RESUMO
Congenital heart disease (CHD) has a strong genetic etiology, making it a likely candidate for therapeutic intervention using genetic editing. Complex genetics involving an orchestrated series of genetic events and over 400 genes are responsible for myocardial development. Cooperation is required from a vast series of genetic networks, and mutations in such can lead to CHD and cardiovascular abnormalities, affecting up to 1% of all live births. Genome editing technologies are becoming better studied and with time and improved logistics, CHD could be a prime therapeutic target. Syndromic, nonsyndromic, and cases of familial inheritance all involve identifiable causative mutations and thus have the potential for genome editing therapy. Mouse models are well-suited to study and predict clinical outcome. This review summarizes the anatomical and genetic timeline of myocardial development in both mice and humans, the potential of gene editing in typical CHD categories, as well as the use of mice thus far in reproducing models of human CHD and correcting the mutations that create them.
Assuntos
Edição de Genes , Redes Reguladoras de Genes , Humanos , Camundongos , Animais , Mutação , Modelos Animais de Doenças , Padrões de HerançaRESUMO
INTRODUCTION: Imprinted genes are preferentially expressed from one parentally inherited allele, and many are crucial to the regulation of placental function and fetal growth. Murine Krüppel-like factor 14 (Klf14) is a maternally expressed imprinted transcription factor that is a component of the Mest imprinted gene cluster on mouse chromosome 6. We sought to determine if loss of Klf14 expression alters the course of normal mouse extraembryonic development. We also used high-throughput RNA sequencing (RNAseq) to identify a set of differentially expressed genes (DEGs) in placentas with loss of Klf14. METHODS: We generated a Klf14 knockout (Klf14null) mouse using recombineering and transgenic approaches. To identify DEGs in the mouse placenta we compared mRNA transcriptomes derived from 17.5dpc Klf14matKO and wild-type littermate placentas by RNAseq. Candidate DEGs were confirmed with quantitative reverse transcription PCR (qPCR) on an independent cohort of male and female gestational age matched Klf14matKO placentas. RESULTS: We found that 17.5dpc placentas inheriting a maternal null allele (Klf14matKO) had a modest overgrowth phenotype and a near complete ablation of Klf14 expression. However, there was no effect on fetal growth. We identified 20 DEGs differentially expressed in Klf14matKO placentas by RNAseq, and subsequently validated five that are highly upregulated (Begain, Col26a1, Fbln5, Gdf10, and Nell1) by qPCR. The most enriched functional gene-networks included those classified as regulating cellular development and metabolism. CONCLUSION: These results suggest that loss of the maternal Klf14 locus in the mouse placenta acts results in changes in gene expression patterns that modulate placental growth.
Assuntos
Fatores de Transcrição Kruppel-Like/metabolismo , Placentação , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Redes Reguladoras de Genes , Impressão Genômica , Fator 10 de Diferenciação de Crescimento/metabolismo , Camundongos Knockout , Gravidez , Proteínas Recombinantes/metabolismoRESUMO
Clinical studies have revealed an increased incidence of growth and genomic imprinting disorders in children conceived using assisted reproductive technologies (ARTs), and aberrant DNA methylation has been implicated. We propose that compromised oocyte quality associated with female infertility may make embryos more susceptible to the induction of epigenetic defects by ART. DNA methylation patterns in the preimplantation embryo are dependent on the oocyte-specific DNA methyltransferase 1o (DNMT1o), levels of which are decreased in mature oocytes of aging females. Here, we assessed the effects of maternal deficiency in DNMT1o (Dnmt1Δ1o/+) in combination with superovulation and embryo transfer on offspring DNA methylation and development. We demonstrated a significant increase in the rates of morphological abnormalities in offspring collected from Dnmt1Δ1o/+ females only when combined with ART. Together, maternal oocyte DNMT1o deficiency and ART resulted in an accentuation of placental imprinting defects and the induction of genome-wide DNA methylation alterations, which were exacerbated in the placenta compared to the embryo. Significant sex-specific trends were also apparent, with a preponderance of DNA hypomethylation in females. Among genic regions affected, a significant enrichment for neurodevelopmental pathways was observed. Taken together, our results demonstrate that oocyte DNMT1o-deficiency exacerbates genome-wide DNA methylation abnormalities induced by ART in a sex-specific manner and plays a role in mediating poor embryonic outcome.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Oócitos/fisiologia , Técnicas Reprodutivas/efeitos adversos , Fatores Etários , Animais , Metilação de DNA , Epigênese Genética , Feminino , Infertilidade Feminina/fisiopatologia , Camundongos , Modelos Animais , Oócitos/patologia , Placenta/metabolismo , Gravidez , Superovulação/genética , Superovulação/fisiologiaRESUMO
Mutations in imprinted genes or their imprint control regions (ICRs) produce changes in imprinted gene expression and distinct abnormalities in placental structure, indicating the importance of genomic imprinting to placental development. We have recently shown that a very broad spectrum of placental abnormalities associated with altered imprinted gene expression occurs in the absence of the oocyte-derived DNMT1o cytosine methyltransferase, which normally maintains parent-specific imprinted methylation during preimplantation. The absence of DNMT1o partially reduces inherited imprinted methylation while retaining the genetic integrity of imprinted genes and their ICRs. Using this novel system, we undertook a broad and inclusive approach to identifying key ICRs involved in placental development by correlating loss of imprinted DNA methylation with abnormal placental phenotypes in a mid-gestation window (E12.5-E15.5). To these ends we measured DNA CpG methylation at 15 imprinted gametic differentially methylated domains (gDMDs) that overlap known ICRs using EpiTYPER-mass array technology, and linked these epigenetic measurements to histomorphological defects. Methylation of some imprinted gDMDs, most notably Dlk1, was nearly normal in mid-gestation DNMT1o-deficient placentas, consistent with the notion that cells having lost methylation on these DMDs do not contribute significantly to placental development. Most imprinted gDMDs however showed a wide range of methylation loss among DNMT1o-deficient placentas. Two striking associations were observed. First, loss of DNA methylation at the Peg10 imprinted gDMD associated with decreased embryonic viability and decreased labyrinthine volume. Second, loss of methylation at the Kcnq1 imprinted gDMD was strongly associated with trophoblast giant cell (TGC) expansion. We conclude that the Peg10 and Kcnq1 ICRs are key regulators of mid-gestation placental function.
Assuntos
Impressão Genômica/fisiologia , Canal de Potássio KCNQ1/fisiologia , Proteínas Nucleares/fisiologia , Placenta/anormalidades , Fatores de Transcrição/fisiologia , Animais , Proteínas Reguladoras de Apoptose , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/deficiência , Metilação de DNA , Proteínas de Ligação a DNA , Feminino , Morte Fetal/etiologia , Idade Gestacional , Canal de Potássio KCNQ1/genética , Tamanho da Ninhada de Vivíparos , Camundongos , Camundongos da Linhagem 129 , Proteínas Nucleares/genética , Fenótipo , Gravidez , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA , Fatores de Transcrição/genética , Triglicerídeos/metabolismoRESUMO
Genome-wide demethylation and remethylation of DNA during early embryogenesis is essential for development. Imprinted germline differentially methylated domains (gDMDs) established by sex-specific methylation in either male or female germ cells, must escape these dynamic changes and sustain precise inheritance of both methylated and unmethylated parental alleles. To identify other, gDMD-like sequences with the same epigenetic inheritance properties, we used a modified embryonic stem (ES) cell line that emulates the early embryonic demethylation and remethylation waves. Transient DNMT1 suppression revealed gDMD-like sequences requiring continuous DNMT1 activity to sustain a highly methylated state. Remethylation of these sequences was also compromised in vivo in a mouse model of transient DNMT1 loss in the preimplantation embryo. These novel regions, possessing heritable epigenetic features similar to imprinted-gDMDs are required for normal physiological and developmental processes and when disrupted are associated with disorders such as cancer and autism spectrum disorders. This study presents new perspectives on DNA methylation heritability during early embryo development that extend beyond conventional imprinted-gDMDs.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Genoma Humano , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA , HumanosRESUMO
BACKGROUND & AIMS: Augmenter of liver regeneration (ALR, encoded by GFER) is a widely distributed pleiotropic protein originally identified as a hepatic growth factor. However, little is known about its roles in hepatic physiology and pathology. We created mice with liver-specific deletion of ALR to study its function. METHODS: We developed mice with liver-specific deletion of ALR (ALR-L-KO) using the albumin-Cre/LoxP system. Liver tissues were collected from ALR-L-KO mice and ALR(floxed/floxed) mice (controls) and analyzed by histology, reverse-transcription polymerase chain reaction, immunohistochemistry, electron microscopy, and techniques to measure fibrosis and lipids. Liver tissues from patients with and without advanced liver disease were determined by immunoblot analysis. RESULTS: Two weeks after birth, livers of ALR-L-KO mice contained low levels of ALR and adenosine triphosphate (ATP); they had reduced mitochondrial respiratory function and increased oxidative stress, compared with livers from control mice, and had excessive steatosis, and hepatocyte apoptosis. Levels of carbamyl-palmitoyl transferase 1a and ATP synthase subunit ATP5G1 were reduced in livers of ALR-L-KO mice, indicating defects in mitochondrial fatty acid transport and ATP synthesis. Electron microscopy showed mitochondrial swelling with abnormalities in shapes and numbers of cristae. From weeks 2-4 after birth, levels of steatosis and apoptosis decreased in ALR-L-KO mice, and numbers of ALR-expressing cells increased, along with ATP levels. However, at weeks 4-8 after birth, livers became inflamed, with hepatocellular necrosis, ductular proliferation, and fibrosis; hepatocellular carcinoma developed by 1 year after birth in nearly 60% of the mice. Hepatic levels of ALR were also low in ob/ob mice and alcohol-fed mice with liver steatosis, compared with controls. Levels of ALR were lower in liver tissues from patients with advanced alcoholic liver disease and nonalcoholic steatohepatitis than in control liver tissues. CONCLUSIONS: We developed mice with liver-specific deletion of ALR, and showed that it is required for mitochondrial function and lipid homeostasis in the liver. ALR-L-KO mice provide a useful model for investigating the pathogenesis of steatohepatitis and its complications.
Assuntos
Carcinoma Hepatocelular/etiologia , Fígado Gorduroso/etiologia , Neoplasias Hepáticas/etiologia , Regeneração Hepática/fisiologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/fisiologia , Animais , Apoptose , Redutases do Citocromo/fisiologia , Humanos , Metabolismo dos Lipídeos , Cirrose Hepática Experimental/etiologia , Camundongos , Camundongos Knockout , Mitocôndrias/fisiologiaRESUMO
The DNMT1 cytosine methyltransferase enzyme contains a large â¼300-aa intrinsically disordered domain (IDD) that we previously showed regulated DNA methylation patterns in mouse ES cells. Here we generated seven mouse lines with different mutations in the IDD. Homozygous mutant mice of five lines developed normally, with normal levels of methylation on both imprinted and nonimprinted DNA sequences. The other two lines, however, had alterations in imprinted and/or nonimprinted (global) DNA methylation appearing during embryonic development. Embryos of one line expressing a DNMT1 variant containing a 6-aa rat orthologous sequence in the IDD maintained imprinted methylation, showed very reduced levels of global methylation and occasionally completed fetal development. These in vivo studies demonstrate that at least two DNMT1-dependent methylation processes can be distinguished during fetal development. One process maintains the bulk of genomic methylation on nonimprinted sequences. The other process maintains methylation on a much smaller class of sequences including but not limited to gametic differentially methylated domains (gDMDs) that transmit essential imprinted parent-specific methylation for embryonic development.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Genoma , Proteínas Intrinsicamente Desordenadas , Domínios e Motivos de Interação entre Proteínas , Sequência de Aminoácidos , Animais , Linhagem Celular , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/química , Células-Tronco Embrionárias/metabolismo , Éxons , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Estudos de Associação Genética , Estudo de Associação Genômica Ampla , Masculino , Camundongos , Dados de Sequência Molecular , Mutação , Fenótipo , Alinhamento de SequênciaRESUMO
The maintenance of key germline derived DNA methylation patterns during preimplantation development depends on stores of DNA cytosine methyltransferase-1o (DNMT1o) provided by the oocyte. Dnmt1o(mat-/-) mouse embryos born to Dnmt1(Δ1o/Δ1o) female mice lack DNMT1o protein and have disrupted genomic imprinting and associated phenotypic abnormalities. Here, we describe additional female-specific morphological abnormalities and DNA hypomethylation defects outside imprinted loci, restricted to extraembryonic tissue. Compared to male offspring, the placentae of female offspring of Dnmt1(Δ1o/Δ1o) mothers displayed a higher incidence of genic and intergenic hypomethylation and more frequent and extreme placental dysmorphology. The majority of the affected loci were concentrated on the X chromosome and associated with aberrant biallelic expression, indicating that imprinted X-inactivation was perturbed. Hypomethylation of a key regulatory region of Xite within the X-inactivation center was present in female blastocysts shortly after the absence of methylation maintenance by DNMT1o at the 8-cell stage. The female preponderance of placental DNA hypomethylation associated with maternal DNMT1o deficiency provides evidence of additional roles beyond the maintenance of genomic imprints for DNA methylation events in the preimplantation embryo, including a role in imprinted X chromosome inactivation.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , Impressão Genômica , Inativação do Cromossomo X/genética , Animais , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/deficiência , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Placenta/anormalidades , Gravidez , RNA Longo não Codificante/genética , Cromossomo X/genéticaRESUMO
The DNA cytosine methyltransferase 1 (DNMT1) is a ubiquitous nuclear enzyme that catalyzes the well-established reaction of placing methyl groups on the unmethylated cytosines in methyl-CpG:CpG base pairs in the hemimethylated DNA formed by methylated parent and unmethylated daughter strands. This activity regenerates fully methylated methyl-CpG:methyl-CpG pairs. Despite the straightforward nature of its catalytic activity, detailed biochemical, genetic, and developmental studies revealed intricate details of the central regulatory role of DNMT1 in governing the epigenetic makeup of the nuclear genome. DNMT1 mediates demethylation and also participates in seemingly wide cellular functions unrelated to maintenance DNA methylation. This review brings together mechanistic details of maintenance methylation by DNMT1, its regulation at transcriptional and posttranscriptional levels, and the seemingly unexpected functions of DNMT1 in the context of DNA methylation which is central to epigenetic changes that occur during development and the process of cell differentiation.
Assuntos
Células/enzimologia , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Animais , DNA (Citosina-5-)-Metiltransferase 1 , Humanos , Biologia MolecularRESUMO
Mitochondrial targeted manganese superoxide dismutase is a major antioxidant enzyme, the levels of which modulate the response of cells, tissues and organs to ionizing irradiation. We developed a Tet-regulated MnSOD mouse (MnSOD(tet)) to examine the detailed relationship between cellular MnSOD concentration and radioresistance and carried out in vitro studies using bone marrow culture derived stromal cell lines (mesenchymal stem cells). Homozygous MnSOD(tet/tet) cells had low levels of MnSOD, reduced viability and proliferation, increased radiosensitivity, elevated overall antioxidant stores, and defects in cell proliferation and DNA strand-break repair. Doxycycline (doxy) treatment of MnSOD(tet/tet) cells increased MnSOD levels and radioresistance from ñ of 2.79 ± 1.04 to 8.69 ± 1.09 (P = 0.0060) and normalized other biologic parameters. In contrast, MnSOD(tet/tet) cells showed minimal difference in baseline and radiation induced mRNA and protein levels of TGF-ß, Nrf2 and NF-κB and radiation induced cell cycle arrest was not dependent upon MnSOD level. These novel MnSOD(tet/tet) mouse derived cells should be valuable for elucidating several parameters of the oxidative stress response to ionizing radiation.
Assuntos
Linhagem Celular/efeitos da radiação , Raios gama/efeitos adversos , Tolerância a Radiação/genética , Células Estromais/efeitos da radiação , Superóxido Dismutase/fisiologia , Animais , Medula Óssea , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular/enzimologia , Células Clonais/enzimologia , Células Clonais/efeitos da radiação , Ensaio de Unidades Formadoras de Colônias , Quebras de DNA/efeitos da radiação , Doxiciclina/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/efeitos da radiação , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/enzimologia , Mutagênese Sítio-Dirigida , Fator 2 Relacionado a NF-E2/biossíntese , Fator 2 Relacionado a NF-E2/genética , NF-kappa B/biossíntese , NF-kappa B/genética , Plasmídeos/genética , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Células Estromais/enzimologia , Superóxido Dismutase/biossíntese , Superóxido Dismutase/genética , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genéticaRESUMO
Teratomas are a unique class of tumors composed of ecto-, meso- and endodermal tissues, all foreign to the site of origin. In humans, the most common teratoma is the ovarian teratoma. Not much is known about the molecular and genetic etiologies of these tumors. Female carriers of the Tgkd transgene are highly susceptible to developing teratomas. Ovaries of Tgkd/+ hemizygous female mice exhibit defects in luteinization, with numerous corpora lutea, some of which contain central trapped, fully-grown oocytes. Genetically, Tgkd teratomas originate from mature oocytes that have completed meiosis I, suggesting that Tgkd teratomas originate from these trapped oocytes. The insertion of Tgkd 3' of the Inpp4b gene is associated with decreased expression of Inpp4b and changes in intracellular PI3 Kinase/AKT signaling in follicular granulosa cells. Because Inpp4b is not expressed in fully-grown wild-type or Tgkd oocytes, these findings suggest that hyperactivation of the PI3K/AKT pathway caused by the decrease in INPP4B in granulosa cells promotes an ovarian environment defective in folliculogenesis and conducive to teratoma formation.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Ovarianas/genética , Monoéster Fosfórico Hidrolases/metabolismo , Transdução de Sinais/fisiologia , Teratoma/genética , Transgenes/genética , Animais , Bromodesoxiuridina , Ensaio de Imunoadsorção Enzimática , Feminino , Genótipo , Células da Granulosa/metabolismo , Immunoblotting , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Folículo Ovariano/crescimento & desenvolvimento , Neoplasias Ovarianas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Estatísticas não Paramétricas , Teratoma/metabolismoRESUMO
Monoallelic expression of imprinted genes, including ones solely expressed in the placenta, is essential for normal placental development and fetal growth. To better understand the role of placental imprinting in placental development and fetal growth, we examined conceptuses developing in the absence of maternally derived DNA (cytosine-5-)-methyltransferase 1o (DNMT1o). Absence of DNMT1o results in the partial loss of methylation at imprinted differentially methylated domain (DMD) sequences in the embryo and the placenta. Mid-gestation E9.5 DNMT1o-deficient placentas exhibited structural abnormalities of all tissue layers. At E17.5, all examined placentas had aberrant placental morphology, most notably in the spongiotrophoblast and labyrinth layers. Abnormalities included an expanded volume fraction of spongiotrophoblast tissue with extension of the spongiotrophoblast layer into the labyrinth. Many mutant placentas also demonstrated migration abnormalities of glycogen cells. Additionally, the volume fraction of the labyrinth was reduced, as was the surface area for maternal fetal gas exchange. Despite these placental morphologic abnormalities, approximately one-half of DNMT1o-deficient fetuses survived to late gestation (E17.5). Furthermore, DNMT1o-deficient placentas supported a broad range of fetal growth. The ability of some DNMT1o-deficient and morphologically abnormal placentas to support fetal growth in excess of wild type demonstrates the importance of differential methylation of DMDs and proper imprinting of discrete gene clusters to placental morphogenesis and fetal growth.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Impressão Genômica/fisiologia , Placenta/embriologia , Placenta/metabolismo , Animais , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/deficiência , DNA (Citosina-5-)-Metiltransferases/genética , Primers do DNA/genética , Feminino , Técnicas Histológicas , Imuno-Histoquímica , Hibridização In Situ , Modelos Lineares , Camundongos , Análise em Microsséries , Gravidez , Estrutura Terciária de Proteína , Estatísticas não ParamétricasRESUMO
High levels of DNA methyltransferase 1 (DNMT1), hypermethylation, and downregulation of GAD(67) and reelin have been described in GABAergic interneurons of patients with schizophrenia (SZ) and bipolar (BP) disorders. However, overexpression of DNMT1 is lethal, making it difficult to assess the direct effect of high levels of DNMT1 on neuronal development in vivo. We therefore used Dnmt1(tet/tet) mouse ES cells that overexpress DNMT1 as an in vitro model to investigate the impact of high levels of DNMT1 on neuronal differentiation. Although there is down-regulation of DNMT1 during early stages of differentiation in wild type and Dnmt1(tet/tet) ES cell lines, neurons derived from Dnmt1(tet/tet) cells showed abnormal dendritic arborization and branching. The Dnmt1(tet/tet) neuronal cells also showed elevated levels of functional N-methyl d-aspartate receptor (NMDAR), a feature also reported in some neurological and neurodegenerative disorders. Considering the roles of reelin and GAD(67) in neuronal networking and excitatory/inhibitory balance, respectively, we studied methylation of these genes' promoters in Dnmt1(tet/tet) ES cells and neurons. Both reelin and GAD(67) promoters were not hypermethylated in the Dnmt1(tet/tet) ES cells and neurons, suggesting that overexpression of DNMT1 may not directly result in methylation-mediated repression of these two genes. Taken together, our results suggest that overexpression of DNMT1 in ES cells results in an epigenetic change prior to the onset of differentiation. This epigenetic change in turn results in abnormal neuronal differentiation and upregulation of functional NMDA receptor.
Assuntos
Diferenciação Celular , DNA (Citosina-5-)-Metiltransferases/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Regulação para Cima , Animais , Células Cultivadas , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , Immunoblotting , Camundongos , Neurônios/citologia , Neurônios/patologia , Proteína Reelina , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
DMAP1 (DNMT1-associated protein 1) is a member of the TIP60-p400 complex that maintains embryonic stem (ES) cell pluripotency and a complex containing the somatic form of DNA methyltransferase 1 (DNMT1s). DMAP1 interacts with DNMT1s through a domain that is absent in Dnmt1(V)(/)(V) mice expressing just the oocyte form (DNMT1o). A Dmap1-null allele was generated to study the role of DMAP1 in development. Consistent with the phenotypes of loss of other members of the TIP60-p400 complex, Dmap1(-/-) mice died during preimplantation in both Dnmt1(+/+) and Dnmt1(V)(/)(V) backgrounds. Unexpectedly, in the Dnmt1(V)(/)(V) background, Dmap1(+/-) parents produced mainly Dmap1(+/-) mice. Most Dmap1(+/+) progeny died during midgestation, with loss of DNA methylation on imprinted genes, suggesting that DMAP1 influences maintenance methylation mediated by DNMT1o. In this regard, a DMAP1-DNMT1o complex was detected in ES cells when DNMT1o was stably expressed but not when transiently expressed, indicating a novel interaction between DMAP1 and DNMT1o. These results suggest that DMAP1-DNMT1s and DMAP1-DNMT1o interactions are essential for normal development and that DMAP1-DNMT1o complexes are not readily formed in the embryo. Therefore, DMAP1 mediates distinct preimplantation epigenetic reprogramming processes: TIP60-p400 nucleosome remodeling and DNMT1 maintenance methylation.
Assuntos
Camundongos/crescimento & desenvolvimento , Proteínas Repressoras/metabolismo , Animais , Células Cultivadas , Metilação de DNA , Embrião de Mamíferos/metabolismo , Deleção de Genes , Impressão Genômica , Homozigoto , Camundongos/genética , Fenótipo , Domínios e Motivos de Interação entre Proteínas , Proteínas Repressoras/genéticaRESUMO
Deletion analysis of mouse DNMT1, the primary maintenance methyltransferase in mammals, showed that most of the N-terminal regulatory domain (amino acid residues 412-1112) is required for its enzymatic activity. Although analysis of deletion mutants helps to identify regions of a protein sequence required for a particular activity, amino acid deletions can have drastic effects on protein structure and/or stability. Alternative approaches represented by rational design and directed evolution are resource demanding, and require high-throughput selection or screening systems. We developed Regional Frame-shift Mutagenesis (RFM) as a new approach to identify portions required for the methyltransferase activity of DNMT1 within the N-terminal 89-905 amino acids. In this method, a short stretch of amino acids in the wild-type protein is converted to a different amino acid sequence. The resultant mutant protein retains the same amino acid length as the wild type, thereby reducing physical constrains on normal folding of the mutant protein. Using RFM, we identified three small regions in the amino-terminal one-third of the protein that are essential for DNMT1 function. Two of these regions (amino acids 124-160 and 341-368) border a large disordered region that regulates maintenance methylation activity. This organization of DNMT1's amino terminus suggests that the borders define the position of the disordered region within the DNMT1 protein, which in turn allows for its proper function.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Animais , DNA (Citosina-5-)-Metiltransferase 1 , DNA Complementar/metabolismo , Células-Tronco Embrionárias/citologia , Mutação da Fase de Leitura , Deleção de Genes , Imuno-Histoquímica/métodos , Camundongos , Mutagênese , Mutagênese Sítio-Dirigida , Mutação , Plasmídeos/metabolismo , Dobramento de Proteína , Estrutura Terciária de Proteína , Transcrição GênicaRESUMO
Reprogramming of DNA methylation patterns during mammalian preimplantation development involves the concurrent maintenance of methylation on differentially methylated domains (DMDs) of imprinted genes and a marked reduction of global (non-DMD) genomic methylation. In the developing mammalian embryo, one allele of a DMD is unmethylated, and the opposite parental allele is methylated, having inherited this methylation from the parental gamete. The maintenance of DMDs is important for monoallelic imprinted gene expression and normal development of the embryo. Because the DNMT1 cytosine methyltransferase governs maintenance methylation in mammals, rearrangements of non-DMD, but not DMD methylation in preimplantation embryos suggest that the preimplantation DNMT1-dependent maintenance mechanism specifically targets DMD sequences. We explored this possibility using an engineered mouse ES cell line to screen for mutant DNMT1 proteins that protect against the loss of DMD and/or global (non-DMD) methylation in the absence of the wild-type endogenous DNMT1 methyltransferase. We identified DNMT1 mutants that were defective in maintenance of either DMD and/or non-DMD methylation. Among these, one mutant maintained non-DMD methylation but not imprinted DMD methylation and another mutant maintained just DMD methylation. The mutated amino acids of these mutants reside in a mammal-specific, disordered region near the amino terminus of DNMT1. These findings suggest that DNMT1 participates in epigenetic reprogramming through its ability to distinguish different categories of methylated sequences.
Assuntos
DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/metabolismo , Impressão Genômica/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Especificidade da EspécieRESUMO
Stable and full differentiation of pluripotent stem cells into functional beta-cells offers the potential to treat type I diabetes with a theoretically inexhaustible source of replacement cells. In addition to the difficulties in directed differentiation, progress toward an optimized and reliable protocol has been hampered by the complication that cultured cells will concentrate insulin from the media, thus making it difficult to tell which, if any, cells are producing insulin. To address this, we utilized a novel murine embryonic stem cell (mESC) research model, in which the green fluorescent protein (GFP) has been inserted within the C-peptide of the mouse insulinII gene (InsulinII-GFP). Using this method, cells producing insulin are easily identified. We then compared four published protocols for differentiating mESCs into beta-cells to evaluate their relative efficiency by assaying intrinsic insulin production. Cells differentiated using each protocol were easily distinguished based on culture conditions and morphology. This comparison is strengthened because all testing is performed within the same laboratory by the same researchers, thereby removing interlaboratory variability in culture, cells, or analysis. Differentiated cells were analyzed and sorted based on GFP fluorescence as compared to wild type cells. Each differentiation protocol increased GFP fluorescence but only modestly. None of these protocols yielded more than 3% of cells capable of insulin biosynthesis indicating the relative inefficiency of all analyzed protocols. Therefore, improved beta-cells differentiation protocols are needed, and these insulin II GFP cells may prove to be an important tool to accelerate this process.
Assuntos
Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Células Secretoras de Insulina/fisiologia , Insulina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Linhagem Celular , Células-Tronco Embrionárias/citologia , Genes Reporter , Proteínas de Fluorescência Verde/genética , Insulina/genética , Células Secretoras de Insulina/citologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Recombinantes de Fusão/genética , Distribuição TecidualRESUMO
Most mouse embryos developing in the absence of the oocyte-derived DNA methyltransferase 1o (DNMT1o-deficient embryos) have significant delays in development and a wide range of anatomical abnormalities. To understand the timing and molecular basis of such variation, we studied pre- and post-implantation DNA methylation as a gauge of epigenetic variation among these embryos. DNMT1o-deficient embryos showed extensive differences in the levels of methylation in differentially methylated domains (DMDs) of imprinted genes at the 8-cell stage. Because of independent assortment of the methylated and unmethylated chromatids created by the loss of DNMT1o, the deficient embryos were found to be mosaics of cells with different, but stable epigenotypes (DNA methylation patterns). Our results suggest that loss of DNMT1o in just one cell cycle is responsible for the extensive variation in the epigenotypes in both embryos and their associated extraembryonic tissues. Thus, the maternal-effect DNMT1o protein is uniquely poised during development to normally ensure uniform parental methylation patterns at DMDs.
Assuntos
DNA (Citosina-5-)-Metiltransferases/fisiologia , Metilação de DNA/fisiologia , Implantação do Embrião/fisiologia , Epigênese Genética/fisiologia , Impressão Genômica/fisiologia , Animais , Linhagem Celular , Cromátides/genética , Cromátides/fisiologia , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , Epigênese Genética/genética , Feminino , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Oócitos/metabolismo , Placenta/metabolismoRESUMO
BACKGROUND: Establishing a suitable level of exogenous gene expression in mammalian cells in general, and embryonic stem (ES) cells in particular, is an important aspect of understanding pathways of cell differentiation, signal transduction and cell physiology. Despite its importance, this process remains challenging because of the poor correlation between the presence of introduced exogenous DNA and its transcription. Consequently, many transfected cells must be screened to identify those with an appropriate level of expression. To improve the screening process, we investigated the utility of the human interleukin 12 (IL-12) p40 cDNA as a reporter gene for studies of mammalian gene expression and for high-throughput screening of engineered mouse embryonic stem cells. RESULTS: A series of expression plasmids were used to study the utility of IL-12 p40 as an accurate reporter of gene activity. These studies included a characterization of the IL-12 p40 expression system in terms of: (i) a time course of IL-12 p40 accumulation in the medium of transfected cells; (ii) the dose-response relationship between the input DNA and IL-12 p40 mRNA levels and IL-12 p40 protein secretion; (iii) the utility of IL-12 p40 as a reporter gene for analyzing the activity of cis-acting genetic elements; (iv) expression of the IL-12 p40 reporter protein driven by an IRES element in a bicistronic mRNA; (v) utility of IL-12 p40 as a reporter gene in a high-throughput screening strategy to identify successful transformed mouse embryonic stem cells; (vi) demonstration of pluripotency of IL-12 p40 expressing ES cells in vitro and in vivo; and (vii) germline transmission of the IL-12 p40 reporter gene. CONCLUSION: IL-12 p40 showed several advantages as a reporter gene in terms of sensitivity and ease of the detection procedure. The IL-12 p40 assay was rapid and simple, in as much as the reporter protein secreted from the transfected cells was accurately measured by ELISA using a small aliquot of the culture medium. Remarkably, expression of Il-12 p40 does not affect the pluripotency of mouse ES cells. To our knowledge, human IL-12 p40 is the first secreted reporter protein suitable for high-throughput screening of mouse ES cells. In comparison to other secreted reporters, such as the widely used alkaline phosphatase (SEAP) reporter, the IL-12 p40 reporter system offers other real advantages.
Assuntos
Bioensaio/métodos , Células-Tronco Embrionárias/fisiologia , Perfilação da Expressão Gênica/métodos , Genes Reporter/genética , Interleucina-12/genética , Interleucina-12/metabolismo , Engenharia de Proteínas/métodos , Animais , Linhagem Celular , Humanos , Camundongos , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: An alteration in the mechanism that maintains the monoallelic, imprinted expression of genes can result in their biallelic expression and lead to disruptions in fetal development. Here, we examined the consequences of a loss of maintenance methylation at one specific stage of preimplantation, induced by a deficiency of the oocyte-derived Dnmt1o protein and known to produce biallelic expression of imprinted genes. METHODS: Phenotypes of mid-gestation Dnmt1o-deficient mouse embryos were assessed by a scoring system based on the developmental stage of 17 anatomical features and by magnetic resonance microscopy. RESULTS: Many mid-gestation embryos developing without Dnmt1o protein exhibited significant developmental delays of multiple organ systems (P < 0.05) and a wide variety of morphologic anomalies compared with wild-type embryos. Most of the remaining mid-gestation Dnmt1o-deficient embryos appeared normal. CONCLUSIONS: These findings indicate that a profound range of gestational phenotypes can be induced by the loss of a single protein at a specific preimplantation developmental stage. This is best explained by the formation of epigenetic mosaic early embryos, composed of somatic cells with different spectra of normal intact genomic imprints. These findings have important implications for understanding the types of embryonic phenotypes related to the disruption of inherited imprints, and thus may provide a model of altered imprinting in humans. In particular, because Dnmt1o functions in the preimplantation embryo, a complete or partial loss of Dnmt1o function may play a role in epigenetic abnormalities seen in assisted reproduction technology births.