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

Abstract

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. Here, we show 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.

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

“…During the development of Drosophila , metamorphosis is also a process that flies use to tolerate starvation stress. Even though our 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 21 , 22 . Together with our 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.…”

“…Three to five adult flies were dissected in PBS and fixed for 20 min in 4% paraformaldehyde at 25 °C. After permeabilization with 0.3% Triton X-100 in PBS, samples were incubated with primary antibodies specific for Atg8a (1:200, Novus Biologicals), dG9a (1:500, produced previously 40 ), or H3K9me2 (1/400, previously produced and used for Drosophila cells 22 , 42 ) at 4 °C for 16 h. After washing with 0.3% Triton X-100 in PBS, samples were incubated with the secondary antibody, Alexa Fluor 594 anti-mouse IgG (1:400, Molecular Probes) at 25 °C for 2 h. Samples were mounted with VECTASHIELD mounting media with DAPI (Vector Laboratories, Inc.). Intensity was only measured from nuclei in interphase and with a z-axis clearly present in their centers.…”

“…In Drosophila melanogaster , G9a (dG9a) has been shown to play an important role in neural development as well as behavioral processes such as learning and memory 20 . A previous study using a dG9a null mutant strain ( dG9a RG 5 ) demonstrated that dG9a was not essential for Drosophila viability or fertility 21 ; however, embryogenesis was delayed 22 . It is important to note that while there are various environmental stresses in the wild, Drosophila is always maintained under optimal conditions in the laboratory.…”

Functional convergence of histone methyltransferases EHMT1 and KMT2C involved in intellectual disability and autism spectrum disorder

“…G9a mutant flies are fully viable [ 17 , 18 ] but do show phenotypic differences compared to wildtype flies. Loss of Drosophila G9a delays embryonic development [ 19 ]. In larval stages, G9a mutants show defects in the morphology of multidendrite neurons and have altered crawling behaviour [ 18 ].…”

Histone methyltransferase G9a is a key regulator of the starvation-induced behaviors in Drosophila melanogaster

“…Canton S was used as the wild-type strain. The dG9a RG5 flies were kindly provided by Dr. P. Spierer and used as the dG9a null mutant 52 . The dG9a RG5 flies show no defects in viability compared to Canton S in 1st instar larvae, pupae and adult stages 52 .…”