Differences in histone modification composition of histones

of apoptosis

Nucleosomal DNA fragmentation in association with chromatin condensation is one of the most important nuclear events occurring during apoptosis (Ajiro, 2000; Ajiro et al., 2004; Lee et al., 1999; Waring et al., 1997). Specific histone modifications have been proposed to affect chromatin structure and functions during both cell cycle events and apoptosis (Kratzmeier et al., 2000; Kratzmeier et al., 1999; Talasz et al., 2002; Widlak et al., 2005). Therefore we have investigated histone modifications which are connected with transcriptional activation like H3-dimethyl K4, K36, H3-trimethyl K4, H3-acetyl K9, K18, K23, those connected with gene silencing like H3-di- and trimethyl K9 and one which is connected with DNA fragmentation and DNA repair, H2AX-phosphate S139. Apoptosis derived nucleosomal fragments of different sizes were analysed for their contents of these histones, to investigate a functional correlation between their modification and DNA fragmentation. Our results demonstrated that dimethylated histone H3 at lysine 9 and phosphorylated histone H2AX at serine 139 were accumulated in fragmented chromatin (chapt. 3.10.1), whereas trimethyl K9 and H3-acetyl K9 were depleted in fragmented chromatin fractions (chap. 3.10.2). All other histone modifications, H3-dimethyl K4, K36, H3-trimethyl K4, H3-acetyl K18 and H3-acetyl K23 appeared to be almost equally distributed in fragmented and non-fragmented chromatin (3.10.3). This agrees with the data already described in chapter 4.5.2.

Most publications dealing with apoptosis-related chromatin condensation, nucleosomal DNA fragmentation and its functional correlation with histone modifications, have up to now only reported about phosphorylation or dephosphorylation, including the particular function of linker histone H1 (Ajiro and Allis, 2002; Enomoto et al., 2003; Waring et al., 1997). Enomoto et al. (Enomoto et al., 2003; Waring et al., 1997) even postulated that phosphorylation of H1, H2A and H3 in particular is supposed to be an early event to trigger DNA fragmentation in thymocytes during apoptosis

The phosphorylation or dephosphorylation of histones H1, H2A and their respective variants have up to now been the most controversially discussed candidates, responsible for triggering apoptosis-related chromatin condensation and DNA fragmentation. Most recent works of Talasz et al. (Talasz et al., 2002) and Widlak et al. (Widlak et al., 2005) assign this crucial role to H1 and its subtypes. Widlak and co-workers analysed histone H1 and subtype interaction with the apoptotic nuclease DNA fragmentation factor (DFF40/CAD). This group

provided strong evidence that the histone H1.2 subtype preferentially initiates and enhances the DFF40 mediated cleavage of apoptotic nucleosomal DNA by binding to this nuclease.

However, all other somatic subtypes of H1 were equally able to stimulate the nuclease activity in vitro. Moreover it could be shown that the H1 C-terminal tail is responsible for the activation of DFF40. As a conclusion, this group suggested that on the basis of the described strong interactions between the C-terminal domain of H1 histones and DFF40, linker DNA cleavage during the final stages of apoptosis was triggered.

With relation to specific histone modifications Kratzmeiser and co-workers (Kratzmeier et al., 2000; Kratzmeier et al., 1999) reported a rapid dephosphorylation of H1 and its subtypes after treatment of HL-60 cells with the topoisomerase I inhibitor topotecan^®. Later Talasz et al. (Talasz et al., 2002) stated the same phenomenon for murine cell line NIH3T3, which were treated with various inducers such as TNF-α, CD95 and cycloheximide. H1 dephosphorylation was accompanied by chromatin condensation and preceding the onset of chromatin oligonucleosomal DNA fragmentation. In contrast hyperphosphorylation of the histone H2A subtype H2AX but not H2A.1 or H2A.2 was found to be strongly correlated with DNA fragmentation. The latter findings are in agreement with our own results. Investigations of topotecan^® induced histone H2AX-phosphorylation of serine 139 in association with apoptosis-related DNA fragmentation, which was analysed via sucrose gradients and quantitative FACS measurements, showed that H2AX-phosphate S139 predominantly accumulated in regions of fragmented chromatin, presenting the highest degree of DNA double strand breaks. These findings were supplemented by TUNEL-assay measurements (chap. 3.11). All data obtained strongly supports the general idea of H2AX-phosphate S139 as an early marker for DNA double strand breaks and an indicator for sites of DNA repair (our results and (Fernandez-Capetillo et al., 2004; Halicka et al., 2005; Huang et al., 2003;

Rogakou et al., 2000b).

Up to now no studies have been published with reference to a correlation of methylated and acetylated histones and their putative function on DNA fragmentation. As already mentioned, in parallel with H2AX phosphorylation, we observed a strong accumulation of H3-dimethyl K9 in the fragmented chromatin. Dimethylated and trimethylated histone H3 at lysine 9 have been generally stated to function in transcription repression and gene silencing (Bryk et al., 2002; Jiang et al., 2004; Rogakou et al., 2000b; Schotta et al., 2004a; Wang et al., 2001a).

An explanation for the possible function of this accumulation might lie in its already assigned role of gene silencing. A silencing pathway, in which H3-dimethyl K9 in co-operation with the heterochromatin protein HP1 form heterochromatin regions, has already been described for yeast and mammalian cells (Bannister et al., 2001; Nakayama et al., 2001). This might well support the assumption that after induction of apoptosis certain chromatin areas are to be marked for preferential cleavage by histone modifications functioning in gene repression.

This might be important to ensure that during the following disintegration of the cell, no uncontrolled gene expression takes place which then might lead to cancer. Therefore, we additionally analysed a possible concomitant accumulation of HP1-α in fragmented chromatin regions. However, such a correlation could not be confirmed (chap. 3.10.5).

Our observations of the accumulation of H3-trimethyl K9 in non-fragmented chromatin regions (depletion in fragmented chromatin) appears to be compatible with the idea that this particular modification has also been described as a mark and enhancer for heterochromatin formation. Wang et al. (Wang et al., 2001a) reported that trimethylated histone H3 at K9 inhibits the acetylation of H3 at K14, K18 and K23 by interfering with acetyltransferases.

Schotta et al. (Schotta et al., 2004a; Schotta et al., 2004b) and Jiang et al. (Jiang et al., 2004) even observed a kind of synergism with two histone modifications functioning together in the formation of constitutive heterochromatin. Both H3-trimethyl K9 and H4-trimethyl K20 emerged as hallmarks of pericentric heterochromatin in mammals. In this respect H3-trimethyl K9 is required to induce H4-H3-trimethyl K20. In association with all these findings and our own results, H3-trimethyl K9 might function almost the same way as H3-dimethyl K9, as a marker for preferential chromatin cleavage sites during apoptotic DNA fragmentation. In addition, besides marking particular chromatin regions for DNA cleavage H3-trimethyl K9 promotes gene silencing by recruiting other modifications to the site of action (e.g. H4-trimethyl K20).

Together with H3-trimethyl K9, H3-acetyl K9 was also found to be accumulating in non-fragmented chromatin (chap. 3.10.2). Generally published data proposes histone acetylation, and thereby acetylation of histones H3 and H4 in particular, to have an overall function in transcription activation and enhancing gene expression (Grunstein, 1997; Turner, 2000). The most well established acetylation sites on the N-terminal tail of histone H3 are K9, K14, K18 and K23 (Roth et al., 2001). Acetylation of histone N-terminal tails can weaken DNA-histone contacts (Allfrey, 1966), they might alter histone-histone interactions between neighbouring nucleosomes (Luger et al., 1997; Richmond et al., 1993; Tse et al., 1998a; Tse et al., 1998b;

Wolffe and Hayes, 1999), as well as interactions between histones and regulatory proteins (Edmondson and Roth, 1996; Hecht et al., 1995). Any or all of these impacts on chromatin higher-order folding will finally lead to a more open and permissive chromatin structure which is accessible for the transcription machinery (Roth et al., 2001). Taking this background information into account including our results, it might be possible that H3-acetyl K9 usually plays a special role in transcription activation but might via histone ‘cross talk’ be marked for transcription repression by modifications like H3-trimethyl K9. We might even suggest a kind of antagonistic effect between those two modifications. This would possibly explain why these two were both found to be accumulating in non-fragmented chromatin during apoptosis. Trimethylated H3 K9 might be induced during apoptosis to indicate or even

replace previously acetylated H3 at K9, finally setting a mark for preferential cleavage of those chromatin regions which used to be active.

Besides the just mentioned histone modifications, we observed a kind of ‘random distribution’

for H3-di- and trimethyl K4, H3-dimethyl K36 and H3-acetyl K18 and K23. As di- and trimethylated H3 at lysine 4 and 36 has been announced to function as useful markers of active genes (Bannister et al., 2005; Santos-Rosa et al., 2004; Santos-Rosa et al., 2002), one might assume that these modifications would also be depleted in fragmented chromatin.

This would also be assumed to be the case with acetylated histone H3 at lysines K18 and K23. These modifications, too, are generally correlated with active chromatin (Richards and Elgin, 2002; Turner, 2002) and were even reported by McKittrick and co-workers (McKittrick et al., 2004) to be enriched on the replication independent histone variant H3.3. This does even more support the idea of a transcription activating function of H3-acetyl K18 and K23, which might have to be removed during apoptosis to reach a final state of heterochromatin formation.

All in all histone modifications in association with apoptosis-related DNA fragmentation on the one hand seem to be markers for hetero- or euchromatin formation; on the other hand some of them apparently function as markers to indicate preferential cleavage sites for subsequent enzymatic activity by endonucleases. Examples for both would be the synergistic effect of H3-trimethyl K9 and H4-trimethyl K20 in forming pericentric heterochromatin regions and the suggested antagonism of H3-trimethyl K9 versus H3-acetyl K9.