Krüppel-like Transcription Factor 7 Is a Causal Gene in Autism Development

Abstract

Background: Autism spectrum disorder (ASD) is a complex neurodevelopmental disease. To date, more than 1000 genes have been shown to be associated with ASD, and only a few of these genes account for more than 1% of autism cases. Klf7 is an important transcription factor of cell proliferation and differentiation in the nervous system, but whether klf7 is involved in autism is unclear.

Methods: We first performed ChIP-seq analysis of klf7 in N2A cells, then performed behavioral tests and RNA-seq in klf7^+/- mice, and finally restored mice with adeno-associated virus (AAV)-mediated overexpression of klf7 in klf7^+/- mice.

Results: Klf7 targeted genes are enriched with ASD genes, and 631 ASD risk genes are also differentially expressed in klf7^+/- mice which exhibited the core symptoms of ASD. When klf7 levels were increased in the central nervous system (CNS) in klf7^+/- adult mice, deficits in social interaction, repetitive behavior and majority of dysregulated ASD genes were rescued in the adults, suggesting transcriptional regulation. Moreover, knockdown of klf7 in human brain organoids caused dysregulation of 517 ASD risk genes, 344 of which were shared with klf7^+/- mice, including some high-confidence ASD genes.

Conclusions: Our findings highlight a klf7 regulation of ASD genes and provide new insights into the pathogenesis of ASD and promising targets for further research on mechanisms and treatments.

Keywords: ASD; human brain organoids; klf7; klf7+/− mice; regulatory gene.

Figure 1

Klf7 targeted genes are enriched for ASD risk genes. (A–C) The klf7 level in the prefrontal cortex (PFC) of ASD patients according to human brain single-cell RNA-seq (n = 16 control, n = 15 ASD patients). Klf7 expression decreased significantly in ODCs (log2FC = −0.34968, adjusted p = 3.89 × 10⁻⁸), astroglia (log2FC = −0.42086, adjusted p = 1.09 × 10⁻⁵) and projection neurons (log2FC = −0.3741, adjust p = 5.94 × 10⁻¹¹). (D) Shared genes between human klf7 target genes and SFARI autism database. The number of ASD risk genes from SFARI database bound by klf7 is shown, and the total number in each subset is presented in parentheses. (E) Functional enrichment analysis of ASD risk genes targeted by human klf7. These ASD risk genes were mainly enriched in processes related to long-term potentiation, circadian rhythm and synapse part. (F) Klf7-binding sites in two biological replicates of N2A cells relative to the IgG control. The peak number of biological replicates is shown in the Venn diagram. (G) Functional annotation of all klf7 target genes. All genes were enriched in processes related to CNS development and neurons. (H) Shared genes between SFARI autism database and klf7 target genes in N2A cells. The number of ASD risk genes in the SFARI database bound by klf7 is given, and the total number in each subset is presented in parentheses. (I) Functional enrichment analysis of ASD risk genes targeted by klf7 in N2A cells. These ASD target genes were enriched for functions related to neurons and synapses. ASD, Autism spectrum disorder; klf7, Krüppel-like transcription factor 7; ODCs, oligodendroglial cells; log2FC, log2(Fold Change); SFARI, Simons Foundation Autism Research Initiative; CNS, central nervous system.

Figure 2

Loss of klf7 causes the mice to develop the core symptoms of ASD. (A–F) Three-chamber experiment. (A) All genotype mice had no preference for either of the two empty cages. (B) Whereas WT mice spent more time in the stranger mouse-containing caged chamber than in the empty cage-containing chamber, klf7^+/− mice had no preference for either chamber. (C) Klf7^+/− mice interacted with the stranger mouse for a shorter amount of time than WT mice. (D) Whereas WT mice spent more time in the chamber containing the novel mouse than in the chamber containing the familiar mouse, klf7^+/− mice had no preference for either chamber. (E) Klf7^+/− mice interacted with the novel mouse for a shorter period of time than WT mice did. (F) Representative tracks show the time spent in different phase of three-chambers social interaction experiment for WT mice and klf7^+/− mice. (G-H) Klf7^+/− mice exhibited stereotypical behaviors. (G) In the self-grooming test, klf7^+/− mice spent more time in self-grooming than WT mice. (H) In the Y maze spontaneous selection experiment, klf7^+/− mice preferred the original target arm, while WT mice preferred to explore the novel arm. The result is indicated by the ratio of entries into the original target arm to the entries into the novel arm in 10 trials. The data are presented as the mean ± SEM. n = 12 for WT and klf7^+/− mice. * p < 0.05, ** p < 0.01, and *** p < 0.001 by two-way ANOVA and unpaired t test; klf7, Krüppel-like transcription factor 7; ASD, autism spectrum disorder; WT, wild type.

Figure 3

ASD risk genes are dysregulated in klf7^+/− mice. (A) Some klf7 target ASD risk genes were strongly dysregulated in klf7^+/− mice compared with WT mice. Of the 228 ASD risk genes targeted by klf7, the expression levels of 43 genes were significantly altered, with 23 being upregulated and 20 being downregulated (log2FC > 1 or log2FC < −1, false discovery rate (FDR) < 0.1). (B) A total of 631 genes were both identified as DEGs in klf7^+/− mice and found in the SFARI database. The number of ASD risk genes in the SFARI database disrupted by klf7 loss is indicated, and the total number in each subset is presented in parentheses. (C) Of the 631 autism genes in (B) that were disrupted by klf7 deletion, 98 were significantly upregulated (log2FC > 1), 116 were significantly downregulated (log2FC < −1), and 417 showed minimal changes (−1 < log2FC < 1). (D) The 631 autistic genes disrupted in klf7^+/− mice shown in (B) were functionally annotated. n = 4 for A; klf7, Krüppel-like transcription factor 7; ASD, autism spectrum disorder; DEGs, differentially expressed gene; SFARI, Simons Foundation Autism Research Initiative; log2FC, log2(Fold Change); TFs, transcription factors.

Figure 4

Increasing klf7 levels rescue the core symptoms of ASD in klf7^+/− mice. (A,B) Histogram showing the amount of time spent in the chamber containing either the stranger mouse or the novel mouse and the amount of time spent interacting with the stranger mouse or novel mouse by AAV-control-injected WT mice, AAV-control-injected klf7^+/− mice, AAV-klf7-injected WT mice and AAV- klf7-injected klf7^+/− mice in the three-chambers sociability test. Whereas klf7^+/− mice in the AAV-control group did not show an obvious social preference, klf7^+/− mice in the AAV-klf7 group showed improved social ability. Notably, elevating klf7 levels rescued the abnormal social ability of klf7^+/− mice. (C) Histogram showing the amount of time the AAV control-injected WT mice, AAV control-injected klf7^+/− mice, AAV- klf7-injected WT mice and AAV-klf7-injected klf7^+/− mice spent grooming in the 10 min self-grooming test. Klf7^+/− mice in the AAV-control group displayed an increased grooming time, indicating repetitive behavior. The abnormal grooming behavior was alleviated in the AAV-klf7 group. (D) Summary graph showing autism-related genes dysregulated after klf7 deletion whose expression was restored by elevating klf7 levels in klf7^+/− mice. The data are the mean ± SEM. n = 12 per group in A-C. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: no significance by one-way ANOVA and two-way ANOVA; klf7, Krüppel-like transcription factor 7; ASD, autism spectrum disorder; AAV, adeno-associated virus; WT, wild type.

Figure 5

Klf7 knockdown in a human induced pluripotent stem cell (hiPSC)-derived organoid model. (A) Klf7 knockdown efficiency measured by qPCR (n = 3) and Western blot (n = 3) in organoids (90 days after differentiation). (B) The morphology of organoids in the control group and klf7 knockdown group after 90 days of differentiation. Klf7 knockdown significantly decreased the diameter of organoids; the average diameter of the control group was 750.8 μm, and the average diameter of the klf7 knockdown group was 734.8 μm. n = 20. Scale bar = 500 μm. (C) Genes shared between the SFARI autism database, klf7^+/− mice and klf7 knockdown organoids. The total number in each subset is noted in parentheses. (D,E) Of the 517 ASD risk genes that were dysregulated after klf7 deletion in hiPSC-derived organoids, 134 showed significantly upregulated expression (log2FC > 1), 362 showed significantly downregulated expression (log2FC < −1), and 21 showed minimal changes (−1 < log2FC < 1). (F) ASD risk genes (including 14 that showed upregulated expression and 45 that showed downregulated expression, adjusted p < 0.01) found to be dysregulated in klf7 knockdown organoids and identified as high-confidence genes in the ASD population by exome sequencing are indicated in the volcano plot. Some high-confidence ASD risk genes are shown. (G) ASD risk genes (including 18 that showed upregulated expression and 40 that showed downregulated expression, adjusted p < 0.01) dysregulated in klf7 knockdown organoids and identified as high-confidence genes by Willsey et al. are indicated in the volcano plot. Some high-confidence ASD risk genes are shown. (H–J) The plot shows ASD-related DEGs in klf7 knockdown organoids whose expression patterns were similar to those the brains of ASD patients. The data are presented as the mean ± SEM. n = 12 for A. * p < 0.05, ** p < 0.01 by unpaired t test. hiPSCs, human induced pluripotent stem cells; klf7, Krüppel-like transcription factor 7; qPCR, quantitative polymerase chain reaction; ASD, autism spectrum disorder; SFARI, Simons Foundation Autism Research Initiative; log2FC, log2(Fold Change); DEGs, differentially expressed genes.