Change Of Gene Expression Induced By Variants Of AT-hook1 In MeCP2

Research Background and purposeRett syndrome(RTT)is a rare genetic disease characterized by neurodevelopmental regression,including cognitive and motor development impairment begins to deteriorate after 6-18 months of normal development,with progressive aggravation.The main manifestations are the decline of language skills and the loss of the ability to use hands purposefully;At the same time,patients often have pain,insensitivity and autism behavior and other accompanying symptoms.MECP2 encodes an epigenetic protein,which contains DNA binding domain and transcriptional inhibition domain with known function,as well as AT-hook1 conserved structure with unknown role.Previously,we found a case of atypical RTT patient with a missense mutation R190H in AT-hookl domain of underdetermined significance.Thus,we further constructed a mutant male mouse line carrying Mecp2 gene with eight conserved amino acid deletions(aa 186-193,RGRPKG)in AT-hookl(aa184-195),which reproduced some RTT phenotypes,such as decreased cognitive function,anxiety,pain abnormalities,and so on.Pathological analysis of the mouse hippocampus showed that the cell layer of CA1 area became thinner,and the mechanism was unclear.QPCR result showed that the expression of suppressor neural related genes was abnormal.The purpose of this study is to further explore the effect of AT-hookl domain on the expression of downstream genes.Research Methods1.In vivo experiment:Mecp2ΔAt-hook1/y mutant male mice were subjected to whole brain transcriptome data analysis and qPCR was used to verify the expression of related differential genes;2.In vitro experiment:SH-SY5Y and HEK-293T cell lines were established which express MeCP2B、MeCP2B AT-hookl point mutation(R202H)and MeCP2B AThookl deletion mutation(ΔAT)were constructed:SH-SY5Y/MeCP2B,SH-SY5Y/MeCP2B-R202H,SH-SY5Y/MeCP2B-ΔAT,HEK-293T/MeCP2B,HEK-293T/MeCP202H and HEK-293T/MeCP2B-ΔAT.The following experiment was performed.1)Subcellular localization of mutated MeCP2;2)The expression level of suppressor neural related genes were verified by qPCR;3)QPCR was used to verify the expression levels of GPCRs related genes suggested by animal transcriptomics;4)Cell viabilities were measured;5)Detection of apoptosis signal pathway:WB was used to detect Cleaved Caspase3;6)The expression level of Htr7 encoded receptor protein 5-HT7R was verified by WB.After treatment with 5-HT7R antagonist and agonist,the cell viability was observed;7)Statistical methods:SPSS 22.0 software was used to analyze the data.The statistical methods were one-way analysis of variance(ANOVA)or student’s t test(P<0.05).Research Results1.Transcriptome analysis of Mecp2ΔAT-hook1/y male mice showed that the expression of GPCRs family was significantly down regulated.Six G protein coupled receptors,Htr1d、Drd3、Tacr2,、Avprlb、Gabra6 and Adra2b,were down-regulated,and Htr7,Chrnb4 and Prdm2 were up-regulated in mutant male mice.2.Eight stable expression cell lines were successfully constructed:SH-SY5Y/MeCP2B,SH-SY5Y/MeCP2B-R202H,SH-SY5Y/MeCP2B-ΔAT,SH-SY5 Y/CN,HEK-293T/MECP2B,HEK-293T/MECP2B-R202H,HEK-293T/MECP2B-ΔAT,HEK-293T/CN.The following analysis was carried out in these stable expression cell lines.1)The nuclear localization of MeCP2 AT-hookl mutation was not change;2)AT-hookl mutation changes the expression of suppressor neural related genes;3)AT-hookl mutation leads to changes in the expression of GPCRs related genes;4)AT-hookl mutation induced cell viability decline and apoptosis;5)Activation of 5-HT7 receptor protects neurons from AT-hookl mutation.Conclusion1.In vivo experiments:MeCP2 AT-hookl mutation resulted in the expression of G protein coupled receptors family related genes and had the most significant effect on the interaction between neuroactive ligand and receptor;2.In vitro experiment:(1)MeCP2 AT-hookl mutation affects the expression of the gene relative to suppressor neurons and GPCRs;(2)MeCP2 AT-hookl mutation did not affect the subcellular localization of MeCP2 protein,but affected cell viability and apoptosis.Activation of 5-HT7 receptor could improve the viability of SH-SY5Y cells stably expressing MeCP2 AT-hookl point mutation and deletion mutation.