chm is expressed in the two epithelia of wing discs, the columnar epithelium, and the peripodial membrane, and by 8 h after puparium formation (APF), when the two contralateral discs meet at the dorsal midline, transcription proceeds in fusion regions only. The expression pattern of chm supports the idea that it functions in migration and/or fusion of wing discs during metamorphosis (Miotto, 2006).
Old age is associated with a progressive decline of mitochondrial function and changes in nuclear chromatin. However, little is known about how metabolic activity and epigenetic modifications change as organisms reach their midlife. This study assessed how cellular metabolism and protein acetylation change during early aging in Drosophila melanogaster. Contrary to common assumptions, it was found that flies increase oxygen consumption and become less sensitive to histone deacetylase inhibitors as they reach midlife. Further, midlife flies show changes in the metabolome, elevated acetyl-CoA levels, alterations in protein-notably histone-acetylation, as well as associated transcriptome changes. Based on these observations, the activity of the acetyl-CoA-synthesizing enzyme ATP citrate lyase (ATPCL) or the levels of the histone H4 K12-specific acetyltransferase Chameau were decreased. These targeted interventions both alleviate the observed aging-associated changes and promote longevity. These findings reveal a pathway that couples changes of intermediate metabolism during aging with the chromatin-mediated regulation of transcription and changes in the activity of associated enzymes that modulate organismal life span (Peleg, 2016).
The process of aging is characterized by a deterioration of multiple interconnected cellular pathways, which makes the identification of molecular mechanisms of phenotypic aging and death particularly difficult. Many molecular analyses have focused on the comparison of young and old organisms, which resulted in the formulation of nine hallmarks of aging ranging from telomere shortening and epigenetic alterations, to differences in nutrient sensing and stem cell depletion. While many of these experiments have identified valuable paths toward life span extension, such studies face the complication that old individuals suffer from the progressive deterioration of multiple cellular systems, which can make it challenging to distinguish primary from secondary effects. To identify changes involved in the onset of aging, this study compared D. melanogaster flies at young age and during midlife at the onset of a premortality plateau, when most individuals of a population are still alive (Peleg, 2016).
Surprisingly, heads from midlife flies consume more oxygen than the young ones. This is in apparent contradiction to the general observation of a reduced metabolism when animals age, which this study also observe in old flies. There are several possible explanations for this unexpected finding. In many studies, the oxygen consumption rate was extrapolated from measurements of isolated mitochondria, which may lack crucial extra-mitochondrial signals when investigated in isolation, whereas this study has measured activity in isolated fly heads. Alternatively, flies may change their feeding behavior when reaching midlife, or switch from an anaerobic to a more aerobic metabolism due to their decreased activity, which is consistent with higher levels of metabolites generated by oxidative processes in midlife flies. Finally, the metabolic changes may be due to a feed-forward activation of metabolic enzymes that become stimulated by hyper-acetylation. The observation that the treatment of isolated fly heads with lysine deacetylase (KDAC) inhibitors increases oxygen consumption rate (OCR) within minutes suggests that such a direct feed-forward mechanism might indeed exist. The finding that midlife flies have a higher ground state of acetylation and are less susceptible to a stimulation by KDAC inhibitors argues for similar acetylation events triggered by KDAC inhibitor treatment and aging (Peleg, 2016).
The increased level of acetyl-CoA in midlife flies correlates with a very specific change in the histone modification pattern as flies reach midlife. As it is not possible to distinguish between mitochondrial and cytosolic acetyl-CoA, the substrate for acetyltransferases, the observed correlation may not be causal. However, an increased activity of the main enzyme was also observed for the synthesis of cytosolic acetyl-CoA, ATPCL, in midlife flies, and therefore it was assumed that the cytosolic acetyl-CoA level is indeed higher when flies reach midlife. Interestingly, this increased activity is not caused by increased protein synthesis of ATPCL, but potentially by posttranslational mechanisms such as a hyper-acetylation. This is also supported by the observation that a fly strain heterozygous for an atpcl mutation shows only a 15% reduction in ATPCL activity, suggesting that there is a substantial degree of posttranscriptional regulation of this enzymatic activity. Such a regulation of ATPCL has also been proposed to stimulate lipid synthesis and tumor growth in rats. The current findings that a fly strain carrying a mutation in the ATPCL gene has an extended life span and a delayed onset of aging further confirm the importance of extra-mitochondrial acetyl-CoA for the regulation of aging. Interestingly, the reduction in ATPCL has a much stronger effect on the metabolism of midlife animals when compared to young animals. The effects observed analyzing head tissue of Drosophila melanogaster are in line with earlier reports that the targeted depletion of an unrelated acetyl-CoA synthase in fly neurons extends life span. It will be interesting to resolve the physiological effects of ATPCL mutation on the metabolome, the histone acetylation, and the transcriptome in isolated neurons (Peleg, 2016).
The ATPCL mutation results in a rather specific change in histone acetylation and does not affect all acetylation sites to the same degree. In midlife animals, the ATPCL mutation has the strongest effect on H4K12ac-an acetylation site that had been implicated in age-dependent memory impairment and transcriptional elongation and which is increased when flies reach their premortality plateau phase. This may be due to modulation of the enzymatic properties of Chameau or of a corresponding deacetylase. An increased activity in several deacetylases has been shown to extend life span in various organisms and higher concentrations of the sirtuin cofactor NAD+ have been shown to be beneficial for life span extension. However, the effect of sirtuins on life span continues to be debated and their effect has so far not been associated with a particular histone modification pattern. The quantitative analysis of specific histone modifications in this study has allowed identification of Chameau as an enzyme responsible for the increased modification in midlife flies (Feller, 2015). It is worth mentioning that the chm mutant allele is homozygous lethal and the beneficial effect on life span is more pronounced in males than in females, suggesting that Chameau has additional function, which are not yet fully understood. However, the fact that a reduction in the activity of the acetyltransferase Chameau robustly promotes longevity in male flies supports the hypothesis that this enzyme has an active role in modulating life span at least in Drosophila males (Peleg, 2016).
Previous studies demonstrated that old flies show an impaired transcriptome surveillance, as manifested in increased transcriptional noise and expression of aberrant or immature mRNAs. This study found substantial changes in the transcriptional profile as flies reach midlife, suggesting that the differential regulation of gene expression is one of the early hallmarks of the aging process. It remains to be explored how specific changes in gene expression integrate with regulatory modifications and metabolic activity. Chameau appears to promote the expression of a large number of genes particularly during the midlife period genes. Conceivably, the enhanced H4K12 acetylation leads to widespread chromatin opening, with positive effects for the transcription of specific genes. A side effect of this loosening of chromatin structure may be the increased transcriptional noise, which might compromise a variety of physiological functions. Considering the localization of H4K12ac at the gene body of highly expressed genes, it will be interesting to investigate whether the increased transcription during midlife is due to a higher rate of transcript elongation or a higher activity of cryptic promoters. It is hypothesized that the attenuation of this effect in chm mutant flies is the cause for their extended life span. A similar effect is also seen in ATPCL mutant flies, and the observation that life span is not further extended if the ATPCL and chm alleles are combined suggests that the two enzymes may act in the same pathway (Peleg, 2016).
These data provide an overview of the metabolic, proteomic, and transcriptomic changes that occur as flies reach the premortality plateau phase. Conceivably, metabolic processes are linked to changes in gene expression through differential protein acetylation, in general, and histone acetylation, in particular. Currently it cannot be unambiguously distinguish whether the shift in metabolic activity upon fly aging precedes the increases in protein/histone acetylation, or whether increases in protein/histone acetylation result in specific metabolic changes. Most likely, both principles affect each other in a complex network of feedback and feed-forward loops. Indeed, many mitochondrial enzymes that have been shown to be acetylated in response to metabolic changes either gain or lose enzymatic activity (Peleg, 2016).
Considering the high conservation of central metabolism, metabolic regulation, and epigenetics between flies and humans, these data raise the possibility that small molecule regulators of acetyl-CoA production or consumption, or changes in the activity of selective acetyltransferase functions, could prolong a healthy midlife also in humans. These model organism data reveal a potential alternative strategy that could extend midlife and delay aging-associated homeostatic decline in humans (Peleg, 2016).
Reversible acetylation of histone tails plays an important role in chromatin remodelling and regulation of gene activity. While modification by histone acetyltransferase (HAT) is usually linked to transcriptional activation, evidence is provided for HAT function in several types of epigenetic repression. Chameau (Chm), a new Drosophila member of the MYST HAT family, dominantly suppresses position effect variegation (PEV), is required for the maintenance of Hox gene silencing by Polycomb group (PcG) proteins, and can partially substitute for the MYST Sas2 HAT in yeast telomeric position effect (TPE). Finally, in vivo evidence is provided that the acetyltransferase activity of Chm is required in these processes, since a variant protein mutated in the catalytic domain no longer rescues either PEV modification, telomeric silencing of SAS2-deficient yeast cells, or lethality of chm mutant flies. These findings emphasize the role of an acetyltransferase in gene silencing, which supports, according to the histone code hypothesis, the observation that transcription at a particular locus is determined by a precise combination of histone tail modifications rather than by overall acetylation levels (Grienenberger, 2002).
Mutations were generated by mobilization of a P element inserted 1.2 kb downstream of the 3′ end of chm. chm14 deletes the last three exons, breaking in the MYST domain; Df(2L)221 deletes most of the coding region. These deficiencies are recessive pupal lethal and genetically behave as null alleles of chm. They do not affect another essential gene, since the associated lethality is rescued by a heat shock construct of chm cDNA in transgenic animals raised at 25°C (Grienenberger, 2002).
To assay for a role in modulating chromatin structure and transcription, PEV, a phenomenon of epigenetic repression mediated by pericentric heterochromatin, was first analyzed. Genes that dominantly modify PEV are thought to encode products that affect chromatin structure, leading when mutated to an increased (suppressors of PEV) or a decreased (enhancers) transcription of neighboring genes. The wm4h inversion leads to a mosaic pattern of eye pigment mutation. Mutation of one chm copy gives significant increase of pigmented area, resulting in a 2.5-fold increase of pigment levels. chm mutation therefore dominantly suppresses variegation of white in wm4h. To assess whether chm also affects other variegating rearrangements, the γ238 minichromosome was used that contains close to centric heterochromatin the yellow (y) gene, responsible for the dark pigmentation of bristles. The number of dark bristles at the wing margin is significantly increased in animals heterozygous for chm, indicating a suppression of y variegation. To rule out a possible effect of the genetic background on PEV, whether Chm overexpression, provided by a HSchm transgene at 25°C, could reverse the y derepression seen in chm heterozygous flies was examined. This demonstrates that chm haploinsufficiency causes PEV suppression, consistent with a role of its product in heterochromatin-mediated gene silencing (Grienenberger, 2002).
Chromatin-mediated transcriptional control during development is best illustrated by the PcG and trxG groups of genes, whose function is required for appropriate maintenance of Hox gene expression. To address whether Chm plays a role in this process, the effect of chm mutation was tested on the activity of Fab-7, a fragment from the Bithorax complex that contains a Pc response element (PRE) and recapitulates many aspects of transcriptional repression mediated by PcG proteins. The strong PcG-dependent repression of mini-white by Fab-7 in 5F24 25.2 flies is impaired upon inactivation of one copy of chm. Heterozygosity for the amorphous PcXT109 allele produces comparable derepression of mini-white, showing that the effect of chm on Fab-7 is as strong as that of Pc. In a parallel control, reducing the dosage of mof fails to modify mini-white expression, indicating that Chm but not any MYST protein provides activity acting at Fab-7. Chm is thus required for transcriptional repression mediated by the Fab-7 PRE, suggesting a role in the formation and/or activity of silencing PcG complexes (Grienenberger, 2002).
To further examine whether Chm and PcG proteins act together to maintain Hox gene repression, the effect of a reduction of chm dosage was tested on homeotic transformations that result from mutations affecting either PcG transregulators or a PRE cis-regulatory element. The first PcG dominant phenotype examined was a T2 into T1 transformation. In the second leg disc of Pc male heterozygotes, derepression of Sex comb reduced leads to the formation on the second leg of a sex comb, a structure normally found on the first leg only. The mutation of one copy of chm significantly enhances this phenotype. chm and PcG gene interactions in the specification of adult abdomen identities were tested. In parasegment 9 (PS9) of males heterozygous for PcXT109 or for the ph410 allele of polyhomeotic (ph), inappropriate expression of Abdominal-B (Abd-B) produces a mild transformation of the fourth abdominal segment into the fifth (A4 into A5), as evidenced by patches of pigmentation in the anterior part of A4. This phenotype, which is never observed in a wild-type context, occurs at low frequency in males heterozygous for chm (less than 1%). Double heterozygotes for chm and for ph or Pc exhibit increased A4 into A5 transformation and/or increased number of transformed individuals, compared to single PcG mutants. Finally, the homeotic transformation induced by a PRE mutation was examined. McpB116 affects Abd-B silencing in PS9, giving rise to incomplete A4 into A5 transformations. This homeotic phenotype is stronger in double heterozygotes for chm14 and McpB116 and becomes further enhanced by the mutation of one copy of Pc. In the various genetic contexts reported here, chm was therefore found to genetically interact with PcG genes and the Mcp element in a positive manner. These synergistic effects strongly suggest that Chm collaborates with PcG proteins for PRE-mediated repression at Hox gene loci (Grienenberger, 2002).
Direct evidence for a role of Chm in Hox gene silencing was obtained from the examination of Ubx expression in imaginal discs. Whereas Ubx is not detected in the columnar epithelium of a wild-type wing disc, derepression is observed in few cells from discs heterozygous for Pc, and a more extended activation occurs in discs heterozygous for both chm and Pc. These results confirm that Chm and PcG proteins act together to repress Hox genes. No misexpression of Ubx could be detected, however, in discs from chm homozygous larvae. Thus, chm can be classified as an enhancer of PcG mutations instead of a novel PcG gene (Grienenberger, 2002).
Recombinant Chm turned out unable to acetylate histones in vitro, as its human homolog HBO1 (Iizuka, 1999). Thus, assays of acetyltransferase activity in vivo were carried out. The heterologous yeast system was used. The rationale was to test, using a three step process, whether the ability of Chm to substitute for a MYST yeast protein is lost upon enzymatic inactivation. In a first step, it was established that chm can replace SAS2 in TPE. As already described, disruption of SAS2 derepresses telomeric silencing and impairs cells carrying a subtelomeric URA3 gene to grow on 5-FOA. Chm overexpression in sas2Δ cells partially restores TPE as shown by the increased colony-forming ability on 5-FOA. As a control, Chm is unable to rescue telomeric silencing defect caused by Set1 deficiency, a SET domain protein devoid of MYST domain, and, reciprocally, Set1 cannot restore defect in silencing associated with loss of Sas2. These experiments indicate that Chm does not act as a general signal in telomere silencing but rather specifically replaces Sas2 in TPE. A Chm variant was generated where the glycine at position 680 was mutated into glutamate. This glycine lies at a central position in the Q/RxxGxG motif and is essential for enzymatic activity. The variant protein fails to restore TPE, indicating that Chm acetyltransferase activity is needed for the assembly of repressive telomeric chromatin in sas2Δ cells (Grienenberger, 2002).
In a second step, PEV modification was examined. As reported above, Chm overexpression can reverse the defect of y repression caused by chm heterozygosity in γ238 flies. Providing ChmG680 instead of wild-type Chm in an otherwise similar background has no effect on y expression. ChmG680 mutation does not impair protein stability, since extracts from HSchm and HSchmG680 animals raised at 25°C react similarly on Western blot. Thus, ChmG680 cannot modify PEV, indicating that heterochromatin-mediated silencing requires Chm acetyltransferase activity (Grienenberger, 2002).
Third, whether ChmG680 could rescue the lethality of chm14 animals was examined. If the acetyltransferase activity is essential for development, then either no or a far less efficient rescue is expected from heat shock constructs providing ChmG680 instead of wild-type Chm. Two lines transgenic for the wild-type and four for the variant were generated. Efficient rescue by HSchm was obtained, raising one line at 25°C and providing the other with a larval heat pulse. In contrast, HSchmG680 does not allow any rescue at 25°C. For two of the four transgenic lines, however, rare escapers were eventually obtained after larval induction. These results indicate that the acetyltransferase activity of Chm is required for normal development. The fact that ChmG680 can rarely rescue lethality suggests either that the variant still possesses residual acetyltransferase activity and/or that other regions of the protein encode distinct essential functions (Grienenberger, 2002).
These results extend previous observations that MYST proteins are important for the definition of silent heterochromatin. Thus, H4 lysine 16 is a likely in vivo target of Sas2, and this HAT activity is required for telomere silencing mediated by a complex formed by Sas2 and the chromatin assembly factors CAF-1 and Asf1. The ability of Chm but not of ChmG680 to partially rescue the sas2Δ silencing defect suggests it may have a similar HAT activity. Other findings have connected MYST acetyltransferases, ORC, and heterochromatin-mediated silencing. HBO1, the human homolog of Chm, interacts with ORC1, and DmOrc2 is, as chm, a dominant suppressor of PEV. Furthermore, ORC subunits and the Su(var) proteins HP1 and HOAP form a complex required for heterochromatin assembly. In this context, the recruitment of HBO1/Chm might provide specific activity needed for the reestablishment of acetylation patterns after DNA replication and for the ORC/HP1 function in the process of heterochromatin formation (Grienenberger, 2002).
The results provide evidence that Chm is required for PcG-mediated silencing during larval development. This is presumably not the case during embryogenesis, since chm mutation has no effect on cuticular identity. Two PcG protein complexes have been isolated, only from embryos so far, and both contain histone modifying activities. The E(z)/Esc complex, partially characterized, contains the RPD3 HDAC. The 30 proteins from PRC1 have been identified, among which are RPD3 and the TAFII250 HAT. This indicates that PRC1 needs some HAT activity to mediate repression and that the silent chromatin state likely results from a steady-state acetylation level defined by the combination of TAFII250 acetylating and RPD3 deacetylating activities. Chm has not been found in PRC1, consistent with the conclusion that it is dispensable for PcG silencing during embryogenesis. It will be interesting to check for the presence of Chm in PcG repressive complexes acting during imaginal development (Grienenberger, 2002).
Further work is needed to elucidate how Chm conveys chromatin-mediated silencing. One possibility that would obey the general correlation between histone acetylation and gene activation is that Chm promotes acetylation and allows transcription at loci required for repression, including PcG and Su(var) genes. The ability of Chm to replace Sas2 in TPE in a process dependent of its acetyltransferase activity rather suggests a direct involvement in silencing. Considering the novel conceptual frame of the histone code, Chm may thus provide acetylation marks required for the definition of histone tail modification patterns allowing the recruitment of silencing complexes, such as ORC/HP1 at heterochromatin or PcG complexes at PREs. Alternatively, Chm might contribute to epigenetic silencing by modifying chromatin proteins other than histones, such as PcG and heterochromatic proteins (Grienenberger, 2002).
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date revised: 2 July 2006
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