SETD5 gene mutations have been identified as a frequent cause of idiopathic intellectual disability. Here we show that Setd5-haploinsufficient mice present developmental defects such as abnormal brain-to-body weight ratios and neural crest defect-associated phenotypes. Furthermore, Setd5-mutant mice show impairments in cognitive tasks, enhanced long-term potentiation, delayed ontogenetic profile of ultrasonic vocalization, and behavioral inflexibility. Behavioral issues are accompanied by abnormal expression of postsynaptic density proteins previously associated with cognition. Our data additionally indicate that Setd5 regulates RNA polymerase II dynamics and gene transcription via its interaction with the Hdac3 and Paf1 complexes, findings potentially explaining the gene expression defects observed in Setd5-haploinsufficient mice. Our results emphasize the decisive role of Setd5 in a biological pathway found to be disrupted in humans with intellectual disability and autism spectrum disorder.
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The data that support the findings of this study are available from the corresponding author upon reasonable request.
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We thank V. Stein and M. Döngi for guidance in setting up LTP experiments, P. Jonas for consultancy on LTP data analysis, M. Kutzer for help in statistical analysis of nonparametric datasets, S. Deixler and A. Lepold for management of the animal colony, T. Asenov and his team in the Machine Shop, F. Marr, H.C. Önal, A. Coll, and M. Schunn and the Preclinical Facility team for technical assistance. We thank the EMBL proteomics, flow cytometry, and genomics core facilities for the sample preparation, data generation, and analysis support. We thank M. van Gerven for helping with the SETD5 human mutation study. This work was supported by the Simons Foundation Autism Research Initiative (grant 401299) to G.N. and the DFG (SPP1738 grant NO 1249) to K.-M.N.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
a, Schematic diagram for Setd5-floxed allele (top) showing exons 3-6 flanked by loxP sites and the resulting Setd5-knockout allele. b, Typical genotyping results of Setd5+/− and Setd5+/+ mice; the results are shown for single experiment and replication experiments were not performed. c, RT-PCR showing reduction of Setd5 gene expression in the brain of Setd5+/− and Setd5+/+ mice (n(mice)=4 per genotype); Hprt was used as control. Pictures in b and c represent part of the gels; see Supplementary Fig. 14 for full-length picture.
a, Doughnut chart indicating that Setd5+/− animals are born at a non-Mendelian ratio (n=585 mice, of which 387 Setd5+/+ and 198 Setd5+/−). b, Kaplan-Meier survival curves showing reduced survival probability of mutant animals (n is 387 for Setd5+/+ and 198 Setd5+/−), which seems to be slightly more pronounced in the females (n is 98 male and 100 female Setd5+/−). c, Increased normalised brain/body weight ratio is observed in P1 male (n=9 controls, n=8 mutants) and female mutant pups (n=6 controls, n=8 mutants) as well as in adult male mutants (>P30, n=10-17) (values normalised to Setd5+/+); *P<0.05, **P<0.01, ***P<0.001; data presented as means and SEM, as well as scatter plots (Supplementary Table 1 contains detailed statistics). d-e, Typical X-ray images of Setd5+/− and Setd5+/+ adult animals; craniofacial maxillary/pre-maxillary abnormalities (d, arrowheads), vertebral fusion abnormalities (d, open arrow) and teeth abnormalities (e, arrowheads) are observed in Setd5+/− mice. The percentage of animals with each of the described phenotype is indicated as doughnut charts (n=13 mice of either genotype; data and pictures (as well as top eye picture in Fig. 1c) taken with permission from International Mouse Phenotyping Consortium, available at http://www.mousephenotype.org/data/genes/MGI:1920145).
a, Schematics of CRISPR/Cas9 strategy to generate the Setd5+/− ESC line; two CRISPR guides were used: one targeting the intronic region upstream of exon 3, the other targeting the intronic region downstream of exon 15. The homologous recombination template carries a Red Fluorescent Protein/Puromycin selection cassette flanked by two 1 kb homology arms identical to the regions upstream or downstream of the CRISPR/Cas9 cut sites (green dashed lines). b, Schematic representation of the differentiation process from ESCs to NPCs; LIF: leukemia inhibitory factor; RA: retinoic acid; c, Heatmaps of RNA-seq counts (z-Score) for up-regulated neurodevelopment-associated genes in Setd5+/− at 3 stages of in vitro differentiation. Setd5+/+;tr, CRISPR/Cas9 control lines (tr., transfected). d, Chord plot illustrating the overlap between the in vivo and in vitro DEG datasets. Ribbons show overlaps between sets and ribbon width scales with the number of overlapping genes. Remaining areas represent genes unique for each set. e, Gene ontology (GO) terms enriched in upregulated (top, bars to the right) and downregulated (bottom, bars to the left) differentially expressed genes (adjusted p-value < 0.05) between wild type and Setd5 heterozygous ESCs, EBs and NPCs. Bars show the negative decimal logarithm of the enrichment p-value (shown as negative for downregulated genes). (n=2 wildtype cell lines, 2 CRISPR control lines, and 2 mutant Setd5+/− cell lines, one-sided fisher`s exact test).
a, Setd5 gene expression in brains of wild type animals at different developmental stages (E16.5, P1, P21, adult) assessed by RT-PCR; Hprt was used as control. b, Setd5-GFP reporter expression in coronal brain sections of E12.5 Setd5GFP embryos and control (Setd5WT), Setd5-GFP is expressed throughout the developing cortex (boxed region and close-up; dashed line: ventricular wall). c, Representative coronal sections of different brain regions of adult control (Setd5WT) and Setd5GFP animals. Broad GFP expression can be observed in several brain regions: cortex (Ctx), hippocampus (Hipp), olfactory bulb (OB) and cerebellum (Crbl). d, Setd5-GFP positive cells co-localize with NeuN-positive neurons, parvalbumin (PV)-positive interneurons and glial fibrillary acidic protein (GFAP)-positive astrocytes in the adult cortex. e, Fluorescent in-situ hybridization (n= one experiment) employing probes against Setd5 exon III-VI and Setd5 exon XIII in adult C57BL/6J sections show nuclear signal and co-localization with Emx1. DAPI nuclear staining (shown in blue) can be seen in (b-e). n=1 Setd5WT and n=3 Setd5GFP. Scale bars: 150 µm in (b); 25 µm in (b, close-up); 50 µm in (c) (Ctx); 200 µm in (c) (Hipp); 25 µm in (c) (OB); 200 µm in (c) (Crbl); 20 µm in (d) (all) and 50 µm in (e). Experiments (a-d) were repeated at least three times, independently, with similar results, using 3 animals per genotype per developmental stage.
a-f, Representative images (a-c) and quantifications (d-f) of coronal forebrain sections and immunofluorescent stainings at E12.5; dashed line: ventricular wall. a,d, Two hour BrdU incorporation is similar in Setd5+/− and their wild type littermate embryos; b,e, Very low numbers of cleaved Caspase3-positive cells can be found in the developing forebrain of both genotypes (n(mouse embryos)=3 per genotype); c,f, The number of Pax6-positive radial glia in the ventricular zone and Tuj1-positive immature neurons in the developing cortical plate is not changed upon heterozygous loss of Setd5 (n(mouse embryos)=5 per genotype). g, Nissl staining on sagittal sections reveals normal morphology of the adult Setd5+/− brain (n=3 animals per genotype); h-i, Representative images and quantification of the lamination in coronal sections of the somatosensory cortex of Setd5+/+ and Setd5+/− adult brain as assessed by staining for Cux1 positive upper layer and Ctip2 positive lower layer neurons (n=3 animals per genotype); data presented as means and SEM, as well as scatter plots; Scale bars: 25 µm in (a-c); 1 mm in (g); 100 µm in (h), wm: white matter; n.s., not significant, unpaired, two-sided t test, exact p-values and detailed statistics provided in Supplementary Table 1.
a, Normal exploratory behaviour in the open field. Representative trajectories (a, left), quantifications (a, centre) of velocity, total distance moved, total time spent in the centre (also as measure of anxiety) as well as the distance moved in 5 min bins (a, right) by wild type (grey, Setd5+/+) and mutant (red, Setd5+/−) mice. b, Lack of repetitive behaviours in the open field (b, left) and in marble burying within 30 min (b, right). c, Likewise to the controls, Setd5+/− mice prefer to interact more with a mouse than with an object (normal sociability; left) and favour sniffing a novel mouse over a familiar one (normal preference for social novelty; right) in the three chamber sociability test (a-c, n=25 age and sex-matched littermate pairs of Setd5+/+ and Setd5+/−); d, Performance of male (upper panels) and female (lower panels) Setd5+/− mice in the elevated plus maze (total duration 6 min); Quantification of the total time spent in the closed and open arm as well as arm entries point out natural levels of anxiety, largely identical to the wild type littermates (n=16 male and 10 female age- and sex-matched littermate pairs of wild type and Setd5 heterozygous animals); e, Nest scores showing normal nesting abilities in Setd5+/− males; n=9; f, Isolation-induced ultrasonic vocalisation profile of Setd5+/+ and Setd5+/− male mouse pups, indicating normal number of calls within the first 6 days, followed by failure of mutants to extinguish this type of communication (n=23 male mouse pups per genotype). Data (a-e) presented as means and SEM and scatter plots; boxplots (f) indicate medians (middle line), 25%-75% interquartile range (box) and minimum and maximum data point (whiskers). ***P<0.001; *P<0.05; n.s., not significant (detailed statistics in the Supplementary Table 1); For behavioural tests, in which no inter-genotypic differences were observed, the results of both sexes are presented together, although initially analysed separately. However, for tests in which inter-genotypic differences were discovered, the results are presented for each sex separately.
a, Setd5+/− female mice in the Intellicage show more nose pokes per visit (left) but perform significantly less unintentional (no licks) visits with nose pokes (middle) while displaying similar number of licks per visit (right) as compared with the controls (means, SEM and individual data points, n=14 females per genotype). b, Similar freezing behaviour curves during fear memory acquisition (training day, 3 shocks) exclude differences in the pain/sensorial perception between the two genotypes (n=18 adult females per genotype). c, Contextual fear conditioning (CFC) in male mice (strong training, 3 shocks, 5 min total exposure to context, n=15 males per genotype) indicates inability of mutants to extinguish the acquired fear memory when the context becomes a safe environment. Boxplots (c, d) indicate medians (middle line), 25%-75% interquartile range (box) and minimum and maximum data point (whiskers). d, Weak training contextual fear conditioning in male mice (1 shock, 150 s total exposure to context, n=7 males per genotype) indicates that mutants learn the context-shock association after sub-threshold training, and further fail to extinguish it (10 min extinction training in both weak and strong CFC); e, Subthreshold training (3 min) in novel object location memory test induces abnormal memory retention in Setd5 heterozygous males (n=13 males per genotype). Data is presented either as means and SEM, as well as scatter plot; *P<0.05; **P<0.01; ***P<0.001; n.s., not significant; f, Averaged traces (mean ± SEM) indicating the dynamics of fEPSP slope before and up to 5h after LTP induction by high frequency stimulation (HFS, min 0): enhanced late-LTP in the mutants; n=6 (5h), 8 (4h), 10 (3h), 10 (1h) slices for each genotype; one slice per mouse; males, P21-24. g, Similar averaged input-output curves (8 dorsal hippocampus slices from 8 animals per genotype, left panel) and likewise paired-pulse ratios (11 dorsal hippocampus slices from 3 animals per genotype), indicating no difference in the presynaptic function at the hippocampal CA3 to CA1 synapses of P21-24 male Setd5+/+ and Setd5+/- mice. For detailed statistics please refer to Supplementary Table 1.
a, Volcano plots highlighting Creb1 target genes in the gene expression changes 1h or 3h after conditioning in wild type and mutant animals. The y-axis shows the negative decimal logarithm of the adjusted p-value and the binary logarithm of the fold change is shown on the x-axis; pink, differentially expressed Creb1 target genes; black, other differentially expressed genes; grey, non-significant genes (n(mice)=4 control and 5 mutant animals, EdgeR, likelihood ratio test). b, Example of a DEG cluster showing the same CFC response in Setd5+/+ and Setd5+/− mice (see Methods). Genes listed in the top right corner are early response genes contained in this cluster. Thick lines, average of all genes per cluster; thin lines, single genes; red, Setd5+/−; grey, Setd5+/+; HC, n=5; 1h, n=4; 3h, n=5 per genotype respectively.
a-b, Immunoblots and quantifications evaluating histone H3 methylation states in Setd5 mutant hippocampal and ESC samples. (a) n(mice)=5, N(independent experiments)=3 (b) n(independent cell lines Setd5+/− and Setd5−/−)=2 and 3 respectively, N(independent experiments)=3. No difference in the levels of H3K4-1me, H3K4-2me, H3K4-3me. H3K9-2me, H3K36-3me or H3K36-2me was observed. Data presented as means and SEM (a) or SD (detailed statistics in the Supplementary Table 1) (b). c, Sequence alignment of human SET domain containing proteins. SETD5 and its closest homolog KMT2E (MLL5) are aligned to 13 characterized histone methyltransferase enzymes. Colour scheme: yellow, amino acids involved in SAM binding; blue, amino acids involved in histone H3 recognition. Amino acids in red are the 5 residues in SETD5 that differ the most from the consensus SAM binding motif. The specific histone H3 lysine residues methylated by the characterised SET domains are indicated on the left. d, Schematics of the endogenously C-terminus tagged Setd5. e, PCR genotyping of a successful insertion of the FLAG-HA tag construct (Setd5+/+;HA) compared to a Setd5+/+ control line; FLAG-HA was inserted only on one allele. Experiments were repeated three times, with similar results. f, Western blot results showing the successful tagging of endogenous Setd5. To show that our CRISPR/Cas9 strategy was capable of knocking out Setd5, the two single RNA guides used to generate Setd5−/− (Supplementary Fig. 11a-b) were used on a Setd5+/HA background line and the western blots show complete loss of the Setd5-HA band. Experiments were repeated four times, with similar results. g, Western blot of Setd5-HA immunoprecipitation in non-tagged Setd5+/+ and tagged Setd5+/HA. Membranes blotted for Setd5-HA (left), Tbl1x (right). Arrowheads: Setd5-HA and Tbl1x respectively; asterisk: non-specific band; IN, input: nuclear extract before immunoprecipitation; IP, immunoprecipitation eluate with triple-FLAG peptide; 0.5% of IN and 25% of IP eluate were loaded. Experiment was performed once. h, Volcano plot of proteins identified by quantitative TMT mass spectrometry from Setd5 immunoprecipitation in ESCs and NPCs. Significant proteins (adjusted P value < 0.05 and log2 Fold Change > 0) in red, not significant proteins in grey. Proteins associated with ASD in underlined italics. (n=2 wild type and 2 Setd5 FLAG/HA knock in line, limma statistical analysis).
a, Schematics of the localization of de novo SETD5 mutations in ID and ASD patients; each mutation is colour-coded based on the study reporting the de novo mutation; mutations used in this study are highlighted in bold. b-d, Representative example of the bulk enrichment sort and single cell sorting strategy. Shape analysis scatter plots (b). To exclude doublets, live cells are gated on forward scatter/ side scatter (FSC/SSC, left) followed by SSC (middle) and FSC (right). Scatter plots (c) of primary bulk enrichment for Setd5-P2A-GFP and CRISPR/Cas9-T2A-Cerulean3 positive cells (right) compared to WT non-transfected control (left). Scatter plots (d) showing enrichment for Setd5-P2A-GFP cells over the course of the secondary bulk enrichment sort (middle) and tertiary single cell sort (right). Auto-fluorescence was measured to ensure GFP positive cells.
a, Schematics of the CRISPR/Cas9 strategy to generate the ΔSET Setd5+/−and Setd5−/− ESC line; two CRISPR guides were used: one targeting the intronic region upstream of exon 7, the other targeting the intronic region downstream of exon 8. b, Schematics of WT and SET domain deleted Setd5 proteins. c, Western blots and quantification of histone post-translational modification in Setd5+/+, Setd5+/− and Setd5−/− ESCs, showing hyperacetylation of histone H4 in Setd5−/−; n(cell lines)= 3 for Setd5+/+ and Setd5−/− and n=2 for Setd5+/−; d-f, Western blot images and quantifications showing H4K8, H4K5, H4K12 and H3 pan acetylation in Setd5+/− CA hippocampal regions dissected from homecage animals (d), 3 hours after CFC training (e) and 1 hour after CFC (f). The band intensity of each acetylated protein residue was normalized to the corresponding total histone level (n(mice)=5 per genotype, N(independent experiments)=3, per time point, respectively). g, H4K8ac dynamics in CA hippocampal samples obtained from homecage and CFC-trained mice showing H4K8 acetylation is significantly higher in samples from homecage (p-value= 0.054) and 3 hours post-training (p-value = 0.009) animals (levels normalized to WT H4K8ac; n(mice)=5 per genotype, n(independent experiments)=3, per timepoint respectively; (two-sided t-test for each timepoint) h, Hdac3 activity assay in brain samples from naïve Setd5+/+ and Setd5+/− mice (n=7). i, Bar plots (means) of de-acetylation activity assay using samples from Hdac3 IP in Setd5+/+ and Setd5−/− ESCs. Dots represent individual technical replicates; n(independent enzymatic reactions)=2 in DMSO and 3 in presence of Trichostatin A (TSA), broad HDAC inhibitor, used as control to show signal specificity towards de-acetylation, data means are shown). Data in c-h presented as means and SEM, **P<0.01; n.s., not significant; detailed statistics in the Supplementary Table 1.
Supplementary Figure 12 ChIP-seq binding profile and differential binding analysis at TSS of Hdac3, Pol II, and H3K27ac.
a-c, Boxplots of input normalised ChIP-seq in Setd5+/+, Setd5+/−, and Setd5−/−. Signal for 4 different genomic regions: Transcription Start Sites (TSS, n=34159) Gene bodies (n=34151), transcription end sites (TES, n=34159), intergenic ESCs specific enhancers (n=4099) for Hdac3 (a), Pol II (b), and H3K27ac (c). d-f, MA plot of differential binding analysis for Setd5+/− (left) and Setd5−/− (right) compared to Setd5+/+ at TSS using peaks of Hdac3 (d), Pol II (e), and H3K27ac (f). EdgeR likelihood ratio test. g, Boxplots of input normalised ChIP-seq RPKM in Setd5+/+, Setd5+/HA, Setd5+/−, and Setd5−/−. Signals of Setd5-HA, Pol II, Hdac3, H3K27ac and H4 pan acetylation for 182 genes that show significant (FDR < 0.05) decrease RNA-Pol II binding in Setd5+/− at TSS. The presence of H3K27ac signal indicates expressed genes. h, Boxplots of ChIP-seq RPKM signal in Setd5+/+, Setd5+/−, and Setd5−/− for 2 different genomic features: TSS having at least one H4 pan acetylation peak (n=3018) and H4 pan acetylation peaks within gene bodies (n=43448 peaks in 9088 gene bodies). TSS is defined as +/- 1.25 kb around gene start position; one-sided Mann–Whitney U test. Boxplots in a, b, c, g, and h represent mean, normalized by input Reads Per Kilobase of genomic regions, per Million mapped reads (RPKM) values of 2 biological replicates from two independent cell lines. Input levels are equal to 1. Outliers, less than 5 or more than 95 percentile of mean RPKM, were removed. The upper, centre, and lower line of the boxplot indicates 75%, 50%, and 25% quantile, respectively. Whiskers extend to the most extreme data point within 1.5-times the interquartile range.
ChIP-seq tracks of Setd5-HA in non-tagged (Setd5+/+) and HA-tagged (Setd5+/HA) ESCs and ChIP-seq tracks of Pol II, Hdac3, and H3K27ac in Setd5+/+, Setd5+/−, Setd5−/− ESCs at the Meis2 and Neurog1 gene locus. Presented signals are input-subtracted, merged replicates, Reads per Genomic Content (RPGC). (n=2 independent ChIP biological replicates and n=3 independent input biological replicates for Pol II, Hdac3, and H3K27ac ChIP-seq and n=1 independent input sample for Setd5-HA ChIP-seq with similar results).
Dashed orange boxes indicate radioactive film-, gel- and western blot-fragments presented in the respective figure panels.
Supplementary Figures 1–14
Differentially expressed gene analysis.
Gene Ontology (GO) term enrichment analysis.
Differentially expressed gene analysis for CFC samples.
GO term cluster analysis.
Setd5 mass spectrometry results.
PolII ChIP-seq analysis.
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Deliu, E., Arecco, N., Morandell, J. et al. Haploinsufficiency of the intellectual disability gene SETD5 disturbs developmental gene expression and cognition. Nat Neurosci 21, 1717–1727 (2018). https://doi.org/10.1038/s41593-018-0266-2
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