Abnormal neocortex arealization and Sotos-like syndrome-associated behavior in Setd2 mutant mice

Abstract

Proper formation of area identities of the cerebral cortex is crucial for cognitive functions and social behaviors of the brain. It remains largely unknown whether epigenetic mechanisms, including histone methylation, regulate cortical arealization. Here, we removed SETD2, the methyltransferase for histone 3 lysine-36 trimethylation (H3K36me3), in the developing dorsal forebrain in mice and showed that Setd2 is required for proper cortical arealization and the formation of cortico-thalamo-cortical circuits. Moreover, Setd2 conditional knockout mice exhibit defects in social interaction, motor learning, and spatial memory, reminiscent of patients with the Sotos-like syndrome bearing SETD2 mutations. SETD2 maintains the expression of clustered protocadherin (cPcdh) genes in an H3K36me3 methyltransferase-dependent manner. Aberrant cortical arealization was recapitulated in cPcdh heterozygous mice. Together, our study emphasizes epigenetic mechanisms underlying cortical arealization and pathogenesis of the Sotos-like syndrome.

Fig. 1. Ablation of Setd2 causes abnormal area patterning of the cerebral cortex.

(A) Representative images of fixed P7 control and Setd2^Emx1-cKO brains. (B) Comparison of cortical area of hemispheres (blue in schematic). (C to E) Analyses of Lmo4 (C), Cad8 (D), and Rorβ (E) expression by whole-mount ISH on P7 control and Setd2^Emx1-cKO brains. Arrowheads indicate expression alterations. (F, H, and J) Lmo4 (F), Cad8 (H), and Rorβ (J) expressions in P7 control and Setd2^Emx1-cKO sagittal brain sections, with boxed somatosensory regions magnified on the right. (G, I, and K) Quantifications of normalized intensities of ISH signals in (F), (H), and (J). n = 3 brains for each genotype. (L and M) Left: 5-HT immunostaining on control (L) and Setd2^Emx1-cKO (M) tangential sections of P7 flattened cortices. Right: 5-HT reveals primary sensory areas. (N to Q) Measurements of 5-HT–stained tangential sections. n = 6 for control [n = 5 in (Q)] and n = 4 for Setd2^Emx1-cKO. Data are represented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Scale bars, 1 mm (A, C to E, F, H, J, L, and M) and 100 μm (magnified views in F, H, and J).

Fig. 2. Setd2 is essential for the formation of corticothalamo cortical circuits.

(A and C) Representative adult sagittal sections showing anterograde labeling of thalamocortical projections by injecting recombinant AAV-EYFP into the VL (ventral lateral nucleus of thalamus) (A) and the VPM (ventral posteromedial nucleus of thalamus) (C). Boxed regions are enlarged in right panels showing the thalamocortical projection terminated in cortices. (B and D) Quantifications of the fluorescence distribution in motor cortices (A) and somatosensory cortices (C). n = 3 brains for each genotype; data are presented as means ± SEM. (E) Schematic illustration showing anterograde labeling of corticothalamic axons to demarcate VL and VP (ventral posterior nucleus of thalamus) by injecting AAV-mCherry and AAV-EYFP into layer VI of M1 and S1 cortex. (F) Representative adult coronal sections indicating corticothalamic projections terminated in VL and VP, respectively, in control brains (left). The enlarged boxes displaying discrete axons in the IC. In Setd2^Emx1-cKO brains (right), corticothalamic axons from M1 and S1 fail to sort into VL and VP, with overlapping tracts in the IC. Cx, cortical cortex; Hp, hippocampus; LV, lateral ventricle; Str, striatum; IC, internal capsule. Scale bars, 100 μm.

Fig. 3. Setd2^Emx1-cKO mice exhibit defects in social interaction, motor learning, and spatial memory.

(A) Representative traces of a control mouse and a Setd2^Emx1-cKO mouse in the open-field arena. (B to D) Quantification of mobility time (B), number of entries into center (C), and time in the center (D) in the open-field test. (E) Representative tracing heatmap of a control mouse and a Setd2^Emx1-cKO mouse during three-chamber sociability test and novel sociability test. (F) Quantification of (E). (G and H) Immobility time in tail suspension (G) and forced swim test (H). (I) Time spent in open arms in elevated plus maze test. (J) Latency to find the hidden platform across training days. (K) Frequencies of platform crossing during a probe trial (without a platform) after training. (L and M) Distance moved (L) and velocity (M) during the probe trial. (N) Latency to fall during the rotarod test. Each point represents data derived from one mouse. Data are represented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; NS, not significant. n = 9 mice for control [n = 7 in (I)] and n = 11 mice for Setd2^Emx1-cKO.

Fig. 4. Pcdhαβγ haploinsufficient cortices display area patterning deficiency.

(A) E13.5 control and Setd2^Emx1-cKO dorsal cortices were subjected to RNA-seq transcriptome analysis. Fold change (log₂) of expressions of cPcdh genes in Setd2^Emx1-cKO cortices relative to control is shown. (B) Validation of Setd2 and cPcdh expression of adult control and Setd2^Emx1-cKO neocortices by qRT-PCR analysis (n = 3 mice for each genotype). (C) Generation and genotyping of Pcdhαβγ heterozygous mice. WT, wild-type; gRNA, guide RNA. (D, F, and H) ISH for Lmo4 (D), Cad8 (F), and Rorβ (H) mRNA expression in P7 control and Pcdhαβγ^+/− sagittal brain sections, with boxed somatosensory regions magnified on the right. (E, G, and I) Quantification of normalized intensity of ISH signals in boxed regions of (D), (F), and (H). The y axes represent relative positions from the pial surface to the ventricle surface, with cortices evenly divided into 500 bins. The intensity values are normalized to background. *P < 0.05, **P < 0.01, and ***P < 0.001. Data are represented as means ± SEM. Scale bars, 1 mm (D, F, and H), 100 μm (magnified views in D, F and H).

Fig. 5. H3K36me3 modifications are reduced in cis-regulatory sites of cPcdh genes in Setd2^Emx1-cKO cortices.

(A) H3K36me3 immunofluorescence on E11.5 (left) coronal and E13.5 (right) sagittal brain sections. Boxed regions were enlarged on the right. (B) ChIP-seq density heatmaps in E13.5 control (n = 2) and Setd2^Emx1-cKO (n = 2) cortices. Plots depicting H3K36me3 signals from 3 kb upstream of the transcription start site (TSS) to 3 kb downstream of the transcription end site (TES). Each line in a heatmap represents one gene. (C) Genomic structure of cPcdh genes on mouse chromosome 18. Deoxyribonuclease (DNase) I hypersensitive sites (HS) are indicated as black arrows. (D) H3K36me3 levels at three HS elements in E13.5 control and Setd2^Emx1-cKO cortices. The green frame shows the HS position and size. (E) ChIP-qPCR analyses showing DNMT3A/B binding to the HS region and promoters of cPcdh locus in E13.5 dorsal cortices. Quantification of enrichment was displayed as fold enrichment over immunoglobulin G (IgG) controls (n = 3 animals for each genotype). *P < 0.05 and **P < 0.01. Data are represented as means ± SEM. Scale bars, 100 μm. CoP, commissural plate.

Fig. 6. The H3K36me3 methyltransferase activity of SETD2 is required for the transcription activation of cPcdh.

(A) Western blots for 293T^SETD2-KO cells transduced with pCIG (vector), the full-length human SETD2 (SETD2^FL), the SET domain–deleted human SETD2 (SETD2^∆SET), and a point mutation of human SETD2 (SETD2^L1815W), and 293T cells transduced with pCIG as the control. Histone H3 is used as loading control. (B) Schematic overview of the in utero electroporation (IUE) strategy for Setd2^fl/fl embryos. Animals were euthanized and analyzed at E18.5. (C) Schematic diagram of experimental setup for measuring genes expressions in electroporated cortical cells. FACS, fluorescence-activated cell sorting; cDNA, complementary DNA. (D) qRT-PCR analyses on sorted EGFP⁺ cells from electroporated E18.5 cortices. Four embryos used for pCAG-Cre-IRES-EGFP, pCAG-Cre-IRES-EGFP/pCAG-SETD2^FL and pCAG-Cre-IRES-EGFP/pCAG-SETD2^∆SET IUE. Three embryos use for pCAG-Cre-IRES-EGFP/pCAG-SETD2^L1815W IUE. Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. (E) Working model. Histone H3 trimethylation at lysine-36 mediated by SETD2 is required for expression of cPcdh genes, cortical area patterning, proper establishment of corticothalamic circuits, and subsequent social behaviors. Data are represented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.