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. Here, we have dissected the neuronal function of the Drosophila euchromatin histone methyltransferase (EHMT), 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, we generated genome-wide H3K9 dimethylation profiles 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. Our 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.
Mis J, Ner S. S, 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: 513–526.
Tachibana M, Sugimoto K, Nozaki M, Ueda J, Ohta T, et al. (2002) G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. Genes Dev 16: 1779–1791.
Tachibana M, Ueda J, Fukuda M, Takeda N, Ohta T, et al. (2005) Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9. Genes Dev 19: 815–826.
Ogawa H, Ishiguro K, Gaubatz S, Livingston D. M, Nakatani Y (2002) A complex with chromatin modifiers that occupies E2F- and Myc-responsive genes in G0 cells. Science 296: 1132–1136.
Stabell M, Eskeland R, Bjorkmo M, Larsson J, Aalen R. B, et al. (2006) The Drosophila G9a gene encodes a multi-catalytic histone methyltransferase required for normal development. Nucleic Acids Res 34: 4609–4621.
Kleefstra T, van Zelst-Stams W. A, Nillesen W. M, Cormier-Daire V, Houge G, et al. (2009) Further clinical and molecular delineation of the 9q Subtelomeric Deletion Syndrome supports a major contribution of EHMT1 haploinsufficiency to the core phenotype. J Med Genet 46: 598–606.
Kleefstra T, Smidt M, Banning M. J, Oudakker A. R, van Esch H, et al. (2005) Disruption of the gene Euchromatin Histone Methyl Transferase1 (Eu-HMTase1) is associated with the 9q34 subtelomeric deletion syndrome. J Med Genet 42: 299–306.
Kleefstra T, Brunner H. G, Amiel J, Oudakker A. R, Nillesen W. M, et al. (2006) Loss-of-function mutations in euchromatin histone methyl transferase 1 (EHMT1) cause the 9q34 subtelomeric deletion syndrome. Am J Hum Genet 79: 370–377.
Stewart D. R, Huang A, Faravelli F, Anderlid B. M, Medne L, et al. (2004) Subtelomeric deletions of chromosome 9q: a novel microdeletion syndrome. Am J Med Genet A 128A: 340–351.
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.
Robinow S, White K (1991) Characterization and spatial distribution of the ELAV protein during Drosophila melanogaster development. J Neurobiol 22: 443–461.
Campbell G, Goring H, Lin T, Spana E, Andersson S, et al. (1994) RK2, a glial-specific homeodomain protein required for embryonic nerve cord condensation and viability in Drosophila. Development 120: 2957–2966.
Grueber W. B, Jan L. Y, Jan Y. N (2003) Different levels of the homeodomain protein cut regulate distinct dendrite branching patterns of Drosophila multidendritic neurons. Cell 112: 805–818.
Ainsley J. A, Pettus J. M, Bosenko D, Gerstein C. E, Zinkevich N, et al. (2003) Enhanced locomotion caused by loss of the Drosophila DEG/ENaC protein Pickpocket1. Curr Biol 13: 1557–1563.
Song W, Onishi M, Jan L. Y, Jan Y. N (2007) Peripheral multidendritic sensory neurons are necessary for rhythmic locomotion behavior in Drosophila larvae. Proc Natl Acad Sci U S A 104: 5199–5204.
Ainsley J. A, Kim M. J, Wegman L. J, Pettus J. M, Johnson W. A (2008) Sensory mechanisms controlling the timing of larval developmental and behavioral transitions require the Drosophila DEG/ENaC subunit, Pickpocket1. Dev Biol 322: 46–55.
Ferveur J. F, Savarit F, O'Kane C. J, Sureau G, Greenspan R. J, et al. (1997) Genetic feminization of pheromones and its behavioral consequences in Drosophila males. Science 276: 1555–1558.
Barski A, Cuddapah S, Cui K, Roh T. Y, Schones D. E, et al. (2007) High-resolution profiling of histone methylations in the human genome. Cell 129: 823–837.
Hoskins R. A, Carlson J. W, Kennedy C, Acevedo D, Evans-Holm M, et al. (2007) Sequence finishing and mapping of Drosophila melanogaster heterochromatin. Science 316: 1625–1628.
Brower-Toland B, Riddle N. C, Jiang H, Huisinga K. L, Elgin S. C (2009) Multiple SET methyltransferases are required to maintain normal heterochromatin domains in the genome of Drosophila melanogaster. Genetics 181: 1303–1319.
Marks H, Chow J. C, Denissov S, Francoijs K. J, Brockdorff N, et al. (2009) High-resolution analysis of epigenetic changes associated with X inactivation. Genome Res 19: 1361–1373.
Martin D, Brun C, Remy E, Mouren P, Thieffry D, et al. (2004) GOToolBox: functional analysis of gene datasets based on Gene Ontology. Genome Biol 5: R101.
Ding N, Zhou H, Esteve P. O, Chin H. G, Kim S, et al. (2008) Mediator links epigenetic silencing of neuronal gene expression with x-linked mental retardation. Mol Cell 31: 347–359.
Roopra A, Qazi R, Schoenike B, Daley T. J, Morrison J. F (2004) Localized domains of G9a-mediated histone methylation are required for silencing of neuronal genes. Mol Cell 14: 727–738.
Mavrich T. N, Ioshikhes I. P, Venters B. J, Jiang C, Tomsho L. P, et al. (2008) A barrier nucleosome model for statistical positioning of nucleosomes throughout the yeast genome. Genome Res 18: 1073–1083.
O'Sullivan J. M, Tan-Wong S. M, Morillon A, Lee B, Coles J, et al. (2004) Gene loops juxtapose promoters and terminators in yeast. Nat Genet 36: 1014–1018.
O'Reilly D, Greaves D. R (2007) Cell-type-specific expression of the human CD68 gene is associated with changes in Pol II phosphorylation and short-range intrachromosomal gene looping. Genomics 90: 407–415.
Tan-Wong S. M, French J. D, Proudfoot N. J, Brown M. A (2008) Dynamic interactions between the promoter and terminator regions of the mammalian BRCA1 gene. Proc Natl Acad Sci U S A 105: 5160–5165.
Tan-Wong S. M, Wijayatilake H. D, Proudfoot N. J (2009) Gene loops function to maintain transcriptional memory through interaction with the nuclear pore complex. Genes Dev 23: 2610–2624.
Wu H, Coskun V, Tao J, Xie W, Ge W, et al. (2010) Dnmt3a-dependent nonpromoter DNA methylation facilitates transcription of neurogenic genes. Science 329: 444–448.
Trimarchi J. R, Schneiderman A. M (1995) Different neural pathways coordinate Drosophila flight initiations evoked by visual and olfactory stimuli. J Exp Biol 198: 1099–1104.
McBride S. M, Giuliani G, Choi C, Krause P, Correale D, et al. (1999) Mushroom body ablation impairs short-term memory and long-term memory of courtship conditioning in Drosophila melanogaster. Neuron 24: 967–977.
Keleman K, Kruttner S, Alenius M, Dickson B. J (2007) Function of the Drosophila CPEB protein Orb2 in long-term courtship memory. Nat Neurosci 10: 1587–1593.
Iliadi K. G, Avivi A, Iliadi N. N, Knight D, Korol A. B, et al. (2008) nemy encodes a cytochrome b561 that is required for Drosophila learning and memory. Proc Natl Acad Sci U S A 105: 19986–19991.
Zhao H, Zheng X, Yuan X, Wang L, Wang X, et al. (2009) ben functions with scamp during synaptic transmission and long-term memory formation in Drosophila. J Neurosci 29: 414–424.
Feng J, Zhou Y, Campbell S. L, Le T, Li E, et al. (2010) Dnmt1 and Dnmt3a maintain DNA methylation and regulate synaptic function in adult forebrain neurons. Nat Neurosci 13: 423–430.
Hagerman R. J, Berry-Kravis E, Kaufmann W. E, Ono M. Y, Tartaglia N, et al. (2009) Advances in the treatment of fragile X syndrome. Pediatrics 123: 378–390.
Berry-Kravis E, Sumis A, Hervey C, Nelson M, Porges S. W, et al. (2008) Open-label treatment trial of lithium to target the underlying defect in fragile X syndrome. J Dev Behav Pediatr 29: 293–302.
Chang S, Bray S. M, Li Z, Zarnescu D. C, He C, et al. (2008) Identification of small molecules rescuing fragile X syndrome phenotypes in Drosophila. Nat Chem Biol 4: 256–263.
McBride S. M, Choi C. H, Wang Y, Liebelt D, Braunstein E, et al. (2005) Pharmacological rescue of synaptic plasticity, courtship behavior, and mushroom body defects in a Drosophila model of fragile X syndrome. Neuron 45: 753–764.
Schenck A, Bardoni B, Langmann C, Harden N, Mandel J. L, et al. (2003) CYFIP/Sra-1 controls neuronal connectivity in Drosophila and links the Rac1 GTPase pathway to the fragile X protein. Neuron 38: 887–898.
Semotok J. L, Westwood J. T, Goldman A. L, Cooperstock R. L, Lipshitz H. D (2008) Measuring mRNA stability during early Drosophila embryogenesis. Methods Enzymol 448: 299–334.
Neal S. J, Gibson M. L, So A. K, Westwood J. T (2003) Construction of a cDNA-based microarray for Drosophila melanogaster: a comparison of gene transcription profiles from SL2 and Kc167 cells. Genome 46: 879–892.
