Excess histone levels mediate cytotoxicity via multiple mechanisms.
We have presented evidence here to suggest that the presence of excess histones negatively impacts numerous cellular processes via multiple mechanisms based largely on potential electrostatic interactions between the highly positively charged histones and negatively charged molecules in the cell. Mutant yeast cells such as rad53
Δ and ubc4
Δ that are defective in the regulation of histone protein levels and harbor excess endogenous histones, are very sensitive to exogenous histone overexpression which is highly toxic for these cells.10,11
All these mutant strains are also sensitive to DNA damaging agents and exhibit genomic instability in the form of elevated chromosome loss rates. The data presented here allows us to finally explain the deleterious effects of excess histones not just in these mutants, but also in wild type cells. Excess histones can swamp the binding sites available on histone chaperones10
and then overload histone modifying enzymes. This can have serious consequences for the normal regulation of gene expression and the formation/maintenance of epigenetic marks on histones. Not surprisingly, cells lacking certain histone modifying enzymes were sensitive to histone overexpression (). Further, the stability of histone posttranslational modifications would depend not only on the enzymes that put them on and remove them, but also on the stability of the histone proteins themselves. For example, until the discovery of the first histone lysine demethylase a few years ago,31
histone methylation marks were considered to be as stable as histone themselves.32,33
There is a constant exchange of histones between the chromatin bound and free states as a result of transcriptional eviction,34,35
the action of chromatin remodelling factors36
and the opposing effects of chromatin assembly and disassembly.37–38
We have recently obtained evidence that the tyrosine 99 residue of histone H3 may be phosphorylated only when histone H3 is not bound to the chromatin and this modification may serve as a mark to target this histone for degradation.11
Hence, our findings regarding the degradation of non-chromatin bound histones10,11
may have wide-ranging ramifications for the stability and maintenance of epigenetic marks on histones in chromatin by providing an additional layer of regulation.
We also found that excess histones can alter the fine structure of chromatin. This could potentially impact all metabolic activities that require access to the DNA. Hence, we have now systematically analyzed the genome wide effects of excess histones on gene expression using microarrays. We found that although overexpression of histone H3 alone is toxic to the cells ()10,11
and alters chromatin fine structure, it did not result in an appreciable alteration of gene expression (). However, overexpression of the H3-H4 gene pair or simultaneous overexpression of all four core histones lead to a significant alteration in the expression levels of 4% of the budding yeast genome, including several genes that are essential for viability. This difference between overexpression of H3 alone or in combination with H4 may be due to the fact that histones are deposited on to the DNA as pairs of H3-H4 and H2A-H2B dimers.39
Hence, although overexpression of H3 alone may lead to its non-specific association with the DNA, this association may not be very strong in vivo and as such it may not prove to be a significant obstacle for the transcriptional machinery. On the other hand, (H3-H4)2
tetramers and the core histone octamer are known to associate tightly with the DNA and so upon the overexpression of the H3-H4 gene pair or all the core histones, it is likely that these excess histones will form chromatin structures that may present a significant challenge to the transcriptional machinery40
and our microarray data appears to support this notion. Overall, our microarray data suggests that although excess histones are capable of altering the expression of a small fraction of yeast genes that probably contributes to their deleterious effects in vivo, the fact that H3 overexpression does not alter gene expression and is still highly toxic strongly argues that the other mechanisms discussed here are likely to be mediating most of the cytotoxic effects of excess histones.
Numerous studies in recent years have revealed the dynamic nature of chromatin structure, particularly in the context of transcription.36–38
Our microarray based analysis of yeast genes with altered expression patterns upon histone overexpression revealed that a significant number of them lie in small clusters of 2–4 genes that are co-ordinately regulated (Sup. Tables 1–3
). These clusters could have unique chromatin features that may facilitate the normal regulated expression of genes within them. Such special chromatin features may include promoter regions with highly positioned nucleosomes or promoters and/or coding sequences that are largely devoid of nucleosomes. A cursory survey of published data on nucleosome occupancy in yeast35
suggests that this may be the case indeed with at least a few of the identified mini clusters. For example, the YNR056C, YNR057C, YNR058W cluster which is downregulated upon histone overexpression (Sup. Table 1
) appears to have poor nucleosome occupancy in yeast based on published data.35
It is possible that histone overexpression results in a higher nucleosome occupancy in this region, thereby resulting in lower levels of transcription consistent with our microarray data. On the other hand, the YOR382W, YOR383C, YOR385W cluster that is also downregulated upon histone overexpression appears to have well-positioned nucleosomes based on published genome wide nucleosome occupancy data.35
How exactly histone overexpression brings about a reduction in the expression levels of ORFs within mini clusters that may have either poorly positioned or highly positioned nucleosomes is unclear. Future studies involving chromatin fine structure mapping will confirm if there are indeed common chromatin structural features within the identified mini clusters and reveal the exact nature of any chromatin structure changes at these loci upon histone overexpression.
Another intriguing feature of the identified mini clusters is their telomere (and occasionally centromere) proximal localization. This is reminiscent of the effect of histone H4 depletion where ~50% of the genes within 20Kb of the telomeres were preferentially derepressed, compared to derepression of just 15% of the genes genome-wide.28
Any special chromatin features in these clusters may be related to their relative proximity to the telomeres (or the centromeres) that are known to have specialized chromatin structures, although these are not normally known to spread over ~20 Kb from the telomeres in the budding yeast and the closest mini clusters are at least that far away from the telomere.41
Another possibility is that histone overexpression may result in their excessive association with certain loci where they may not be present normally and this in turn may lead to their silencing by the recruitment of Sir proteins, perhaps via subsequent localization of these loci to the nuclear periphery.42
Future studies will reveal whether the relative telomere proximal location of the mini clusters affected by histone overexpression has any functional significance.
As opposed to the potential alterations in transcription caused by excess histones, the process of transcription itself may generate excess histones if the reassembly of the transcriptionally evicted histones onto the chromatin is blocked, as in conditional spt16
mutants, where the evicted histones are presumably subjected to Rad53 mediated histone proteolysis.43
In fact, during the G1
phase of the cell cycle, the presence of excess transcriptionally evicted histones in the spt16
mutants or exogenously expressed histones in wild type cells triggers the downregulation of the G1
, leading to a delay in S-phase entry. Contrary to a situation of histone excess, it has also been suggested that a lack of adequate histones due to sub-optimal histone pre-mRNA processing may also trigger a similar G1
Hence, eukaryotic cells may have evolved surveillance mechanisms similar to known cell cycle checkpoints to protect them from the harmful effects of both excess histone accumulation as well as a scarcity of histones. These mechanisms delay S-phase entry by prolonging the G1
phase of the cell cycle, presumably to provide cells with additional time to either degrade excess histones or increase histone mRNA production as needed prior to S-phase entry and start of replication, when excess or inadequate amounts of histones could potentially be highly detrimental for the cells.
Additional problems due to the presence of excess histones may arise from the translational defects occurring as a result of these histones binding to coding RNAs () and interfering with their translation. Further, since the majority of the RNAs in the cell are structural ribosomal RNAs (rRNAs) that are crucial for ribosome function, their non-specific interaction with excess histones would serve as a double setback for ongoing protein synthesis in the cell. Hence, it is not surprising that overexpression of histones is highly toxic and confers lethality in mutants such as rad53
Δ that cannot efficiently degrade excess histones.10
This lethality probably represents the cumulative effects of excess histones by the various mechanisms discussed above. In fact, even wild type yeast cells experience ~20% lethality upon histone overexpression (data not shown) and exhibit genomic instability in the form of enhanced rates of chromosome loss.10,11,20
Genomic instability is characterized by the increased rate of acquisition of alterations in the genome and is associated with most human cancers.44,45
Taken together, these data suggest that improper histone stoichiometry and aberrant chromatin structure may contribute to genomic instability and carcinogenesis. As such, our studies highlight the potentially crucial role of proper histone stoichiometry and chromatin structure as well as multiple histone regulatory mechanisms in maintaining viability and genomic stability.