin blocs due to Cot-1 DNA suppression in FISH-experiments or depletion of repetitive sequences in probe generation (Bolzer et al., 1999) it is not surprising that overlap occurs merely to a minor extent. This observation was confirmed by experiments where signals of centromeres and constitutive heterochromatin marked by H4K20me3 were found in “holes” of chromosome paints. Therefore co-localization data are mainly obtained by overlapping regions not associated with pericentromeric heterochromatin. A large number of small foci