The Histone Demethylase KDM5 Activates Gene Expression by Recognizing Chromatin Context through Its PHD Reader Motif

Abstract

KDM5 family proteins are critically important transcriptional regulators whose physiological functions in the context of a whole animal remain largely unknown. Using genome-wide gene expression and binding analyses in Drosophila adults, we demonstrate that KDM5 (Lid) is a direct regulator of genes required for mitochondrial structure and function. Significantly, this occurs independently of KDM5's well-described JmjC domain-encoded histone demethylase activity. Instead, it requires the PHD motif of KDM5 that binds to histone H3 that is di- or trimethylated on lysine 4 (H3K4me2/3). Genome-wide, KDM5 binding overlaps with the active chromatin mark H3K4me3, and a fly strain specifically lacking H3K4me2/3 binding shows defective KDM5 promoter recruitment and gene activation. KDM5 therefore plays a central role in regulating mitochondrial function by utilizing its ability to recognize specific chromatin contexts. Importantly, KDM5-mediated regulation of mitochondrial activity is likely to be key in human diseases caused by dysfunction of this family of proteins.

Keywords: H3K4me3; KDM5; Lid; PHD motif; histone; mitochondria; transcription.

Figure 1. Genes directly regulated by KDM5 in adults

(A) Analyses of RNA-seq data to identify enriched Biological Process and Cellular Component categories for genes with altered expression in kdm5 mutant flies (up and downregulated genes). (B) Overlap between anti-HA (two replicates) and KDM5 ChIP-seq datasets (q<0.05). (C) Genomic distribution of KDM5 ChIP-seq peaks using anti-HA. (D) Comparison of RNA-seq and ChIP-seq data showing directly repressed genes. (E) Directly activated genes. (F) Gene ontology analysis of directly regulated genes. (G) KDM5 ChIP-seq read density relative to the transcriptional start site (TSS) at differentially expressed genes. (H) H3K4me3 ChIP-seq read density relative to the TSS of KDM5 bound and regulated genes. (I) IGV genome browser view of Nf1 showing overlapping KDM5 and H3K4me3 peaks. Middle (blue) H3K4me3 trace is data from our analyses. Bottom (orange) H3K4me3 is published data (GSM400670). (J) Heat map showing distribution of KDM5 and H3K4me3 relative to TSS (clustered using k-means). (K) Transcription factor binding sites enriched within KDM5 peaks using MEME-ChIP. Abbreviations are Mad (Mothers against decapentaplegic), Lola (Longitudinals lacking), Da (Daughterless), Bowl (brother of odd with entrails limited), Ato (Atonal), Hnf4 (Hepatocyte nuclear factor 4), Ttk (Tramtrack), Kr (Krüppel).

Figure 2. KDM5 directly regulates genes required for mitochondrial function

(A) Real-time PCR of genes indicated in kdm5^K6801/10424 adults shown relative to wildtype (w¹¹¹⁸). Genes were normalized to rp49 and shown as relative expression in kdm5 mutants. * p<0.05. (B) Heatmap showing overlapping KDM5 and H3K4me3 peaks at target genes. Red bars show regions used for ChIP-PCR validation shown in (C) and (D). (C) ChIP-PCR using anti-HA (KDM5) and IgG control showing KDM5 enrichment at targets. (D) ChIP-PCR using anti-H3K4me3 or control IgG from wildtype (w¹¹¹⁸) and kdm5^K6801/10424 mutant adults.

Figure 3. kdm5 mutant adults have mitochondrial and metabolic defects

(A, B) TEM images showing 5K and 10K magnifications of mitochondria within thoracic muscles from wildtype (w¹¹¹⁸) flies. (C, D) TEM images from kdm5^K6801/10424 mutant flies. (E) ATP levels in kdm5^K6801/10424 mutant flies shown as percentage of wildtype and normalized to the total protein levels. * p<0.01. (F) Lactic acid levels in kdm5^K6801/10424 adults relative to w¹¹¹⁸. (G) Quantitation the number of wildtype (w¹¹¹⁸) or kdm5^K6801/10424 flies that climb 7cm in 20 seconds after being tapped to the bottom of the vial. * p<0.001. (H) Citrate levels (ng/μl) in wildtype and kdm5^K6801/10424 mutant flies. * p<0.01. (I) Levels of distinct lipid subtypes in kdm5^K6801/10424 mutant flies relative to wildtype. LysoPC is lysophosphatidylcholine acyls (fatty acids generated as intermediate products), PCaa is phosphatidylcholine acyl molecules and PCae is phosphatidylcholine acyl-alkyl molecules. Numbers (<38 and >38) indicate number of carbon atoms in fatty acid chain. * p<0.01. (J) Ratio of NAD+ to NADH in wildtype (w¹¹¹⁸) and kdm5^K6801/10424 mutant flies. * p<0.001. (K) Levels of superoxide as assayed by MitoSOX in kdm5^K6801/10424 mutant flies relative to wildtype. * p<0.05. (L) Levels of the antioxidant carnosine (pmol/mg tissue) in wildtype and kdm5^K6801/10424 mutant flies. * p≪0.001. (M) Detection of oxidized proteins in wildtype and kdm5^K680/104241 mutant fly heads using oxyblot (2-fold increase).

Figure 4. KDM5-mediated regulation of mitochondrial function genes is independent of its demethylase activity

(A) Real-time PCR analyses of the genes indicated in demethylase inactive adults (described in (Li et al., 2010); kdm5^JmjC*) relative to wildtype (w¹¹¹⁸). Each gene was normalized to rp49 and shown as relative expression in kdm5^JmjC mutants. * p<0.05. (B, C) TEM images showing 5K magnifications of mitochondria within thoracic muscles from kdm5^K6801 mutant flies rescued by a wildtype KDM5 transgene (KDM5^WT; B) and kdm5^JmjC mutant flies (C). (D) ChIP-PCR showing levels of histone H3 acetylation using a pan-acetyl antibody in wildtype and kdm5^K6801/10424 flies. IgG is a negative control. ChIP-PCR fragments are indicated in Figure 2B. * p<0.05.

Figure 5. kdm5 mutant flies unable to bind H3K4me2/3 retain demethylase activity

(A) Schematic of the PHD3 region of KDM5. Amino acids required for KDM5A binding to H3K4me2/3 are underlined and for zinc coordination in red (Wang et al., 2009). (B) in vitro binding between biotinylated histone H3 peptides di- or trimethylated at lysine 4 (H3K4me2 and H3K4me3) and GST-PHD3. The interaction between KDM5 and H3K4me2 and H3K4me3 is abolished by GST-PHD3^W1771A and reduced by GST-PHD3^EW1780AA. (C) Western from adult heads showing levels of KDM5, H3K4me3 and histone H3. H3 and H3K4me3 are two different channels from the same Western blot. The H3K4me3:H3 ratio is increased in kdm5^K6801/10424 flies (1.6^+/−0.3-fold). (D) Number of kdm5^K6801 homozygous flies observed without a genomic rescue transgene (kdm5^K6801), with a wildtype rescue transgene (kdm5^wt) and two independent insertions of a kdm5^W1771A transgene (1M and 5M). * p<0.01. (E–H) Clones of fat body cells marked by the presence of GFP that express wildtype KDM5 or KDM5^W1771A. hs-FLP; UAS-KDM5 (or UAS-KDM5^W1771A) females were crossed to actin>CD2>Gal4, UAS-GFP males. Transgene-expressing cells are shown by the co-expression of GFP (E, F, G, H). Anti-KDM5 is shown in E′ and G′ and anti-H3K4me3 in F′ and H′. (I) ChIP-PCR analyses of H3K4me3 levels at KDM5 target genes in wildtype flies (kdm5^K6801 mutant flies rescued by a wildtype genomic rescue construct (wt)) and kdm5^W1771A mutant flies. No significant changes were observed. (J) ChIP-PCR analyses of H3K4me2 levels in wildtype and kdm5^W1771A mutant flies.

Figure 6. The PHD3 motif of KDM5 is important for gene activation

(A) Real-time PCR comparing mRNA levels of direct KDM5 genes in flies lacking H3K4me2/3 binding (kdm5^W1771A; W1771A) and wildtype (kdm5 mutant flies rescued by a wildtype transgene). All genes shown are significantly downregulated in kdm5^W1771A flies (p<0.05). (B) anti-KDM5 ChIP-PCR analyses of binding at the target genes indicated in wildtype flies and kdm5^W1771A mutant flies. * p<0.05. (C, D) TEM images showing 5K magnifications of mitochondria within thoracic muscles from wildtype flies (KDM5^WT; C) and kdm5^W1771A mutant flies (KDM5^W1771A; D). (E) Model for KDM5 function. KDM5 recruitment to its target mitochondrial function genes is regulated in part through recognition of the chromatin mark H3K4me3, but PHD3-mediated interaction(s) with as yet unidentified transcription factor(s) (TF) and transcriptional co-factors are also likely to be crucial.