EHMT/G9a Is the Solitary Drosophila Ortholog of EHMT1 and EHMT2/G9a
We set out to study EHMT/
G9a in Drosophila
. In contrast to mammals, which have two EHMT
genes, flies possess a single ortholog 
that we will subsequently refer to as EHMT throughout our manuscript. Phylogenetic analysis of the EHMT protein family in Drosophila
, human, and mouse shows that Drosophila
EHMT is equally similar to both EHMT1 and EHMT2/G9a ().
EHMT phylogeny and expression in the nervous system.
EHMT Is Expressed in the Fly Nervous System
We first examined expression and subcellular localization of the EHMT protein in the fly nervous system using an anti-EHMT antibody 
. In the adult brain EHMT staining is widely abundant, in a pattern resembling nuclear DAPI staining (). Analysis at single cell resolution in the ventral nerve cord of third instar larvae demonstrates that EHMT is localized in the nuclei of neurons as revealed by colocalization with the neuronal nuclear marker elav () 
. Weaker EHMT staining colocalized with repo (), a nuclear glial marker 
. EHMT staining was also observed in the nuclei of the larval multiple dendrite (md) sensory neurons of the peripheral nervous system labeled using the 109(2)80-Gal4
to express memGFP (). In addition, EHMT staining was observed at low levels in non-neuronal tissues such as the muscle and epidermis (Figure S1c
). The anti-EHMT immunolabeling is specific, as it is absent in EHMT
mutant embryos, md neurons, adult brains, and larval body-walls ( and S1
). These data reveal that EHMT is widely expressed in the Drosophila
Molecular characterization of EHMT alleles.
Generation of EHMT Mutant Flies
In order to uncover the functions of EHMT we generated deletions in the EHMT
gene by excision of a P-element, KG01242
, located in the 5′ UTR. We screened 80 independent excision lines and identified two downstream deletions (DD) resulting from imperfect excisions of KG01242
. Both deletion strains are viable to adulthood, which is consistent with a viable EHMT knock-out allele generated by homologous recombination in Drosophila 
lack 870 and 1473 base pairs of DNA downstream of the original P-element insertion site, respectively, including the EHMT translational start site (). We also isolated a precise transposon excision line that represents the same genetic background as our deletion lines and served as a control in all subsequent experiments (referred to as EHMT+
). Western blot analysis revealed a band of the expected size (180 kDa) in EHMT+
embryonic protein extracts, which was absent in extracts from both deletion lines (). No extra bands were detected by the C-terminally directed EHMT antibody 
that would point to expression of an N-terminally truncated protein. EHMT protein was also undetectable by immunohistochemistry in EHMT
mutant embryos, md neurons, and adult brains, while showing a nuclear staining pattern in EHMT+
animals (, , and S1
). Expression of the neighbor gene, CG3038
, was not affected by the deletions (Figure S2
). These data show that EHMTDD1
are strong and specific loss of function mutants, most likely complete null alleles.
EHMT Regulates Dendrite Branching in Type 4 Multiple Dendrite Neurons
Next, we examined several aspects of neuronal development in EHMT
mutant flies. Analysis of adult mushroom body architecture, synaptic morphology of the larval neuromuscular junction, and adult photoreceptor function (assessed by electroretinography) (Figure S3
) as well as analysis of embryonic nervous system integrity (unpublished data) did not reveal significant differences in mutant versus control conditions, indicating that general nervous system development and neuronal function is not affected.
Loss of EHMT
did however result in altered dendrite development in multidendrite (md) neurons, which are sensory neurons that tile the larval body wall. We specifically examined dendritic arbors of type 4 md neurons, which were highlighted using the 477-Gal4
and the UAS-Gal4
(). These neurons are highly stereotyped in their number, position, and morphology, thus allowing for quantitative analysis of dendritic arbors of identical neurons in different animals and genotypes. While the basic organization of these arbors is maintained in EHMT
mutants (primary branches labeled I, II, III, and IV in ), reduction of higher order branching resulted in dendritic fields of appreciably reduced complexity (). We quantitatively assessed this defect by counting the number of dendrite ends per standardized field of view in stacked confocal images. This analysis confirmed that EHMTDD1
had a consistent and statistically significant decrease in the total number of dendrite ends, showing 16 and 17.5 percent reduction, respectively, when compared to EHMT+
EHMT regulates dendrite branching.
To address whether this phenotype results cell-autonomously from EHMT deficiency in neurons, we generated UAS-EHMT transgenic flies and performed cell-specific rescue experiments (). Re-expression of EHMT in mutant type 4 md neurons (using 477-Gal4) did indeed rescue dendrite branching towards wild-type levels (, red and orange bars). This reversal is specific, since expression of EHMT in the EHMT+ genetic background did not increase branching (, black bars). Rather, EHMT overexpression appeared to reduce dendrite branching as compared to controls expressing Gal4 and GFP in the absence of UAS-EHMT, although this reduction was not statistically significant (, black bars). These data show that EHMT is cell-autonomously required in type 4 md neurons to establish normal dendrite complexity.
EHMT Affects Larval Locomotory Behavior
md neurons are important in the regulation of larval locomotion behavior 
. We therefore examined larval locomotory patterns during the early third instar using an established larval foraging assay () 
. Larval crawling paths were analyzed for total path length over a 5 min period and for specific crawling patterns, such as branched versus straight paths. The total path length covered by foraging larvae was not different between mutants and EHMT+
controls (), indicating that crawling ability is not hindered in these larvae. However, striking differences in larval locomotory patterns were observed between mutant and wild-type. Foraging EHMT
mutant larvae often stop, retract, and turn, causing increased branching in their crawling paths (). Quantitative analysis of the length of the resulting side branches revealed an increase of approximately 4-fold and 2-fold, respectively, in EHMTDD1
(). Thus, the dendrite phenotype of EHMT mutant larvae is associated with an altered crawling behavior. In contrast, other innate behaviors, such as adult phototaxis and negative geotaxis, were normal in EHMT
mutants (Figure S4
). To address whether the dendrite phenotype of type 4 md neurons alone is sufficient to cause the abnormal crawling pattern, we attempted to rescue this phenotype by re-expression of UAS-EHMT
in type 4 md neurons using 477-Gal4
. This was not sufficient to restore normal larval locomotor behavior, indicating that dendritic defects in type 4 md neurons and abnormal locomotory behavior might arise independently.
EHMT mutants show altered larval locomotory behavior.
EHMT Regulates Habituation, a Form of Non-Associative Learning
Next, we analyzed the role of Drosophila EHMT
in learning. Habituation is a form of non-associative learning where an initial response to a repeated stimulus gradually wanes 
. In the light-off jump reflex habituation assay 
flies were exposed to a sudden light-off pulse and measured for a jump response over the course of 100 trials with a 1 s inter-trial interval. show the proportion of flies that do show a jump response over the course of 100 trials. Hemizygous EHMT
mutant males (genotypes: EHMTDD1/Y
) and transheterozygous EHMT
mutant females (genotype: EHMTDD1/EHMTDD2
) both displayed a drastically slower response decrement during the habituation procedure as compared to wild-type EHMT+
flies (). Individual flies were deemed to have habituated when they failed to jump in five consecutive trials (no-jump criterion). Habituation was scored as the number of trials required to reach the no-jump criterion (trials to criterion). The mean number of trials to criterion for mutants, EHMTDD1/Y
, and EHMTDD1/EHMTDD2
, was significantly higher (12-, 8-, and 6-fold, respectively) than for EHMT+
wild-type flies (p
<0.001) (). These experiments establish a role for EHMT in regulating non-associative learning.
EHMT affects non-associative learning and courtship memory.
EHMT Is Required for Courtship Memory
Having established a role for EHMT in habituation, a simple learning process, we asked whether EHMT is also involved in more complex forms of learning and/or memory using the courtship conditioning paradigm. This assay is based on the conditioning of male courtship behavior by exposure to a non-receptive female, which in presence of normal learning and memory capacities results in suppression of courtship 
. Male flies were paired with a non-receptive pre-mated female for appropriate time intervals (see Experimental Procedures) and tested for courtship suppression immediately following the training period, after 30 min or after 24 h, to assess learning, short-, and long-term memory, respectively. The mean Courtship Index (CI, the percentage of time spent on courtship during a 10 min interval) of trained males and of socially naïve males was assessed to calculate a Learning Index (LI), which is defined as the percent reduction in mean courtship activity in trained males compared to naïve males; LI
. We found that EHMT mutant flies are perfectly capable of this form of learning, as they efficiently suppressed courtship immediately following the training period (). Strikingly, the Learning Index of EHMTDD1
males was reduced by 50% at 30 min after training (STM-short term memory), and even more dramatically, to 17% of the wild-type value at 24 h after training (LTM-long term memory) (). These results indicate that EHMT is dispensable for courtship learning but necessary for both short- and long-term courtship memory.
The Requirement for EHMT-Based Courtship Memory Maps to 7B-Gal4 Positive Neurons
To provide evidence for the specificity of the courtship conditioning phenotype and to roughly map where EHMT is required to control learning and memory in this paradigm, we performed rescue experiments in the EHMTDD2
background using tissue specific expression of UAS-EHMT
and short-term memory (30 min after training) as a read-out. The elav-Gal4
driver was used to express EHMT in all neurons, and the 7B-Gal4
promoter for more selective expression. Indeed, pan-neuronal expression of EHMT
in the mutant background restored the Learning Index to normal levels (, orange bars, pan neuronal versus EHMT mutants), providing evidence that EHMT is required cell-autonomously in neurons to achieve normal memory. Elav-driven expression of EHMT
in the EHMT+
genetic background had no significant effect on Learning Index (, black bars, pan neuronal versus EHMT mutant). 7B-Gal4 is predominantly expressed in the mushroom bodies of adult brains but is also expressed and at lower levels in some other brain regions, including the antennal lobe (Figure S5
. Expression of EHMT with this driver in the EHMT mutant background was able to rescue the Learning Index (, orange bars, 7B-Gal4 versus EHMT mutant), revealing that EHMT function in 7B-Gal4 neurons is sufficient for normal memory. We also observed that overexpression of EHMT
in the EHMT+
background significantly reduced the Learning Index (, black bars, 7B-Gal4 versus EHMT mutants). Since the Learning Index was normal in EHMT
mutants containing both 7B-Gal4
, we conclude that there is no deleterious effect due to the expression of Gal4
or the 7B-Gal4
P-element insertion itself. We therefore asked whether the presence of endogenous EHMT could make a significant difference to the absolute protein levels in the mushroom body upon 7B-Gal4
-mediated overexpression. We observe a very high and uniform EHMT staining in all mushroom body Kenyon cells upon UAS-EHMT
expression with 7B-Gal4
in the EHMT+
background (Figure S6
). A similar staining pattern was observed using this driver in the EHMT
mutant background, although staining intensity was noticeably lower, likely due to the absence of endogenous EHMT (Figure S6
). In contrast, the elav-Gal4
driver resulted in a non-uniform staining pattern, with high EHMT levels in only a small proportion of Kenyon cells (Figure S6
). Thus, overexpression of EHMT in 7B-Gal4
neurons appears to be deleterious when above a certain threshold. These results suggest that appropriate levels of EHMT in the Drosophila
nervous system are critical for courtship memory and indicate that the requirement for EHMT in this process is confined to 7B-Gal4
Taken together with the defect in jump reflex habituation, these data reveal an important role for EHMT not only in a simple form of learning but also in a more complex cognitive process such as courtship memory.
EHMT-Mediated Memory Can Be Rescued in Adulthood
Recently, it has been reported that postnatal loss of Ehmt1 and G9a in mice causes cognitive defects in the absence of obvious developmental abnormalities 
. We therefore asked whether the memory defects of EHMT mutants in the courtship conditioning paradigm can be rescued by expression of EHMT in adulthood. Indeed, induced expression of EHMT using hs-Gal4
after eclosion (see Experimental Procedures) completely restored memory defects shown by siblings of the same genotype that had not undergone the heat-shock procedure (). This demonstrates that EHMT is required for memory in adult flies and highlights that cognitive defects are reversible in EHMT
Generation of Genome Wide H3K9me2 Methylation Profiles
The reversible memory defects in EHMT mutant flies suggest a critical role for EHMT in neuronal function in addition to its role in dendrite development. We therefore wanted to determine the molecular mechanisms through which EHMT regulates neuronal processes. EHMT proteins mediate histone 3 lysine 9 dimethylation (H3K9me2) in euchromatic regions of the mammalian and fly genomes 
. Therefore, we investigated EHMT target sites by generating genome-wide H3K9me2 profiles for EHMT mutant and wild-type larvae using chromatin immunoprecipitation (ChIP) with an H3K9me2 antibody followed by massive parallel sequencing of the co-precipitated DNA (ChIP-seq technology). Mapping of the sequenced tags to the Drosophila
genome revealed a genome-wide profile that is consistent with known H3K9me2 patterns 
. High H3K9me2 is a known characteristic of heterochromatin 
. Accordingly, we find high H3K9me2 levels in both wild-type and EHMT mutant strains in annotated heterochromatic regions that are contiguous with the assembled euchromatic chromosome arms (Chr2Lh, Chr2Rh, Chr3Lh, Chr3Rh, and ChrXh) (Figure S7
. This was expected, since EHMT is known to have no effect on heterochromatin formation and heterochromatic H3K9me2 levels are known to be unaffected by loss of EHMT/G9a in fly and mouse 
. The generated H3K9me2 profiles also follow expected patterns in euchromatin. H3K9me2 is known to dip immediately before the transcriptional start site (tss) and near the polyadenylation site (polyA) of genes 
either due to nucleosome depletion or decreased H3K9me2 in these regions. We indeed observe a dip in H3K9 dimethylation in these regions (s, black lines), thus demonstrating the accuracy and reliability of our H3K9me2 profiles.
EHMT methylates discrete regions of the euchromatic genome.
EHMT Affects H3K9me2 Levels in Discrete Regions of the Euchromatic Genome
Since the global pattern of H3K9me2 appeared to be normal in EHMT mutants, we reasoned that EHMT must affect discrete regions within the genome. To identify these regions we divided the euchromatic genome into 300 bp bins and compared the number of sequenced tags per bin in wild-type versus mutant samples. For each of the 384,944 bins in the euchromatic genome we calculated a methylation ratio by dividing the number of tags in wild-type by the number of tags in the mutant. Thus, a ratio greater than 1 identifies regions where methylation is decreased in EHMT mutant flies. We have plotted the log of these ratios (log(2)wt/mt) in a histogram, in which Loss of Methylation Bins (LOMBs) are found in the area of positive log values. The histogram roughly follows a normal distribution but is asymmetric, with 19,258 bins outside two-times the standard deviation of the mean on the positive side, while only 50 bins outside two-times the standard deviation on the negative side (). The 19,258 LOMBs constitute about 5% of the euchromatic genome and provide an unbiased confirmation for the role of EHMT in H3K9 dimethylation. Loss of methylation (LOM) can also be visualized in the USCS genome browser as areas where H3K9me2 levels are depleted in the mutant but remain high in wild-type (two examples given in ). Interestingly, we find that LOMBs are not randomly distributed in the genome but are enriched in the areas 1 kb upstream of the tss and 1 kb downstream of the polyA site by 1.6-fold and 3.3-fold, respectively (). As mentioned above, we observe a local depletion of H3K9me2 in these regions in wild-type animals (, black line). In EHMT mutants, this local depletion is strongly augmented both upstream of the tss and near the polyA site (, orange line), providing further evidence that EHMT deposits H3K9me2 marks in discrete euchromatic loci, with a bias towards the 5′ and 3′ ends of genes.
Loss of H3K9me2 in EHMT Mutants Can Affect Gene Transcription
H3K9me2 is a marker for condensed, transcriptionally repressive chromatin 
, however the modification itself does not strongly correlate with transcription levels on a genome wide scale as is seen for some other histone modifications, like H3K4me3 and H3K27me3 
. To determine whether H3K9me2 can contribute to transcriptional repression in Drosophila
, we performed microarray expression analysis to compare mRNA levels in EHMT
wild-type and mutant larvae. We then analyzed H3K9me2 levels upstream of the tss and downstream of the polyA site for genes that were up- and downregulated in EHMT mutants. Genes that are activated by EHMT (i.e. greater that 2.5-fold downregulated in mutants, Table S1
) showed no difference in H3K9me2 profiles upstream of the tss or downstream of the polyA site when comparing EHMT wild-type and mutant strains (, middle panels). In contrast, genes that are repressed by EHMT (i.e. greater that 2.5-fold upregulated in mutants, Table S2
) have a clearly augmented dip in methylation both at the tss and polyA sites (s) when compared to the wild-type profile and to the average methylation profiles of all genes. These data indicate that EHMT-mediated H3K9 dimethylation immediately up and downstream of genes can affect transcriptional repression in Drosophila.
EHMT Target Loci
Next, we investigated which genes were affected by loss of methylation in EHMT mutants by associating each LOMB with its closest gene. In total, LOMBs were found in or near 5,136 genes; 1,229 genes had LOMBs upstream of the tss (upstream LOMB) and 1,712 genes had LOMBs downstream of the polyA site (downstream LOMB) (Table S3
). The two groups overlap by 255 genes ().
Bioinformatic analysis of EHMT target genes.
To assess the function of LOMB-associated genes, we analyzed their gene ontology for enrichment of specific biological processes using GOToolBox 
. Genes associated with LOMBS are highly enriched in terms related to the nervous system (, for lists of genes associated with selected terms see Table S4
). The broad term nervous system development, associated to more than 350 LOMB genes, reaches the highly significant p
value of 2.3×10−28
and is the most enriched tissue-specific term. Consequently, the term is highly depleted from the pool of genes with unaltered H3K9me2 in EHMT mutants; i.e. in genes that are not associated with LOMBs (, no LOMBs). Strikingly, all GO terms that describe EHMT
mutant phenotypes (e.g. short- and long-term memory, non-associative learning, dendrite morphogenesis, and larval locomotory behavior) show significant enrichment when considering all LOMB-associated genes and genes associated with downstream LOMBs (, observed phenotypes). Other neuronal terms that show high enrichment are also shown (, neuronal terms). Signal transduction is also amongst the most strongly enriched terms, with a p
value of 6.2×10−48
. Many specific signaling pathway terms are highly overrepresented amongst LOMB-associated genes, with G-protein coupled receptor protein signaling pathway and small GTPase mediated signal transduction being the top terms (, signaling pathways). We also note significant enrichment of pathway terms that directly relate to EHMT mutant phenotypes, such as cAMP signaling, a major pathway involved in learning and memory.
Notably, there is a stark contrast in enriched terms when comparing genes associated with either upstream or downstream LOMBs (, Downstream LOMBs and Upstream LOMBs). Downstream LOMBs are associated with genes that are enriched for neuronal terms, signaling pathways, and terms describing observed EHMT mutant phenotypes, while upstream LOMBs are associated with genes involved in biological processes requiring a high transcriptional plasticity, such as stress response and actin cytoskeleton remodeling (, enriched in upstream LOMBs). The contrast between these two groups in their gene ontology illustrates the importance of H3K9me2 position at target gene loci and provides further support as to the biological relevance of these data.
Finally, genes involved in regulatory processes such as translation, chromatin assembly/disassembly, and chromosome organization are highly depleted from LOMB-associated genes (, depleted), which contrasts the striking enrichment of nervous system and phenotype-relevant terms amongst LOMB-associated genes.