| ObjectiveIntellectual disability(ID)is a neurological disorder with considerable genetic heterogeneity,which is mainly manifested as mental retardation,persistent cognitive dysfunction,followed by abnormal mobility and continuous abnormal mental activity.Among the mental disorders caused by genetic factors,2p15-p16.1deletion is a specific type of ID.Studies have reported that several cases in which only the BCL11 A locus is deleted was able to show signs of mental retardation.These reports confirm that BCL11 A gene plays an important yet underappreciated role in the development of neurons.In our study,we will explore the expression pattern of BCL11 A and decipher the molecular mechanism how BCL11 A regulates the essential cortical development factors.Above all,our approach holds promise for illustrating the role of BCL11 A in cortical development.Materials and Methods1.The expression of BCL11 A in human tissues was analyzed and confirmed by GTEx database and ENCODE gene array assays.2.Western blot and qPCR were used to verify the expression of BCL11 A in mouse organs,and to confirm that BCL11 A was highly expressed in mouse cortex at E13.5days.3.Use immunofluorescence to label the specific expression of BCL11 A,NeuN with DAPI in the cerebral cortex of fetal murine at E13.5d.4.Transfect SH-SY5 Y cell line with siRNA meanwhile build BCL11 A overexpressed SH-SY5 Y cells with specifically decreased and overexpressed BCL11 A DNA products.Western blot and immunofluorescence were used to detect the expression of BCL11 A in SH-SY5 Y.5.Downregulated BCL11 A disrupted the expression of neural transcription factor Sox6,Tbr2 and NeuroD2,as well as pPolII,meanwhile upregulated histone deacetylases HDAC1/2.6.ChIP-qPCR were adopted to elucidate histone modification H3K4me3 and H3K27me3 changes in BCL11 A deprived SH-SY5 Y cells.Results1.Comparative analysis of GTEx(Genotype-Tissue Expression)gene database showed that BCL11 A was mainly expressed in human fetal brain,frontal cortex,spleen,B lymphocytes and their progenitor cells;Gene array results from ENCODE(Encyclopedia of DNA Elements)project showed that BCL11 A was found in human whole brain cortex and frontal lobe,and the cortex contains highly expressed BCL11 A,which is consistent with GTEx results.2.Western blot and qPCR results showed BCL11 A was expressed in the cerebral cortex,spleen,and lungs of adult mice,and in E11.5d,E13.5d,E15.5d,and E17.5 days of fetal murine brain,E13.5d had the highest fetal brain expression amongst them all.3.The results of immunofluorescence elucidated that in the cerebral cortex of E13.5d fetal mice,BCL11 A was highly expressed,and highly consistent with neuron-specific transacting factor NeuN.4.Western blot and immunofluorescence assay confirmed the transfection efficacy and efficiency of siRNA and overexpression plasmid in SH-SY5 Y cells,meanwhile knockdown and overexpressed BCL11 A SH-SY5 Y cells were obtained separately.5.qPCR and Western blot results revealed that downregulated BCL11 A disrupted the expression of neural transcription factor Sox6,Tbr2 and NeuroD2,as well as pPolII,meanwhile upregulated histone deacetylases HDAC1/2.6.ChIP-qPCR experiments confirmed that knockdown of BCL11 A in SH-SY5 Y cells would lead to a decrease of enrichment of histone-specific active marker H3K4me3,and an increase of H3K27me3 accumulation in the promoter of neuron-related factors Sox6,Tbr2,NeuroD2.Conclusion1.The gene expression pattern of murine mbcl11 a was consistent with the human gene BCL11 A in various tissues and organs.2.During the critical period of embryonic murine neural development,on E13.5d,BCL11 A and key factors of neural development shows simultaneously high expression activity.3.Knocking down the expression of BCL11 A in SH-SY5 Y cells will lead to adecreased expression level of neural TFs,while downregulate the enrichment of histone-specific active marker H3K4me3,and an increase of H3K27me3 accumulation in the promoter of key factors essential to neuronal development. |
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