BIOLOGICAL OVERVIEW

Yeast SIR2 (Silent Information Regulator 2) is a nicotinamide adenine dinucleotide (NAD)⁺-dependent histone deacetylase required for heterochromatic silencing at telomeres, rDNA, and mating-type loci. The Drosophila Sir2 also encodes deacetylase activity and is required for heterochromatic silencing, but unlike ySir2, is not required for silencing at telomeres. Drosophila Sir2 interacts genetically and physically with members of the Hairy/Deadpan/E(Spl) family of bHLH euchromatic repressors, key regulators of Drosophila development. Drosophila Sir2 is an essential gene whose loss of function results in both segmentation defects and skewed sex ratios, associated with reduced activities of the Hairy and Deadpan bHLH repressors. These results indicate that Sir2 in higher organisms plays an essential role in both euchromatic repression and heterochromatic silencing (Rosenberg, 2002).

Histone deacetylases (HDACs) act as cofactors that are recruited to promoters by sequence-specific DNA binding factors resulting in the local modification of histones to promote chromatin compaction with subsequent inhibition of gene transcription. HDACs have been divided into classes based on their similarity to known yeast factors: class I HDACs are similar to yRPD3, while class II HDACs are related to yHDA1. Class III HDACs, exemplified by ySIR2, have NAD⁺-dependent HDAC activity and are not sensitive to inhibitors of class I HDACs, such as trichostatin A (TSA; Bernstein, 2000). SIR2 also has ADP-ribosylase activity (Tanny, 1999). While SIR2's HDAC activity is essential for silencing in yeast, its ADP-ribosylase activity is not essential for silencing (Imai, 2000), and no biological function has yet been assigned to this activity. ySIR2 acts as a dedicated silencing protein that deacetylates histones at heterochromatic targets, including the mating-type loci, telomeres, and rDNA repeats (reviewed in Gottschling, 2000; Guarente, 2000). ySIR2 plays an important role in aging, but is not an essential gene. There are four other SIR2-like proteins (or sirtuins) in yeast, however, none can compensate for all of the functions of ySIR2 and the yeast quintuple mutant is viable (Brachmann, 1995). Like other HDACs, ySIR2 is recruited to DNA by DNA binding factors (Rosenberg, 2002 and references therein).

Cofactor recruitment by DNA bound factors is an important feature of transcriptional repression mechanisms used to establish complex patterns of gene expression during development. A number of developmentally regulated repressors are transcription factors that recruit HDACs as cofactors to bring about repression. In Drosophila, the sequence-specific DNA binding repressor Hairy has been studied extensively in this context. hairy is a member of the pair-rule class of genes that is essential for the proper establishment of segmentation in the developing embryo. hairy encodes a bHLH transcription factor that belongs to the Hairy/Enhancer of Split/Deadpan (or HES) family of proteins. Drosophila HES family proteins are key repressors in the developmental processes of segmentation, neurogenesis, and sex determination. All members of this repressor family possess (1) a highly conserved bHLH domain, required for protein dimerization and DNA binding; (2) an adjacent Orange domain, which confers specificity among family members, and (3) a C-terminal tetrapeptide motif, WRPW, which has been shown to be necessary and sufficient for the recruitment of the corepressor, Groucho. Groucho has in turn been proposed to recruit the class I HDAC, Rpd3, suggesting a mechanism by which HES repressors use Groucho and Rpd3 to create a chromatin environment that is repressive of transcription. In some assays, however, Hairy can function in the absence of its WRPW motif, indicating that in the absence of Groucho and presumably Rpd3, Hairy can still repress transcription. This repression may be achieved through other mechanisms, such as the recruitment of other cofactors (Rosenberg, 2002).

A Drosophila homolog of the yeast histone deacetylase SIR2 was identified by sequence homology (CG5216). There are five sirtuins in Drosophila, with Sir2 (CG5216) sharing the highest degree of sequence similarity to ySir2. Drosophila Sir2 is an essential gene that is dynamically expressed throughout development. Sir2 is required for position effect variegation, suggesting a role for Sir2 similar to that of its yeast counterpart in maintaining heterochromatic silencing. Sir2 also has a strong maternal component such that progeny from mothers with reduced Sir2 exhibit segmentation defects. A direct physical and genetic interaction is observed between Sir2 and Hairy, suggesting this as a basis for the segmentation defects. In addition, a direct physical interaction has been detected between Sir2 and Deadpan (Dpn), but not with other HES family proteins. Consistent with this and with a role for Sir2 in Dpn repression, progeny with altered gene dosage of Sir2 exhibit skewed sex ratios. These results indicate that Sir2 interacts directly with members of the HES bHLH class of euchromatic transcriptional repressors in mediating processes essential for the early development of the embryo (Rosenberg, 2002).

In addition to its conserved role in heterochromatic silencing, examination of loss-of-function mutants reveals a significant role for Sir2 in Drosophila embryogenesis. Sir2 is essential for zygotic function since progeny that are homozygous for Sir2⁰⁵³²⁷ die as embryos. Loss of zygotic Sir2 function does not affect early embryo patterning since the dead homozygous mutant embryos exhibit cuticle phenotypes indistinguishable from wild-type. Sir2 also has a strong maternal effect. A Sir2 mutation was initially identified in a genetic screen for maternal genes essential for embryonic development. In this screen, a change-of-function mutation (called wimp) in the second largest subunit of RNA polymerase II was used to reduce, but not eliminate, maternal Sir2 contribution. Embryos derived from mothers trans-heterozygous for wimp and the Sir2 allele die and exhibit anterior-posterior patterning defects, including loss of segments and pairwise denticle band fusions, as compared to wild-type or wimp/+, demonstrating a role for maternally contributed Sir2 in the establishment of body pattern (Rosenberg, 2002).

To identify the earliest stage at which segmentation is affected, the expression of genes at different tiers of the segmentation gene hierarchy were examined. Protein expression patterns of the gap genes Krüppel and knirps are unaffected in progeny from females with reduced maternal Sir2 (Sir2⁰⁵³²⁷/+;wimp/+ or Sir2^ex10/+;wimp/+ transheterozygous mothers). Sir2 is first required for regulation of segmentation at the level of pair rule gene expression. Pair rule genes can be separated into two classes: primary pair rule genes establish double segment periodicity, whereas secondary pair rule genes respond to this pattern. Pair rule gene products are expressed as a series of seven transverse stripes in wild-type or wimp/+ embryos. Stripes of the secondary pair rule gene fushi tarazu (ftz) are severely derepressed (stripes are broadened) in embryos from mothers with reduced Sir2 expression. Aberrant regulation of Ftz stripe expression in Sir2 mutant embryos is consistent with reduced function of the primary pair rule gene, Hairy, which behaves genetically as a repressor of ftz. Hairy expression was examined in Sir2 mutant embryos: in contrast to Ftz expression, which is significantly altered, Hairy is largely unaffected in these embryos (Rosenberg, 2002).

The Ftz derepression phenotype in Sir2 embryos is reminiscent of the Ftz expression pattern seen in hairy mutants. Sir2 was examined for genetic interaction with hairy; these mutations exhibit a dominant genetic interaction. Progeny from either hairy heterozygous mothers or Sir2 heterozygous mothers mated to wild-type males are viable and exhibit wild-type cuticle phenotypes. In contrast, embryos derived from mothers heterozygous for both Sir2 and hairy (Sir2/+; hairy/+ trans-heterozygous mothers) mated to wild-type males exhibit moderate to severe cuticle abnormalities. Consistent with this segmentation cuticle phenotype, Ftz is derepressed in these embryos, with a reduction in expression of stripes 4, 6, and 7, suggesting that these segmentation defects are largely mediated by interaction of Sir2 with Hairy. Interestingly, Hairy stripes 3 and 4 are also affected in progeny from mothers trans-heterozygous for Sir2 and hairy (Sir2/+; hairy/+ females), suggesting interactions between Sir2 and other developmental regulators. Sir2 was tested for interaction with repression cofactors groucho (gro) and dCtBP, as well as the other primary pair rule genes, even skipped (eve), and runt (run). No dominant synthetic lethal interactions were detected between Sir2 and any of these mutations. Hairy was tested for genetic interaction with the class I HDAC, Rpd3, which has been proposed to be recruited to Hairy via the corepressor Groucho. However, no genetic interaction was detected between Rpd3 and hairy (Rosenberg, 2002).

In yeast, SIR2 is required for silencing at heterochromatic loci (including mating-type loci, rDNA arrays, and telomeres; Rine, 1987), as well as for silencing of an auxotrophic marker inserted within heterochromatin (Gottschling, 1990). Using reporter lines carrying w⁺ insertions, it has been found that Sir2 affects heterochromatic silencing of pericentric markers and markers inserted within repeated DNA arrays (Rosenberg, 2002).

In contrast, Sir2 does not appear to be involved in telomeric position effect. Reduction of Sir2 function suppresses position effect variegation (PEV) at telomere 4, but not at telomeres 2L or 3R. Studies by Cryderman (1999) have shown that subtelomeric and pericentric hsp 70-w⁺ transposon insertions are suppressed by different mutations, indicating regulation of heterochromatic and telomeric PEV by distinct sets of proteins. This study also showed that telomere 4 is unique among telomere insertions in that it responds to suppressors of heterochromatic silencing, but not to suppressors of telomeric silencing (Cryderman, 1999). That Sir2 is required for silencing of telomere 4 and not other telomeres tested suggests that Sir2 has a role in specific types of heterochromatic silencing which are distinct from telomeric silencing, although a role for Sir2 at telomeres cannot be ruled out. Since Drosophila has four additional sirtuins which all bear the same conserved catalytic core region, the roles of ySIR2 that are not shared by Sir2 may be regulated by these other sirtuins (Rosenberg, 2002).

While ySir2 has generally been described as a dedicated heterochromatic silencing protein, Drosophila Sir2 can also interact with specific euchromatic transcription factors. Sir2 interacts with the euchromatic bHLH repressor Hairy, both genetically and physically. This interaction requires Hairy's basic domain that is highly similar among members of the HES family. However, despite their extensive conservation, Sir2 binds to only a subset of Hairy/E(Spl) family members. Since the four amino acids necessary for mediating Sir2 binding are invariant within this family, there must be additional recognition features within the basic domain or elsewhere within the proteins. Previous reports have shown the basic domain to be an essential domain for DNA binding and dimerization among bHLH proteins. Sir2 binding to this region represents a novel domain for Hairy cofactor binding (Rosenberg, 2002).

The basic domains of bHLH proteins have been shown to undergo a disordered-to-ordered transition upon binding to DNA, making cofactor binding in this region an interesting paradox. Since no supershift has been detected upon addition of Sir2 protein and Sir2 does not appear to affect the ability of Hairy to bind DNA, Sir2 and Hairy may not be in a stable complex with DNA. The simplest explanation for these observations is that the interaction between Sir2 and Hairy in the presence of DNA is weak, requiring other proteins to stabilize the complex in vitro. However, there are several other ways in which Sir2 could affect Hairy function. Upon binding to Hairy, Sir2 may alter chromatin structure, affect distally bound factors on Hairy, or alter Hairy's ability to recruit cofactors required for other Hairy functions. In addition, Sir2 may deacetylate Hairy, altering either its DNA binding or activity, similar to altered p53 activity following deacetylation by human SIRT1 (Vaziri, 2001; Luo, 2001). This would not have been detected by gel shift assays, since bacterially expressed Hairy is not acetylated. Alternatively, other repressors, such as Polycomb Group complexes, can initiate silencing by transient recruitment of repression cofactors during brief interactions of distinct repressor complexes. The ability of Hairy to recruit distinct histone deacetylase containing complexes may represent a mechanism through which Hairy can both initiate and maintain a repressed state of chromatin through distinct and transiently interacting complexes. Such a complex containing Sir2 and DNA-bound Hairy would not have to be very stable, since a short-lived interaction may be sufficient (Rosenberg, 2002).

It is interesting that Hairy has been linked to two distinct histone deacetylases. Hairy and other HES family members recruit Groucho, which in turn has been proposed to recruit the class I HDAC, Rpd3. While Rpd3 mutant embryos exhibit segmentation defects, they involve only minor disruption of the Eve and Engrailed segmentation gene products, leading to the conclusion that Rpd3 is involved in segmentation but cannot represent a major pathway of repression in the early embryo. No dominant interaction has been detected between hairy and Rpd3; however, Sir2 is thought to be required for the processes of segmentation and sex determination in which Groucho is also required by bHLH factors. In contrast to the Rpd3 loss of function phenotypes, the segmentation defects observed in Sir2 loss-of-function embryos are severe (Rosenberg, 2002).

It is proposed that Hairy uses different deacetylases in different contexts. Phenotypic analysis of different hairy mutants suggests that the requirements for Sir2 and Groucho are overlapping but not redundant. Hairy has never been found to activate transcription, in contrast to other factors, such as the bHLHZip protein Myc, which has been shown in different contexts to either activate or repress transcription. Perhaps the requirement for Sir2 in processes that likely involve a separate HDAC complex represents a mechanism of repression by HES proteins that enable them to be dedicated repressors. Alternatively, the ability of Hairy to recruit two distinct histone deacetylases may allow it to independently regulate distinct processes (Rosenberg, 2002).

In light of the requirement for Sir2 throughout embryogenesis, the dynamic subcellular changes in Sir2 expression are intriguing. Dynamic localization of HDACs has been shown to be important for the activity of other bHLH factors, such as myocyte enhancer factor-2 (MEF-2). The class II mammalian HDAC5, which interacts with MEF2, must be removed from the nucleus to permit myocyte differentiation. Phosphorylation of HDAC5 alters its subcellular localization, allowing its export from the nucleus and subsequent progression of myoblast differentiation. Since the early Drosophila embryo is a closed system, it is possible that some developmental programs in the early embryo require removal of Sir2 from the nucleus. It is worth noting that at the times at which Sir2 plays a role in developmental processes (nuclear cycle 9-10 for sex determination and nuclear cycle 14 for segmentation), Sir2 is detectable in the nucleus, while at times in between (nuclear cycle 13), Sir2 is excluded from the nucleus. Future studies that characterize Sir2 localization and its developmental regulation will be informative about the requirements for Sir2 for diverse processes in the early embryo (Rosenberg, 2002).

The results show that Sir2 plays an important role in Drosophila euchromatic gene regulation through its interaction with bHLH repressors. Consistent with this, Sir2 localizes to both distinct euchromatic sites and generally to the centric heterochromatin on polytene chromosomes. van Steensel (2000) has also identified multiple euchromatic targets of Sir2 recruitment using an in vitro chromatin assay. ySIR2 has not been thought to interact with euchromatic repressor complexes. However, a euchromatic role for Sir2 may in fact exist in yeast. Lieb (2001) reported that the ySIR2-interactor RAP1 binds to 5% of yeast genes including intergenic regions that may correspond to promoters. Together, these results suggest that the ability of Sir2 to act as a euchromatic repressor represents a widespread and conserved function for Sir2. The finding that Sir2 is required for PEV also highlights the notion that mechanisms of repression are shared in part by heterochromatin and euchromatin (Rosenberg, 2002).

It is interesting to consider that the role of Sir2 in development may involve additional functions ascribed to the yeast enzyme. ySir2 has been shown by its gene dosage-dependent effects on lifespan (Kaeberlein, 1999) and by its NAD⁺-dependence (Landry 2000) to be linked to metabolism, perhaps by monitoring redox states in the cell. Sir2 may act during development to coordinate the progression of developmental programs by sensing the metabolic needs and outputs of the embryo and modifying key regulators of development to act accordingly. This may be an important aspect of Drosophila development where much of the early developmental program is executed in a closed system, consisting of maternally contributed factors in the absence of de novo transcription. bHLH factors that are key regulators of circadian rhythms were shown to have altered DNA binding affinity and heterodimerization preferences in response to changing cellular ratios of NAD⁺:NADH (Rutter, 2001). The bHLH domain mediates responsiveness of these factors to NAD⁺, which itself can respond both by altered heterodimerization and by altered DNA binding affinity (Rosenberg, 2002).

The finding that Sir2 is required for heterochromatic gene silencing and euchromatic repression represents a common link between the mechanisms of repression utilized by heterochromatin and euchromatin. Future studies on the precise molecular nature of Sir2 activity will likely uncover exciting new roles for it in both euchromatic and heterochromatic silencing.

dSir2 deficiency in the fatbody, but not muscles, affects systemic insulin signaling, fat mobilization and starvation survival in flies

Sir2 is an evolutionarily conserved NAD+ dependent protein. Although, SIRT1 has been implicated to be a key regulator of fat and glucose metabolism in mammals, the role of Sir2 in regulating organismal physiology, in invertebrates, is unclear. Drosophila has been used to study evolutionarily conserved nutrient sensing mechanisms, however, the molecular and metabolic pathways downstream to Sir2 (dSir2) are poorly understood. This study has knocked down endogenous dSir2 in a tissue specific manner using gene-switch gal4 drivers. Knockdown of dSir2 in the adult fatbody leads to deregulated fat metabolism involving altered expression of key metabolic genes. The results highlight the role of dSir2 in mobilizing fat reserves and demonstrate that its functions in the adult fatbody are crucial for starvation survival. Further, dSir2 knockdown in the fatbody affects dilp5 (insulin-like-peptide) expression, and mediates systemic effects of insulin signaling. This report delineates the functions of dSir2 in the fatbody and muscles with systemic consequences on fat metabolism and insulin signaling. In conclusion, these findings highlight the central role that fatbody dSir2 plays in linking metabolism to organismal physiology and its importance for survival (Banerjee, 2012).

This study reports that dSir2 is a critical factor that regulates metabolic homeostasis and mediates organismal physiology. Using genetic tools (inducible RNAi) that negate background effects, concrete results are provided that highlight the importance of endogenous dSir2 in the whole body, and in metabolically relevant tissues, such as fatbody and muscle. The findings point out the importance of nutrient signaling in eliciting dSir2-dependent molecular changes, which play an important role in tissue specific metabolic functions that affect systemic outputs in flies. By describing a metabolic phenotype in flies that lack dSir2, this study reiterates that Drosophila can be used to study sirtuin biology, but also highlight the evolutionary conservation of dSir2/SIRT1 functions in regulating organismal physiology (Banerjee, 2012)

Until now, the conservation of molecular mechanisms underlying Sir2 biology was poorly addressed in invertebrates. It is only in mammals that a functional interplay between metabolic flux, SIRT1 and its downstream molecular factors has been addressed, thus far (Longo, 2006; Canto, 2009; Finkel, 2009). Results from backcrossed dSir2 mutant and whole body dSir2 knockdown flies indicated that absence or down-regulation of dSir2 expression results in gross metabolic defects. Interestingly, it was observed that the effects on glucose levels were different in these two cases. The differences in glucose levels might reflect the systemic alterations in response to a complete absence of the protein in the case of mutants and down-regulation of expression in the case of knockdowns. It is interesting to note that studies in Sirt1^+/-, liver specific Sirt1 knockout and knockdown micehave also yielded seemingly conflicting results. Specifically, with respect to glucose metabolism, these differences indicate that the manifestation of functions of Sir2/SIRT1 might be dependent upon the extent to which its expression is altered. Importantly, this underpins the need to further investigate the molecular interactions that bring about such varied phenotypes, in both mammals and flies (Banerjee, 2012).

It is important to note that consistent phenotypic, metabolic and molecular readouts were obtained with respect to fat metabolism in dSir2-mutant and -RNAi flies. A decrease (or absence) of dSir2 expression was found to result in increased fat storage in the fatbodies, as determined by oil red staining and biochemical analyses. This fat accumulation is due to altered expression of genes involved in fat metabolism. Importantly, it was shown that genes involved in fat breakdown are downregulated in the dSir2 knockdown flies, in addition to an upregulation of genes involved in fat synthesis. These findings are not only in accordance with the results obtained from dSir2 mutant larvae but also implicate dSir2 as a key player in fat metabolism in adult flies (Banerjee, 2012).

A role for dSir2 was uncovered in regulating systemic insulin signaling in flies. To investigate if the ability of dSir2 to mediate insulin signaling emanates from a specific tissue, dilp5 expression was assayed in fatbody and muscle specific dSir2^RNAi flies. Interestingly, it was found that knocking down dSir2 only in the fatbody, but not muscles led to increased dilp5 expression, and mimicked dSir2 mutants and whole body dSir2^RNAi flies. Specifically, this study addressed the role of dSir2 in the fatbody to mediate systemic effects on insulin signaling. Further investigations should help understand the dSir2-dependent molecular and physiological links between the fatbody and medial secretory neurons (MSNs). Very recently, hepatic SIRT1 was shown to mediate peripheral insulin signaling in mice. Importantly, the current findings underpin the importance of dSir2/SIRT1 in the homologous metabolic tissues, fatbody and liver, on systemic insulin signaling (Banerjee, 2012).

Efforts to link the molecular functions of dSir2 and organismal physiology led to the implication of dSir2 in starvation survival. dSir2 mutants and whole body dSir2^RNAi flies succumb to starvation earlier than the controls and interestingly, are phenocopied by fatbody dSir2^RNAi flies. Moreover it was shown that this is due to an inability to mobilize fat reserves from the fatbody, and a resultant of decreased expression of lipid breakdown genes, both under fed and starved conditions. The importance of dSir2 in the fatbody and fat mobilization is corroborated by an absence of deregulated fat metabolism in muscle specific dSir2^RNAi flies. Further, a lack of starvation phenotype when dSir2 is knocked down from the muscles highlights the physiological relevance of fatbody (Banerjee, 2012).

In summary, this study has elucidated the significance of the functions of dSir2 in the fatbody in mediating central and peripheral effects on metabolic homeostasis and insulin signaling. Therefore, it is concluded that dSir2 is a key component that links dietary inputs with organismal physiology and survival. Most importantly, this study highlights the functions of dSir2 in the fatbody as a deterministic factor in governing fly physiology. This study delineates the functions of dSir2 in two metabolic tissues in affecting organismal survival. Metabolic homeostasis and the ability to utilize stored energy reserves are also crucial for mediating the effects of calorie restriction. These results, which emphasize the importance of dSir2 in maintaining homeostasis, reiterates its role in calorie restriction. Finally, this report highlights the need to further investigate the functions of dSir2, and should motivate future studies to understanding Sir2's interactions with other pathways and importance during aging (Banerjee, 2012).

Regulation of expression of autophagy genes by Atg8a-interacting partners Sequoia, YL-1, and Sir2 in Drosophila

Autophagy is the degradation of cytoplasmic material through the lysosomal pathway. One of the most studied autophagy-related proteins is mammalian LC3. Despite growing evidence that LC3 is enriched in the nucleus, its nuclear role is poorly understood. This study shows that Drosophila Atg8a protein, homologous to mammalian LC3, interacts with the transcription factor Sequoia in a LIR motif-dependent manner. Sequoia depletion induces autophagy in nutrient-rich conditions through the enhanced expression of autophagy genes. Atg8a interacts with YL-1, a component of a nuclear acetyltransferase complex, and it is acetylated in nutrient-rich conditions. Atg8a interacts with the deacetylase Sir2, which deacetylates Atg8a during starvation to activate autophagy. These results suggest a mechanism of regulation of the expression of autophagy genes by Atg8a, which is linked to its acetylation status and its interaction with Sequoia, YL-1, and Sir2 (Jacomin, 2020).

Autophagy is a fundamental, evolutionary conserved process in which cytoplasmic material is degraded through the lysosomal pathway. It is a cellular response during nutrient starvation; yet, it is also responsible in basal conditions for the removal of aggregated proteins and damaged organelles and therefore plays an important role in the maintenance of cellular homeostasis. There are three main types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. Macroautophagy, referred to as autophagy, is the best-described type of autophagy. During macroautophagy, cytoplasmic material is isolated into double-membrane vesicles called autophagosomes. Autophagosomes eventually fuse with lysosomes, allowing for the degradation of cargoes by lysosomal hydrolases. The products of degradation are transported back into the cytoplasm through lysosomal membrane permeases and can be reused by the cell (Jacomin, 2020).

One of the most important and well-studied autophagy-related proteins is LC3 (microtubule-associated protein 1 light chain 3, called Atg8 in yeast and Drosophila), which participates in autophagosome formation. LC3 interacts with LIR (LC3-interacting region) motifs also known as AIM (Atg8-interacting motifs) on selective autophagy receptors that carry cargo for degradation, and is one of the most widely used markers of autophagy. Despite growing evidence that LC3 is enriched in the nucleus, little is known about the mechanisms involved in targeting LC3 to the nucleus and the nuclear components with which it interacts (Jacomin, 2020).

This study shows that Drosophila Atg8a protein, homologous to mammalian LC3 and yeast Atg8, interacts with the transcription factor Sequoia in a LIR motif-dependent manner that is not responsible for the degradation of Sequoia. This study shows that Sequoia depletion induces autophagy in nutrient-rich conditions through the enhanced expression of autophagy genes. Atg8a was found to be acetylated and interacts with YL-1, a component of the NuA4/Tip60 nuclear acetyltransferase complex. This study show that Atg8a interacts with the deacetylase Sir2, which deacetylates Atg8a during starvation to activate autophagy. These results suggest a novel mechanism of regulation of autophagy gene expression by Atg8a, which is linked to its acetylation status and its interaction with Sequoia, YL-1, and Sir2 (Jacomin, 2020).

Atg8 family proteins have been extensively described for their implications in autophagosome formation and cargo selection in the cytoplasm. Although Atg8 family proteins also localize in the nucleus, their role in this compartment remains largely unexplored. This study has uncovered a nuclear role for Drosophila Atg8a in the regulation of autophagy gene expression and induction of autophagy via a LIR motif-dependent mechanism, regulated by Atg8a acetylation. The transcription factor Sequoia interacts with Atg8a in the nucleus to control the transcriptional activation of autophagy genes. It is suggestd that the acetylation status of Atg8a at position K48 contributes to the modulation of the interaction between Sequoia and Atg8a in the nucleus. This study also identified that YL-1, a component of a nuclear acetyltransferase complex, and deacetylase Sir2 interact with Atg8a, and that they act as regulators of Atg8a acetylation (Jacomin, 2020).

A working model is proposed in which in fed conditions, histone-interacting protein YL-1 contributes to the acetylation of Atg8a, while Sequoia resides at the promoter regions of autophagy genes to repress their expression. In such conditions, the interaction between Sequoia and Atg8a contributes to the sequestration of Atg8a in the nucleus. Atg8a cannot therefore translocate to the cytoplasm to take part in the formation of autophagosomes. This hypothesis is supported by the observation that mutation of the Sequoia LIR motif results in an increased accumulation of autophagosomes and autolysosomes in the fed condition. This is observed alongside a reduction in the enrichment of Sequoia at the promoter region of autophagy genes, resulting in their increased expression. Hence, in the absence of an interaction between Atg8a and Sequoia, the subsequent translocation of Atg8a to the cytoplasm may also play a key role in relieving the repressive abilities of Sequoia at the promoter regions of autophagy genes. Upon starvation, Sir2 interacts with and deacetylates Atg8a. Deacetylated Atg8a interacts more strongly with Sequoia, which cannot be maintained at the promoter regions of autophagy genes, leading to the activation of their transcription. Deacetylated Atg8a is then able to translocate to the cytoplasm and contribute to the formation of autophagosomes. It is proposed that Atg8a plays an essential role in relieving Sequoia from the promoter regions of autophagy genes specifically during starvation-induced autophagy as Atg8a loss of function results in the repression of the expression of autophagy genes (Jacomin, 2020).

The results support previous findings about the yeast and mammalian homologs of Sequoia, Rph1, and KDM4A, respectively, which have been shown to negatively regulate the transcription of autophagy genes (Bernard, 2015). The current study elucidates how the LIR-dependent interaction between Sequoia and Atg8a is involved in modulating the expression of autophagy genes during starvation. Mammalian KDM4A also directly interacts with GABARAP-L1; however, the interaction does not require a functional LIR motif. This may be related to the loss of the functionality of the LIR motif during evolution, as it has been shown for Kenny, another LIR-motif containing protein in Drosophila, and its mammalian homolog inhibitor of nuclear factor κB kinase (NF-κB) subunit γ/NF-κB essential modulator (IKKγ/NEMO). The current study also supports previous reports about the role of acetylation and deacetylation of LC3 in mammals and the regulation of autophagy by acetylation (Jacomin, 2020).

Higher eukaryotes express YL-1, a highly conserved Swc2 homolog, which has specific H2A.Z-binding properties. Drosophila YL-1 has been shown to have a H2A.Z-binding domain that binds H2A.Z-H2B dimer (Liang, 2016). The current study reports a novel role for YL-1 in the regulation of acetylation of non-histone proteins and the regulation of autophagy induction (Jacomin, 2020).

In conclusion, these results unveil a novel nuclear role for Atg8a in the regulation of autophagy gene expression in Drosophila, which is linked to its acetylation status and its ability to interact with transcription factor Sequoia. This study highlights the physiological importance of the non-degradative role of LIR motif-dependent interactions of Atg8a with a transcription factor and provide novel mechanistic insights on an unanticipated nuclear role of a protein that controls cytoplasmic cellular self-eating (Jacomin, 2020).

Acetyl-CoA-mediated autoacetylation of fatty acid synthase as a metabolic switch of de novo lipogenesis in Drosophila

De novo lipogenesis is a highly regulated metabolic process, which is known to be activated through transcriptional regulation of lipogenic genes, including fatty acid synthase (FASN). Unexpectedly, this study found that the expression of FASN protein remains unchanged during Drosophila larval development from the second to the third instar larval stages (L2 to L3) when lipogenesis is hyperactive. Instead, acetylation of FASN is significantly upregulated in fast-growing larvae. This study further showed that lysine K813 residue is highly acetylated in developing larvae, and its acetylation is required for elevated FASN activity, body fat accumulation, and normal development. Intriguingly, K813 is autoacetylated by acetyl-CoA (AcCoA) in a dosage-dependent manner independent of acetyltransferases. Mechanistically, the autoacetylation of K813 is mediated by a novel P-loop-like motif (N-xx-G-x-A). Lastly, this study found that K813 is deacetylated by Sirt1, which brings FASN activity to baseline level. In summary, this work uncovers a previously unappreciated role of FASN acetylation in developmental lipogenesis and a novel mechanism for protein autoacetylation, through which Drosophila larvae control metabolic homeostasis by linking AcCoA, lysine acetylation, and de novo lipogenesis (Miao, 2022).

De novo lipogenesis (DNL) is a complex yet highly regulated metabolic process which converts excess carbohydrates into fatty acids that are then esterified to storage triglyceride (TAG). Abnormal upregulation of DNL is a vital contributor to increased fat mass in the pathogenesis of various metabolic disorders involving non-alcoholic fatty liver disease and diabetes and in the progression of tumors. DNL is known to be transcriptionally regulated via sterol regulatory element-binding protein 1 (SREBP1) and carbohydrate-responsive element-binding protein (ChREBP) in response to metabolic and hormonal cues. However, it has been recently proposed that allosteric regulation and post-translational modifications (PTMs) are indispensable to control metabolic flux since the timescale of the gene expression is too long to balance the quick turnover of metabolites. Lipid anabolism is active during Drosophila larval development, and the excessive TAG storage in larvae is an essential reservoir for surviving from starvation in the post-feeding pupa stage of metamorphosis. The fatty acids stockpiled in TAG are either derived from the diet or DNL. Regulated by hormonal and transcriptional programs, the mRNA expressions of multiple enzymes in DNL pathways, including acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN), correlate with the dynamic changes in TAG levels in fly embryonic and larval development. However, whether PTMs are involved in the regulation of developmental DNL is poorly studied (Miao, 2022).

Lysine acetylation has recently risen as a novel player that links metabolites [e.g., acetyl coenzyme A (acetyl-CoA)], cell signaling, and gene regulation. Previous acetylome studies found that almost all metabolic enzymes are acetylated, including FASN . FASN, an essential cytosolic enzyme in DNL pathway, catalyzes the biosynthesis of saturated fatty acids from acetyl-CoA (AcCoA) and malonyl coenzyme A (malonyl-CoA). Recently, FASN has emerged as a novel therapeutic target for the treatment of obesity, diabetes, fatty liver diseases, and cancers. Although FASN is known to be regulated through SREBP1-mediated transcriptional activation, several conflicting results show little correlation between FASN expression and its enzymatic activity. These findings suggest a possible involvement of PTMs in the regulation of FASN function. Phosphorylation and acetylation have been proposed as alternative mechanisms of FASN regulation. Nevertheless, how PTMs regulate FASN activity and lipogenesis remains largely unknown (Miao, 2022).

Although protein acetylation is mainly catalyzed by lysine acetyltransferases (KATs), it has been recently reported that acetylation also arises from a nonenzymatic reaction with AcCoA in eukaryotes. AcCoA is the acetyl donor for protein acetylation and is a reactive metabolic intermediate involved in various metabolic pathways. The levels of AcCoA fluctuate in response to both intracellular and extracellular cues (e.g., growth signals and nutrient conditions), which consequently impacts chromatin modifications and transcriptional reprogramming. Previously, it was thought that nonenzymatic acetylation only occurs to mitochondria proteins, as high concentrations of AcCoA and alkaline environment inside mitochondrial matrix favor lysine nucleophilic attack on the carbonyl carbon of AcCoA. In recent years, the capability of enzyme-independent acetylation of cytosolic proteins was also determined. Yet, the mechanism of nonenzymatic acetylation, especially of cytosolic proteins, remains elusive (Miao, 2022).

This study shows that the expression of Drosophila FASN (dFASN or FASN1) protein remains unchanged during larval development, the stages when lipogenesis is hyperactive. In contrast, acetylation of dFASN at K813 is significantly induced in response to increased cellular AcCoA levels, which elevates dFASN enzymatic activity and lipogenesis in fast-growing larvae. Strikingly, acetylation of K813 is controlled through a unique KAT-independent mechanism that involves a novel motif "N-xx-G-x-A." In summary, these findings uncover a novel AcCoA-mediated self-regulatory module that regulates developmental lipogenesis via autoacetylation of dFASN (Miao, 2022).

Metabolic homeostasis plays an important role in animal development and growth. One novel mechanism underlying the coordination of metabolic homeostasis and growth is the interplay between metabolic intermediates and PTMs. This study uncover a novel role of AcCoA-mediated autoacetylation of dFASN in lipogenesis during Drosophila larval development. On the one hand, AcCoA fuels dFASN as the carbon donor for the growing fatty acid chain. On the other hand, AcCoA, as the acetyl-group donor, directly modulates dFASN enzymatic activity through acetylation of the critical lysine residue K813. Surprisingly it was found that acetylation of dFASN does not require KATs; instead, it is mediated by a conserved P-loop-like motif N-xx-G-x-A neighboring K813. Lastly, Sirt1 was identied as the primary deacetylase for dFASN, which acts as a negative regulatory mechanism (Miao, 2022).

TAG levels are tightly controlled during Drosophila development, and the massive buildup of TAG storage is a feature of larval growth. TAG synthesis is catalyzed by isoenzymes and competes with pathways that consume fatty acids, such as fatty acid oxidation and membrane lipid synthesis. During the entire embryonic and larval development, the mRNA expressions of multiple lipogenic enzymes, including FASN, correlate with TAG levels. Moreover, flies with mutations in FASN1 and FASN2 store less TAG in both larval and adult stages of Drosophila, suggesting that DNL is a vital contributor to TAG storage throughout development (Miao, 2022).

Under conditions like excess nutrition, growth factor stimulation, obesity, diabetes, fatty liver diseases, or cancer, DNL is significantly elevated, and the mRNA expression of FASN positively correlates with elevated lipogenesis. However, the protein levels of FASN are rarely characterized. Interestingly, several conflicting results show little correlation between FASN protein expression and its enzymatic activity. Consistently, it was found that the protein levels of dFASN remain unchanged from L2 to L3 and do not match the pattern of its enzymatic activity. In contrast, acetylation of dFASN at lysine K813 is positively associated with dFASN activity, developmental lipogenesis, and TAG accumulation. However, although dFASN level is not upregulated with elevated lipogenesis and TAG accumulation at L3 larvae, it correlates with TAG levels when the entire embryonic and larval development stages are considered. These findings suggest that despite the well-established transcriptional program controlling the expression of dFASN at different stages of development, lysine acetylation of dFASN plays a crucial role in accelerating dFASN activity and fine-tuning dFASN-mediated lipogenesis in fast-growing L3 animals (Miao, 2022).

Indeed, genetic and biochemical analysis further demonstrates that K813R substitution reduces dFASN enzymatic activity and lipogenesis, while AcCoA-mediated acetylation of recombinant dFASN proteins increases its enzyme activity. Although acetylation of FASN has been reported in several previous global acetylome studies, the functional roles of FASN acetylation remain largely unknown. A recent study investigated the role of FASN acetylation in DNL in human cell culture. The study shows that treatment of KDAC inhibitors induces the acetylation of hFASN, promotes FASN degradation, and reduces lipogenesis. However, it remains to be determined whether the regulation of lipogenesis by KDAC inhibition is due to global acetylation, or if it is directly through FASN acetylation. In addition, the functional lysine residues of hFASN that are responsible for altered lipogenesis are not identified. Because of the high conservation between K813 of dFASN and K673 of hFASN, it is possible that K673 is the key lysine residue mediating DNL in human (Miao, 2022).

Apart from K813, other three lysine residues of dFASN (K926, K1800, and K2466) are highly conserved among animal species, and their homologs are also found to be acetylated in other animal species. Since these lysine residues are located on different domains, it is not hard to imagine that acetylation of each lysine may play distinct roles related to their associated domains. The present study shows that acetylation of K813, but not K926, modulates dFASN activity, body fat accumulation, and Drosophila developmental timing. It is likely that acetylation of K926 affects other aspects of enzyme properties that are less important for larval development. K813 is at the substrate docking pocket of the MAT domain. This unique localization suggests that acetylation of K813 might introduce conformational changes to the docking site and modulate dFASN catalytic activity in response to substrate availability during larval development (Miao, 2022).

Another surprising finding from this study is that acetylation of dFASN at K813 does not require a KAT; rather, it is autoacetylated by AcCoA in a dosage-dependent manner. The cytosolic pool of AcCoA increases under feeding or excess nutrient conditions. Consistently, these studies reveal that the amount of AcCoA elevates in fast-growing larvae, which could modulate dFASN activity by promoting both the biosynthesis of MalCoA and autoacetylation of K813 for the conformational changes of MalCoA docking pocket (Miao, 2022).

It was previously thought that only mitochondria proteins were nonenzymatically acetylated since no KATs have been identified in mitochondria. Besides, the high AcCoA concentration and relatively high pH of the mitochondrial matrix facilitate the lysine nucleophilic attack on the carbonyl carbon of AcCoA. Recently, KAT-independent acetylation of cytosolic proteins has been reported. Yet, the underlying mechanism for nonenzymatic acetylation remains largely unknown. When investigating how dFASN is autoacetylated by AcCoA, this study uncovered a novel motif N-xx-G-x-A near acetylated K813. Substituting any of the three key amino acids largely blocks AcCoA-mediated dFASN autoacetylation. The N-xx-G-x-A motif resembles the signature P-loop sequence (Q/R-xx-G-x-A/G) of KATs, which is required for AcCoA recognition and binding. It is predicted that the N-xx-G-x-A motif of dFASN performs a similar function as the P-loop of KATs for AcCoA binding. Moreover, the N-xx-G-x-A motif is highly conserved, pointing out a conserved mechanism for autoacetylation of FASN (Miao, 2022).

In addition to the well-established KATs of the MYST, p300/CBP, and GCN5 families, there are over 15 proteins that have been reported to possess KATs activity, such as CLOCK and Eco1. Since FASN may contain an AcCoA binding motif of KATs, it is possible that FASN, particularly MAT domain, possesses KATs activity and acetylates other proteins, especially those in DNL pathways. This possibility may be further explored through acetylome analysis in the future. In summary, this study uncovered a previously unappreciated role of FASN acetylation in developmental lipogenesis and a novel mechanism for lysine autoacetylation. These findings provide new insights into AcCoA-mediated metabolic homeostasis during animal development. In addition, rhwaw studies underscore a promising therapeutic strategy to combat metabolic disorders by targeting autoacetylation of FASN (Miao, 2022).

GENE STRUCTURE

cDNA clone length - 3782

Bases in 5' UTR - 396

Exons - 2

Bases in 3' UTR - 913

PROTEIN STRUCTURE

Amino Acids - 823

Structural Domains

An alignment of the Sir2 proteins from yeast, Drosophila, and human reveals remarkable conservation within the catalytic core of the enzyme, including the regions that encode the histone deacetylase and ADP-ribosylase functions of the yeast protein (Tanny, 1999; Imai, 2000; Landry, 2000). To test Sir2 for histone deacetylase activity, the ability of recombinant Sir2 to deacetylate labeled histone peptides in vitro was examined (Bedalov, 2001). Sir2 exhibits NAD⁺-dependent histone deacetylase activity. A small molecule inhibitor of ySir2, Splitomicin, has recently been shown to fully inhibit ySIR2 activity in vivo and recombinant ySIR2 by roughly 20%. Splitomicin also inhibits recombinant Drosophila Sir2 activity, to levels comparable to that of recombinant ySIR2 (20% inhibition; Bedalov, 2001). Together, these data suggest that Sir2 shares considerable functional homology with its yeast and human counterparts within this region (Rosenberg, 2002).

The predicted amino acid sequence of Sir2 is 823 aa in length, making it the longest known Sir2 homolog. The core domain, which encodes the deacetylase activity, is the only region of Sir2 that is conserved with ySir2. Bacterially expressed Sir2 has an intrinsic NAD⁺-dependent deacetylase activity. Outside the core domain, there is no homology to any known proteins, making it difficult to speculate on the function of these domains. Within the Drosophila genome, five genes contain significant homology to the yeast Sir2. Of the five genes, Sir2 is the most similar to yeast Sir2. This is an important fact when comparing mutant phenotypes across phylogenetic lines (Newman, 2002).

date revised: 20 August 2023

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