Molecular Mechanism Of CTCF Variant In Short Stature

Backgroud:Mental retardation,autosomal dominant 21(MRD21,OMIM#615502),a very rare autosomal dominant disease caused by CTCF gene variants,is characterized with growth retardation,intellectual disability and special facial features.One of the main clinical features of this syndrome is short stature,for which the molecular mechanism leading to short stature is unclear.Recent studies have shown that CTCF protein plays an important role in regulating the spatio-temporal expression of other genes during limb development.For example,CTCF can act as a transcriptional regulator to activate the transcription of IGF2which is critical for growth regulation.The data suggested that CTCF is involved in the regulation of insulin-like growth factors(IGFs)signal pathway.In our initial study,we detected a pathogenic CTCF variant(c.1699C>T,p.Arg567Trp)in a child with short stature.Objective:This study aimed to explore the effect of CTCF^R567Wvariant on IGFs signaling pathway,which might explain the short stature feature of this condition.Methods:1.Bioinformatics analysis:the conservation and pathogenicity of CTCF amino acid sequence were analyzed by online websites(Con Surf,WEBLOGO,PREDICTSNP,et al).The structural changes of CTCF^R567Wvariants were analyzed by constructing the three-dimensional structure model of CTCF^R567Wprotein.2.Cell model construction:Immortalized lymphocytes(LCLs)were generated from the patient carrying CTCF^R567Wvariant.The wild-type CTCF carrier plasmid(CTCF-WT)was constructed by homologous recombination technique using the p CMV-Flag-N plasmid as the blank plasmid.The CTCF^R567Wvariant carrier plasmid(CTCF-MUT)was constructed by gene site-directed mutagenesis using the CTCF-WT as the template.3.Experimental groups:the plasmids above were transfected into human embryonic kidney HEK293T cell line(HEK293T)and human normal liver cell lines(L-O2)by liposome transfection technique,and the experiment was divided into three groups:blank group(p CMV),wild group(CTCF-WT)and mutant group(CTCF-MUT).4.Gene expressions:the expression of CTCF and IGFs genes(IGF1,IGF2,IGFBP3,IGF1R)were analyzed by real-time quantitative PCR(qPCR),enzyme-linked immunosorbent assay(ELISA)and Western blotting(WB).5.Interaction of CTCF and the promoter of IGF1 or IGF2:the occupation of CTCF^R567Wvariant with IGF1 and IGF2 gene promoters was analyzed by chromosome immunoprecipitation combined with qPCR(CHIP-qPCR).The effect of CTCF^R567Wvariant on the promoter activity of IGF1 and IGF2 genes were evaluated using Luciferase assay.6.Transcriptome sequencing:high-throughput RNA transcriptome sequencing(RNA-seq)was performed to identify differentially expressed genes on the LCLs from patients and healthy controls.These data were analyzed by Deseq software and then subjected to GO and KEGG enrichment pathway analysis.Results:1.p.Arg567 was highly conserved in vertebrates.The online software Dynamut predicted that the amino acid residue Arg567 replaced by Trp567 might decrease the stability of CTCF protein(??SVib ENCo M:-4.186kcal/mol/K).Three-dimensional structural analysis showed that hydrogen bonding between Arg567and DC21 was absent,which might affect alpha helix folding and the CTCF-DNA interaction.2.The results of qPCR assay showed that the m RNA expression levels of IGF1,IGF2 and IGFBP3 genes were significantly decreased compared to the wild-type group,and the m RNA levels of CTCF^R567Wvariant and IGF1R gene had almost no change compared to the wild-type group.3.Western Blot showed no obvious abnormality of CTCF protein expression.ELISA assay showed that the protein expression of IGF1,IGF2 and IGFBP3 genes decreased significantly compared to the wild-type group.4.CHIP-qPCR showed the binding rate of CTCF^R567Wvariant to IGF1 and IGF2 gene promoters was significantly decreased.5.The luciferase assay showed that the transcriptional activity of IGF1 and IGF2 gene promoters in the mutant group decreased significantly compared to the wild-type.6.RNA-seq result showed several differentially expressed genes were associated with IGFs signaling pathway.GO analysis showed that differentially expressed genes were enriched in the sequence-specific DNA binding region.KEGG analysis showed that those genes were involved in the cytokines signaling mechanism.Conclusion:Our data provides a new insight into the pathogenesis of CTCF variants leading to short stature.The CTCF^R567Wvariant resulted in a loss of function and can decrease the activation of IGFs signaling pathway,which in turn leading to short stature.The results of RNA-seq suggested that other genes might be involved in the mechanism of pathogenic CTCF gene variants.This study provides a new insight into the pathogenesis of short stature caused by CTCF gene mutation,and offers a theoretical basis for exploring the therapeutic target of MRD21 short stature.