InteractiveFly: GeneBrief

G9a: Biological Overview | References

BIOLOGICAL OVERVIEW

Organisms have developed behavioral strategies to defend themselves from starvation stress. Despite of their importance in nature, the underlying mechanisms have been poorly understood. This study shows that Drosophila G9a (dG9a), one of the histone H3 Lys 9-specific histone methyltransferases, functions as a key regulator for the starvation-induced behaviors. RNA-sequencing analyses utilizing dG9a null mutant flies revealed that the expression of some genes relating to gustatory perception are regulated by dG9a under starvation conditions. Reverse transcription quantitative-PCR analyses showed that the expression of gustatory receptor genes for sensing sugar are up-regulated in starved dG9a null mutant. Consistent with this, proboscis extension reflex tests indicated that dG9a depletion increased the sensitivity to sucrose under starvation conditions. Furthermore, the locomotion activity was promoted in starved dG9a null mutant. It was also found that dG9a depletion downregulates the expression of insulin-like peptide genes that are required for the suppression of starvation-induced hyperactivity. Furthermore, refeeding of wild type flies after starvation conditions restores the hyperactivity and increased sensitivity to sucrose as well as dG9a expression level. These data suggest that dG9a functions as a key regulator for the decision of behavioral strategies under starvation conditions (Shimaji, 2017).

Behavioral epigenetics has attracted attentions for the last decade, because epigenetic regulation can induce rapid and long-lasting effects on gene expression in response to environmental changes. Although a numerous studies have revealed the biological roles of epigenetic factors over the last 40 years, how epigenetic regulation affects behaviors of organisms and how behaviors affect epigenetic regulation have just started to be investigated. Previous studies reported that several epigenetic factors are relevant to behaviors included in learning/memory, neurodevelopmental disorders, drug addiction, parenting and stress responses in mammals. Some of these relationships have also been found in Drosophila melanogaster, the model organism extensively used for genetic studies because of its short life span and the high homology to human genes. For example, some Drosophila epigenetic factors like Ash1 and Suppressor of variegation 4–20-homolog 1 are associated with autism spectrum disorders, one of the neurodevelopmental disorders characterized by the impaired communication, restricted interests and hyperactivity. Especially it is revealed that the dG9a, a Drosophila homolog of mammalian G9a functions as the key regulator of learning and memory through alteration of histone modification (Mis, 2006; Kramer, 2011; Shimaji, 2017 and references therein).

G9a has been identified in mammals as one of the histone H3 Lysine 9 (H3K9) specific methyltransferases (HMTases) that catalyzes both H3K9 mono-methylation and H3K9 di-methylation (Tachibana, 2001; 2002). G9a has various biological roles including DNA replication (Esteve, 2006; Nagano, 2008; Wagschal, 2008), developmental reprogramming (Myant, 2012) and substance addiction. Especially, G9a plays a critical role in embryogenesis, since G9a knockout mice show embryonic lethality in early stages due to severe growth defects. Generally, G9a functions to suppress expression of its target genes through H3K9 methylation, although from some reports it may act as a co-activator to positively regulate some genes such as targets of the hormone-activated glucocorticoid receptor. However, studies on in vivo functions of G9a utilizing the mouse model have not advanced efficiently because of the embryonic lethality of the G9a knockout mice. On the other hand, dG9a depletion exerted no effect on fly viability at all (Seum, 2007). Therefore, Drosophila melanogaster is suitable for the functional analyses of G9a in adults. dG9a can catalyze the methylation of H3K9 in euchromatin regions and the methylated H3K9 contributes to heterochromatin formation and transcriptional repression of specific genes in vivo. Although the dG9a depleted flies show no viability defect (Seum, 2007; Kato, 2008; Lee, 2010) previous reports utilizing dG9a knockdown flies or knockout flies revealed that dG9a has a regulatory role in the developmental process of germ cell line like spermatogenesis and oocyte specification (Lee, 2010; Ushijima, 2012) as well as in learning and memory (Kramer, 2011). In contrast to the mammalian G9a, dG9a was not essential for Drosophila viability. Because of this unexpected finding, one can consider that the epigenetic regulation through H3K9 methylation is not developed to be critical for Drosophila viability and therefore dG9a plays no important role for Drosophila viability under laboratory conditions. However, it was important to note that while there are various environmental stresses in the wild, Drosophila was always maintained under optimal conditions in the laboratory. Therefore, this study focused on the analyses under stressed conditions and recently revealed that dG9a has a critical role in acquisition of tolerance under starvation stress in adult stage through regulating the activity of autophagy (Shimaji, 2017).

In terms of behavioral changes under starvation stress, Drosophila developed two behavioral strategies to increase the possibility that they can find new food sources. Firstly, they increase responsiveness for a sugar taste by means of up-regulating the expression of Gustatory receptor (Gr) 64a, a well-known gustatory receptor for sensing sugar. Secondary, starvation stress induces hyperactivity through activating octopaminergic neurons whose functions can be regulated by glucagon and insulin signals. However, it remains totally unknown whether there is a key regulator for the decision of these starvation-induced behaviors in spite of its importance in nature (Shimaji, 2017).

In this study, RNA-sequencing (RNA-seq) analyses followed by gene ontology (GO) analyses revealed that the expression of genes encoding gustatory receptors and odorant binding proteins are altered in dG9a mutants under starvation conditions. Further genetic analyses revealed that dG9a depletion increases the expression levels of gustatory receptor genes for sensing sucrose. Behavioral analyses revealed that dG9a depletion up-regulates the sucrose sensitivity in response to starvation stress. These data suggest that dG9a regulates the starvation-induced shift of locomotion activity through controlling the expression of insulin-like peptide (Ilp) genes that are required for the suppression of starvation-induced hyperactivity. Refeeding of wild type flies after starvation conditions restored the hyperactivity and increased sensitivity to sucrose as well as dG9a expression level. These data suggest that dG9a functions as a key regulator for the decision of behavioral strategies under starvation conditions (Shimaji, 2017).

Prior to this study, dG9a null mutant flies were found to be sensitive to starvation and the underlying mechanism were explored. dG9a is functional for saving energy through recycling cellular components by regulating the expression of genes required for autophagy (An, 2017). In addition to this process, it was further found that dG9a functions as a suppressor of starvation-induced hyperactivity. This is also preferable for saving energy under starvation conditions. In nature, animals are exposed to starvation frequently, however, foraging require the costs of food-seeking energy as well as the threats from predation and environmental changes along with their migration. Therefore, this foraging strategy requires assumption that the nutrient-poor conditions do not last long. Moreover, the strategy appears to be not effective under the conditions that there is no food available near them. Another way to survive under starvation is saving energy without moving like the hibernation associated with seasonal fluctuations of food availability, which can be observed in a wide range of animals including Drosophila. The current data indicated that dG9a suppresses the starvation-induced hyperactivity and that wild type flies exhibit the hyperactivity along with reduction of dG9a expression. These data suggest that the wild type flies save energy without moving at the early phase of starvation and they become active to seek foods with risks along with the reduction of dG9a expression at the late phase of starvation. Therefore, these data suggest that dG9a functions as a key regulator for flies to decide these strategies depending on the time course of starvation and has an adaptive advantage to survive starvation conditions (Shimaji, 2017).

RNA-seq analyses was performed to identify which genes are regulated by dG9a under starvation stress. This analyses indicated that the most enriched term was 'innate immune response' and the second was 'response to bacterium', which suggests that there is strong relationship between dG9a and innate immune responses. In Drosophila, starved conditions and the following disruption of insulin signaling induce the expression of four antimicrobial peptides (AMPs), Metchnikowin (Mtk), Drosocin (Dro), Drosomycin (Drs) and Attacin-A (AttA). RNA-seq analyses detected the significant increase in the expression of all of these four antimicrobial genes under 12-h starved dG9a null mutant. The expression of these representative antibacterial genes by was examined by RT-qPCR using Canton S and dG9a^RG5 kept under same conditions. The expression of three representative genes, Mtk, Dro and Drs, showed similar expression pattern to that observed in the results of RNA-seq analyses. These results indicate that the expression pattern of antibacterial genes was not due to accidental infection of one set of the flies, but truly due to dG9a mutation. These observations suggest a link between activation of innate immune system and starvation. Previous reports suggested that the induction of AMPs may help maintaining and enhancing the defense activity in particular when organisms are exposed to poor conditions like starvation. The data indicates the possibility that dG9a null mutant acquires the excess defense activity to bacterial infection under starvation conditions (Shimaji, 2017).

This study revealed that the dG9a depletion increases taste sensitivity to sucrose under 24-h starved conditions. Our RT-qPCR results indicated that dG9a depletion significantly increased the expression levels of genes encoding gustatory receptors for sensing sugar taste under 24-h starved conditions. These results suggest that the sucrose sensitivity under 24-h starved conditions is dependent on the expression levels of gustatory receptor genes regulated by dG9a. However, the dG9a depletion does not increase sucrose sensitivity under earlier (8-h) starved conditions, although the significant increases were detected in gustatory receptor genes under 0-h and 6-h starved dG9a null mutant. Moreover, none of these genes in wild type flies are up-regulated under 12-h and 24-h starvation conditions in compared to non-starved conditions. These results suggest that the starvation-induced increase of the sucrose sensitivity in wild type flies as well as increase of the sucrose sensitivity under non-starved conditions are dependent on underlying mechanisms other than expression changes of gustatory receptor genes, including the alterations of translation levels of Gr mRNAs, localization of Gr mRNA/proteins and the responses of higher order gustatory circuits in the brain like the feeding behavior control by neuropeptides like dRYamides. Further analyses are required to clarify these mechanisms (Shimaji, 2017).

Epigenetic regulation of starvation-induced autophagy in Drosophila by histone methyltransferase G9a

Epigenetics is now emerging as a key regulation in response to various stresses. This study identified the Drosophila histone methyltransferase G9a (dG9a) as a key factor to acquire tolerance to starvation stress. The depletion of dG9a led to high sensitivity to starvation stress in adult flies, while its overexpression induced starvation stress resistance. The catalytic domain of dG9a was not required for starvation stress resistance. dG9a plays no apparent role in tolerance to other stresses including heat and oxidative stresses. Metabolomic approaches were applied to investigate global changes in the metabolome due to the loss of dG9a during starvation stress. The results obtained indicated that dG9a plays an important role in maintaining energy reservoirs including amino acid, trehalose, glycogen, and triacylglycerol levels during starvation. Further investigations on the underlying mechanisms showed that the depletion of dG9a repressed starvation-induced autophagy by controlling the expression level of Atg8a, a critical gene for the progression of autophagy, in a different manner to that in cancer cells. These results indicate a positive role for dG9a in starvation-induced autophagy (An, 2017).

Previous studies revealed that G9a is important for early embryogenesis and essential for viability in mice. G9a is also highly conserved among various metazoans including Drosophila, frogs (Xenopus tropicalis), fish (Danio rerio, Tetraodon nigroviridis, and Takifugu rubripes), and mammals. In Drosophila, although G9a is not essential for viability, the results of the present study suggest that G9a is conserved from the fly to mammals because of its importance in starvation stress tolerance, to which organisms are often exposed in the wild. This is also the first indication that epigenetic regulator-like G9a plays an essential role in the acquisition of starvation tolerance (An, 2017).

In order to clarify the underlying mechanisms by which the dG9a null mutant is more susceptible to starvation stress, 'bottom up' approaches have been used. Non-targeted GC-MS-based and targeted LC-MS/MS-based metabolic profiling was performed to investigate changes in the metabolome due to the loss of dG9a. The results obtained from metabolic profiles showed that dG9a played important roles in maintaining energy homeostasis, the key factor for nutrient stress tolerance. dG9a modulated energy reservoirs including amino acid, trehalose, glycogen, and TAG levels during starvation via the autophagic process. One of the unique features of the adult dG9a^RG5 mutant is its higher content of glycogen under non-starved normal conditions than that of the wild-type. A previous study reported that the deletion of G9a in mouse adipose tissues promotes adipogenesis and increases body weight (Wang, 2013). These findings and the present results suggest that dG9a is also responsible for the suppression of adipogenesis, similar to mammalian G9a. Further analyses are needed in order to clarify this point (An, 2017).

The results of the present study also indicated that dG9a controlled starvation-induced autophagy by activating the expression of Atg8a; however, dG9a generally represses gene expression by dimethylating H3K9. Previous studies reported that histone and non-histone protein methylation by G9a either activated or inhibited gene expression. This study also found that the catalytic activity of dG9a was not required for the acquisition of starvation stress resistance by dG9a. This is consistent with the results of immunostaining showing that H3K9me2 levels in the nuclei of fat body cells under starvation were not significantly affected by the loss of dG9a. G9a has also been reported to activate gene expression as a molecular scaffold for the assembly of transcriptional co-activators, and the catalytic domain of G9a is not required for this function (Bittencourt, 2012). Further studies are needed in order to clarify the mechanisms by which dG9a regulates the expression of Atg8a (An, 2017).

Similar Atg8a mRNA levels were observed after 6h of fasting between wild-type and dG9a^RG5 mutant flies; however, Atg8a immunostaining signals was weaker in the dG9a^RG5 mutant than in the wild-type . Therefore, the loss of dG9a may repress the expression of genes that control Atg8a protein stability. Further studies are needed in order to elucidate the underlying mechanisms. During the development of Drosophila, metamorphosis is also a process that flies use to tolerate starvation stress. Even though this study demonstrated that dG9a is important for starvation stress tolerance, the viability of the dG9a^RG5 mutant was not significantly less than that of the wild-type during the pupal stage. Together with the current results showing that the viability of the dG9a^RG5 mutant at the larval stage was not affected by fasting conditions, the function of dG9a for starvation stress appears to be specific to the adult stage. Since programmed autophagy during the 3^rd instar larval and pupal stages is well-known to be regulated by ecdysone through the PI3K pathway, starvation-induced autophagy by dG9a in the adult stage may be operated by other pathways (An, 2017).

G9a is suggested to play a positive role in the promotion of tumorigenesis in various human cancer cells such as prostate, leukemia, lung, breast, and aggressive ovarian carcinoma. The inhibition of G9a activity in cancer cells significantly inhibited cell proliferation by triggering cell cycle arrest, inducing apoptosis, or activating autophagic cell death. The novel results obtained in this study on the role of dG9a to acquire starvation tolerance may also make it possible to explain the positive role of G9a in the promotion of tumorigenesis. Cells inside a tumor mass are exposed to starvation conditions because nutrients are not fully supplied to these cells. In order to overcome starvation stress, autophagy is induced in these cells. Therefore, G9a may play a role in the acquisition of starvation tolerance in cells in the tumor mass. The present study found that the loss of dG9a led to the inactivation of starvation-induced autophagy due to a decrease in Atg8a levels. In contrast, a previous study on cancer cells showed that the loss of G9a during starvation activated the transcription of LC3B (the Atg8a ortholog in mammals) and triggered autophagy (Martinez de Narvajas, 2013). Taken together, these results suggest that the epigenetic gene regulation of G9a depends on cell/tissue types (An, 2017).

Epigenetic mechanisms modulate differences in Drosophila foraging behavior

Little is known about how genetic variation and epigenetic marks interact to shape differences in behavior. The foraging (for) gene regulates behavioral differences between the rover and sitter Drosophila melanogaster strains, but the molecular mechanisms through which it does so have remained elusive. This study shows that the epigenetic regulator G9a interacts with for to regulate strain-specific adult foraging behavior through allele-specific histone methylation of a for promoter (pr4). Rovers have higher pr4 H3K9me dimethylation, lower pr4 RNA expression, and higher foraging scores than sitters. The rover-sitter differences disappear in the presence of G9a null mutant alleles, showing that G9a is necessary for these differences. Furthermore, rover foraging scores can be phenocopied by transgenically reducing pr4 expression in sitters. This compelling evidence shows that genetic variation can interact with an epigenetic modifier to produce differences in gene expression, establishing a behavioral polymorphism in Drosophila (Anreiter, 2017).

Rovers and sitters have a natural difference in adult foraging behavior that is caused by differences in G9a-dependent expression of the for pr4 transcripts. pr4 is differentially methylated by G9a in rovers and sitters, and this study demonstrates that pr4 is solely responsible for the rover-sitter behavioral polymorphism in adult foraging behavior. Nevertheless, G9a is not the sole transcriptional regulator, or the sole H3K9 methyltransferase, regulating pr4 expression. The results show that the loss of G9a can result in more or less H3K9me2 at pr4, depending on the for allele present. This dual function of G9a has been previously shown in mice, where G9a is able to both repress and activate gene expression through interactions with other proteins in its regulatory complex. While pr4 is responsible for regulating the rover-sitter difference in adult foraging behavior, other for promoters likely regulate other for-related phenotypes. In fact, expression data show that other for promoters are differentially expressed in rovers and sitters. For example, pr2 is highly expressed in sitters and not expressed at all in rovers. The pr2 expression difference also correlates with H3K9me2, but cannot be explained solely by G9a. pr2 and pr4 transcribe different isoforms of for (P1 and P4, respectively) that might differ in function. These results suggest that the expression of for's four promoters might be regulated by distinct regulatory complexes, and that each promoter might influence distinct behavioral phenotypes (Anreiter, 2017).

The difference in pr4 expression is tissue-specific, being driven by the brain and ovaries. The central nervous system and ovaries might be linked in regulating feeding behavior, since reproduction constitutes the major energy expenditure of female flies, and sex peptide signaling in the reproductive organs affects the feeding behavior of female flies. This work highlights the complex epigenetic architecture that underlies behavioral regulation (Anreiter, 2017).

The lack of a DNA-binding domain suggests that G9a is targeted to specific DNA regions through interactions with DNA-binding proteins, such as transcription factors. SNPs in the promoter region could lead to differential binding of DNA-binding proteins that recruit G9a. For instance, the SNP in pr4 lies within a conserved site of a putative Mad binding motif, and potentially could affect Mad binding. If Mad is one of the elements in the G9a complex, then less binding of Mad in the rover strain (which would be predicted from the SNP) potentially could explain the lower pr4 H3K9me2 levels in rovers. Like G9a, Mad has been shown to act as both a repressor and an activator of gene transcription, depending on context. Although Mad is best known for its role in development, some studies suggest that it might have regulatory functions in the mature nervous system (Anreiter, 2017).

In conclusion, the mechanisms by which epigenetic regulation influences behavioral differences are poorly understood. Epigenetic regulation has been shown to be a mechanism through which animals adjust their behavior and physiology to the environment in which they live. Not all individuals respond similarly to the same environmental cue, however. In this case, epigenetic-by-genetic interaction would be an important but neglected component of gene-by-environment interactions. The deposition of epigenetic marks can depend on underlying genetic differences, and genetic variation likely plays an important role in moderating epigenetic differences between individuals. Importantly, epigenetic-by-genetic interactions present an avenue through which genetic variation outside of gene coding regions can modulate phenotypic variability. Two other noteworthy studies in humans and prairie voles have reported associations among genetic variation, DNA methylation, and behavior. This study used the fruit fly to establish molecular causality, and provide definitive evidence for how the complex interactions among genetics, epigenetics, and isoform-specific gene regulation causes variation in naturally occurring behavioral polymorphisms (Anreiter, 2017).

The epigenetic regulator g9a mediates tolerance to RNA virus infection in Drosophila

Little is known about the tolerance mechanisms that reduce the negative effects of microbial infection on host fitness. This study demonstrates that the histone H3 lysine 9 methyltransferase G9a regulates tolerance to virus infection by shaping the response of the evolutionary conserved Jak-Stat pathway in Drosophila. G9a-deficient mutants are more sensitive to RNA virus infection and succumb faster to infection than wild-type controls, which was associated with strongly increased Jak-Stat dependent responses, but not with major differences in viral load. Genetic experiments indicate that hyperactivated Jak-Stat responses are associated with early lethality in virus-infected flies. These results identify an essential epigenetic mechanism underlying tolerance to virus infection (Merkling, 2015).

Genomewide identification of target genes of histone methyltransferase dG9a during Drosophila embryogenesis

Post-translational modification of the histone plays important roles in epigenetic regulation of various biological processes. Among the identified histone methyltransferases (HMTases), G9a is a histone H3 Lys 9 (H3K9)-specific example active in euchromatic regions. Drosophila G9a (dG9a) has been reported to feature H3K9 dimethylation activity in vivo. This study shows that the time required for hatching of a homozygous dG9a null mutant and heteroallelic combination of dG9a null mutants is delayed, suggesting that dG9a is at least partially responsible for progression of embryogenesis. Immunocytochemical analyses of the wild-type and the dG9a null mutant flies indicated that dG9a localizes in cytoplasm up to nuclear division cycle 7 where it is likely responsible for di-methylation of nucleosome-free H3K9. From cycles 8-11, dG9a moves into the nucleus and is responsible for di-methylating H3K9 in nucleosomes. RNA-sequence analysis utilizing early wild-type and dG9a mutant embryos showed that dG9a down-regulates expression of genes responsible for embryogenesis. RNA fluorescent in situ hybridization analysis further showed temporal and spatial expression patterns of these mRNAs did not significantly change in the dG9a mutant. These results indicate that dG9a controls transcription levels of some zygotic genes without changing temporal and spatial expression patterns of the transcripts of these genes (Shimaji, 2015).

This study shows that the time required for hatching is extended by the lack of dG9a. Immunocytochemical studies suggested that cytoplasmic dG9a is responsible for di-methylation of nucleosome-free H3K9 in cytoplasm up to nuclear division cycle 7, whereas from cycle 8 to cycle 11, it moves into nucleus and di-methylates H3K9 in the nucleosome. Across these cycles, dG9a could be responsible for regulating the expression level of many genes required for embryogenesis and transcription (Shimaji, 2015).

Human G9a localizes in nuclei of HEK293 cells and is also reported to be present in nuclei in salivary glands and in cultured Drosophila Kc cells. However, this study found that dG9a almost exclusively localizes in cytoplasm of early embryos up to nuclear division cycle 7 and is likely for a determinant of H3K9me2 levels in cytoplasm. To support rapid progression of nuclear division cycles in Drosophila, large amounts of DNA replication enzymes are maternally stored, along with histone mRNAs and histone proteins. Therefore, the available results indicate that dG9a methylates nucleosome-free histone H3K9 stored in cytoplasm during early stages of nuclear division cycles. It should be noted that dG9a can di-methylate histone H3K9 that is free from nucleosomes in vitro (Stabell, 2006). Some of the di-methylated H3K9 may be selectively transported into nuclei from cycle 8, although further analysis is necessary to clarify this point (Shimaji, 2015).

In later stages, dG9a signals rather accumulate in nuclei from nuclear division cycles 8 to 12 and appear to be responsible for di-methylation of nuclear H3K9. Nuclear localization signals that are distinct from those of mammalian G9a in their positions and amino acid sequences may be responsible for the nuclear localization of dG9a during cycle 8 (Kato, 2011). It is well known that a first wave of ZGA occurs around cycle 8 and activates genes required for cellularization. Nuclear localized dG9a may confer specificity to the zygotic gene transcription by di-methylating H3K9 at some specific genomic regions during these stages (Shimaji, 2015).

In embryos at nuclear division cycle 13 and at cellular blastoderm stage, dG9a mainly localizes in cytoplasm although its accumulation in some nuclei was observed. Furthermore, dG9a appears to be not responsible for H3K9me2 in either cytoplasm or nuclei from cycle 12. Drosophila has three HMTases specific to H3K9: SU(VAR)3-9, DmSETDB1 and dG9a. SU(VAR)3-9 is reported to catalyze H3K9me2 at the chromocenter and tri-methylation of H3K9 (H3K9me3) at the core of the chromocenter, but is not functional for mono-methylation, either at the chromocenter or along chromosome arms, nor for di-methylation at chromosome arms and telomeres of the salivary gland polytene chromosomes. DmSETDB1 catalyzes H3K9me2 and H3K9me3 on chromosome 4, and dG9a acts for H3K9me1 and H3K9me2 in euchromatic regions of the polytene chromosomes (Kato, 2008; Lee, 2010). However, despite dG9a is localized in nuclei, it appears to be not responsible for methylation of H3K9 in elongation stages of Drosophila spermatogenesis. In contrast, DmSETDB1 is responsible for H3K9me1 and H3K9me3 and SU(VAR)3-9 for H3K9me1 in these stages. RNA-sequence transcriptome profiles collected at 2-h intervals for embryogenesis of Drosophila showed that expression of dG9a reaches a peak in 0–2 h embryos, although those of DmSETDB1 and Su(var)3-9 are at 2–4 and 4–6 h, respectively. Therefore, in cycle 13 and cellular blastoderm stages, SU(VAR)3-9 and DmSETDB1 instead of dG9a may be responsible for H3K9 methylation. It has in fact been reported that SU(VAR)3-9 functions prominently during cellular blastoderm stages. However, to address contribution of each of these three H3K9-specific HMTases during early embryogenesis, further analyses with double or triple mutants would be necessary (Shimaji, 2015).

This study determined expression of genes affected by the lack of dG9a in embryogenesis. Previous research focused on differentially expressed genes during the larval stage in dG9a^DD1, most likely a dG9a null mutant, by microarray analysis (Kramer, 2011). Among the genes differentially expressed 2.5-fold or more in dG9a^DD1 larvae, no example was detected that was differentially expressed in dG9a^RG5 embryos.The expression of dG9a in whole body shows its peak during 0–2 h embryo and reduces to approximately 10% by the larval stages (Graveley, 2011). In contrast, expression of other H3K9-specific methyltransferases such as DmSETDB1 and Su(var)3-9 increases by the larval stages. These results suggest that the target genes of dG9a vary greatly between embryonic and larval stages, although the possibility cannot be excluded that the difference may be caused by allelic difference (Shimaji, 2015).

Gene ontology analyses in the present study indicate that up-regulated genes in dG9a^RG5 are highly involved in embryogenesis and transcription, in line with the conclusion that dG9a regulates expression of zygotically activated genes from cycle 8 which are important for processes like cellularization. However, RNA FISH analysis showed that spatial and temporal expression patterns of mRNAs of representative up-regulated genes in dG9a-depleted embryos were not significantly changed. Maternal depletion of Zelda, a key activator of the early ZGA, changes expression patterns of genes required for cellular blastoderm formation and causes lethal phenotype during embryogenesis. In contrast, dG9a regulates the amount of expression of specific genes without affecting spatial and temporal expression patterns of their mRNAs, which may be the reason why depletion of dG9a does not affect viability during embryogenesis. Among up-regulated genes in dG9a-depleted embryos, there are four genes, ci, glass bottom boat, patched and wntD, involved in Hedgehog signaling pathway. Among these genes, patched is a negative regulator of Wnt signaling pathway and wntD is a target and also an inhibitor of the Dorsal/Twist/Snail network that functions for ventral cell invagination. In the dG9a null mutant, over-expression of such negative regulators may be responsible for delay of embryogenesis, although further analyses are necessary to more precisely address molecular mechanisms (Shimaji, 2015).

Epigenetic regulation of learning and memory by Drosophila EHMT/G9a

The epigenetic modification of chromatin structure and its effect on complex neuronal processes like learning and memory is an emerging field in neuroscience. However, little is known about the 'writers' of the neuronal epigenome and how they lay down the basis for proper cognition. This study has dissected the neuronal function of the Drosophila euchromatin histone methyltransferase (EHMT; G9a), a member of a conserved protein family that methylates histone 3 at lysine 9 (H3K9). EHMT is widely expressed in the nervous system and other tissues, yet EHMT mutant flies are viable. Neurodevelopmental and behavioral analyses identified EHMT as a regulator of peripheral dendrite development, larval locomotor behavior, non-associative learning, and courtship memory. The requirement for EHMT in memory was mapped to 7B-Gal4 positive cells, which are, in adult brains, predominantly mushroom body neurons. Moreover, memory was restored by EHMT re-expression during adulthood, indicating that cognitive defects are reversible in EHMT mutants. To uncover the underlying molecular mechanisms, genome-wide H3K9 dimethylation profiles were generated by ChIP-seq. Loss of H3K9 dimethylation in EHMT mutants occurs at 5% of the euchromatic genome and is enriched at the 5' and 3' ends of distinct classes of genes that control neuronal and behavioral processes that are corrupted in EHMT mutants. This study identifies Drosophila EHMT as a key regulator of cognition that orchestrates an epigenetic program featuring classic learning and memory genes. Our findings are relevant to the pathophysiological mechanisms underlying Kleefstra Syndrome, a severe form of intellectual disability caused by mutations in human EHMT1, and have potential therapeutic implications. Our work thus provides novel insights into the epigenetic control of cognition in health and disease (Kramer, 2011).

This study demonstrates that Drosophila EHMT, a histone methyltransferase, regulates sensory dendrite development, larval locomotory behavior, simple learning (habituation), and complex memory (courtship conditioning). Notably, EHMT mutants are viable, appear healthy, and many other aspects of neuronal development and function are normal, highlighting the selectivity with which EHMT regulates specific aspects of neuronal development and function. Genome-wide molecular analysis of EHMT mutant flies supports this idea, revealing altered histone methylation at target loci encompassing a selection of neuronal genes that control learning, memory, and other phenotype-relevant processes (Kramer, 2011).

The EHMTs are an evolutionarily conserved family of proteins that regulate H3K9 methylation at euchromatic DNA (Tachibana, 2006; Tachibana, 2007; Stabell, 2006; Schaefer, 2009). Previous studies have shown that EHMTs affect transcription through H3K9 dimethylation in the promoters of certain genes. This study provides the first genome-wide overview of EHMT function with respect to its role in post-translational histone modifications. Evidence is provided that Drosophila EHMT induces H3K9 dimethylation at a proportion (about 5%) of the euchromatic genome, with a preference for discrete regions at the 5' and 3' ends of genes. Genes with differential H3K9me2 levels at the 5' end (within 1 kb upstream of the transcriptional start site) are predominantly involved in biological processes related to stress response (e.g. heat shock response and actin cytoskeleton remodeling), which require rapid and frequent changes in transcription. This observation is consistent with studies in yeast and humans, which show that chromatin structure immediately upstream of transcriptional start sites directly correlates with transcriptional plasticity. In contrast, genes that are differentially methylated at the 3' end are highly enriched in genes that control neuronal processes that are disrupted in EHMT mutants. The general view is that gene expression is regulated through interactions at the promoter, or 5' end. However, recent studies have revealed that 3' gene ends also play an important and complex role in the regulation of transcription by: (1) mediating gene looping, which is necessary for transcriptional memory, i.e. the altered transcriptional responsiveness of genes after a previous cycle of activation and repression; (2) serving as an initiation site for antisense transcripts; and (3) regulating transcript termination, a process that also affects transcript levels. Currently there is no evidence linking H3K9me2 to any of these processes, however it is conceivable that differential histone methylation at the 3' end of neuronal genes may act as a mechanism to control their expression. In line with this idea a recent study has reported that the DNA methyltransferase, Dnmt3a, also targets neuronal genes in 'non-promoter' regions, including 3' ends. Thus, it appears that epigenetic alterations to non-promoter regions is emerging as a general theme for the regulation of neuronal gene expression (Kramer, 2011).

EHMT mutants show a decrease in dendrite branching in sensory neurons of the Drosophila peripheral nervous system. Type 4 md neurons are known to provide the sensory input that they receive via their dendrites as an essential functional component to the neuronal circuitry governing larval movement. The current analysis of larval locomotion in EHMT mutants revealed a behavioral phenotype characterized by an increased performance of stops, retractions, and turns. It has been reported that such a phenotype can directly arise from dysfunction of type 4 md neurons, which raised the possibility that decreased dendrite branching and altered locomotory behavior are connected traits. Re-expression of EHMT in type-4 md neurons did, however, not rescue larval locomotion defects, suggesting that larval locomotion and type 4 md neuron development are controlled independently by EHMT. Thus, this lack of rescue may be due to requirements for EHMT in additional peripheral or central neurons relevant to the crawling pattern. Unspecific secondary effects or that precise levels of re-expressed EHMT may be crucial for turning behavior cannot be excluded. Ultimately, the relevance of EHMT in both dendrite development and crawling is illustrated by the observation that EHMT mutants show loss of H3K9me2 at 65 of 147 genes annotated to be involved in dendrite development and 15 of 16 genes involved in larval locomotory behavior (Kramer, 2011).

This study has shown that EHMT is required for light-off jump reflex habituation, a simple form of non-associative learning that is known to require classic learning and memory genes such as rutabaga. In this paradigm a sequence of leg extension and flight initiation is induced by sudden darkness. This behavioral response is mediated by the giant fiber interneurons, which receive sensory input from the visual system in the brain and relay this information through the thoracic ganglion where efferent neurons descending from the giant fiber to thoracic muscles are stimulated. Only a few genes are known to control jump reflex habituation and most of these are ion channels, or are involved in cAMP and cGMP second messenger signaling pathways. EHMT is the first histone modifying enzyme to be implicated in this simple form of learning. Jump-reflex habituation is not an official gene ontology term, but significantly, seven of the eight genes known to be involved in jump-reflex habituation show loss of H3K9 dimethylation in EHMT mutants (Kramer, 2011).

This study has also identified a role for EHMT in courtship memory. This is a complex form of memory that allows male flies to discriminate between receptive and non-receptive females, presumably to optimize the energy that they spend on courtship. Loss of EHMT leads to impaired short- and long-term memory while the learning capacity of the EHMT mutants was unaffected. Moreover, this study shows that normal courtship memory is restored upon re-expression of EHMT in the whole nervous system and in a subset of neurons labeled by 7B-Gal4, which is predominantly expressed in the mushroom body neurons of the adult brain. Although further work is required to map the specific circuits required for EHMT-dependent courtship memory, the mushroom body is known to be crucial for courtship memory, but not learning, pointing towards a deficit in this area of the brain. Significantly, EHMT affects histone methylation in 22 of 36 genes that were annotated at the time of this analysis to be involved in memory . Other relevant memory genes, such as Orb2 , nemy, and ben, that were not yet included in gene ontology databases are also affected by loss of EHMT. Together, these data suggest that EHMT targets two-thirds of all currently known memory genes (Kramer, 2011).

Importantly, it was possible to fully restore memory deficits by re-expression of EHMT during adulthood. Thus, although EHMT can affect neuronal hardwiring (dendrite development in the peripheral nervous system), it appears that adult cognitive defects do not arise from neurodevelopmental defects occurring prior to eclosion. This is consistent with a recently reported impairment in fear conditioning that has been observed in mice with postnatal loss of Ehmt1 in the brain and with the current observation that mushroom body morphology appears unaffected in EHMT mutant flies. Thus, EHMT-mediated H3K9 dimethylation of specific loci is required in adult post-mitotic neurons to consolidate or retrieve consolidated memories. Interestingly, other epigenetic regulators, such as the DNA methyltransferases Dnmt1 and Dnmt3a, are also required in post-mitotic neurons for normal memory. These studies support the idea that the process of learning induces reprogramming of the neuronal epigenome, which crucially underlies memory. Such 'stable' chromatin modifications, including DNA and histone methylation, appear to be good candidates for 'writing' long-term memory, however these marks must also remain dynamic allowing for memories to be modified. Understanding of this stable versus dynamic state of epigenetics in neurons and its consequences are highly limited. It will thus be important to dissect the extent of epigenetic plasticity during the different phases of learning, memory consolidation, and memory retrieval, and to determine how these alterations to the epigenetic landscape translate into transcriptional changes required for information processing and storage (Kramer, 2011).

A recent study of mRNA levels in mice with brain region-specific loss of Ehmt1 has identified 56 genes that are consistently misregulated in the mutant mouse brain (Schaefer, 2009). Of these 56 genes, 18 are non-neuronal, which led to the interpretation that EHMT proteins control cognition through repression of non-neuronal genes in neuronal tissues. In contrast to this view, the current data show that Drosophila EHMT mediates H3K9 dimethylation at more than 350 neuronal gene loci with proven critical roles in nervous system development and function. Does this apparent discrepancy reflect evolutionary differences? Of the 56 differentially expressed genes identified by Schaefer (2010), 30 are conserved in flies and 20 show loss of H3K9me2 in EHMT mutants. This correlation is very unlikely to occur by chance, suggesting that EHMT target genes are, at least in part, evolutionarily conserved. The great number of highly enriched neuronal genes amongst Drosophila EHMT targets, their striking match with EHMT mutant phenotypes, and the reversibility of cognitive defects argue that EHMT orchestrates an epigenetic program that directly regulates a battery of neuronal players underlying the molecular basis of cognition. It is also noteworthy that EHMT targets include fly orthologs of NF1, FMR1, FMR2, CNTNAP2, GDI, DLG3, and of many more genes underlying syndromic and non-syndromic forms of intellectual disability. Also, the major signaling pathways known to underlie intellectual disability, Rho and Ras GTPase pathways, are highly enriched in the ontology analysis (GO term: small GTPase mediated signal transduction) carried out for this study (Kramer, 2011).

This study complements a number of reports on post-embryonic rescue of cognitive phenotypes in disease models of intensively studied disorders such as Fragile X syndrome, Neurofibromatosis I, Tuberous sclerosis, Rubinstein-Taybi, Angelman, and Rett syndrome. The growing number of such examples provides an argument for reappraisal of the traditional view that genetic forms of intellectual disability are largely due to irreversible neurodevelopmental defects, findings which open prospects for therapeutic intervention. Currently, clinical trials are underway to treat Fragile X patients with compounds that have initially been identified to rescue phenotypes in fly models of Fragile X syndrome. The EHMT mutant fly has provided novel insights into the epigenetic regulation of cognition and will be a valuable tool to work further towards such translational approaches. Furthermore, a better understanding of the epigenetic mechanisms regulating cognitive processes is relevant to the wider medical community, considering the increased awareness of the epigenetic contributions to neurodevelopmental and psychiatric disorders in general (Kramer, 2011).

Identification of nuclear localization signals of Drosophila G9a histone H3 methyltransferase

G9a is one of the well-characterized histone methyltransferases. G9a regulates H3K9 mono- and dimethylation at euchromatic region and consequently plays important roles in euchromatic gene regulation. Mammalian G9a contains several distinct domains, such as GHD (G9a homology domain), ANK, preSET, SET and PostSET. These domains are highly conserved between mammals and Drosophila. Although mammalian G9a has nuclear localization signal (NLS) in its N-terminal region, the amino acid sequences of this region are not conserved in Drosophila. This study examined the subcellular localization of a series of truncated forms of Drosophila G9a (dG9a). The identified region (aa337-aa470) responsible for nuclear localization of dG9a contains four short stretches of positively charged basic amino acids (NLS1, aa334-aa345; NLS2, aa366-aa378; NLS3, aa407-aa419; NLS4, aa461-aa472). Each of NLS1, NLS3 and NLS4 is sufficient for the nuclear localization when they are fused with the enhanced green fluorescent protein. These NLSs of dG9a are distinct from those of mammalian G9a in their positions and amino acid sequences (Kato, 2011).

Drosophila G9a is implicated in germ cell development

In Drosophila ovaries, germline stem cells (GSCs) divide asymmetrically in the germaria to produce daughter GSCs and cystoblasts. Single cystoblasts differentiate to form germline cysts with 16 germline cells, all of which are connected by the fusome, a vesiculated structure critical for oocyte specification. This study shows that histone H3K9 methyltransferase G9a is associated with spectrosome/fusome formation in the germarium; dG9^a13414 mutant ovaries have disorganized spectrosome/fusome in about half the germaria, with reduced levels of Hu-li tai shao and alpha-Spectrin proteins. The amount of germline cells within cysts was reduced and oocyte determination often failed in egg chambers of the dG9^a13414 mutant ovaries. These results suggest that a mutation in G9a gene gives rise to anomalous spectrosome/fusome structures, which in turn lead to faulty germ-cell development in Drosophila ovaries (Lee, 2010).

Characterization of Drosophila G9a in vivo and identification of genetic interactants

In mammals, G9a is a histone H3 lysine 9 (H3-K9)-specific histone methyltransferase (HMTase), known to be essential for murine embryogenesis. It has been reported that Drosophila G9a (dG9a) is a dominant suppressor of position effects of variegation, has HMTase activity in vitro, and is important for Drosophila development. This study shows that dG9a has H3-K9 dimethylation activity in vivo and is important for the recruitment of HP1 in the euchromatic region. Over-expression in eye imaginal discs inhibited the differentiation of pupal ommatidial cells and resulted in abnormal eye morphology (rough eye phenotype) in the adults, although a methylase defective mutant did not demonstrate such effects. These results suggest that HMTase activity of dG9a affects transcription of genes involved in pupal eye formation. The dG9a-induced rough eye phenotype was enhanced by a half-dose reduction of the Polycomb group (PcG) gene. In contrast, mutants for little imaginal discs (lid), encoding histone H3-K4 demethylase, demonstrated suppression of the rough eye phenotype induced by dG9a. Furthermore co-expression of Lid in eye imaginal discs enhanced the rough phenotype induced by dG9a. The results suggest that the function of dG9a is negatively regulated by the PcG complex and positively regulated by Lid in vivo (Kato, 2008).

Identification of three histone methyltransferases in Drosophila: dG9a is a suppressor of PEV and is required for gene silencing

Organization of chromatin structure and regulation of gene transcription are contingent on histone tail modifications. Regions of the genome packaged with nucleosomes that contain methyl histone H3 at lysine 9 (Me K9H3) strongly correlate with regions that are silenced for transcription. To date Su(var)3-9 is the only K9H3 specific enzyme characterized in Drosophila melanogaster. This study describes the identification of three additional Drosophila genes that potentially encode K9H3 specific methyltransferases (HMTase) with homology to known mammalian proteins. By several criteria, including sequence alignments, phylogenic analyses, and enzyme activity of the protein, one of these is a homologue of the human G9a and hence it was named dG9a. dG9a catalyzes the transfer of methyl groups to full-length histone H3 and to N-terminal H3 peptides that contain lysine 9, suggesting that the major target for dG9a is K9H3. Chromatin extracts prepared from a P-element insert mutation in dG9a display an altered K9H3 methylation profile. In addition, the dG9a mutant is a dominant suppressor of position-effect variegation (PEV), a heterochromatin-associated gene silencing phenomenon. Su(var)3-9 also suppresses PEV. The combined Su(var)3-9 and dG9a mutations have severe developmental defects suggesting an overlapping role for dG9a and Su(var)3-9 in the packaging of heterochromatin and gene silencing via a K9H3 methylation pathway (Mis, 2006).

The Drosophila G9a gene encodes a multi-catalytic histone methyltransferase required for normal development

Mammalian G9a is a histone H3 Lys-9 (H3-K9) methyltransferase localized in euchromatin and acts as a co-regulator for specific transcription factors. G9a is required for proper development in mammals as g9a-/g9a- mice show growth retardation and early lethality. This study describes the cloning, the biochemical and genetical analyses of the Drosophila homolog dG9a. dG9a shares the structural organization of mammalian G9a, and it is a multi-catalytic histone methyltransferase with specificity not only for lysines 9 and 27 on H3 but also for H4. Surprisingly, it is not the H4-K20 residue that is the target for this methylation. Spatiotemporal expression analyses reveal that dG9a is abundantly expressed in the gonads of both sexes, with no detectable expression in gonadectomized adults. In addition a low but clearly observable level of dG9a transcript was observen in developing embryos, larvae and pupae. Genetic and RNAi experiments reveal that dG9a is involved in ecdysone regulatory pathways (Stabell, 2006).

Jmjd2c/Kdm4c facilitates the assembly of essential enhancer-protein complexes at the onset of embryonic stem cell differentiation

Jmjd2/Kdm4 H3K9-demethylases (see Drosophila Kdm4A) cooperate in promoting mouse embryonic stem cell (ESC) identity. However, little is known about their importance at the exit of ESC pluripotency. This study uncovered that Jmjd2c facilitates this process by stabilizing the assembly of Mediator-Cohesin complexes at lineage-specific enhancers. Functionally, Jmjd2c is required in ESCs to initiate appropriate gene expression programs upon somatic multi-lineage differentiation. In the absence of Jmjd2c, differentiation is stalled at an early post-implantation epiblast-like stage, while Jmjd2c-knockout ESCs remain capable of forming extra-embryonic endoderm derivatives. Dissection of the underlying molecular basis revealed that Jmjd2c is re-distributed to lineage-specific enhancers during ESC priming for differentiation. Interestingly, Jmjd2c-bound enhancers are co-occupied by the H3K9-methyltransferase G9a/Ehmt2, independently of its H3K9-modifying activity. Loss of Jmjd2c abrogates G9a recruitment and furthermore destabilizes loading of the Mediator and Cohesin components Med1 and Smc1a (see Drosophila Smc1) at newly activated and poised enhancers in ESC-derived epiblast-like cells. These findings unveil Jmjd2c-G9a as novel enhancer-associated factors, and implicate Jmjd2c as a molecular scaffold for the assembly of essential enhancer-protein complexes with impact on timely gene activation (Tomaz, 2017).

Histone H3K9 methyltransferase G9a represses PPARgamma expression and adipogenesis

PPARgamma promotes adipogenesis while Wnt proteins inhibit adipogenesis. However, the mechanisms that control expression of these positive and negative master regulators of adipogenesis remain incompletely understood. By genome-wide histone methylation profiling in preadipocytes, this study found that among gene loci encoding adipogenesis regulators, histone methyltransferase (HMT) G9a-mediated repressive epigenetic mark H3K9me2 is selectively enriched on the entire PPARgamma locus. H3K9me2 and G9a levels decrease during adipogenesis, which correlates inversely with induction of PPARgamma. Removal of H3K9me2 by G9a deletion enhances chromatin opening and binding of the early adipogenic transcription factor C/EBPbeta to PPARgamma promoter, which promotes PPARgamma expression. Interestingly, G9a represses PPARgamma expression in an HMT activity-dependent manner but facilitates Wnt10a expression independent of its enzymatic activity. Consistently, deletion of G9a or inhibiting G9a HMT activity promotes adipogenesis. Finally, deletion of G9a in mouse adipose tissues increases adipogenic gene expression and tissue weight. Thus, by inhibiting PPARgamma expression and facilitating Wnt10a expression, G9a represses adipogenesis (Wang, 2013).

Epigenetic regulation of autophagy by the methyltransferase G9a

Macroautophagy is an evolutionarily conserved cellular process involved in the clearance of proteins and organelles. Although the cytoplasmic machinery that orchestrates autophagy induction during starvation, hypoxia, or receptor stimulation has been widely studied, the key epigenetic events that initiate and maintain the autophagy process remain unknown. This study shows that the methyltransferase G9a coordinates the transcriptional activation of key regulators of autophagosome formation by remodeling the chromatin landscape. Pharmacological inhibition or RNA interference (RNAi)-mediated suppression of G9a induces LC3B expression and lipidation that is dependent on RNA synthesis, protein translation, and the methyltransferase activity of G9a. Under normal conditions, G9a associates with the LC3B, WIPI1, and DOR gene promoters, epigenetically repressing them. However, G9a and G9a-repressive histone marks are removed during starvation and receptor-stimulated activation of naive T cells, two physiological inducers of macroautophagy. Moreover, this study shows that the c-Jun N-terminal kinase (JNK) pathway is involved in the regulation of autophagy gene expression during naive-T-cell activation. Together, these findings reveal that G9a directly represses genes known to participate in the autophagic process and that inhibition of G9a-mediated epigenetic repression represents an important regulatory mechanism during autophagy (Artal-Martinez de Narvajas, 2013).

G9a functions as a molecular scaffold for assembly of transcriptional coactivators on a subset of glucocorticoid receptor target genes

Histone H3 lysine-9 methyltransferase G9a/EHMT2/KMT1C is a key corepressor of gene expression. However, activation of a limited number of genes by G9a (independent of its catalytic activity) has also been observed, although the precise molecular mechanisms are unknown. By using RNAi in combination with gene expression microarray analysis, this study found that G9a functions as a positive and a negative transcriptional coregulator for discrete subsets of genes that are regulated by the hormone-activated Glucocorticoid Receptor (GR). G9a is recruited to GR-binding sites (but not to the gene body) of its target genes and interacts with GR, suggesting recruitment of G9a by GR. In contrast to its corepressor function, positive regulation of gene expression by G9a involves G9a-mediated enhanced recruitment of coactivators CARM1 and p300 to GR target genes. Further supporting a role for G9a as a molecular scaffold for its coactivator function, the G9a-specific methyltransferase inhibitor UNC0646 does not affect G9a coactivator function but selectively decreases G9a corepressor function for endogenous target genes. Overall, G9a functions as a coactivator for hormone-activated genes and as a corepressor in support of hormone-induced gene repression, suggesting that the positive or negative actions of G9a are determined by the gene-specific regulatory environment and chromatin architecture. These findings indicate distinct mechanisms of G9a coactivator vs. corepressor functions in transcriptional regulation and provide insight into the molecular mechanisms of G9a coactivator function. These results also suggest a physiological role of G9a in fine tuning the set of genes that respond to glucocorticoids (Bittencourt, 2012).

Search PubMed for articles about Drosophila G9a

An, P. N. T., Shimaji, K., Tanaka, R., Yoshida, H., Kimura, H., Fukusaki, E. and Yamaguchi, M. (2017). Epigenetic regulation of starvation-induced autophagy in Drosophila by histone methyltransferase G9a. Sci Rep 7(1): 7343. PubMed ID: 28779125

Anreiter, I., Kramer, J. M. and Sokolowski, M. B. (2017). Epigenetic mechanisms modulate differences in Drosophila foraging behavior. Proc Natl Acad Sci U S A 114(47): 12518-12523. PubMed ID: 29078350

Artal-Martinez de Narvajas, A., Gomez, T. S., Zhang, J. S., Mann, A. O., Taoda, Y., Gorman, J. A., Herreros-Villanueva, M., Gress, T. M., Ellenrieder, V., Bujanda, L., Kim, D. H., Kozikowski, A. P., Koenig, A. and Billadeau, D. D. (2013). Epigenetic regulation of autophagy by the methyltransferase G9a. Mol Cell Biol 33(20): 3983-3993. PubMed ID: 23918802

Bittencourt, D., Wu, D. Y., Jeong, K. W., Gerke, D. S., Herviou, L., Ianculescu, I., Chodankar, R., Siegmund, K. D. and Stallcup, M. R. (2012). G9a functions as a molecular scaffold for assembly of transcriptional coactivators on a subset of glucocorticoid receptor target genes. Proc Natl Acad Sci U S A 109(48): 19673-19678. PubMed ID: 23151507

Esteve, P. O., Chin, H. G., Smallwood, A., Feehery, G. R., Gangisetty, O., Karpf, A. R., Carey, M. F. and Pradhan, S. (2006). Direct interaction between DNMT1 and G9a coordinates DNA and histone methylation during replication. Genes Dev 20(22): 3089-3103. PubMed ID: 17085482

Kato, Y., Kato, M., Tachibana, M., Shinkai, Y. and Yamaguchi, M. (2008). Characterization of Drosophila G9a in vivo and identification of genetic interactants. Genes Cells 13(7): 703-722. PubMed ID: 18498352

Kato, Y., Ushijima, Y. and Yamaguchi, M. (2011). Identification of nuclear localization signals of Drosophila G9a histone H3 methyltransferase. Cell Struct Funct 36(1): 121-129. PubMed ID: 21512259

Kramer, J. M., Kochinke, K., Oortveld, M. A., Marks, H., Kramer, D., de Jong, E. K., Asztalos, Z., Westwood, J. T., Stunnenberg, H. G., Sokolowski, M. B., Keleman, K., Zhou, H., van Bokhoven, H. and Schenck, A. (2011). Epigenetic regulation of learning and memory by Drosophila EHMT/G9a. PLoS Biol 9(1): e1000569. PubMed ID: 21245904

Lee, K. S., Yoon, J., Park, J. S. and Kang, Y. K. (2010). Drosophila G9a is implicated in germ cell development. Insect Mol Biol 19(1): 131-139. PubMed ID: 20002223

Merkling, S. H., Bronkhorst, A. W., Kramer, J. M., Overheul, G. J., Schenck, A. and Van Rij, R. P. (2015). The epigenetic regulator g9a mediates tolerance to RNA virus infection in Drosophila. PLoS Pathog 11: e1004692. PubMed ID: 25880195

Mis, J., Ner, S. S. and Grigliatti, T. A. (2006). Identification of three histone methyltransferases in Drosophila: dG9a is a suppressor of PEV and is required for gene silencing. Mol Genet Genomics 275(6): 513-526. PubMed ID: 16622709

Myant, K., Termanis, A., Sundaram, A. Y., Boe, T., Li, C., Merusi, C., Burrage, J., de Las Heras, J. I. and Stancheva, I. (2011). LSH and G9a/GLP complex are required for developmentally programmed DNA methylation. Genome Res 21(1): 83-94. PubMed ID: 21149390

Nagano, T., Mitchell, J. A., Sanz, L. A., Pauler, F. M., Ferguson-Smith, A. C., Feil, R. and Fraser, P. (2008). The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin. Science 322(5908): 1717-1720. PubMed ID: 18988810

Schaefer, A., Sampath, S. C., Intrator, A., Min, A., Gertler, T. S., et al. (2009) Control of cognition and adaptive behavior by the GLP/G9a epigenetic suppressor complex. Neuron 64: 678-691

Seum, C., Bontron, S., Reo, E., Delattre, M. and Spierer, P. (2007). Drosophila G9a is a nonessential gene. Genetics 177(3): 1955-1957. PubMed ID: 18039887

Shimaji, K., Konishi, T., Tanaka, S., Yoshida, H., Kato, Y., Ohkawa, Y., Sato, T., Suyama, M., Kimura, H. and Yamaguchi, M. (2015). Genomewide identification of target genes of histone methyltransferase dG9a during Drosophila embryogenesis. Genes Cells 20(11): 902-914. PubMed ID: 26334932

Shimaji, K., Tanaka, R., Maeda, T., Ozaki, M., Yoshida, H., Ohkawa, Y., Sato, T., Suyama, M. and Yamaguchi, M. (2017). Histone methyltransferase G9a is a key regulator of the starvation-induced behaviors in Drosophila melanogaster. Sci Rep 7(1): 14763. PubMed ID: 29116191

Stabell, M., Eskeland, R., Bjørkmo, M., Larsson, J., Aalen, R.B., Imhof, A. & Lambertsson, A. (2006). The Drosophila G9a gene encodes a multi-catalytic histone methyltransferase required for normal development. Nucleic Acids Res. 34: 4609-4621. PubMed ID: PubMed Central

Tachibana, M., Sugimoto, K., Fukushima, T. and Shinkai, Y. (2001). Set domain-containing protein, G9a, is a novel lysine-preferring mammalian histone methyltransferase with hyperactivity and specific selectivity to lysines 9 and 27 of histone H3. J Biol Chem 276(27): 25309-25317. PubMed ID: 11316813

Tachibana, M., Sugimoto, K., Nozaki, M., Ueda, J., Ohta, T., Ohki, M., Fukuda, M., Takeda, N., Niida, H., Kato, H. and Shinkai, Y. (2002). G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. Genes Dev 16(14): 1779-1791. PubMed ID: 12130538

Tachibana, M., Nozaki, M., Takeda, N. and Shinkai, Y. (2007). Functional dynamics of H3K9 methylation during meiotic prophase progression. EMBO J 26(14): 3346-3359. PubMed ID: 17599069

Tomaz, R. A., et al. (2017). Jmjd2c/Kdm4c facilitates the assembly of essential enhancer-protein complexes at the onset of embryonic stem cell differentiation. Development [Epub ahead of print]. PubMed ID: 28087629

Ushijima, Y., Inoue, Y. H., Konishi, T., Kitazawa, D., Yoshida, H., Shimaji, K., Kimura, H. and Yamaguchi, M. (2012). Roles of histone H3K9 methyltransferases during Drosophila spermatogenesis. Chromosome Res 20(3): 319-331. PubMed ID: 22476432

Wagschal, A., Sutherland, H. G., Woodfine, K., Henckel, A., Chebli, K., Schulz, R., Oakey, R. J., Bickmore, W. A. and Feil, R. (2008). G9a histone methyltransferase contributes to imprinting in the mouse placenta. Mol Cell Biol 28(3): 1104-1113. PubMed ID: 18039842

Wang, L., Xu, S., Lee, J. E., Baldridge, A., Grullon, S., Peng, W. and Ge, K. (2013). Histone H3K9 methyltransferase G9a represses PPARgamma expression and adipogenesis. EMBO J 32(1): 45-59. PubMed ID: 23178591

date revised: 25 October 2023

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