EGR1 Transcriptional Regulation Of PARP1 Induced Photoreceptor Cell Death In RD Mice Postgraduate:Wei Chunling Supervisor:Prof.Hu Zhulin

Purpose:Retinitis pigmentosa(RP)is an inherited retinal degeneration disease that can lead to the death of photoreceptor cells and vision loss.PARP1-dependent cell death(Parthanatos)is the main cause of the death of RP photoreceptor cells.Meanwhile,our previous study found that EGR1 is expressed in the outer nucleus layer of the retina in RP mice,and the expression of EGR1 can induce the decrease of photoreceptor cell layers and the decrease of photoreceptor cell function amplitude.Therefore,we further study the mechanism of EGR1-induced photoreceptor cell death to find important target molecules for the occurrence and development of the disease,and provide new targets and ideas for the delay and even treatment of RP.Methods:Part 1 High-throughput sequencing was performed on the retinal tissues of RD mice(rd1,rd2,rd10),with C3H mice of the same genetic origin as the control,to screen the different genes.EGR1 binding signals near the PARP1 transcription start site were analyzed by Dnase-seq,CHIP-seq and MEME.Part 2 Chromatin-immunoprecipitation,Dual-Luciferase reporter assay and immunofluorescence staining were used to verify the binding of EGR1 to PARP1 and its loci.Part 3 In vivo and in vitro experiments were proformed to demonstrate that EGR1 upregulates PARP1,causes PAR aggregation and promotes photoreceptor cell death.First,the expression levels of EGR1,PARP1 and PAP in retinal tissues of C3H and rd1 mice were detected by immunofluorescence,western blot and qPCR.Secondly,lentivirus overexpression and interference of Egr1 gene in cultured retinal explants of C3H and rd1 mice were divided into four groups:Ctrl-sh(EGR1 interference control group),sh-EGR1(EGR1 interference group),Ctrl-m(EGR1 overexpression control group)and m-EGR1(EGR1 overexpression group).TUNEL assay detected the rate of TUNEL positive cells,the number of photoreceptor cells and the thickness of the outer nuclear layer of the retina.Meanwhile,western blot and qPCR were used to detect the expression changes of EGR1,PARP1 and PAR.Part 4 The in vitro culture of C3H and rd1 mouse retinal explants were treated with MAPK pathway inhibitors PD98059(ERK inhibitors)and Tanzisertib(JNK inhibitors).TUNEL assay detected the rate of TUNEL positive cells,the number of photoreceptor cells and the thickness of the outer nuclear layer of the retina.Meanwhile,western blot was used to detect the phosphorylation levels of ERK,JNK and c-Jun,as well as the expression levels of EGR1.qPCR was used to detect the expression changes of ERK,JNK,c-Jun and EGR1.Result:Part 1 The peak of photoreceptor cell death in rd1,rd2 and rd10 mice was different,which was Postnatals 13,P29 and P23,respectively,while the C3H photoreceptor cell death in the control group was at a low level at the same time.Compared with C3H,rd1 has 1926 differentially expressed genes(DEGs).rd10 has 3096 DEGs and rd1 has 375 DEGs.By using Dnase-seq,H3K4me3 and CHIP-seq data of mouse retinal tissue,a DNase hypersensitive site was found on the Parp1 gene promoter,and EGR1 binding signal was further found in the Parp1 gene promoter region.Part 2 The results of CHIP and Dual-Luciferase reporter in the retinal tissues of C3H and rd1 mice showed that EGR1 was bound to the Parpl DHS at the binding site of AACGCCCACGGAAC.Immunofluorescence detection showed that EGR1 and PARP1 were co-expressed in the photoreceptor nucleus,indicating co-localization.Part 3 Immunofluorescence staining of retinal tissues of rd1 mice showed that the expression of EGR1 and PARP1 reached the peak at P13,which coincided with the peak of photoreceptor cell death,while immunofluorescence staining of retinal tissues of C3H mice showed no significant expression of EGR1 and PARP1.Meanwhile,western blot and qPCR showed the same expression trend of EGR1,PARP1 and PAR.The overexpression of Egr1 gene in the retinal explants of C3H and rd1 mice significantly increased the rate of TUNEL positive cells in the outer nucleus layer of the retina,especially P9,accompanied by a significant decrease in the number of photoreceptor cells and the thickness of the outer nucleus layer of the retina of P13.At the same time,the overexpression of Egr1 gene in the retinal explants resulted in the significant increase of EGR1,PARP1,PAR protein levels and mRNA expression levels.Inhibition of Egr1 gene in cultured retinal explants significantly reduced the rate of TUNEL positive cells in the outer nuclear layer of the retina of rd1 mice,accompanied by a decrease in the number of photoreceptor cells and the thickness of the outer nuclear layer of the retina of P13.At the same time,the inhibition of Egr1 gene in retinal explants resulted in the decrease of EGR1,PARP1 and PAR protein levels and mRNA expression.Part 4 Treatment with PD98050 and Tanzisertib reduced the rate of TUNEL positive cells and the number of photoreceptor cell layers and the thickness of outer nuclear layer in rd1 mice.Western blot analysis showed that PD98059 and Tanzisertib inhibited the phosphorylation of ERK and JNK,and then inhibited the phosphorylation of c-Jun and down-regulated the expression of EGR1.qPCR showed that PD98059 and Tanzisertib down-regulated the expression of ERK,JNK,c-Jun and EGR1 mRNA.Conclusion:In rd1 mice,the MAPK/ERK/JNK pathway was activated and c-Jun phosphorylation increased,thus mediating the increased level of transcription factor EGR1,resulting in the increased expression level of PARP1 and aggregation of substrate PAR,which ultimately led to the death of photoreceptor cells through the Parthanatos pathway.