has recently been reported (Shi et al., 2006). The authors showed that members of the inhibitor of growth family (ING) of tumor suppressor proteins bind to H3K4me3 via a PHD- domain and thus stabilize a HDAC-complex at the promoters of proliferation genes.
The DNA dye TO-PRO-3 has been shown to be a specific and sensitive nuclear counterstaining dye in human cells (Matsuzaki 1997) and in Xenopus laevis (de Maziere et al., 1996) embryos as well as on frozen sections of rat (Suzuki et al., 1997). Its affinity to ds DNA makes it comparable to DAPI (Bink et al., 2001). The latter however has an affinity to AT-rich sequences which was not reported for TO-PRO-3 (Petty 2000). Areas of low TO- PRO-3 staining intensity represent less compacted or decondensed DNA, a chromatin state indicative for high gene density and genetic activity (Gilbert et al., 2004). On the light microscopic level both dyes stained similar areas namely regions containing dense chromatin.
It was shown recently that H4K20me1 is involved in cell cycle regulation (Karachentsev et al., 2005). The observation during the present thesis that two “populations” of antibody staining signals exist supports the idea of a connection of H4K20me1 to cell cycle progression. Other processes where H4K20me1 was reported to play a role are chromosome segregation during mitosis in Drosophila (Julien and Herr, 2004), DNA repair
mechanisms in yeast (Nakamura et al., 2004; Sanders et al., 2004) and the initiation of X- inactivation (Kohlmaier et al., 2004). Controversal data have been published assigning H4K20me1 to promotor regions on the one hand (Talasz et al., 2005) and to silent chromatin one the other hand (Karachentsev et al., 2005; Nishioka et al., 2002). The striking similarity in the nuclear arrangement and the dot like appearance of both antibody signals H3K4me3 and H4K20me1 suggests that the latter modification is also associated with active gene loci or at least chromatin with a transcriptional permissive state. The still large fraction of separate signals of the two antibodies after co-immunostaining indicates that chromatin segments exist which were spatially excluded from each other and therefore mark distinct types of chromatin.
A connection of H4K20me1 to repetitive sequences was shown in the mouse model, where in ChIP experiments H4K20me1 was merely found to be enriched in SINEB1 (correspond to the human Alu family) repeats (Martens et al., 2005). However this modification could not be assigned to centromeres as evaluated by co-localization analysis. The observation that H4K20me1 is apparently only to a minor extent associated with nascent RNA leads to the conclusion that the detected pattern mirrors a very special type of chromatin that cannot simply be assigned to either actively transcribed or repressed chromatin regions.
Assessment of H3K9me1 in human led to the suggestion that this modification reflects temporarily silent chromain domains (Rice et al., 2003). Tests by ChIP analysis in mouse revealed low H3K9me1 enrichment at repetitive sequences as well as in transcriptionally active regions (Martens et al., 2005). Comparable to H4K20me1 also H3K9me1 is characterized by a punctual pattern. Both modifications showing staining foci distributed throughout the nucleus (except nucleoli and the nuclear rim) are alike regarding their low level of co-localization with centromeres and nascent RNA. The immunostaining experiments produced often signals in TO-PRO-3 rich areas which favors the idea of H3K9me1 reflecting rather silent domains. The discrepancy of information about this modification is probably due to varieties between the species human and mouse (Martens et al., 2005; Rice et al., 2003).
H3K27me3 is predominantly mentioned in the context of imprinted facultative heterochromatin formation and initiation of X-chromosome inactivation (Okamoto et al., 2004; Plath et al., 2003). Additionally a function in maintaining X inactivation has recently beeen described (Kohlmaier et al., 2004). The presence of H3K27me3 at unexpressed autosomal genes suggests that this mark is an ubiqitary label for heterochromatin rather than a X- specific marker (Brinkman et al., 2006). The staining pattern of H3K27me3 in DLD-1 and MCF-7 cells resembles the pattern for mid (to-late) replicating chromatin in a very striking manner. This pattern is found around the nucleoli and at the nuclear periphery thereby sustaining the idea of an association with gene repressing chromatin. Mid-replicating chromatin and facultative heterochromatin (Craig and Bickmore, 1993) corresponds to a large extent to G-dark bands which contain tissue-specific genes that are transcribed only in selected cell types (Manuelidis, 1990). These genes have a high content of LINE elements but no enrichment of H3K27me3 with LINE elements could be uncovered in mouse (Martens et al., 2005). The question if such an association is also existant in humans remains unanswered at the moment.
In neither cell type an association of H3K27me3 with centromeres was observed and also co-localization analysis with nascent RNA revealed low overlapping volume. Taken into account that H3K27me3 was never brought into connection with actively transcribed genes and the co-localization data of this modification with centromeres and nascent RNA obtained in this work, chromatin visualized by H3K27me3 seems to represent a discrete type of heterochromatin.
H3K9me3 is the probably best known and characterized epigenetic marker for constitutive heterochromatin in a variety of species (Lehnertz et al., 2003; Martin and Zhang, 2005; Rea et al., 2000; Rice et al., 2003). After it was shown that this epitope serves as a binding site for HP1-alpha it became evident that H3K9me3 plays a key role in nuclear formation of