RESUMO
AIMS: Epigenetic signatures of germline cells are dynamically reprogrammed to induce appropriate differentiation, development and sex specification. We investigated sex-specific epigenetic changes in mouse fetal germ cells (FGCs) and neonatal germ cells. MATERIALS & METHODS: Six histone marks in mouse E13.5 FGCs and P1 neonatal germ cells were analyzed by chromatin immunoprecipitation and sequencing. These datasets were compared with transposase-accessible chromatin sites, DNA methylation and transcriptome. RESULTS: Different patterns of each histone mark were detected in female and male FGCs, and H3K4me3/H3K27me3 bivalent marks were enriched in different chromosomal regions of female and male FGCs. CONCLUSION: Our results suggest that histone modifications may affect FGC gene expression following DNA methylation erasure, contributing to the differentiation into female and male germ cells.
Assuntos
Células Germinativas/metabolismo , Histonas/metabolismo , Animais , Animais Recém-Nascidos , Epigenômica , Feminino , Feto/citologia , Células Germinativas/citologia , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , TranscriptomaRESUMO
The fertility of sex-reversed XY female mice is severely impaired by a massive loss of oocytes and failure of meiotic progression. This phenomenon remains an outstanding mystery. We sought to determine the molecular etiology of XY oocyte dysfunction by generating sex-reversed females that bear genetic ablation of Sry, a vital sex determination gene, on an inbred C57BL/6 background. These mutant mice, termed XYsry- mutants, showed severe attrition of germ cells during fetal development, resulting in the depletion of ovarian germ cells prior to sexual maturation. Comprehensive transcriptome analyses of primordial germ cells (PGCs) and postnatal oocytes demonstrated that XYsry- females had deviated significantly from normal developmental processes during the stages of mitotic proliferation. The impaired proliferation of XYsry- PGCs was associated with aberrant ß-catenin signaling and the excessive expression of transposable elements. Upon entry to the meiotic stage, XYsry- oocytes demonstrated extensive defects, including the impairment of crossover formation, the failure of primordial follicle maintenance, and no capacity for embryo development. Together, these results suggest potential molecular causes for germ cell disruption in sex-reversed female mice, thereby providing insights into disorders of sex differentiation in humans, such as "Swyer syndrome," in which patients with an XY karyotype present as typical females and are infertile.
Assuntos
Disgenesia Gonadal 46 XY/fisiopatologia , Oócitos/crescimento & desenvolvimento , Proteína da Região Y Determinante do Sexo/genética , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Ligados ao Cromossomo Y , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mitose , Mutação , TranscriptomaRESUMO
In mice, primordial germ cells migrate into the genital ridges by embryonic day 13.5 (E13.5), where they are then subjected to a sex-specific fate with female and male primordial germ cells undergoing mitotic arrest and meiosis, respectively. However, the sex-specific basis of primordial germ cell differentiation is poorly understood. The aim of this study was to investigate the sex-specific features of mouse primordial germ cells. We performed RNA-sequencing (seq) of E13.5 female and male mouse primordial germ cells using next-generation sequencing. We identified 651 and 428 differentially expressed transcripts (>2-fold, P < 0.05) in female and male primordial germ cells, respectively. Of these, many transcription factors were identified. Gene ontology and network analysis revealed differing functions of the identified female- and male-specific genes that were associated with primordial germ cell acquisition of sex-specific properties required for differentiation into germ cells. Furthermore, DNA methylation and ChIP-seq analysis of histone modifications showed that hypomethylated gene promoter regions were bound with H3K4me3 and H3K27me3. Our global transcriptome data showed that in mice, primordial germ cells are decisively assigned to a sex-specific differentiation program by E13.5, which is necessary for the development of vital germ cells.
Assuntos
Biomarcadores/metabolismo , Diferenciação Celular/genética , Metilação de DNA , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Animais , Imunoprecipitação da Cromatina , Feminino , Células Germinativas/citologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Histonas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Componente Principal , Fatores Sexuais , Análise de Célula Única/métodosRESUMO
The distal region of mouse chromosome 12 harbors the Dlk1-Dio3 domain, is essential for normal development and encodes maternally expressed noncoding RNAs (ncRNAs), including Gtl2 as well as paternally expressed proteins.Gtl2 works as a tumor suppressor in several types of human cancer cell lines; however, whether this reflects its function in vivo is unknown. Deleting Gtl2 from the maternal allele (Gtl2((-/+))) results in loss of expression of Gtl2 and decreased expression of downstream ncRNAs, including many miRNAs. To determine the role of ncRNAs in tumorigenesis, we induced teratomas by engrafting E6.5 embryos (wildtype or Gtl2((-/+))) under the kidney capsule of scid mice. Some teratomas derived from the Gtl2((-/+)) embryos exhibited hypertrophic growth, suggesting that ncRNAs, including Gtl2, may act as tumor suppressors in vivo. Microarray analysis of miRNAs expressed by Gtl2((-/+)) teratomas revealed decreased expression of 28 miRNAs encoded by the Dlk1-Dio3 domain, low expression of embryonic stem cell-specific miRNAs and dysregulation of miRNAs involved in tumorigenesis. This study suggests that downregulation of ncRNAs in the Dlk1-Dio3 domain leads to enhanced teratoma growth and repression of stem cell markers.
Assuntos
Deleção de Genes , RNA Longo não Codificante/metabolismo , Células-Tronco/citologia , Teratoma/metabolismo , Células 3T3 , Alelos , Animais , Proteínas de Ligação ao Cálcio , Proliferação de Células , Feminino , Perfilação da Expressão Gênica , Impressão Genômica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Iodeto Peroxidase/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , RNA não Traduzido/metabolismoRESUMO
Four expression plasmids for recombinant human aryl hydrocarbon receptor (hAhR) consisting of a ligand binding domain of hAhR, a DNA-binding domain of LexA and a transactivation domain of VP16 as well as ß-glucuronidase (GUS) reporter genes were constructed. All the expression plasmids were transformed into tobacco plants. The selected transgenic tobacco plants were used to assay. PCB congeners showed GUS activity in a TEF-dependent manner. The selected transgenic tobacco plant XhD4V17 was compared with the transgenic tobacco plants XmD4V26 and XgD2V23 containing recombinant mouse (m) AhR-mediated GUS reporter gene expression cassette and recombinant guinea pig (g) AhR-mediated GUS reporter gene expression cassette for PCB congener-inducible GUS activity. The data revealed that the tobacco plant XgD2V23 was the most active in PCB congener-inducible GUS activity. In a 1:1 mixture of PCB126 and PCB80 a reduced PCB126-induced GUS activity was observed in plant XgD2V23, which could possibly be due to interaction between PCB126 and PCB80.
Assuntos
Monitoramento Ambiental/métodos , Poluentes Ambientais/metabolismo , Genes Reporter , Glucuronidase/genética , Nicotiana/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Bifenilos Policlorados/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Clonagem Molecular , Poluentes Ambientais/química , Expressão Gênica , Glucuronidase/metabolismo , Cobaias , Humanos , Camundongos , Plantas Geneticamente Modificadas/genética , Bifenilos Policlorados/química , Receptores de Hidrocarboneto Arílico/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Nicotiana/genética , Ativação TranscricionalRESUMO
Limb-girdle muscular dystrophy type 2A (LGMD2A) is a genetic disease that is caused by mutations in the calpain 3 gene (CAPN3), which encodes the skeletal muscle-specific calpain, calpain 3 (also known as p94). However, the precise mechanism by which p94 functions in the pathogenesis of this disease remains unclear. Here, using p94 knockin mice (termed herein p94KI mice) in which endogenous p94 was replaced with a proteolytically inactive but structurally intact p94:C129S mutant protein, we have demonstrated that stretch-dependent p94 distribution in sarcomeres plays a crucial role in the pathogenesis of LGMD2A. The p94KI mice developed a progressive muscular dystrophy, which was exacerbated by exercise. The exercise-induced muscle degeneration in p94KI mice was associated with an inefficient redistribution of p94:C129S in stretched sarcomeres. Furthermore, the p94KI mice showed impaired adaptation to physical stress, which was accompanied by compromised upregulation of muscle ankyrin-repeat protein-2 and hsp upon exercise. These findings indicate that the stretch-induced dynamic redistribution of p94 is dependent on its protease activity and essential to protect muscle from degeneration, particularly under conditions of physical stress. Furthermore, our data provide direct evidence that loss of p94 protease activity can result in LGMD2A and molecular insight into how this could occur.
Assuntos
Adaptação Fisiológica , Calpaína/fisiologia , Músculo Esquelético/fisiologia , Distrofia Muscular do Cíngulo dos Membros/etiologia , Envelhecimento/fisiologia , Animais , Calpaína/análise , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Musculares , Distrofia Muscular do Cíngulo dos Membros/fisiopatologia , Miofibrilas/fisiologia , Proteínas Nucleares/fisiologia , Condicionamento Físico Animal , Regeneração , Proteínas Repressoras/fisiologia , Estresse MecânicoRESUMO
p94/calpain 3 is a Ca(2+)-binding intracellular protease predominantly expressed in skeletal muscles. p94 binds to the N2A and M-line regions of connectin/titin and localizes in the Z-bands. Genetic evidence showing that compromised p94 proteolytic activity leads to muscular dystrophy (limb-girdle muscular dystrophy type 2A) indicates the importance of p94 function in myofibrils. Here we show that a series of p94 splice variants is expressed immediately after muscle differentiation and differentially change localization during myofibrillogenesis. We found that the endogenous N-terminal (but not C-terminal) domain of p94 was not only localized in the Z-bands but also directly bound to sarcomeric alpha-actinin. These data suggest the incorporation of proteolytic N-terminal fragments of p94 into the Z-bands. In myofibrils localization of exogenously expressed p94 shifted from the M-line to N2A as the sarcomere lengthens beyond approximately 2.6 and 2.8 microm for wild-type and proteaseinactive p94, respectively. These data demonstrate for the first time that p94 proteolytic activity is involved in responses to muscle conditions, which may explain why p94 inactivation causes limb-girdle muscular dystrophy.
Assuntos
Calpaína/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Desenvolvimento Muscular/fisiologia , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiologia , Peptídeo Hidrolases/metabolismo , Sarcômeros/fisiologia , Actinina/metabolismo , Processamento Alternativo , Animais , Western Blotting , Calpaína/genética , Diferenciação Celular , Células Cultivadas , Conectina , Primers do DNA/química , Imunofluorescência , Biblioteca Gênica , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , Músculo Esquelético/citologia , Proteínas Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Técnicas do Sistema de Duplo-HíbridoRESUMO
p94/calpain 3 is a skeletal muscle-specific member of the Ca(2+)-regulated cytosolic cysteine protease family, the calpains. Defective p94 protease activity originating from gene mutations causes a muscular dystrophy called calpainopathy, indicating the indispensability of p94 for muscle survival. Because of the existence of the p94-specific regions IS1 and IS2, p94 undergoes very rapid and exhaustive autolysis. To elucidate the physiological relevance of this unique activity, the autolytic profiles of p94 and the effect of the p94 binding protein, connectin/titin, on this process were investigated. In vitro analysis of p94 autolysis showed that autolysis in IS1 proceeds without immediate disassembly into fragments and that the newly identified cryptic autolytic site in IS2 is critical for disassembling autolyzed fragments. As a genetic system to assay p94 autolysis semiquantitatively, p94 was expressed in yeast as a hybrid protein between the DNA binding and activation domains of the yeast transcriptional activator Gal4. Transcriptional activation by the Gal4-p94:WT hybrid protein is precluded by p94 autolysis. Complete or partial loss of autolytic activity by C129S active site mutation, limb girdle muscular dystrophy type 2A pathogenic missense mutations, or PCR-based random mutagenesis could be detected by semiquantitative restoration of Gal4-dependent beta-galactosidase gene expression. Using this system, the N2A connectin fragment that binds to p94 was shown to suppress p94 autolytic disassembly. The proximity of the IS2 autolytic and connectin-binding sites in p94 suggested that N2A connectin suppresses IS2 autolysis. These data indicate the importance of p94-connectin interaction in the control of p94 functions by regulating autolytic decay of p94.
Assuntos
Calpaína/metabolismo , Proteínas Musculares/metabolismo , Proteínas Quinases/metabolismo , Animais , Sítios de Ligação/genética , Células COS , Calpaína/química , Catálise , Chlorocebus aethiops , Conectina , Ativação Enzimática , Humanos , Camundongos , Modelos Moleculares , Proteínas Musculares/química , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Proteínas Quinases/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiaeRESUMO
Variation in the calpain 10 gene has recently been shown to be associated with type 2 diabetes by positional cloning. Since then, studies on calpain 10 have been started in correlation with diabetes and insulin-mediated signaling. In this review, the activation mechanism of calpain by calcium ions, which is essential to understand its physiological functions, is discussed on the basis of recent X-ray structural analyses. Further, special features of the structure of calpain 10 that differ from those of typical micro - or m-calpain used in most studies are summarized together with discussion of the physiological function of calpain with respect to type 2 diabetes.
Assuntos
Calpaína/fisiologia , Diabetes Mellitus Tipo 2/genética , Cálcio , Calpaína/química , Calpaína/genética , Clonagem Molecular , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
There are two classes of an intracellular 'modulator protease', calpain: ubiquitous and tissue-specific. p94/calpain 3 is an example of the latter, predominantly expressed in muscle. A defect in the p94 gene causes muscular dystrophy. Here we report that human and mouse p94 genes have a possible novel alternative promoter expressing p94 variants in all tissues examined including human lens epithelial cells. The possible promoter region and the following novel exons overlap the 3' region of the neutral alpha-glucosidase C gene. Unlike p94, the novel p94 variants expressed in COS7 cells do not undergo rapid autolysis, suggesting basic functions different from p94.
Assuntos
Calpaína/biossíntese , Calpaína/genética , Éxons/genética , alfa-Glucosidases/genética , Sequência de Aminoácidos , Animais , Células COS , Primers do DNA/genética , Perfilação da Expressão Gênica , Genes , Humanos , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas , Estrutura Terciária de Proteína , RatosRESUMO
Calpain, a Ca(2+)-requiring cytoplasmic cysteine protease, plays indispensable roles in various cellular functions such as signal transduction, cell growth and differentiation, apoptosis, necrosis, and so on. Although most of the detailed physiological functions of calpains have not yet been elucidated, the importance of calpain is obvious from the increasing numbers of papers describing relationships between human disease states (such as Alzheimer's disease, cataract, and muscular dystrophies) and malfunction of calpain. One of the recent remarkable topics of calpain is that a single nucleotide polymorphism of CAPN10, the gene for calpain 10, is related to type 2 diabetes. However, physiological functions of calpain 10 and its relation to diabetes are still unclear. Among 14 human calpain genes, mutations in CAPN3, the gene for p94/calpain 3a and Lp82/calpain 3b, are the only example that genetically connects the calpain gene and human disease, in this case, limb-girdle muscular dystrophy type 2A (LGMD2A). p94 has unique characteristics such as apparent Ca(2+)-independent activation and very rapid autolytic activity, which are dependent on p94-specific regions, NS, IS1, and IS2. Based on the 3D structures of micro - and m-calpain, molecular functions of p94 in relation to LGMD2A are discussed, with the hope of providing us with some clues to understand calpain functions and its relationships to human diseases.