Fig. 6
PA1 together with JUN regulates downstream gene expression. a PA1 bound motifs identified by MEME in Sertoli cells. The motif sequence is shown in the left column, the P value is shown in middle column and the corresponding transcription factors (TFs) are shown in the right column. The most significant motif sequence was labeled with asterisk. b Bubble plot of the upstream transcription factor analysis of 1045 downregulated genes in Amh-pa1−/− Sertoli cells. The components of AP-1 complex were labeled with asterisks. c PA1 was shown to be able to interact with JUN in TM4 Sertoli cells. Lane 1 indicated the input of the pCS2-Myc-Jun transfected TM4 cells. Lane 2 represented the input of the TM4 cells co-transfected with pCS2-Myc-Jun and pRK-Flag-Pa1. Lane 3 are the immunoprecipitated products of pCS2-Myc-Jun transfected TM4 cells using anti-FLAG antibody. Lane 4 are the immunoprecipitated products of the TM4 cells co-transfected with pCS2-Myc-Jun and pRK-Flag-Pa1 using anti-FLAG antibody. All the lanes were analyzed with anti-MYC and anti-FLAG antibodies in immunoblot experiment. d The endogenous PA1 could interact with JUN. TM4 cells in 6-well plate were transfected with 0.5ug pCS2-Myc-Jun plasmid and then the proteins were further immunoprecipitated with PA1 antibody and analyzed with anti-PA1 and anti-JUN antibodies in immunoblot experiment. e Immunofluorescence images of JUN in adult mouse testis. Seminiferous tubule sections of wild-type testes were co-stained for Sertoli cell marker SOX9 (red) and JUN (green) and the nuclei was stained with DAPI (blue). Arrows indicated positive cells for positive signals for SOX9 and JUN. Dashed boxes showed localization of the enlarged images. Dashed curve indicated basal membrane of seminiferous tubules. f Venn diagrams displaying the overlap of PA1 bound genes, JUN bound genes in MCF-7 cells [38] and downregulated and upregulated genes from the transcriptome analysis. g KEGG pathway analysis of 35 overlapped genes among PA1 bound genes in Sertoli cells, JUN bound genes in MCF-7 cells and downregulated genes in Amh-pa1−/− Sertoli cells. Term of gap junction was labeled with asterisk. h Immunoblot analysis and statistical analysis of the protein level of Cx43 in Pa1F/F and Amh-pa1−/− testis. (n = 3). Data are presented as mean ± SEM. **p < 0.01. i Cx43 signal was diminished in Amh-pa1−/− mouse testis compared with Pa1F/F mouse testis. Seminiferous tubule sections of Pa1F/F mice (upper) and Amh-pa1−/− mice (lower) were stained for Cx43 (green), and the nuclei was stained with DAPI (blue). Dashed boxes showed localization of the enlarged images. Dashed curve indicated basal membrane of seminiferous tubules. Arrows indicate Cx43 signals at the basal compartment of seminiferous tubules. j The overexpression of PA1 together with JUN could promote the promoter activity of Cx43. Luciferase reporter assays in TM4 cells were transfected with empty pCS2 vector, pCS2-Myc-Pa1, pCS2-Myc-Jun or pCS2-Myc-Pa1 and pCS2-Myc-Jun plasmids (n = 3). Data are presented as mean ± SEM. **p < 0.01. ***p < 0.001, ****p < 0.001. k PA1 is required for the construction and maintenance of the BTB in mouse Sertoli cells. PA1 functions as a cofactor of JUN to regulate the downstream gene expression in the nuclei of Sertoli cells, such as Cx43, and participate in maintaining the functional BTB. Moreover, PA1 may also regulate downstream gene expression associated with BTB regulation, such as basal ES, contributing to the integrity of the BTB