Genome-wide Kdm4 histone demethylase transcriptional regulation in Drosophila

Abstract

The histone lysine demethylase 4 (Kdm4/Jmjd2/Jhdm3) family is highly conserved across species and reverses di- and tri-methylation of histone H3 lysine 9 (H3K9) and lysine 36 (H3K36) at the N-terminal tail of the core histone H3 in various metazoan species including Drosophila, C.elegans, zebrafish, mice and humans. Previous studies have shown that the Kdm4 family plays a wide variety of important biological roles in different species, including development, oncogenesis and longevity by regulating transcription, DNA damage response and apoptosis. Only two functional Kdm4 family members have been identified in Drosophila, compared to five in mammals, thus providing a simple model system. Drosophila Kdm4 loss-of-function mutants do not survive past the early 2nd instar larvae stage and display a molting defect phenotype associated with deregulated ecdysone hormone receptor signaling. To further characterize and identify additional targets of Kdm4, we employed a genome-wide approach to investigate transcriptome alterations in Kdm4 mutants versus wild-type during early development. We found evidence of increased deregulated transcripts, presumably associated with a progressive accumulation of H3K9 and H3K36 methylation through development. Gene ontology analyses found significant enrichment of terms related to the ecdysteroid hormone signaling pathway important in development, as expected, and additionally previously unidentified potential targets that warrant further investigation. Since Kdm4 is highly conserved across species, our results may be applicable more widely to other organisms and our genome-wide dataset may serve as a useful resource for further studies.

Keywords: Development; Drosophila; Epigenetics; Histone; Histone methylation; Kdm4.

Fig. 1

Kdm4 double mutants appear to have progressive accumulation of differentially regulated genes a Hierarchical clustering was performed using the top 2000 expressed probe sets among wild-type and Kdm4A,B double mutant larval samples at early 1st, late 1st and early 2nd instar larval stages to assess overall changes in gene expression. b The number of probe sets with above 2-fold change down- and upregula-tion compared to the wild-type was quantified and overlaps among the different stages were quantified. c RT-qPCR was performed to validate a subset of genes (spok, Eip71CD, ImpE2, Eip78C, scu, forked, Cpr12A) and found to be downregulated at least 2-fold in Kdm4A,B mutants compared to the wild-type during the 2nd instar larval stage, but not in the early 1st instar in the microarray. Expression values normalized to rp49 are plotted. d ChlP-qPCR of the downregulated gene subset was performed to assess H3K9me3 in the early 2nd instar larval stage and relative H3K9me3 enrichment normalized to actin5C proximal promoter is shown

Fig. 2

Gene ontology analyses found significant enrichment in biological processes terms relevant to ecdysone signaling and other developmental processes and signaling cascades in Kdm4 double mutants. GO enrichment analyses were performed using probe sets with at least 2-fold down- and upregulation in the a early 1st, b late 1st and c early 2nd instar larval stages, d common across the three stages, and biological process terms with top 20-fold change are shown in descending order

Fig. 3

Commonly altered transcripts and putative H3K9/K36me3 developmentally altered target regions are enriched with “ecdysone-related” genes. We classified a the background pool of all probe sets prior to analyses, b those commonly altered in the early 1st, late 1st and early 2nd instar larval stages, and c those representing potential H3K9me3 and/or H3K36me3 2nd instar stage target regions into “ecdysone-related” vs. “non ecdysone-related” genes. Statistical significance of enrichment of “ecdysone-related” genes was calculated using the hypergeometric test

Fig. 4

Model of the transcriptional basis of lethality in Kdm4 double mutants. In wild-type animals, Kdm4A and/or Kdm4B remove H3K9me2,3 at developmental gene loci to regulate transcriptional activation, whereas in the Kdm4A,B double mutants, H3K9me2,3 hypermethylation of direct target genes results in gene silencing by HP1a recruitment. In the gene body of target genes, in wild-type animals, Kdm4 removes H3K36me2,3 at appropriate exons to ensure correct splice site choice; however, Kdm4A,B double mutants are hypermethylated, resulting in improper and/or over-splicing events that result in mRNA without all the correct exons. Considering cellular events other than transcriptional control and alternative splicing regulated by Kdm4 that have been reported previously, it is also likely that additional mechanisms including apoptosis, DNA damage and defects in replication may also contribute to lethality