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. 2023 Apr 19;146(4):1403-1419.
doi: 10.1093/brain/awac352.

Genetic regulatory and biological implications of the 10q24.32 schizophrenia risk locus

Junyang Wang  1 , Jiewei Liu  1 , Shiwu Li  1 , Xiaoyan Li  1 , Jinfeng Yang  1 , Xinglun Dang  1 , Changgai Mu  2 , Yifan Li  1 , Kaiqin Li  1 , Jiao Li  1 , Rui Chen  1 , Yixing Liu  1 , Di Huang  1 , Zhijun Zhang  3   2   4 , Xiong-Jian Luo  3   2
Affiliations

Genetic regulatory and biological implications of the 10q24.32 schizophrenia risk locus

Junyang Wang et al. Brain. .

Abstract

Genome-wide association studies have identified 10q24.32 as a robust schizophrenia risk locus. Here we identify a regulatory variant (rs10786700) that disrupts binding of transcription factors at 10q24.32. We independently confirmed the association between rs10786700 and schizophrenia in a large Chinese cohort (n = 11 547) and uncovered the biological mechanism underlying this association. We found that rs10786700 resides in a super-enhancer element that exhibits dynamic activity change during the development process and that the risk allele (C) of rs10786700 conferred significant lower enhancer activity through enhancing binding affinity to repressor element-1 silencing transcription factor (REST). CRISPR-Cas9-mediated genome editing identified SUFU as a potential target gene by which rs10786700 might exert its risk effect on schizophrenia, as deletion of rs10786700 downregulated SUFU expression. We further investigated the role of Sufu in neurodevelopment and found that Sufu knockdown inhibited proliferation of neural stem cells and neurogenesis, affected molecular pathways (including neurodevelopment-related pathways, PI3K-Akt and ECM-receptor interaction signalling pathways) associated with schizophrenia and altered the density of dendritic spines. These results reveal that the functional risk single nucleotide polymorphism rs10786700 at 10q24.32 interacts with REST synergistically to regulate expression of SUFU, a novel schizophrenia risk gene which is involved in schizophrenia pathogenesis by affecting neurodevelopment and spine morphogenesis.

Keywords: SUFU; dendritic spine density; functional risk variant; rs10786700; schizophrenia.

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Conflict of interest statement

The authors report no biomedical financial interests or potential conflicts of interest.

Figures

Figure 1
Figure 1
rs10786700 shows robust association with SCZ. (A and B) The locus zoom plots show the associations between variants near rs10786700 (500 kb) and SCZ in European and East Asian populations. rs10786700 shows genome-wide significant association with SCZ in both European and East Asian populations.
Figure 2
Figure 2
Validation of the enhancer regulatory effect of rs10786700. (A) rs10786700 is located in the last intron of the Sufu gene. (B) rs10786700 is located in an enhancer element (marked with H3K27ac, H3K4me1 and H3K9ac signals) in human foetal brain. Data were from Roadmap (http://www.roadmapepigenomics.org/). (C) rs10786700 is located in an open chromatin region (i.e. actively transcribed) marked with strong DNase-seq and histone H3K27ac signals in human neuroblastoma cells (SK-N-SH). (D) Position weight matrix (PWM) and FIMO analyses showed that rs10786700 affects REST binding in SK-N-SH cells. (E–G) Dual-luciferase reporter gene assays validated the enhancer activity of rs10786700. The genomic sequence containing rs10786700 exhibited enhancer activity compared with control vector in SH-SY5Y and SK-N-SH cells (but not HEK293T). Of note, the risk allele (C allele) of rs10786700 conferred significant lower luciferase activity compared with the T allele in SH-SY5Y and SK-N-SH cells. Values on the y-axis represent relative reporter gene activity (i.e. the luciferase activity of pGL3 promoter vector containing the cloned sequence relative to the pRL-TK (internal control) vector). n = 8 for control group, n = 16 for experimental groups. (H) REST overexpression significantly repressed the luciferase activity of the genomic fragment containing rs10786700. n = 8. (I) EMSA showed the preferential binding of nuclear extracts to C allele of rs10786700. The loaded amounts of nuclear extracts were 0, 1.5, 3.0 and 4.5 μg, respectively (from left to right). Four binding bands were detected, and the risk allele (C allele) of rs10786700 shows stronger binding affinity to nuclear extracts than the T allele for binding band 3. (J) Super-shift and competitive experiments for EMSA. The binding specificity of rs10786700 to transcription factors was verified by competitive experiments (using unlabelled probes). Super shift experiments were performed to detect the binding of rs10786700 to REST by adding 0.8 μg REST antibody; 6.0 μg total nuclear extracts were added to each lane. The amount of labelled probe was 50 fmol per lane. (I and J) Unpaired two-tailed Student’s t-test; data are presented as mean ± SD; NS, not significant.
Figure 3
Figure 3
rs10786700 regulates SUFU expression by dynamically modulating the activity of the SE in neurodevelopment. (A) DNase-seq and histone modifications for genomic region surrounding rs10786700 (1 Mb centred on rs10786700) showed that rs10786700 is located in an actively transcribed genomic region. Of note, ChIP-Seq data showed that EP300 and REST bind to the genomic sequence containing rs10786700 in a development-dependent manner. In early neurodevelopment stage, the ChIP-seq signals of EP300 and REST were high. As development progresses, the ChIP-seq signals decrease gradually. (B) Position weight matrix (PWM) and FIMO analyses showed that rs10786700 disrupts binding of EP300 in SK-N-SH cells. (C) The amounts of nuclear extracts were 0, 3 and 6 μg, respectively (from left to right). Four binding bands were observed. The risk allele (C allele) of rs10786700 showed stronger binding affinity to nuclear extracts than the T allele for binding band 3. (D) Super-shift experiments validated the binding of rs10786700 to EP300. (E) A 379 bp fragment containing rs10786700 (located in the 11th intron of SUFU gene) was knocked out by CRISPR/Cas9-mediated editing in SH-SY5Y and U251 cells. (F) qPCR showed that rs10786700 knock-out resulted in significant downregulation of SUFU expression in both SH-SY5Y and U251 cells. (G) qPCR validation of SUFU expression in EP300 knocked-down SH-SY5Y cells. n = 3 for F and G. Unpaired two-tailed Student’s t-test; data are presented as mean ± SD. (H) Expression patterns of SUFU in the developing and adult human frontal cortex. Expression level of SUFU across the entire developing stages (from 8 pcw to 40 years) were depicted in different areas of the frontal cortex. The expression data (42 human subjects) were from BrainSpan (http://www.brainspan.org/). Pcw, post-conception weeks; yrs, years; DFC, dorsolateral prefrontal cortex; MFC, medial prefrontal cortex; OFC, orbital prefrontal cortex; VFC, ventrolateral prefrontal cortex. (I–L) The Pearson expression correlations between SUFU and EP300 in the human brain. (I) Correlations in prenatal human brain (LIBD dataset). (J) Correlations in prenatal human brain (expression data from Walker et al.). (K) Correlations in childhood human brain. (L) Correlations in adulthood human brain. Sample group: prenatal (age < 0), child (0 < age < 18), adult (age > 18), non-prenatal (age > 0).
Figure 4
Figure 4
Sufu knockdown inhibits proliferation and neurogenesis of mNSCs. (A) Immunofluorescence staining results of three well-characterized markers (SOX2, PAX6 and NESTIN) for neural stem cells confirmed the identity of the isolated mNSCs. (B) Immunofluorescence staining for BrdU incorporation assay. Red indicates BrdU-positive cells undergoing DNA amplification and DAPI was used to stain the nucleus (blue). (C) Sufu expression was efficiently knocked-down by the shRNA in mNSCs. (D) The quantification results of the BrdU incorporation assay. (E) The results of CCK-8 assay. Data were collected at 0, 1, 2 and 3 days after plating. (F) Immunofluorescence staining images for astrocyte cells (GFAP-positive cells) differentiated from mNSCs. (G) Quantification data for the ratio of GFAP-positive cells. Sufu knockdown led to a significant increase of GFAP-positive cells. (H) Immunofluorescence staining images for mature neurons (MAP2-positive cells) differentiated from mNSCs. (I) Quantification data for the percentage of MAP2-positive cells in total cells. Sufu knockdown resulted in a significant decrease of MAP-positive cells. n = 3 for B–D, F–I; n = 9 for E. mNSCs, mouse neural stem cells. Unpaired two-tailed Student’s t-test; data are presented as mean ± SD. NS, not significant.
Figure 5
Figure 5
Sufu regulates SCZ-associated biological processes and signalling pathways. (A) Expression heat map of 860 DEGs identified in Sufu knockdown mNSCs compared with control mNSCs. (B) Heat map plot of the top 30 DEGs. Five genes (marked by red colour), including Gpr17, Anks1b, Ppp2r2s, Col5a3 and Cbs, were selected for qPCR verification (C). (D) GO analysis based on biological process for the 860 DEGs. Terms marked in red indicate schizophrenia-associated biological processes. (E) KEGG analysis of the 860 DEGs. Terms marked in red indicate schizophrenia-associated signalling pathways. (F, G) Validation of PTCH1 expression in Sufu knockdown mNSCs with Western blot. n = 3 for C and G; unpaired two-tailed Student’s t-test; data are presented as mean ± SD.
Figure 6
Figure 6
Sufu regulates dendritic spine density. (A) Representative immunofluorescence staining images for the density and morphology of dendritic spines in Sufu knockdown neuron compared with control neuron. (B) Sufu expression in rat primary neurons was significantly knocked-down by shRNAs. (C) The result of dendritic spine density analysis for stubby, thin and mushroom spines. n = 36 for control, n = 34 for Sufu-Rat-shRNA#1, n = 40 for Sufu-Rat-shRNA#2. Unpaired two-tailed Student’s t-test; data are presented as mean ± SD.
Figure 7
Figure 7
The working model of rs10786700 in SCZ pathogenesis. (A) rs10786700 is located at 10q24.32 schizophrenia risk locus, a genomic region with multiple SNPs showing robust associations with SCZ. We confirmed the genetic association between rs10786700 and SCZ in a large Chinese cohort (3 718 cases, 7 829 controls; P = 1.56 × 10−04). Trans-ancestry meta-analysis indicated that rs10786700 was robustly associated with SCZ (66 467 cases, 701 733 controls; P = 1.25 × 10−25). (B) Molecular regulatory mechanisms of rs10786700. rs10786700 modulates the activity of the SE (where rs10786700 is located) by dynamically binding EP300 and REST during neurodevelopment. During early neurodevelopmental stages, the genomic region containing rs10786700 exhibits strong SE activity and rs10786700 regulates SUFU expression through affecting REST binding. As development progresses, the activity of the SE containing rs10786700 decreased gradually. The risk allele (C allele) of rs107867000 conferred significant lower SUFU expression by enhancing REST binding. (C) The function of Sufu in the CNS and the potential role of Sufu in SCZ pathogenesis. As a negative regulator of sonic hedgehog signalling pathway, Sufu plays an important role in brain development. Lower Sufu expression inhibits proliferation of mNSCs, promotes gliogenesis and represses neurogenesis. Of note, Sufu knockdown also affected dendritic spine density. Collectively, these results indicated that the functional variant rs10786700 may confer risk of SCZ by dynamically regulating expression of SUFU in the developing human brain, and dysregulation of SUFU contributes to SCZ pathogenesis by affecting neurodevelopment and spine morphogenesis (two characteristic features of schizophrenia pathophysiology).
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