The Mechanism Study Of Chromatin Remodeler Brg1 On Regulating PDGFRα+ OPC Differentiation And Remyelination

Objective:In the central nervous system,neural stem cells(NSCs)can be differentiated into oligodendrocyte precursor cells(OPC)and further differentiate into myelin-forming mature oligodendrocytes,which can support and wraps axons.The developmental OPC is very important in the process of oligodendrocyte maturation and myelination,and the adult OPC also plays a key role in the repair of myelin injury.Therefore,the regulation network of OPC differentiation can be the theoretical basis for the strategy of treating demyelinating diseases with OPC as the target cell,as well as the research direction of specific drug development under the guidance of this strategy.Recent studies have shown that epigenetic modification plays an important role in the fate determination of cells and the proliferation and differentiation of cells.Among them,ATP-dependent chromatin remodeling family SWI/SNF complex component Brgl is very important in mouse neural development.Brgl can regulate the specification of NSC to pri-OPC by binding to the Olig2 promoter,knockout Brgl in the pri-OPC stage can cause severe myelination,however,knockout Brgl in the late mitotic CNP+OPC have little effect on oligodendrocyte differentiation.Therefore,the role of Brgl in OPC differentiation,especially in the differentiation of early PDGFRa+OPC,remains unclear.In addition,the role of Brgl in OPC regeneration process after demyelination is also not clear.Based on these,this study is to explore the regulation of chromatin remodeling factor Brgl in early PDGFRa+OPC and late CNP+OPC differentiation,and to discover the effect of Brgl on adult OPC remyelination after injury,and to clarify its specific molecular mechanism regulating OPC differentiation.Methods:1 Brgl regulates the differentiation and regeneration of PDGFRa+OPC1.1 Brgl specifically regulates the differentiation and maturation of PDGFRα+early OPC(1)The early OPC marker PDGRα and late OPC marker CNP were identified by single-cell transcripto,ne database of oligodendrocytes.(2)The specific knockout of Brg1 mice(Brgl cKO)in the late CNP+ OPC was generated,and the differentiation and maturation of oligodendrocytes in the spinal cord were investigated by in situ hybridization experiments.(3)Genration of early PDGFRα+OPC specifically knocked out Brgl mice(Brgl iKO),and detected the differentiation and maturation of oligodendrocytes in the brain and spinal cord by immunofluorescence assay.Transmission electron microscopy was used to detect myelination in the brain and spinal cord of Brgl iKO mice.(4)Immunostaining were performed to investigate the ef fects of Brgl deletion in OPC on cell prolife ration,cell death,astrocyte and microglia activation in vivo.1.2 The regulation of Brglin adult PDGFRa+ OPC on oligodendrocyte remyelination(1)’The dynamic expression of Smarca4/Brgl in spinal cord injury was analyzed by transcriptome database of spinal cord injury.(2)LPC induced-demyelination mouse model was generated,and the expression level of-Brgl in the lesion sites was detected by immunostaining.(3)LPC was injected into the spinal cord of Control and Brgl iKO adult mice to induce demyelination.In situ hybridization,immunostaining and tranmission electron microscopy experiments were used to investigate the effect of Brgl deficiency on OPC regeneration and remyelination after myelin injury.In situ hybridization was carried out to detect the expression of PDGFRα after Brgl knockout in adult OPC,and investigate the effect of Brg1 on adult OPC repopulation.2 The mechanism of Brgl regulation on PDGFRα+ OPC differentiation and regeneration2.1 Brgl promotes OPC differentiation by regulating H3K27me3 expression(1)Isolation and purification of Control OPCs and Brgl knockout OPCs by flow cytometry and submit to ATAC-seq and RNA-seq analysis.(2)Compare the ATAC-seq data of NSC and OPC to investigate the chromatin opening of Smarca4/Brgl;combine the ATAC-seq data with the Chip-seq data of Brgl in OPC to find the possible regulation network of Brg1.(3)GSEA analysis was used to find out the oligodendrocyte differentiation gene enrichment,and then qPCR and immunostaining experiments were performed to investigate the changes of oligodendrocyte markers after Brglknockout or inhibition in OPC.(4)RNA-seq analysis to find out the change of H3K27me3-related pathways,then qPCR and cell staining experiments confirmed the inhibition of H3K27me3 expression after Brglknockout or inhibition.(5)Combined analysis of ATAC-seq and Chip-seq data to determine the direct regulation of Brgl on H3K27me3 expression.(6)The effect of H3K27me3 inhibition on OPC differentiation was investigated by immunostaining.2.2 Brgl promotes OPC differentiation by specifically regulating the activation of oligodendrocyte,stemness and neuronal associated transcription factors(1)Compare the ATAC-seq data of Control and Brgl knockout OPC to investigate the chromatin opening of oligodendrocyte differentiation-related genes.(2)Motif analysis was used to find specific Motif and transcription factors in Control and Brgl knockout OPC.(3)By analyzing ATAC-seq data and Chip-seq data,it was investigated whether Brg1 can bind to oligodendrocyte-associated transcription factors to regulate chromatin opening of oligodendrocyte differentiation-related genes in OPC.(4)Combined analysis of the specific Motif-predicted transcription factor and differentially upregulated genes in Brg1 knockout OPC to find out the specific upregulated sternness and neuronal associated transcription factors.(5)RNA-seq analysis of enrichment of stem cell markers and neuronal markers.(6)Overexpression of sternness associated transcription factors in OPC to examine the regulation of OPC differentiation-related genes.Results:1 Brgl regulates the differentiation and regeneration of PDGFRa+OPC1.1 Brgl specifically regulates the differentiation and maturation of PDGFRa+early OPC(1)By analyzing the single-cell transcriptome database of oligodendrocytes,OPC can be divided into different stages,in which Pdgfra is mainly expressed in early OPC,Cnp is mainly expressed in late OPC,and different expression patterns of Pdgfra and Cnp suggest that they can be markers for different stages of OPC.(2)Specific knockout of Brg1 in CNP+OPC in mice,in situ hybridization experiments showed that Brg1 deficiency in CNP+OPC had no significant effect on OPC differentiation.(3)Specific knockout of Brg1 in PDGFRa+OPC in mice(Brgl iKO),immunofluorescence and electron microscopy experiments showed that the OPC differentiation and myelination in the brain of’ Brgl iKO mice were significantly inhibited.Immunostaining and transmission electron microscopy experiments showed that OPC differentiation and myelination were also significantly inhibited in the spinal cord of Brgl iKO mice,but quite weaker than in the brain.(4)Immunostaining showed that the effect of Brgl on OPC differentiation in PDGFRα+OPC was cell-autonomous,which was not interfered by astrocytes or microglia.In conclusion,it can be inferred that Brgl can selectively regulate the differcntiation and maturation of PDGFRα+ early OPC,but the regulation of CNP+late OPC differentiation is weak,suggesting that Brgl has a phase-specific regulation of OPC.1.2 The regulation of Brgl in adult PDGFRa+OPC on oligodendrocyte remyelination(1)RNA-seq data analysis of spinal cord injury reported by Kenian Chen in 2013 showed that the expression of Smarca4/Brgl showed dynamic changes during the repair of spinal cord injury.(2)Immunofluorescence results showed that the expression of Brgl in OPC was significantly increased in LPC-induced spinal cord demyelination,suggesting that Brgl may play a role in the process of remyelination.(3)LPC induced demyelination injury was carried out in Control and Brgl iKO adult mice.The results of in situ hybridization,immunostaining and transmission electron microscopy showed that OPC re-differentiation and remyelination were significant inhibited in the lesion sites of Brgl iKO mice,suggesting that Brgl in OPC can regulate the differentiation of adult PDGFRα+OPC,thereby promoting remyelination after injury.2 The mechanism of Brgl regulation on PDGFRα+ OPC differentiation and regeneration2.1 Brgl promotes OPC differentiation by regulating H3K27me3 expression(1)The Control and Brgl KO OPCs marked by PDGFRa+Tomato+were obtained by FACS,and submit to ATAC-seq and RNA-seq.(2)ATAC-seq data analysis showed that the chromatin structure of Brgl in NSC and OPC was open,indicating that transcription factors can be easily binded to the chromatin of Brgl gene,thus promoting transcription.In addition,combined analysis of ATAC-seq and Chip-seq data revealed that the chromatin-opening gene that binds to Brgl in OPC is enriched in some GO gene clusters related to cell proliferation and differentiation.Subsequent GSEA analysis of RNA-seq also showed that Brgl is involved in cell differentiation-associated pathways.The above results suggest that Brgl in OPC is involved in the regulation network related to cell differentiation and maturation.(3)Using GSEA analysis,it was found that Brgl knockout in OPC inhibits OPC differentiation and activates negative regulator Wnt signaling pathway;qPCR and immunostaining also showed oligodendrocyte markers decreased after Brgl knockout or inhibition,suggesting that Brgl regulates the expression of genes involved in OPC differentiation.(4)RNA-seq analysis showed that Brgl knockout inhibited the expression of H3K27me3-related pathway,qPCR and immunostaining confirmed that H3K27me3 expression was decreased after OPC knockdown or inhibition.(5)Combined analysis of ATAC-seq and Chip-seq data,it was found that Brgl regulates H3K27me3 expression by regulating the expression of PRC2 complex.(6)Immunostaining results showed that the reduction of H3K27me3 expression in OPC could inhibit the expression of OPC differentiation genes.In summary,Brgl can promote the differentiation of OPC by regulating H3K27me3 expression.2.2 Brgl promotes OPC differentiation by specifically regulating the activation of oligodendrocyte,stemness and neuronal associated transcription factors(1)Analysis of the ATAC-seq and Chip-seq data of Control and Brg1 knockout OPCs revealed that Brgl can bind to the promoter/enhancer region of the relevant gene.(2)Motif analysis was used to find specific Motif and transcription factors in Control and Brgl knockout OPCs.In Control OPC,mainly oligodendrocyte-associated transcription factors,while in Brgl knockout OPC,mainly stem cell characteristics and Neuron-related transcription factors.(3)By analyzing the ATAC-seq data and the Chip-seq data,it was found that in the presence of Brg1,Brg1 directly regulates the expression of OPC-differentiated genes by binding to OPC-related transcription factors(Olig2,Sox 10),thereby promoting OPC differentiation.(4)When Brg1 is deleted,the expression of stemness-specific transcription factors(Klf4,Sox2,Sox4)and neuronal-associated transcription factors(Cux1,Pbxl)are increased,which promote the expression of stem cell and neuronal marker genes,thereby inhibiting OPC.Differentiation.(5)RNA-seq analysis showed that stem cell markers and neuron marker-related genes and pathways were significantly upregulated after Brgl knockout in OPC.(6)The sternness-associated transcription factor Klf4 was overexpressed in Oli-neu cell,and qPCR results showed that overexpression of K1f4 inhibited the expression of OPC differentiation-related genes.Taken together.Brg1 promotes OPC differentiation by specifically regulating oligodendrocyte,stem cell properties,and activation of neuronal-associated transcription factors.Conclusion:This study found that Brgl has a phase-specific regulation of OPC differentiation,especially for the differentiation of PDGFRa+early OPC.However,Brgl has no significant effect on the differentiation process of CNP+late OPC.Secondly,in the regeneralion process after demyelination,Brgl in adult OPC can also regulate its differentiation and promote remyelination.In addition,Brgl can regulate OPC differentiation through two ways,one is to promote OPC differentiation by regulating the expression of H3K27me3;the second is to specifically regulate the activation of oligodendrocyte,sternness and neuronal associated transcription factors,thereby regulating OPC differentiation.Our findings not only improve the regulatory network of Brgl,but also enrich the molecular mechanism of oligodendrocyte differentiation regulation mediated by SWI/SNF chromatin remodeling complex.In addition,our findings can clarify the possible mechanism of white matter defects from Smarca4 mutated patients with Coffin-Siris syndrome,and provide a certain research basis for the treatment.