RESUMEN
X-linked ATR-X (alpha thalassemia, mental retardation, X-linked) syndrome in males is characterized by mental retardation, facial dysmorphism, alpha thalassemia and urogenital abnormalities, including small testes. It is unclear how mutations in the chromatin-remodeling protein ATRX cause these highly specific clinical features, since ATRX is widely expressed during organ development. To investigate the mechanisms underlying the testicular defects observed in ATR-X syndrome, we generated ScAtrxKO (Sertoli cell Atrx knockout) mice with Atrx specifically inactivated in the supporting cell lineage (Sertoli cells) of the mouse testis. ScAtrxKO mice developed small testes and discontinuous tubules, due to prolonged G2/M phase and apoptosis of proliferating Sertoli cells during fetal life. Apoptosis might be a consequence of the cell cycle defect. We also found that the onset of spermatogenesis was delayed in postnatal mice, with a range of spermatogenesis defects evident in adult ScAtrxKO mice. ATRX and the androgen receptor (AR) physically interact in the testis and in the Sertoli cell line TM4 and co-operatively activate the promoter of Rhox5, an important direct AR target. We also demonstrate that ATRX directly binds to the Rhox5 promoter in TM4 cells. Finally, gene expression of Rhox5 and of another AR-dependent gene, Spinlw1, was reduced in ScAtrxKO testes. These data suggest that ATRX can directly enhance the expression of androgen-dependent genes through physical interaction with AR. Recruitment of ATRX by DNA sequence-specific transcription factors could be a general mechanism by which ATRX achieves tissue-specific transcriptional regulation which could explain the highly specific clinical features of ATR-X syndrome when ATRX is mutated.
Asunto(s)
Proliferación Celular , ADN Helicasas/genética , Proteínas Nucleares/genética , Receptores Androgénicos/metabolismo , Células de Sertoli/metabolismo , Animales , Apoptosis , Ciclo Celular , Células Cultivadas , Ensamble y Desensamble de Cromatina , ADN Helicasas/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Inmunoprecipitación , Masculino , Discapacidad Intelectual Ligada al Cromosoma X/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Testículo/anomalías , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína Nuclear Ligada al Cromosoma X , Talasemia alfa/genéticaRESUMEN
Mutations in ATRX (alpha-thalassaemia and mental retardation on the X-chromosome) can give rise to ambiguous or female genitalia in XY males, implying a role for ATRX in testicular development. Studies on ATRX have mainly focused on its crucial role in brain development and α-globin regulation; however, little is known about its function in sexual differentiation and its expression in the adult testis. Here we show that the ATRX protein is present in adult human and rat testis and is expressed in the somatic cells; Sertoli, Leydig, and peritubular myoid cells, and also in germ cells; spermatogonia and early meiotic spermatocytes. The granular pattern of ATRX staining is consistent with that observed in other cell-types and suggests a role in chromatin regulation. The findings suggest that ATRX in humans may play a role in adult spermatogenesis as well as in testicular development.
Asunto(s)
Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , ADN Helicasas/metabolismo , Proteínas Nucleares/metabolismo , Testículo/enzimología , Animales , Proteínas Cromosómicas no Histona/genética , ADN Helicasas/genética , Trastornos del Desarrollo Sexual/genética , Femenino , Genitales Femeninos/enzimología , Humanos , Células Intersticiales del Testículo/enzimología , Masculino , Discapacidad Intelectual Ligada al Cromosoma X/embriología , Proteínas Nucleares/genética , Ratas , Células de Sertoli/enzimología , Diferenciación Sexual/genética , Diferenciación Sexual/fisiología , Espermatocitos/enzimología , Espermatogénesis/genética , Espermatogonias/enzimología , Proteína Nuclear Ligada al Cromosoma X , Talasemia alfa/embriologíaRESUMEN
In the mammalian embryo, SRY and SOX9 are key Sertoli cell proteins that drive the development of the bipotential gonad into a testes rather than an ovary, leading ultimately to the male phenotype. Clinical SRY and SOX9 mutations causing disorders of sex development (DSD) highlight defective protein-protein interactions between SRY or SOX9, and carrier proteins required for nuclear import (importin-b and calmodulin) and nuclear export (CRM-1). The fine balance between import and export determines the levels of transcriptionally active SRY and SOX9 in the nucleus. Recently, post-translational modifications of SRY and SOX9 have been identified which affect nuclear transport. It is therefore timely that the consequences of sex-reversal mutation upon nuclear transport be reviewed. SRY and SOX9 mutations in DSD have uncovered regulatory sites for sumoylation, ubiquitination, acetylation and phosphorylation, many of which are essential for their transport and sex determining functions.
Asunto(s)
Factor de Transcripción SOX9/metabolismo , Proteína de la Región Y Determinante del Sexo/metabolismo , Animales , Femenino , Humanos , Masculino , Modelos Biológicos , Unión Proteica , Procesamiento Proteico-Postraduccional , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/fisiología , Procesos de Determinación del Sexo , Proteína de la Región Y Determinante del Sexo/genética , Proteína de la Región Y Determinante del Sexo/fisiologíaRESUMEN
Despite 12 yr since the discovery of SRY, little is known at the molecular level about how SRY and the SRY-related protein, SOX9 [SRY-related high-mobility group (HMG) box 9], initiate the program of gene expression required to commit the bipotential embryonic gonad to develop into a testis rather than an ovary. Analysis of SRY and SOX9 clinical mutant proteins and XX mice transgenic for testis-determining genes have provided some insight into their normal functions. SRY and SOX9 contain an HMG domain, a DNA-binding motif. The HMG domain plays a central role, being highly conserved between species and the site of nearly all missense mutations causing XY gonadal dysgenesis. SRY and SOX9 are architectural transcription factors; their HMG domain is capable of directing nuclear import and DNA bending. Whether SRY and SOX9 activate testis-forming genes, repress ovary-forming genes, or both remains speculative until downstream DNA target genes are identified. However, factors that control SRY and SOX9 gene expression have been identified, as have a dozen sex-determining genes, allowing some of the pieces in this molecular genetic puzzle to be connected. Many genes, however, remain unidentified, because in the majority of cases of XY females and in all cases of XX males lacking SRY, the mutated gene is unknown.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas del Grupo de Alta Movilidad/fisiología , Proteínas Nucleares , Diferenciación Sexual , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Animales , Proteínas de Unión al ADN/genética , Trastornos del Desarrollo Sexual , Femenino , Regulación de la Expresión Génica , Dominios HMG-Box/genética , Dominios HMG-Box/fisiología , Proteínas del Grupo de Alta Movilidad/genética , Masculino , Ratones , Datos de Secuencia Molecular , Mutación Missense , Organogénesis/genética , Organogénesis/fisiología , Factor de Transcripción SOX9 , Alineación de Secuencia , Proteína de la Región Y Determinante del Sexo , Factores de Transcripción/genética , Cromosoma Y/genética , Cromosoma Y/fisiologíaRESUMEN
BACKGROUND: ATRX is a tightly-regulated multifunctional protein with crucial roles in mammalian development. Mutations in the ATRX gene cause ATR-X syndrome, an X-linked recessive developmental disorder resulting in severe mental retardation and mild alpha-thalassemia with facial, skeletal and genital abnormalities. Although ubiquitously expressed the clinical features of the syndrome indicate that ATRX is not likely to be a global regulator of gene expression but involved in regulating specific target genes. The regulation of ATRX expression is not well understood and this is reflected by the current lack of identified upstream regulators. The availability of genomic data from a range of species and the very highly conserved 5' regulatory regions of the ATRX gene has allowed us to investigate putative transcription factor binding sites (TFBSs) in evolutionarily conserved regions of the mammalian ATRX promoter. RESULTS: We identified 12 highly conserved TFBSs of key gene regulators involved in biologically relevant processes such as neural and testis development and alpha-globin regulation. CONCLUSIONS: Our results reveal potentially important regulatory elements in the ATRX gene which may lead to the identification of upstream regulators of ATRX and aid in the understanding of the molecular mechanisms that underlie ATR-X syndrome.
RESUMEN
The transcription factor sex-determining region of the Y chromosome (SRY) plays a key role in human sex determination, because mutations in SRY cause disorders of sex development in XY individuals. During gonadal development, Sry in pre-Sertoli cells activates Sox9 gene transcription, committing the fate of the bipotential gonad to become a testis rather than an ovary. The high-mobility group domain of human SRY contains two independent nuclear localization signals, one bound by calmodulin (CaM) and the other by importin-ß. Although XY females carry SRY mutations in these nuclear localization signals that affect SRY nuclear import in transfected cells, it is not known whether these transport mechanisms are essential for gonadal development and sex determination. Here, we show that mouse Sry protein binds CaM and that a CaM antagonist reduces CaM binding, nuclear accumulation, and transcriptional activity of Sry in transfected cells. CaM antagonist treatment of cultured, sexually indifferent XY mouse fetal gonads led to reduced expression of the Sry target gene Sox9, defects in testicular cord formation, and ectopic expression of the ovarian markers Rspondin1 and forkhead box L2. These results indicate the importance of CaM for SRY nuclear import, transcriptional activity, testis differentiation, and sex determination.
Asunto(s)
Transporte Activo de Núcleo Celular/efectos de los fármacos , Antígenos de Diferenciación/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Calmodulina/antagonistas & inhibidores , Proteína de la Región Y Determinante del Sexo/metabolismo , Espermatogénesis , Testículo/fisiología , Animales , Células COS , Calmodulina/metabolismo , Proteínas de Unión a Calmodulina/genética , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Chlorocebus aethiops , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Proteína Forkhead Box L2 , Factores de Transcripción Forkhead/metabolismo , Masculino , Ratones , Técnicas de Cultivo de Órganos , Proteínas Recombinantes/metabolismo , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Procesos de Determinación del Sexo/efectos de los fármacos , Proteína de la Región Y Determinante del Sexo/genética , Cordón Espermático/efectos de los fármacos , Testículo/ultraestructura , Trombospondinas/metabolismo , Activación Transcripcional/efectos de los fármacosRESUMEN
The chromatin-associated protein ATRX was originally identified because mutations in the ATRX gene cause a severe form of syndromal X-linked mental retardation associated with alpha-thalassemia. Half of all of the disease-associated missense mutations cluster in a cysteine-rich region in the N terminus of ATRX. This region was named the ATRX-DNMT3-DNMT3L (ADD) domain, based on sequence homology with a family of DNA methyltransferases. Here, we report the solution structure of the ADD domain of ATRX, which consists of an N-terminal GATA-like zinc finger, a plant homeodomain finger, and a long C-terminal alpha-helix that pack together to form a single globular domain. Interestingly, the alpha-helix of the GATA-like finger is exposed and highly basic, suggesting a DNA-binding function for ATRX. The disease-causing mutations fall into two groups: the majority affect buried residues and hence affect the structural integrity of the ADD domain; another group affects a cluster of surface residues, and these are likely to perturb a potential protein interaction site. The effects of individual point mutations on the folding state and stability of the ADD domain correlate well with the levels of mutant ATRX protein in patients, providing insights into the molecular pathophysiology of ATR-X syndrome.
Asunto(s)
Cromatina/metabolismo , ADN Helicasas/química , ADN Helicasas/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Mutación Puntual/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Transformación Celular Viral , Herpesvirus Humano 4 , Humanos , Linfocitos/virología , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Electricidad Estática , Relación Estructura-Actividad , Propiedades de Superficie , Proteína Nuclear Ligada al Cromosoma XRESUMEN
During mammalian sex determination, SOX9 is translocated into the nuclei of Sertoli cells within the developing XY gonad. The N-terminal nuclear localization signal (NLS) is contained within a SOX consensus calmodulin (CaM) binding region, thereby implicating CaM in nuclear import of SOX9. By fluorescence spectroscopy and glutaraldehyde cross-linking, we show that the SOX9 HMG domain and CaM interact in vitro. The formation of a SOX9.CaM binary complex is calcium-dependent and is accompanied by a conformational change in SOX9. A CaM antagonist, calmidazolium chloride (CDZ), was observed to block CaM recognition of SOX9 in vitro and inhibit both nuclear import and consequent transcriptional activity of SOX9 in treated cells. The significance of the SOX9-CaM interaction was highlighted by analysis of a missense SOX9 mutation, A158T, identified from a XY female with campomelic dysplasia/autosomal sex reversal (CD/SRA). This mutant binds importin beta normally despite defective nuclear import. Fluorescence and quenching studies indicate that in the unbound state, the A158T mutant shows a similar conformation to that of the WT SOX9, but in the presence of CaM, the mutant undergoes unusual conformational changes. Furthermore, SOX9-mediated transcriptional activation by cells expressing the A158T mutant is more sensitive to CDZ than cells expressing WT SOX9. These results suggest first that CaM is involved in the nuclear transport of SOX9 in a process likely to involve direct interaction and second, that CD/SRA can arise, at least in part, from a defect in CaM recognition, ultimately leading to reduced ability of SOX9 to activate transcription of cartilage and testes-forming genes.