The silencing of specific chromosomal regions is dynamically changing during development (Ebert et al. 2004
; Ebert et al. 2006
; Talbert & Henikoff 2006
), but less is known about changes in chromatin state during aging. Since changes in chromatin state can alter gene transcription, resulting in expression of genes otherwise silenced (Elgin & Grewal 2003
; Berger 2007
; Grewal & Jia 2007
; Sedivy et al. 2008
; Dang et al. 2009
) or repression of genes otherwise expressed, it has the potential to significantly alter cellular physiology. During aging in yeast, loss of silencing at the mating locus leads to re-expression of the silenced mating type gene, causing sterility, and loss of silencing near telomeres results in expression of previously silenced genes in the subtelomeric region (Kim et al. 1996
; Smeal et al. 1996
; Berger 2007
; Dang et al. 2009
). These and other examples have suggested that changes in chromatin with age are related to, and may be responsible for, some elements of the aging process.
We used Drosophila
to examine the changes in chromatin state globally and locally with age. An HMM analysis confirmed the expectation that the activation marks, including RNA pol II, H3K4me3 (associated with transcript initiation) and H3k36me3 (associated with transcript extension), are almost exclusively associated with the transcriptional start site (TSS) or gene regions. Consistent with the nearly ubiquitous nature of RNA pol II binding to TSS in other organisms (Barski et al. 2009
), only a small but statistically significant decrease was seen in the enrichment of RNA pol II at the TSS and over genes in older flies. A larger statistically significant decrease in the average enrichment of histone modifications associated with active transcription, H3K4me3 and H3K36me3 at TSS and over genes, was seen in older flies ().
Comparisons between ChIP-chip for total RNA pol II (active and inactive) and whole genome RNA transcriptional microarrays shows the same weak correlation between RNA pol II binding and gene expression that has been reported by others (Kim et al. 2005
; Lee et al. 2006
; Muse et al. 2007
). The addition of ChIP-chip information from two other histone activation marks (H3K4me3 and H3K36me3) improves the correlation between activation marks and gene expression, although the improved correlation remains weak and is insufficient to confidently make definitive predictions. The poor correlation seen between activation marks and gene expression is probably not due to the inherent “noise” in the experimental procedures of using whole genome microarrays. Similar difficulties of correlating activation marks with gene expression continue to be seen with deep-sequencing methodologies that should significantly improve the signal-to-noise ratio (Barski et al. 2009
Unlike activation marks, repressive marks such as H3K9me3 and HP1 are not found closely associated with the TSS of individual genes. In general, repressive marks were seen enriched in broader regions than activation marks and in areas devoid of known transcriptional units. It is thought that HP1 is recruited to a chromosomal region in response to addition of the H3K9me3 mark, resulting in an increase in chromatin packing and gene silencing. This is consistent with our finding of a stronger association between enrichment of HP1 and decreased gene expression, than for H3K9me3 (Fig. , S8
With age we found that the relative enrichment of both H3K9me3 and HP1 is greatly diminished in the pericentric heterochomatin regions, the heterochromatic 4th
chromosome and in islands of facultative heterochromatin in comparison to euchromatin (Fig. , S5, S6
). For H3K9me3, more than for HP1, the large difference in enrichment in these heterochromatin regions in young flies is reduced to being virtually equivalent to the euchromatin regions in older flies (Fig. , S5, S6
). However, since ChIP-chip is not able to measure absolute values, and because of the need of inter-array normalization, these data alone are not able to definitively state whether H3K9me3 or HP1 is lost from these “heterochromatin” regions with age. The observed change with age could be due to a decrease in H3K9me3 and HP1 in the heterochromatin-like regions or an increase in H3K9me3 and HP1 ubiquitously throughout euchromatin. Either scenario would lead to the apparent equilibration of signal between the euchromatin and heterochromatin regions seen in older flies.
Western blots show a significant increase in the level of H3K9me3 and a small increase in the level of HP1 in older flies as compared with total levels of histone H3 (). This absolute increase in H3K9me3 and HP1 with age suggests that either repressive marks increase throughout euchromatin or are sequestered to the ~30% of the genome of Drosophila made up of repetitive heterochromatic DNA which is not examined by ChIP-chip methodology. Single cell immunohistochemistry clearly shows evidence of H3K9me3’s altered distribution to more extensive chromosomal regions with age, although it does not distinguish between the change in distribution being targeted preferentially to euchromatin or repetitive chromosomal regions. The extent of the spread of the H3K9me3 signal throughout most of the nuclear region in older cells suggests the change in H3K9me3 is not limited to repetitive chromosomal regions and likely includes an increase of H3K9me3 in euchromatin.
The finding of extensive changes in repressive H3K9me3 and HP1 marks with age in the absence of a corresponding large-scale change in gene expression suggests that if there is an increase in repressive marks throughout euchromatin it is ineffective in modifying global gene expression. Alternatively, if the level of H3K9me3 and HP1 does not change significantly in euchromatin with age, then many of the changes seen in the level and distribution of repressive marks may reflect a redistribution or sequestration of repressive marks to the repetitive regions of the fly genome. It is possible that there may be significant changes with age in RNAs from repetitive DNA regions or transposons, which we were not able to detect using transcriptional microarrays.
Taken together these studies demonstrate that there are dramatic changes in chromatin structure with age in Drosophila. Although the specific functional consequences of these age-related changes have not been directly determined, it is likely that they alter gene expression and have important effects on cell physiology. Such changes are expected to directly or indirectly impinge upon important cell functions such as gene expression, DNA repair and DNA replication. The combination of genome-wide approaches such as whole genome ChIP and transcriptional studies in conjunction with single cell immunohistochemistry as shown here provides a first step toward defining how changes in chromatin may contribute to the process of aging in metazoans.