Model for KDM5 function.

<p>(A) KDM5-Foxo co-regulated genes are a subset of all Foxo regulated genes. We propose that at these stress response-related promoters, KDM5 facilitates the deacetylation of Foxo (and activation of its DNA binding activity) by binding to HDAC4. (B) Because HDAC4 is required at additional Foxo-regulated genes that are not affected by loss of KDM5, we propose that HDAC4-Foxo complexes that lack KDM5 regulate different targets than KDM5-HDAC4-Foxo complexes (e.g. <i>InR</i> and <i>puc</i>). Additional, as yet uncharacterized, proteins (shown as unlabeled shapes in A and B) are likely to be present in both KDM5-HDAC4-Foxo and HDAC4-Foxo complexes. See text for details.</p

KDM5 directly regulates oxidative stress resistance genes.

<p>(A) Real-time PCR analyses of <i>4E-BP</i>, <i>l(2)efl</i>, <i>CG5316</i>, <i>spirit</i>, <i>Prx2540-2</i> and <i>CG10211</i> from wildtype (<i>w¹¹¹⁸</i>), <i>kdm5^K6801</i> mutant and <i>foxo²¹</i> mutant 3^rd instar larvae in 5% sucrose or 20 mM paraquat/5% sucrose for six hours (oxidative stress). (B) Schematic of the promoters of <i>4E-BP</i>, <i>l(2)efl</i>, <i>CG5316</i>, <i>spirit</i>, <i>Prx2540-2</i> and <i>CG10211</i> showing the position of the Foxo binding site (FHREs; black boxes). Primers surrounding these sites were used for ChIP analyses shown in parts C and D. (C) ChIP analyses of <i>4E-BP</i>, <i>l(2)efl</i>, <i>CG5316</i>, <i>spirit</i>, <i>Prx2540-2</i> and <i>CG10211</i>. Anti-Foxo ChIP is shown in black bars while control anti-Foxo ChIP from <i>foxo²¹</i> homozygous mutant larvae is shown in grey. Data are shown as % of input DNA. All six genes tested show significant attenuation of anti-Foxo ChIP signal (<i>p</i><<0.01). (D) ChIP analyses of <i>4E-BP</i>, <i>l(2)efl</i>, <i>CG5316</i>, <i>spirit</i>, <i>Prx2540-2</i> and <i>CG10211</i>. Anti-KDM5 ChIP is shown in black bars while control anti-KDM5 ChIP from <i>kdm5^K6801</i> homozygous mutant larvae is shown in grey. Data are shown as % of input DNA. All six genes tested show significant attenuation of anti-KDM5 ChIP signal (<i>p</i><<0.01).</p

Reducing KDM5 affects cellular levels of oxidative stress.

<p>(A) Survival of wildtype control (<i>w¹¹¹⁸</i>) and <i>kdm5^10424/K06801</i> female larvae in 5% sucrose (left) or in 20 mM paraquat/5% sucrose (right) for six hours. * <i>p</i><0.01. (B) Western blot showing levels of KDM5 and the loading control histone H3 in wildtype (<i>w¹¹¹⁸</i>) and <i>kdm5^10424/K6801</i> adult female heads (three heads per lane). (C) Survival curve of <i>w¹¹¹⁸</i> and <i>kdm5^10424/K06801</i> adult females fed 20 mM paraquat in 5% sucrose. Adults were one to three days old at the start of the experiment. Error bars represent standard error. The two survival curves are significantly different from one another (<i>p</i><0.05). (D) Detection of oxidized proteins in wildtype and <i>kdm5^K6801</i> homozygous mutant 3^rd instar larvae using oxyblot. Molecular weight markers are shown on the left. – indicates negative control lanes in which carbonyl groups were not derivatized. <i>kdm5</i> mutants show ∼2-fold increase in total levels of oxidized proteins. Histone H3 is shown as a loading control. (E) Schematic of <i>lacZ</i> mutation reporter assay. Adapted from Garcia et al <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004676#pgen.1004676-Garcia1" target="_blank">[51]</a>. (F) <i>lacZ</i> mutation frequency of wildtype control (<i>w¹¹¹⁸</i>) and <i>kdm5¹⁰⁴²⁴</i> homozygous mutant 3^rd instar larvae. Genotypes are <i>w¹¹¹⁸</i>; #9lacZ; + (labeled <i>wt</i>) and <i>w¹¹¹⁸</i>; <i>kdm5¹⁰⁴²⁴</i>, #9lacZ; + (labeled <i>kdm5^−/−</i>. * <i>p</i><0.01. (G) Generation of wildtype clones in a wildtype background in larval wing imaginal disc. Genotype is hs-FLP¹²²/+; FRT40A/FRT40A ubi-GFP. Areas circled with dashed line have two copies of ubi-GFP (twin spots). Areas shown with a solid line have no GFP. (H) Generation of <i>kdm5^K6801</i> mutant clones in wing imaginal disc. Genotype is hs-FLP¹²²/+; <i>kdm5^K6801</i> FRT40A/FRT40A ubi-GFP. Areas shown with a dashed line have two copies of GFP (twin spots). Areas circled with sold line are homozygous mutant for <i>kdm5^K6801</i>. (I) Quantitation of clone area in E (<i>wt</i>; N = 6) and F (<i>kdm5</i> mutant; N = 8). ns = not significantly different.</p

KDM5 mutants show reduced expression of genes required to reduce oxidative stress.

<p>A) Western blot showing levels of KDM5 and the loading control γ-tubulin in wildtype (<i>w¹¹¹⁸</i>) and <i>kdm5¹⁰⁴²⁴</i> homozygous mutant wing imaginal discs used for microarrays. * indicates a non-specific band. (B) Hierarchical clustering analysis of 901 genes that are differentially expressed 1.5-fold or more in <i>kdm5</i> mutant wing discs compared to wildtype (<i>w¹¹¹⁸</i>) (<i>p</i><0.05). Up-regulated genes are shown in red; down-regulated genes in green. (C) Gene ontology analyses showing biological process enrichment analysis of down- and up-regulated genes (<i>p</i>≤0.01). (D) GO David analyses of the Molecular function of the 84 stress response and oxidation reduction class genes (<i>p</i>≤0.01). (E) Real-time PCR showing levels of oxidative stress response genes in <i>kdm5^10424/K6801</i> 3^rd instar female larvae relative to wildtype. Levels of gene expression within each sample were normalized to <i>rp49</i> and shown relative to wildtype. * <i>p</i><0.05, ** <i>p</i><0.01.</p

KDM5 Interacts with Foxo to Modulate Cellular Levels of Oxidative Stress

<div><p>Increased cellular levels of oxidative stress are implicated in a large number of human diseases. Here we describe the transcription co-factor KDM5 (also known as Lid) as a new critical regulator of cellular redox state. Moreover, this occurs through a novel KDM5 activity whereby it alters the ability of the transcription factor Foxo to bind to DNA. Our microarray analyses of <i>kdm5</i> mutants revealed a striking enrichment for genes required to regulate cellular levels of oxidative stress. Consistent with this, loss of <i>kdm5</i> results in increased sensitivity to treatment with oxidizers, elevated levels of oxidized proteins, and increased mutation load. KDM5 activates oxidative stress resistance genes by interacting with Foxo to facilitate its recruitment to KDM5-Foxo co-regulated genes. Significantly, this occurs independently of KDM5's well-characterized demethylase activity. Instead, KDM5 interacts with the lysine deacetylase HDAC4 to promote Foxo deacetylation, which affects Foxo DNA binding.</p></div

KDM5 is required for efficient recruitment of Foxo to a subset of its target promoters.

<p>(A) ChIP using anti-Foxo <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004676#pgen.1004676-Kanao1" target="_blank">[86]</a> to the FHRE region of the <i>InR</i>, <i>puc</i>, <i>4E-BP</i>, <i>l(2)efl</i>, <i>CG5316</i>, <i>spirit</i>, <i>Prx2540-2</i> and <i>CG10211</i> promoters in wildtype (<i>w¹¹¹⁸</i>) and <i>kdm5^10424/K6801</i> mutant larvae. * <i>p</i><0.05. (B) KDM5 ChIP to the FHRE in the <i>InR</i>, <i>puc</i>, <i>4E-BP</i>, <i>l(2)efl</i>, <i>CG5316</i>, <i>spirit</i>, <i>Prx2540-2</i> and <i>CG10211</i> promoters in wildtype (<i>w¹¹¹⁸</i>) and <i>foxo²¹</i> mutant 3^rd instar larvae. * <i>p</i><0.05. IgG is included as an additional control for non-specific binding in A and B. (C) (top) Levels of KDM5, HDAC4, Foxo, acetylated Foxo and the loading control γ-tubulin in wildtype and <i>kdm5^K6801</i> mutant larvae (anterior half of 3^rd instar larvae). Levels of acetylated Foxo were determined by immunoprecipitating Foxo from larval extract and probing Western blot with an anti-acetyl-lysine antibody. * indicates non-specific band in KDM5 blot. (bottom) Levels of Foxo and acetylated Foxo in <i>HDAC4^KG09091</i> mutant larvae. (D) Immunoprecipitating HDAC4 from larval extracts efficiently pulls down HDAC4 and also co-precipitates KDM5. Lysate used was from 3^rd instar larvae. (E) Real time PCR showing levels of <i>HDAC4</i>, <i>InR</i>, <i>puc</i>, <i>4E-BP</i>, <i>l(2)efl</i>, <i>CG5316</i>, <i>spirit</i>, <i>Prx2540-2</i> and <i>CG10211</i> mRNA in <i>HDAC4^KG09091</i> homozygous 3^rd instar larvae showing that all of these genes are significantly downregulated. All genes shown were significantly downregulated (<i>p</i><0.05).</p

KDM5 and Foxo regulate a common set of genes and genetically and physically interact.

<p>(A) Real-time PCR showing levels of gene expression in <i>foxo²¹</i> or foxo^Δ94 homozygous 3^rd instar female larvae relative to wildtype controls (<i>w¹¹¹⁸</i>). <i>foxo²¹</i> was maintained as a homozygous stock while <i>foxo^Δ94</i> was generated by intercrossing heterozygous parents, so potentially contains some maternally derived Foxo. * <i>p</i><0.05. (B) Survival of wildtype (<i>w¹¹¹⁸</i>) and <i>foxo²¹</i> female larvae in 5% sucrose (left) or with 20 mM paraquat in 5% sucrose (right) for six hours. (C) <i>In vitro</i> binding assay showing that S³⁵-labeled full length KDM5 binds to GST-Foxo but not to GST alone. (D) <i>in vivo</i> interaction between Foxo and KDM5. Foxo protein was immunoprecipitated from S2 cell nuclear extract from cells grown in vehicle or oxidative stress conditions (20 mM paraquat for six hours). Levels of KDM5 in Foxo immunoprecipitates were then examined by Western blot and compared to beads alone. (E) Quantitation of genetic interaction between <i>kdm5</i> and <i>foxo</i> using total eye area (>10 eyes per genotype) as measured in pixels using ImageJ. Error bars indicate standard deviation. Significance was determined using a Student's t-test. ** <i>p</i><0.001, *** <i>p</i><0.0001. ns = not significant. (F–J) Representative images of the genetic interaction between Foxo and KDM5 in 15 day old flies. (F) Control flies are GMR-Gal4 crossed to wildtype (<i>w¹¹¹⁸</i>) and (G) GMR-Gal4 combined with UAS-Foxo showing rough eye phenotype. (H) GMR-Gal4 combined with UAS-Foxo and heterozygous for <i>kdm5^K6801</i> showing suppression of GMR>Foxo eye phenotype. (I) GMR-Gal4 combined with UAS-Foxo and UAS-KDM5 showing eye phenotype enhancement. (J) GMR-Gal4 crossed to UAS-KDM5.</p

The H3K4me3 demethylase activity of KDM5 is not required for it to regulate KDM5-Foxo target genes.

<p>(A) Western blot showing levels of KDM5, Foxo and the loading control histone H3 in wildtype (<i>w¹¹¹⁸</i>) and <i>kdm5</i> mutant (<i>kdm5^K6801</i>) 3^rd instar wing imaginal discs (10 discs per lane). (B) Western blot showing levels of KDM5, Foxo and histone H3 in wildtype (<i>w¹¹¹⁸</i>), and <i>foxo²¹</i> mutant 3^rd instar wing discs. (C) H3K4me3 ChIP to the FHRE region of <i>puc</i>, <i>4E-BP</i>, <i>l(2)efl</i>, <i>CG5316</i>, <i>spirit</i>, <i>Prx2540-2</i> and <i>CG10211</i> promoters in <i>lid¹⁰⁴²⁴</i>/<i>lid¹⁰⁴²⁴</i>; gKDM5/gKDM5 (blue) and <i>lid¹⁰⁴²⁴</i>/<i>lid¹⁰⁴²⁴</i>; gKDM5^JmjC*/gKDM5^JmjC* (red) larvae. * <i>p</i><0.05. IgG is included as a negative control. (D) Real-time PCR showing expression levels in wildtype (<i>lid¹⁰⁴²⁴</i>/<i>lid¹⁰⁴²⁴</i>; gKDM5/gKDM5 (black) and <i>lid¹⁰⁴²⁴</i>/<i>lid¹⁰⁴²⁴</i>; gKDM5^JmjC*/gKDM5^JmjC* (grey) 3^rd instar larvae. Data are normalized to <i>rp49</i> expression and changes to expression in demethylase inactive larvae are shown relative to <i>lid¹⁰⁴²⁴</i>/<i>lid¹⁰⁴²⁴</i>; gKDM5/gKDM5 control larvae. ** <i>p</i><0.01.</p

Demethylase inactive male flies are short-lived and sensitive to paraquat.

<p>(A, B) Lifespan of wildtype and demethylase inactive Lid males and females, respectively showing that males are short-lived whereas females have a normal lifespan. (C, D). Survival in response to 20 mM paraquat of wildtype and demethylase inactive males and females, respectively. Like lifespan, males are sensitive to paraquat whereas females are not.</p

<i>lid</i> and <i>dKDM2</i> genetically interact.

<p><i>dKDM2</i> homozygous mutants survive until adulthood at 62% of expected frequency and a majority of <i>lid^K6801</i> homozygotes die during pupal development. In contrast, animals homozygous for <i>dKDM2</i> and <i>lid^K6801</i> die during the 1^st and 2^nd larval instar stages. The lethality associated with <i>lid</i>, <i>dKDM2</i> double mutants can be rescued by a wildtype, but not a demethylase inactive, genomic rescue transgene.</p