ABSTRACT
Objective Transcription factor forkhead box L 2 (FOXL2) is a key regulator of granulosa cells (GCs) estrogen syn-thesis and function maintenance .However, the FOXL2 protein expres-sion and function regulation mechanism are unknown .We explored how DNAJB11 regulates estrogen synthesis of granulosa cells with immunoprecipitation , immunofluorescent staining and luciferase re-porter gene. Methods The expression and localization of DNAJB 11 was detected by immunohistochemistry staining in isolated mouse ovary tissues .we use immunoprecipitation , immunofluorescence staining and luciferase reporter gene assay to investigate the mechanism of DNAJB11, a member of the endoplasmic reticulum Hsp 40 /DnaJ family, regulating the estrogen synthesis in granulosa cells . Results DNAJB11 is expressed in the mouse ovary and granulosa cells .Follicle-stimulating hormone (FSH) promotes DNAJB11 ex-pression in a time and concentration dependent manner and induces endogenous DNAJB 11 protein translocation from the ER to the nu-cleus in KGN cells.Moreover, Adenovirus-mediated overexpression of DNAJB11 did not affect the proliferation of granulosa cells .How-ever, the concentration of estrogen in granulosa cells was affected by concentration -dependent and subcellular localization-dependent manner (10749±801.7 pg/mL vs 14217±1218.0 pg/mL P<0.01).Immunoprecipitation assay confirmed that DNAJB11 binds to FOXL2 in granulosa cells .When overexpressed in the nucleus of granulosa cells , DNAJB11 could significantly enhance the stability of FOXL2 (P<0.05) and promote FOXL2-mediated activity of Cyp19A1 promoter (P<0.01), while the expression of DNAJB11 in the nucleus increased the expression of Cyp 19A1 protein by 1.5 times ( P<0.05) . Conclusion These results demonstrate that DNAJB 11 was a new binding molecule of transcription factor FOXL 2 and regulated FOXL 2 protein stability and transcription activity .
ABSTRACT
Objective Transcription factor forkhead box L 2 (FOXL2) is a key regulator of granulosa cells (GCs) estrogen syn-thesis and function maintenance .However, the FOXL2 protein expres-sion and function regulation mechanism are unknown .We explored how DNAJB11 regulates estrogen synthesis of granulosa cells with immunoprecipitation , immunofluorescent staining and luciferase re-porter gene. Methods The expression and localization of DNAJB 11 was detected by immunohistochemistry staining in isolated mouse ovary tissues .we use immunoprecipitation , immunofluorescence staining and luciferase reporter gene assay to investigate the mechanism of DNAJB11, a member of the endoplasmic reticulum Hsp 40 /DnaJ family, regulating the estrogen synthesis in granulosa cells . Results DNAJB11 is expressed in the mouse ovary and granulosa cells .Follicle-stimulating hormone (FSH) promotes DNAJB11 ex-pression in a time and concentration dependent manner and induces endogenous DNAJB 11 protein translocation from the ER to the nu-cleus in KGN cells.Moreover, Adenovirus-mediated overexpression of DNAJB11 did not affect the proliferation of granulosa cells .How-ever, the concentration of estrogen in granulosa cells was affected by concentration -dependent and subcellular localization-dependent manner (10749±801.7 pg/mL vs 14217±1218.0 pg/mL P<0.01).Immunoprecipitation assay confirmed that DNAJB11 binds to FOXL2 in granulosa cells .When overexpressed in the nucleus of granulosa cells , DNAJB11 could significantly enhance the stability of FOXL2 (P<0.05) and promote FOXL2-mediated activity of Cyp19A1 promoter (P<0.01), while the expression of DNAJB11 in the nucleus increased the expression of Cyp 19A1 protein by 1.5 times ( P<0.05) . Conclusion These results demonstrate that DNAJB 11 was a new binding molecule of transcription factor FOXL 2 and regulated FOXL 2 protein stability and transcription activity .
ABSTRACT
PURPOSE: The purpose of this paper is to identify the forkhead transcription factor gene (FOXL2) mutations in Korean patients with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES). METHODS: We have analyzed the mutations of FOXL2 gene in genomic DNAs extracted from 16 BPES patients and their families by PCR, PCR-SSCP, and sequencing. RESULTS: No deletion in exon 1 to 3 of the FOXL2 gene was observed by PCR. The PCR products were subjected to SSCP analysis and 9 patients showed SSCP shifts. The PCR products showing SSCP shifts were subcloned into plasmid vectors and sequenced to confirm the FOXL2 mutation. In total, 7 mutations (1 nonsense mutation, 1 deletion, and 5 duplications) in exon 2 were identified. CONCLUSIONS: The FOXL2 gene mutations were identified in the Korean BPES patients. Some of the mutations were previously reported and some were new mutations. This study will contribute to the molecular analysis and clinical counseling of BPES patients.
Subject(s)
Humans , Codon, Nonsense , Counseling , DNA , Exons , Plasmids , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Transcription FactorsABSTRACT
Objective To identify the genetic mutation of the gene FOXL2 in a big Chinese family with 4 generations,8 patients and 11 health members which have blepharophimosis-ptosis-epicanthus-inversus syndrome(BPES) type Ⅰassociated with ovarian dysfunction,and demonstrate the correlation between clinical phenotype and genotype.Methods The peripheral blood samples,with 5ml EDTA as decoagulant,from 8 patients and 11 health people in a big Chinese family affected with BPES typeⅠwere collected.The genomic DNA from peripheral blood leukocytes was extracted.7 pairs of primers were designed by the Oligo6 according to the sequence of the FOXL2 gene(GenBank ID AF301906).The exons and the putative core promoter of the FOXL2 gene were amplified using polymerase chain reaction(PCR),and mutation was analyzed by sequencing DNA fragments.Results A novel nonsense mutation at nucleotides in the FOXL2 gene was found in the eight affected patients of the big Chinese family with BPES typeⅠ.No mutation was found in any of the health members in the big Chinese family.Conclusion This is the first reported mutation of the FOXL2 gene in a big Chinese family with BPES typeⅠ.This nonsense mutation in the FOXL2 gene may be an important pathogenesis for the BPES typeⅠin this big Chinese family.It is the first time that a novel nonsense mutation in the FOXL2 gene has ever been found to demonstrate a close correlation between genotype and BPES typeⅠ.