RESUMEN
Sex determination in mammals is governed by antagonistic interactions of two genetic pathways, imbalance in which may lead to disorders/differences of sex development (DSD) in human. Among 46,XX individuals with testicular DSD (TDSD) or ovotesticular DSD (OTDSD), testicular tissue is present in the gonad. Although the testis-determining gene SRY is present in many cases, the etiology is unknown in most SRY-negative patients. We performed exome sequencing on 78 individuals with 46,XX TDSD/OTDSD of unknown genetic etiology and identified seven (8.97%) with heterozygous variants affecting the fourth zinc finger (ZF4) of Wilms' tumor 1 (WT1) (p.Ser478Thrfs*17, p.Pro481Leufs*15, p.Lys491Glu, p.Arg495Gln [x3], p.Arg495Gly). The variants were de novo in six families (P = 4.4 × 10-6), and the incidence of WT1 variants in 46,XX DSD is enriched compared to control populations (P < 1.8 × 10-4). The introduction of ZF4 mutants into a human granulosa cell line resulted in up-regulation of endogenous Sertoli cell transcripts and Wt1Arg495Gly/Arg495Gly XX mice display masculinization of the fetal gonads. The phenotype could be explained by the ability of the mutated proteins to physically interact with and sequester a key pro-ovary factor ß-CATENIN, which may lead to up-regulation of testis-specific pathway. Our data show that unlike previous association of WT1 and 46,XY DSD, ZF4 variants of WT1 are a relatively common cause of 46,XX TDSD/OTDSD. This expands the spectrum of phenotypes associated with WT1 variants and shows that the WT1 protein affecting ZF4 can function as a protestis factor in an XX chromosomal context.
Asunto(s)
Trastornos Testiculares del Desarrollo Sexual 46, XX/metabolismo , Testículo/metabolismo , Proteínas WT1/metabolismo , Trastornos Testiculares del Desarrollo Sexual 46, XX/genética , Trastornos Testiculares del Desarrollo Sexual 46, XX/patología , Animales , Preescolar , Femenino , Humanos , Lactante , Masculino , Ratones , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Testículo/crecimiento & desarrollo , Testículo/patología , Proteínas WT1/química , Proteínas WT1/genética , Dedos de Zinc , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Cell growth requires a high level of protein synthesis and oncogenic pathways stimulate cell proliferation and ribosome biogenesis. Less is known about how cells respond to dysfunctional mRNA translation and how this feeds back into growth regulatory pathways. The Epstein-Barr virus (EBV)-encoded EBNA1 causes mRNA translation stress in cis that activates PI3Kδ. This leads to the stabilization of MDM2, induces MDM2's binding to the E2F1 mRNA and promotes E2F1 translation. The MDM2 serine 166 regulates the interaction with the E2F1 mRNA and deletion of MDM2 C-terminal RING domain results in a constitutive E2F1 mRNA binding. Phosphorylation on serine 395 following DNA damage instead regulates p53 mRNA binding to its RING domain and prevents the E2F1 mRNA interaction. The p14Arf tumour suppressor binds MDM2 and in addition to preventing degradation of the p53 protein it also prevents the E2F1 mRNA interaction. The data illustrate how two MDM2 domains selectively bind specific mRNAs in response to cellular conditions to promote, or suppress, cell growth and how p14Arf coordinates MDM2's activity towards p53 and E2F1. The data also show how EBV via EBNA1-induced mRNA translation stress targets the E2F1 and the MDM2 - p53 pathway.
Asunto(s)
Factor de Transcripción E2F1/genética , Neoplasias/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteína p53 Supresora de Tumor/genética , Carcinogénesis/genética , Ciclo Celular/genética , Proliferación Celular/genética , Daño del ADN/genética , Genes Supresores de Tumor , Herpesvirus Humano 4/genética , Humanos , Neoplasias/virología , Oncogenes/genética , Fosforilación/genética , Dominios Proteicos/genética , Proteínas con Motivos de Reconocimiento de ARN/genética , ARN Mensajero/genética , Proteína p14ARF Supresora de Tumor/genéticaRESUMEN
SOX8 is an HMG-box transcription factor closely related to SRY and SOX9. Deletion of the gene encoding Sox8 in mice causes reproductive dysfunction but the role of SOX8 in humans is unknown. Here, we show that SOX8 is expressed in the somatic cells of the early developing gonad in the human and influences human sex determination. We identified two individuals with 46, XY disorders/differences in sex development (DSD) and chromosomal rearrangements encompassing the SOX8 locus and a third individual with 46, XY DSD and a missense mutation in the HMG-box of SOX8. In vitro functional assays indicate that this mutation alters the biological activity of the protein. As an emerging body of evidence suggests that DSDs and infertility can have common etiologies, we also analysed SOX8 in a cohort of infertile men (n = 274) and two independent cohorts of women with primary ovarian insufficiency (POI; n = 153 and n = 104). SOX8 mutations were found at increased frequency in oligozoospermic men (3.5%; P < 0.05) and POI (5.06%; P = 4.5 × 10-5) as compared with fertile/normospermic control populations (0.74%). The mutant proteins identified altered SOX8 biological activity as compared with the wild-type protein. These data demonstrate that SOX8 plays an important role in human reproduction and SOX8 mutations contribute to a spectrum of phenotypes including 46, XY DSD, male infertility and 46, XX POI.
Asunto(s)
Trastornos del Desarrollo Sexual 46, XX/genética , Trastorno del Desarrollo Sexual 46,XY/genética , Mutación Missense , Oligospermia/genética , Insuficiencia Ovárica Primaria/genética , Factores de Transcripción SOXE/genética , Adolescente , Niño , Femenino , Humanos , MasculinoRESUMEN
The p53 tumor suppressor and its key regulator MDM2 play essential roles in development, ageing, cancer, and cellular stress responses in mammals. Following DNA damage, MDM2 interacts with p53 mRNA in an ATM kinase-dependent fashion and stimulates p53 synthesis, whereas under normal conditions, MDM2 targets the p53 protein for degradation. The peptide- and RNA motifs that interact with MDM2 are encoded by the same conserved BOX-I sequence, but how these interactions have evolved is unknown. Here, we show that a temperature-sensitive structure in the invertebrate Ciona intestinalis (Ci) p53 mRNA controls its interaction with MDM2. We also show that a nonconserved flanking region of Ci-BOX-I domain prevents the p53-MDM2 protein-protein interaction. These results indicate that the temperature-regulated p53 mRNA-MDM2 interaction evolved to become kinase regulated in the mammalian DNA damage response. The data also suggest that the negative regulation of p53 by MDM2 via protein-protein interaction evolved in vertebrates following changes in the BOX-I flanking sequence.
Asunto(s)
Proteínas Proto-Oncogénicas c-mdm2/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/genética , Secuencia de Bases , Línea Celular Tumoral , Ciona intestinalis , Daño del ADN , Cartilla de ADN , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Relación Estructura-ActividadRESUMEN
NR2F2 encodes COUP-TFII, an orphan nuclear receptor required for the development of the steroidogenic lineages of the murine fetal testes and ovaries. Pathogenic variants in human NR2F2 are associated with testis formation in 46,XX individuals, however, the function of COUP-TFII in the human testis is unknown. We report a de novo heterozygous variant in NR2F2 (c.737G > A, p.Arg246His) in a 46,XY under-masculinized boy with primary hypogonadism. The variant, located within the ligand-binding domain, is predicted to be highly damaging. In vitro studies indicated that the mutation does not impact the stability or subcellular localization of the protein. NR5A1, a related nuclear receptor that is a key factor in gonad formation and function, is known to physically interact with COUP-TFII to regulate gene expression. The mutant protein did not affect the physical interaction with NR5A1. However, in-vitro assays demonstrated that the mutant protein significantly loses the inhibitory effect on NR5A1-mediated activation of both the LHB and INSL3 promoters. The data support a role for COUP-TFII in human testis formation. Although mutually antagonistic sets of genes are known to regulate testis and ovarian pathways, we extend the list of genes, that together with NR5A1 and WT1, are associated with both 46,XX and 46,XY DSD.
Asunto(s)
Factor de Transcripción COUP II , Testículo , Humanos , Factor de Transcripción COUP II/metabolismo , Factor de Transcripción COUP II/genética , Testículo/metabolismo , Masculino , Factor Esteroidogénico 1/metabolismo , Factor Esteroidogénico 1/genética , Mutación , Hipogonadismo/genética , Hipogonadismo/metabolismoRESUMEN
Cellular stress conditions activate p53-dependent pathways to counteract the inflicted damage. To achieve the required functional diversity, p53 is subjected to numerous post-translational modifications and the expression of isoforms. Little is yet known how p53 has evolved to respond to different stress pathways. The p53 isoform p53/47 (p47 or ΔNp53) is linked to aging and neural degeneration and is expressed in human cells via an alternative cap-independent translation initiation from the 2nd in-frame AUG at codon 40 (+118) during endoplasmic reticulum (ER) stress. Despite an AUG codon in the same location, the mouse p53 mRNA does not express the corresponding isoform in either human or mouse-derived cells. High-throughput in-cell RNA structure probing shows that p47 expression is attributed to PERK kinase-dependent structural alterations in the human p53 mRNA, independently of eIF2α. These structural changes do not take place in murine p53 mRNA. Surprisingly, PERK response elements required for the p47 expression are located downstream of the 2nd AUG. The data show that the human p53 mRNA has evolved to respond to PERK-mediated regulation of mRNA structures in order to control p47 expression. The findings highlight how p53 mRNA co-evolved with the function of the encoded protein to specify p53-activities under different cellular conditions.
Asunto(s)
Estrés del Retículo Endoplásmico , Proteína p53 Supresora de Tumor , Humanos , Animales , Ratones , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estrés del Retículo Endoplásmico/genética , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismo , Procesamiento Proteico-Postraduccional , Isoformas de Proteínas/metabolismoRESUMEN
The tumor suppressor protein p53 orchestrates cellular responses to a vast number of stresses, with DNA damage and oncogenic activation being some of the best described. The capacity of p53 to control cellular events such as cell cycle progression, DNA repair, and apoptosis, to mention some, has been mostly linked to its role as a transcription factor. However, how p53 integrates different signaling cascades to promote a particular pathway remains an open question. One way to broaden its capacity to respond to different stimuli is by the expression of isoforms that can modulate the activities of the full-length protein. One of these isoforms is p47 (p53/47, Δ40p53, p53ΔN40), an alternative translation initiation variant whose expression is specifically induced by the PERK kinase during the Unfolded Protein Response (UPR) following Endoplasmic Reticulum stress. Despite the increasing knowledge on the p53 pathway, its activity when the translation machinery is globally suppressed during the UPR remains poorly understood. Here, we focus on the expression of p47 and we propose that the alternative initiation of p53 mRNA translation offers a unique condition-dependent mechanism to differentiate p53 activity to control cell homeostasis during the UPR. We also discuss how the manipulation of these processes may influence cancer cell physiology in light of therapeutic approaches.