Figure 1.
Identification of dKDM4A targets by H3K36me3 ChIP-chip analysis.
(A) P-element insertion abrogates the expression of dKDM4A and results in increased levels of bulk histone H3K36me3. Precise excision of P-element restores the expression level of dKDM4A and the bulk level of H3K36me3. Nuclear extracts and acid-extracted histones from embryos of OreR, dkdm4a mutant and precise excision rescued fly lines were analyzed by western blot using indicated antibodies. Quantitation of the signal intensity is shown below each blot. (B) H3K36me3 profiles of chromosome 2 Lh genes. H3K36me3 ChIP-chip results are displayed on the UCSC genome browser for one representative biological replicate. The profile of H3K36me3 ChIP-chip in wild type embryos is shown in blue and the profile in dkdm4a mutant embryos is shown in red. The profile of increased H3K36me3 levels (mt/WT) is shown in green. Peaks called on the ratio track (mt/WT) are marked with black bars. y-axis shows log2 intensity ratio values; x-axis shows protein-coding genes annotated by Flybase.
Figure 2.
Identification of Common Target Genes of dKDM4A and HP1a.
The Venn diagram analysis of heterochromatic genes (A) or euchromatic genes (B) bound by HP1a in wild type and genes with increased H3K36me3 levels in dkdm4a mutant embryos. Genes assigned to the chromosome U are not included in the analysis. The P value was calculated by the hypergeometric test.
Figure 3.
Profiles of H3K36me3 and HP1a ChIP-chip at heterochromatin.
H3K36me3 and HP1a profiles of chromosome 2 Lh (A) and 2 RHet (B) genes. Profiles of H3K36me3 ChIP-chip are shown as in Figure 1B. The profile of HP1a ChIP-chip is shown in brown. Primers used in Figure 4B are indicated in the panel of qPCR primers.
Figure 4.
dKDM4A-mediated H3K36me3 demethylation requires HP1a binding at a subset of heterochromatic genes.
(A) The expression level of endogenous dKDM4A and FLAG-tagged dKDM4A or dKDM4A-V423A. Nuclear extracts from embryos of yw, dkdm4a mutants and mutants rescued by FLAG-dKDM4A were analyzed by western blot. The doublet band is likely to represent two isoforms of dKDM4A. Asterisk indicates non-specific signal. Quantitation of the signal intensity is shown below the blot. (B) The enrichment of H3K36me3 at HP1a-bound heterochromatic genes was observed by ChIP-qPCR. Genes with lower H3K36me3 levels (Scp1, CG17374 and CG40006) are also shown in a separate panel. The primer set amplifying an intergenic region at chromosome 2 L was used as a negative control. The error bars represent standard deviation from 3 biological repeats.
Figure 5.
Model for HP1a targeting dKDM4A activity to a subset of heterochromatic genes.
Demethylation of H3K36me3 by dKDM4A requires binding to HP1a at a subset of heterochromatic loci. Other mechanisms might exist to target dKDM4A activity to euchromatin, as well as heterochromatin. For example, dKDM4A might be targeted to euchromatin through other protein factors.