The Role Of Transcription Factor Klf7 On The Regulation Of Autism Risk Genes

Autism spectrum disorder（ASD）is a complex neurological disease which affects approximately 1 in 68 children.ASD is a heterogeneous disorder with a heritability of 60%-90%.To date,more than 1000 genes have been shown to be associated with ASD,but the mechanism remains poorly understood.A major challenge is the polygenic nature of ASD.Therefore,it is necessary to identify the core gene networks associated with the disease,as well as possible network master regulators,and if they exist,they are more likely to be used for therapeutic interventions in ASD.ASD risk genes are mainly preferentially expressed in fetal brain or developing cortex and are interrelated at the transcriptional level,which indicates that transcriptional regulatory factors that are active during fetal development may be associated with ASD.Klf7 is a mammalian zinc finger transcription factor（TFs）that is highly active in the developing central nervous system.Klf7 was proposed as a candidate gene for ASD,and it may be an important gene for ASD.We may be getting close to the core of ASD.In this study,we first analyzed the expression pattern of klf7 in mouse,human and human brain organoids,and found that klf7 was highly expressed in the embryonic brain stage and significantly decreased after birth which suggests that klf7 is closely related to brain development.To confirm whether klf7 was involved in ASD,single-cell RNA-sequencing（RNA-seq）data from ASD patient brains were used.Klf7 expression was significantly decreased in ODCs,astroglia and projection neurons,indicating that klf7 is involved in ASD.In addition,Ch IP-Seq results in293T cells and N2A cells showed that klf7 can target ASD risk genes,further confirming that klf7 is a drive gene for ASD.Based on the characteristics that klf7 can target ASD risk genes,we constructed Nestin-Cre conditional knockout mice.ASD-related behavior test showed that loss of klf7 causes the mice to exhibit abnormal social interaction and stereotypical behaviors which are the core features of ASD.We also found that klf7-deficient mice performed worse in nest building test,novel recognition experiment and morris water maze test,but greater motor performance in open field experiment.Especially,klf7^-/-mice exhibited a long time jumping during daily feeding.These results suggest that loss of klf7 leads to autism-like behavior in the mice.To determine the effect of klf7 on adult mice,immunofluorescence staining was performed.Neurons in cortex and Purkinje cells in the cerebellum were lost in klf7^+/-mice.We also compared the transcriptome of the whole brains of WT and klf7^+/-mice by RNA-seq,634 ASD risk genes in klf7^+/-mice were dysregulated.215genes of these genes changed significantly,and the remaining 419 genes showed small changes.Considering the large number of genes involved,the cumulative effect of their small changes should not be ignored.In addition,we constructed a regulatory network using klf7 target genes identified by Ch IP-seq,the altered ASD genes in klf7^+/-mice,as well as regulatory relationships in TRUST database.It was clear that klf7 can directly target ASD risk genes and other TFs to further affect more ASD genes in the resulting network.We also explored whether ASD symptoms can be rescued in adults.We overexpressed klf7 in the central nervous system by intravenously administered adeno-associated virus.Behavioral tests showed that the social interaction ability of klf7^+/-mice was improved and the repetitive behaviors were alleviated.At the same time,the expression levels of 427 of the 634 ASD risk genes previously disrupted by klf7 deletion were reversed,further verifying the role of klf7 in regulating ASD risk genes.We also knocked down klf7 levels with a lentiviral short hairpin RNA in a human brain organoid model.548 ASD genes were disrupted,and that 366 ASD genes were shared between klf7 knockdown organoids and klf7^+/-mice.These observations verify that klf7 play the same role in both human and mouse brains,suggesting that our research has the potential to transform therapeutic strategies.In conclusion,this study successfully clarifies that klf7 is an upstream regulator of ASD genes and lays a foundation for the research and development of ASD targeted drugs in future.