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1.
Eur J Hum Genet ; 27(1): 49-60, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30206355

RESUMO

We identified, through a genome-wide search for new imprinted genes in the human placenta, DSCAM (Down Syndrome Cellular Adhesion Molecule) as a paternally expressed imprinted gene. Our work revealed the presence of a Differentially Methylated Region (DMR), located within intron 1 that might regulate the imprinting in the region. This DMR showed a maternal allele methylation, compatible with its paternal expression. We showed that DSCAM is present in endothelial cells and the syncytiotrophoblast layer of the human placenta. In mouse, Dscam expression is biallelic in foetal brain and placenta excluding any possible imprinting in these tissues. This gene encodes a cellular adhesion molecule mainly known for its role in neurone development but its function in the placenta remains unclear. We report here the first imprinted gene located on human chromosome 21 with potential clinical implications.


Assuntos
Moléculas de Adesão Celular/genética , Cromossomos Humanos Par 21/genética , Impressão Genômica , Placenta/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Metilação de DNA , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Gravidez
2.
JCI Insight ; 3(10)2018 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-29769440

RESUMO

Excessive hepatic glucose production (HGP) contributes significantly to the hyperglycemia of type 2 diabetes; however, the molecular mechanism underlying this dysregulation remains poorly understood. Here, we show that fasting temporally increases the expression of H19 long noncoding RNA (lncRNA) in nondiabetic mouse liver, whereas its level is chronically elevated in diet-induced diabetic mice, consistent with the previously reported chronic hepatic H19 increase in diabetic patients. Importantly, liver-specific H19 overexpression promotes HGP, hyperglycemia, and insulin resistance, while H19 depletion enhances insulin-dependent suppression of HGP. Using genome-wide methylation and transcriptome analyses, we demonstrate that H19 knockdown in hepatic cells alters promoter methylation and expression of Hnf4a, a master gluconeogenic transcription factor, and that this regulation is recapitulated in vivo. Our findings offer a mechanistic explanation of lncRNA H19's role in the pathogenesis of diabetic hyperglycemia and suggest that targeting hepatic H19 may hold the potential of new treatment for this disease.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hiperglicemia/metabolismo , Fígado/metabolismo , RNA Longo não Codificante/genética , Animais , Western Blotting , Metilação de DNA , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas , Transcriptoma
3.
Cell Stress ; 1(1): 37-54, 2017 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31225433

RESUMO

The long non-coding RNA (lncRNA) H19 represents a maternally expressed and epigenetically regulated imprinted gene product and is discussed to have either tumor-promoting or tumor-suppressive actions. Recently, H19 was shown to be regulated under inflammatory conditions. Therefore, aim of this study was to determine the function of H19 in hepatocellular carcinoma (HCC), an inflammation-associated type of tumor. In four different human HCC patient cohorts H19 was distinctly downregulated in tumor tissue compared to normal or non-tumorous adjacent tissue. We therefore determined the action of H19 in three different human hepatoma cell lines (HepG2, Plc/Prf5, and Huh7). Clonogenicity and proliferation assays showed that H19 overexpression could suppress tumor cell survival and proliferation after treatment with either sorafenib or doxorubicin, suggesting chemosensitizing actions of H19. Since HCC displays a highly chemoresistant tumor entity, cell lines resistant to doxorubicin or sorafenib were established. In all six chemoresistant cell lines H19 expression was significantly downregulated. The promoter methylation of the H19 gene was significantly different in chemoresistant cell lines compared to their sensitive counterparts. Chemoresistant cells were sensitized after H19 overexpression by either increasing the cytotoxic action of doxorubicin or decreasing cell proliferation upon sorafenib treatment. An H19 knockout mouse model (H19Δ3) showed increased tumor development and tumor cell proliferation after treatment with the carcinogen diethylnitrosamine (DEN) independent of the reciprocally imprinted insulin-like growth factor 2 (IGF2). In conclusion, H19 suppresses hepatocarcinogenesis, hepatoma cell growth, and HCC chemoresistance. Thus, mimicking H19 action might be a potential target to overcome chemoresistance in future HCC therapy.

4.
Development ; 143(6): 962-71, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26980793

RESUMO

The H19 locus controls fetal growth by regulating expression of several genes from the imprinted gene network (IGN). H19 is fully repressed after birth, except in skeletal muscle. Using loss-of-function H19(Δ3) mice, we investigated the function of H19 in adult muscle. Mutant muscles display hypertrophy and hyperplasia, with increased Igf2 and decreased myostatin (Mstn) expression. Many imprinted genes are expressed in muscle stem cells or satellite cells. Unexpectedly, the number of satellite cells was reduced by 50% in H19(Δ3) muscle fibers. This reduction occurred after postnatal day 21, suggesting a link with their entry into quiescence. We investigated the biological function of these mutant satellite cells in vivo using a regeneration assay induced by multiple injections of cardiotoxin. Surprisingly, despite their reduced number, the self-renewal capacity of these cells is fully retained in the absence of H19. In addition, we observed a better regeneration potential of the mutant muscles, with enhanced expression of several IGN genes and genes from the IGF pathway.


Assuntos
Redes Reguladoras de Genes , Impressão Genômica , Músculos/fisiologia , RNA Longo não Codificante/metabolismo , Regeneração/genética , Animais , Cardiotoxinas/toxicidade , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Deleção de Genes , Redes Reguladoras de Genes/efeitos dos fármacos , Impressão Genômica/efeitos dos fármacos , Hiperplasia , Hipertrofia , Masculino , Camundongos Endogâmicos C57BL , Músculos/efeitos dos fármacos , Músculos/patologia , Mioblastos/efeitos dos fármacos , Mioblastos/patologia , RNA Longo não Codificante/genética , Regeneração/efeitos dos fármacos , Células Satélites de Músculo Esquelético/patologia
5.
Nucleic Acids Res ; 42(22): 13799-811, 2014 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-25399420

RESUMO

The H19 lncRNA has been implicated in development and growth control and is associated with human genetic disorders and cancer. Acting as a molecular sponge, H19 inhibits microRNA (miRNA) let-7. Here we report that H19 is significantly decreased in muscle of human subjects with type-2 diabetes and insulin resistant rodents. This decrease leads to increased bioavailability of let-7, causing diminished expression of let-7 targets, which is recapitulated in vitro where H19 depletion results in impaired insulin signaling and decreased glucose uptake. Furthermore, acute hyperinsulinemia downregulates H19, a phenomenon that occurs through PI3K/AKT-dependent phosphorylation of the miRNA processing factor KSRP, which promotes biogenesis of let-7 and its mediated H19 destabilization. Our results reveal a previously undescribed double-negative feedback loop between sponge lncRNA and target miRNA that contributes to glucose regulation in muscle cells.


Assuntos
Glucose/metabolismo , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Regulação para Baixo , Retroalimentação Fisiológica , Humanos , Hiperinsulinismo/genética , Hiperinsulinismo/metabolismo , Insulina/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Proteínas de Ligação a RNA/fisiologia , Transdução de Sinais , Transativadores/fisiologia
6.
Proc Natl Acad Sci U S A ; 110(51): 20693-8, 2013 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-24297921

RESUMO

The H19 gene controls the expression of several genes within the Imprinted Gene Network (IGN), involved in growth control of the embryo. However, the underlying mechanisms of this control remain elusive. Here, we identified the methyl-CpG-binding domain protein 1 MBD1 as a physical and functional partner of the H19 long noncoding RNA (lncRNA). The H19 lncRNA-MBD1 complex is required for the control of five genes of the IGN. For three of these genes--Igf2 (insulin-like growth factor 2), Slc38a4 (solute carrier family 38 member 4), and Peg1 (paternally expressed gene 1)--both MBD1 and H3K9me3 binding were detected on their differentially methylated regions. The H19 lncRNA-MBD1 complex, through its interaction with histone lysine methyltransferases, therefore acts by bringing repressive histone marks on the differentially methylated regions of these three direct targets of the H19 gene. Our data suggest that, besides the differential DNA methylation found on the differentially methylated regions of imprinted genes, an additional fine tuning of the expressed allele is achieved by a modulation of the H3K9me3 marks, mediated by the association of the H19 lncRNA with chromatin-modifying complexes, such as MBD1. This results in a precise control of the level of expression of growth factors in the embryo.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Redes Reguladoras de Genes/fisiologia , Impressão Genômica/fisiologia , RNA Longo não Codificante/metabolismo , Ribonucleoproteínas/metabolismo , Alelos , Animais , Metilação de DNA/fisiologia , Proteínas de Ligação a DNA/genética , Camundongos , Camundongos Knockout , Ribonucleoproteínas/genética
7.
Development ; 140(6): 1231-9, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23406902

RESUMO

The myogenic regulatory factor Myod and insulin-like growth factor 2 (Igf2) have been shown to interact in vitro during myogenic differentiation. In order to understand how they interact in vivo, we produced double-mutant mice lacking both the Myod and Igf2 genes. Surprisingly, these mice display neonatal lethality due to severe diaphragm atrophy. Alteration of diaphragm muscle development occurs as early as 15.5 days post-coitum in the double-mutant embryos and leads to a defect in the terminal differentiation of muscle progenitor cells. A negative-feedback loop was detected between Myod and Igf2 in embryonic muscles. Igf2 belongs to the imprinted H19-Igf2 locus. Molecular analyses show binding of Myod on a mesodermal enhancer (CS9) of the H19 gene. Chromatin conformation capture experiments reveal direct interaction of CS9 with the H19 promoter, leading to increased H19 expression in the presence of Myod. In turn, the non-coding H19 RNA represses Igf2 expression in trans. In addition, Igf2 also negatively regulates Myod expression, possibly by reducing the expression of the Srf transcription factor, a known Myod activator. In conclusion, Igf2 and Myod are tightly co-regulated in skeletal muscles and act in parallel pathways in the diaphragm, where they affect the progression of myogenic differentiation. Igf2 is therefore an essential player in the formation of a functional diaphragm in the absence of Myod.


Assuntos
Diafragma/embriologia , Epistasia Genética/fisiologia , Fator de Crescimento Insulin-Like II/genética , Proteína MyoD/genética , RNA Longo não Codificante/genética , Animais , Animais Recém-Nascidos , Diafragma/crescimento & desenvolvimento , Diafragma/metabolismo , Embrião de Mamíferos , Feminino , Loci Gênicos , Fator de Crescimento Insulin-Like II/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Desenvolvimento Muscular/genética , Proteína MyoD/fisiologia , Organogênese/genética , Gravidez , RNA Longo não Codificante/fisiologia
9.
FASEB J ; 27(4): 1511-8, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23271057

RESUMO

As a result of advances in medical treatment, almost 80% of children who are diagnosed with cancer survive long-term. The adverse consequences of cancer treatments include impaired puberty and fertility. In prepubertal girls, the only therapeutic option is the cryopreservation of an ovary. To date, a dozen births have been reported after reimplantation of cryopreserved mature ovaries. To analyze ovarian function after immature grafts, we performed ovarian grafting in a ewe model. Fresh or cryopreserved ovaries from immature ewes were autografted in prepubertal or adult ewes. Cyclic hormonal activity was recovered 3 mo after grafting. Histological analysis demonstrated the presence of all follicle populations and corpora lutea not affected by cryopreservation. After 3 reproductive seasons, births had been observed in all groups, and the follicle-stimulating hormone status was under the limit, which indicated an exhausted ovary. As an indicator of potential imprinting default, the methylation status of the Igf2r gene was analyzed and did not show significant alteration compared with that of nonmanipulated animals. Taken together, these results demonstrate that immature ovarian grafting is able to restore spontaneous puberty and fertility and could guide the reimplantation of immature cortex in women.


Assuntos
Fertilidade/fisiologia , Hormônio Foliculoestimulante/metabolismo , Folículo Ovariano/transplante , Ovário/transplante , Animais , Criopreservação , Feminino , Ovário/fisiologia , Ovinos , Transplante Autólogo/métodos
10.
FASEB J ; 26(11): 4584-91, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22859371

RESUMO

Brown fat or brown adipose tissue (BAT), found in newborn mammals as small depots localized in the interscapular region, plays a prominent role in regulating thermogenesis perinatally. The physiological importance of functional BAT has been recently reasserted in human adults. Because myoblasts and adipoblasts emerge from a common mesodermal precursor, we investigated developmental determination and the reciprocal relationship between muscle and adipocyte commitment. Here we show that a mutant mouse defective for both Igf2 and Myod genes exhibits massive BAT hypertrophy compared with wild-type and single-mutant newborns. The increased adipocyte proliferation in BAT of double-mutant newborns was associated with overexpression of the brown fat-specific marker Ucp1. More strikingly, expression of the master key gene Prdm16 involved in the switch between myogenic and brown adipogenic lineages was drastically enhanced. We further demonstrate that concomitant Myod and Igf2 inactivation accelerates differentiation of a brown preadipocyte cell line and induces lipid accumulation and increased Ucp1 and Prdm16 expression. This in vitro approach brings additional support for the implication of both Myod and Igf2 in BAT development. These results provide the first in vivo evidence that a myogenic regulator together with a growth factor act simultaneously but through independent pathways to repress Prdm16, which opens potential therapeutic perspectives for human metabolic disorders.


Assuntos
Tecido Adiposo Marrom/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/fisiologia , Fator de Crescimento Insulin-Like II/metabolismo , Proteína MyoD/metabolismo , Fatores de Transcrição/metabolismo , Adipócitos Marrons/citologia , Adipócitos Marrons/fisiologia , Animais , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos , Heterozigoto , Homozigoto , Fator de Crescimento Insulin-Like II/genética , Canais Iônicos/genética , Canais Iônicos/metabolismo , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteína MyoD/genética , RNA Interferente Pequeno , Fatores de Transcrição/genética , Proteína Desacopladora 1
11.
Epigenetics ; 7(9): 1079-90, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22894909

RESUMO

Genomic imprinting characterizes genes with a monoallelic expression, which is dependent on the parental origin of each allele. Approximately 150 imprinted genes are known to date, in humans and mice but, though computational searches have tried to extract intrinsic characteristics of these genes to identify new ones, the existing list is probably far from being comprehensive. We used a high-throughput strategy by diverting the classical use of genotyping microarrays to compare the genotypes of mRNA/cDNA vs. genomic DNA to identify new genes presenting monoallelic expression, starting from human placental material. After filtering of data, we obtained a list of 1,082 putative candidate monoallelic SNPs located in more than one hundred candidate genes. Among these, we found known imprinted genes, such as IPW, GRB10, INPP5F and ZNF597, which contribute to validate the approach. We also explored some likely candidates of our list and identified seven new imprinted genes, including ZFAT, ZFAT-AS1, GLIS3, NTM, MAGI2, ZC3H12Cand LIN28B, four of which encode zinc finger transcription factors. They are, however, not imprinted in the mouse placenta, except for Magi2. We analyzed in more details the ZFAT gene, which is paternally expressed in the placenta (as ZFAT-AS1, a non-coding antisense RNA) but biallelic in other tissues. The ZFAT protein is expressed in endothelial cells, as well as in syncytiotrophoblasts. The expression of this gene is, moreover, downregulated in placentas from complicated pregnancies. With this work we increase by about 10% the number of known imprinted genes in humans.


Assuntos
Genoma Humano/genética , Impressão Genômica/genética , Placenta/metabolismo , Animais , Bovinos , Feminino , Perfilação da Expressão Gênica , Loci Gênicos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Gravidez , RNA Mensageiro/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
12.
PLoS One ; 7(5): e37923, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22662250

RESUMO

It was recently shown that a long non-coding RNA (lncRNA), that we named the 91H RNA (i.e. antisense H19 transcript), is overexpressed in human breast tumours and contributes in trans to the expression of the Insulin-like Growth Factor 2 (IGF2) gene on the paternal chromosome. Our preliminary experiments suggested that an H19 antisense transcript having a similar function may also be conserved in the mouse. In the present work, we further characterise the mouse 91H RNA and, using a genetic complementation approach in H19 KO myoblast cells, we show that ectopic expression of the mouse 91H RNA can up-regulate Igf2 expression in trans despite almost complete unmethylation of the Imprinting-Control Region (ICR). We then demonstrate that this activation occurs at the transcriptional level by activation of a previously unknown Igf2 promoter which displays, in mouse tissues, a preferential mesodermic expression (Pm promoter). Finally, our experiments indicate that a large excess of the H19 transcript can counteract 91H-mediated Igf2 activation. Our work contributes, in conjunction with other recent findings, to open new horizons to our understanding of Igf2 gene regulation and functions of the 91H/H19 RNAs in normal and pathological conditions.


Assuntos
Regulação da Expressão Gênica , Fator de Crescimento Insulin-Like II/genética , Mioblastos/metabolismo , Regiões Promotoras Genéticas , RNA Antissenso/metabolismo , RNA Longo não Codificante/genética , Ativação Transcricional , Animais , Sequência de Bases , Metilação de DNA , Ordem dos Genes , Inativação Gênica , Impressão Genômica , Camundongos , Dados de Sequência Molecular , Sítio de Iniciação de Transcrição , Transcrição Gênica
13.
Nat Cell Biol ; 14(7): 659-65, 2012 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-22684254

RESUMO

The H19 large intergenic non-coding RNA (lincRNA) is one of the most highly abundant and conserved transcripts in mammalian development, being expressed in both embryonic and extra-embryonic cell lineages, yet its physiological function is unknown. Here we show that miR-675, a microRNA (miRNA) embedded in H19's first exon, is expressed exclusively in the placenta from the gestational time point when placental growth normally ceases, and placentas that lack H19 continue to grow. Overexpression of miR-675 in a range of embryonic and extra-embryonic cell lines results in their reduced proliferation; targets of the miRNA are upregulated in the H19 null placenta, including the growth-promoting insulin-like growth factor 1 receptor (Igf1r) gene. Moreover, the excision of miR-675 from H19 is dynamically regulated by the stress-response RNA-binding protein HuR. These results suggest that H19's main physiological role is in limiting growth of the placenta before birth, by regulated processing of miR-675. The controlled release of miR-675 from H19 may also allow rapid inhibition of cell proliferation in response to cellular stress or oncogenic signals.


Assuntos
Proliferação de Células , MicroRNAs/metabolismo , Placenta/metabolismo , RNA não Traduzido/metabolismo , Receptor IGF Tipo 1/metabolismo , Animais , Linhagem Celular , RNA Helicases DEAD-box/metabolismo , Regulação para Baixo , Proteínas ELAV/genética , Proteínas ELAV/metabolismo , Éxons , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Transgênicos , Placentação , Gravidez , Interferência de RNA , RNA Longo não Codificante , Receptor IGF Tipo 1/genética , Ribonuclease III/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção
14.
Bioessays ; 32(6): 473-80, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20486133

RESUMO

The H19 gene produces a non-coding RNA, which is abundantly expressed during embryonic development and down-regulated after birth. Although this gene was discovered over 20 years ago, its function has remained unclear. Only recently a role was identified for the non-coding RNA and/or its microRNA partner, first as a tumour suppressor gene in mice, then as a trans-regulator of a group of co-expressed genes belonging to the imprinted gene network that is likely to control foetal and early postnatal growth in mice. The mechanisms underlying this transcriptional or post-transcriptional regulation remain to be discovered, perhaps by identifying the protein partners of the full-length H19 RNA or the targets of the microRNA. This first in vivo evidence of a functional role for the H19 locus provides new insights into how genomic imprinting helps to control embryonic growth.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Impressão Genômica/fisiologia , RNA não Traduzido/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Impressão Genômica/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante , RNA não Traduzido/genética
15.
PLoS One ; 5(2): e9218, 2010 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-20169163

RESUMO

BACKGROUND: Assisted Reproductive Technologies (ART) are increasingly used in humans; however, their impact is now questioned. At blastocyst stage, the trophectoderm is directly in contact with an artificial medium environment, which can impact placental development. This study was designed to carry out an in-depth analysis of the placental transcriptome after ART in mice. METHODOLOGY/PRINCIPAL FINDINGS: Blastocysts were transferred either (1) after in vivo fertilization and development (control group) or (2) after in vitro fertilization and embryo culture. Placentas were then analyzed at E10.5. Six percent of transcripts were altered at the two-fold threshold in placentas of manipulated embryos, 2/3 of transcripts being down-regulated. Strikingly, the X-chromosome harbors 11% of altered genes, 2/3 being induced. Imprinted genes were modified similarly to the X. Promoter composition analysis indicates that FOXA transcription factors may be involved in the transcriptional deregulations. CONCLUSIONS: For the first time, our study shows that in vitro fertilization associated with embryo culture strongly modify the placental expression profile, long after embryo manipulations, meaning that the stress of artificial environment is memorized after implantation. Expression of X and imprinted genes is also greatly modulated probably to adapt to adverse conditions. Our results highlight the importance of studying human placentas from ART.


Assuntos
Técnicas de Cultura Embrionária/métodos , Fertilização in vitro/métodos , Perfilação da Expressão Gênica , Placenta/metabolismo , Animais , Análise por Conglomerados , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Modelos Animais , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
Hum Mol Genet ; 19(9): 1779-90, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20150233

RESUMO

Genomic imprinting regulates the expression of a group of genes monoallelically expressed in a parent-of-origin specific manner. Allele-specific DNA methylation occurs at differentially methylated regions (DMRs) of these genes. We have previously shown that in vitro fertilization and embryo culture result in methylation defects at the imprinted H19-Igf2 locus at the blastocyst stage. The current study was designed to evaluate the consequences of these manipulations on genomic imprinting after implantation in the mouse. Blastocysts were produced following three experimental conditions: (i) embryos maintained in culture medium after in vivo fertilization or (ii) in vitro fertilization and (iii) a control group with embryos obtained after in vivo fertilization and timed mating. Blastocysts were all transplanted into pseudopregnant females. Embryos and placentas were collected on day 10.5 of development. DNA methylation patterns of the H19, Igf2, Igf2r and Dlk1-Dio3 DMRs were analyzed by quantitative pyrosequencing. In contrast to blastocyst stage, methylation profiles were normal both in embryonic and placental tissues after in vitro fertilization and culture. Expression of a selected set of imprinting genes from the recently described imprinted gene network (IGN) (including Igf2 and H19) was analyzed in placental tissues by quantitative RT-PCR. Placentas obtained after in vitro fertilization and embryo culture displayed significantly disturbed levels of H19 and Igf2 mRNA, as well as of most other genes from the IGN. As embryos were phenotypically normal, we hypothesize that the modulation of a coordinated network of imprinted genes results in a compensatory process capable of correcting potential dysfunction of placenta.


Assuntos
Metilação de DNA/fisiologia , Desenvolvimento Embrionário/fisiologia , Redes Reguladoras de Genes/fisiologia , Impressão Genômica/fisiologia , Placenta/embriologia , Animais , Feminino , Fertilização in vitro , Componentes do Gene , Redes Reguladoras de Genes/genética , Impressão Genômica/genética , Técnicas In Vitro , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Camundongos , Gravidez , RNA Longo não Codificante , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
Epigenomics ; 2(3): 365-75, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22121898

RESUMO

AIMS: The identification of specific biomarkers for colorectal cancer is of primary importance for early diagnosis. The aim of this study was to evaluate if methylation changes at the IGF2/H19 locus could be predictive for individuals at high risk for developing sporadic or hereditary colorectal cancer. MATERIALS & METHODS: Quantitative methylation analysis using pyrosequencing was performed on three differentially methylated regions (DMRs): IGF2 DMR0 and DMR2 and the H19 DMR in DNA samples from sporadic colorectal cancer (n = 26), familial adenomatous polyposis (n = 35) and hereditary nonpolyposis colorectal cancer (n = 19) patients. RESULTS: We report in this article for the first time, that in sporadic colorectal cancer tumor DNA both the IGF2 DMR0 and DMR2 are hypomethylated, while the H19 DMR retains its monoallelic methylation pattern. In lymphocyte DNA, a striking hypomethylation of nine contiguous correlated CpGs was found in the IGF2 DMR2 but only in familial adenomatous polyposis patients. CONCLUSION: Methylation alterations at the IGF2 locus are more extensive than previously reported and DMR2 hypomethylation in lymphocyte DNA might be a specific epigenetic biomarker for familial adenomatous polyposis patients.


Assuntos
Polipose Adenomatosa do Colo/metabolismo , Biomarcadores/metabolismo , Neoplasias Colorretais/metabolismo , Ilhas de CpG/genética , Metilação de DNA/genética , Fator de Crescimento Insulin-Like II/genética , Análise por Conglomerados , França , Humanos , Linfócitos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA/métodos , Sulfitos
18.
Development ; 136(20): 3413-21, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19762426

RESUMO

The imprinted H19 gene produces a non-coding RNA of unknown function. Mice lacking H19 show an overgrowth phenotype, due to a cis effect of the H19 locus on the adjacent Igf2 gene. To explore the function of the RNA itself, we produced transgenic mice overexpressing H19. We observed postnatal growth reduction in two independent transgenic lines and detected a decrease of Igf2 expression in embryos. An extensive analysis of several other genes from the newly described imprinted gene network (IGN) was performed in both loss- and gain-of-function animals. We found that H19 deletion leads to the upregulation of several genes of the IGN. This overexpression is restored to the wild-type level by transgenic expression of H19. We therefore propose that the H19 gene participates as a trans regulator in the fine-tuning of this IGN in the mouse embryo. This is the first in vivo evidence of a functional role for the H19 RNA. Our results also bring further experimental evidence for the existence of the IGN and open new perspectives in the comprehension of the role of genomic imprinting in embryonic growth and in human imprinting pathologies.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , RNA não Traduzido/genética , Sequências Reguladoras de Ácido Nucleico , Animais , Feminino , Fator de Crescimento Insulin-Like II/genética , Masculino , Camundongos , Camundongos Transgênicos , Fenótipo , RNA Longo não Codificante
19.
Proc Natl Acad Sci U S A ; 105(34): 12417-22, 2008 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-18719115

RESUMO

The H19 locus belongs to a cluster of imprinted genes that is linked to the human Beckwith-Wiedemann syndrome. The expression of H19 and its closely associated IGF2 gene is frequently deregulated in some human tumors, such as Wilms' tumors. In these cases, biallelic IGF2 expression and lack of expression of H19 are associated with hypermethylation of the imprinting center of this locus. These observations and others have suggested a potential tumor suppressor effect of the H19 locus. Some studies have also suggested that H19 is an oncogene, based on tissue culture systems. We show, using in vivo murine models of tumorigenesis, that the H19 locus controls the size of experimental teratocarcinomas, the number of polyps in the Apc murine model of colorectal cancer and the timing of appearance of SV40-induced hepatocarcinomas. The H19 locus thus clearly displays a tumor suppressor effect in mice.


Assuntos
Genes Supressores de Tumor/fisiologia , RNA não Traduzido/fisiologia , Animais , Carcinoma Hepatocelular/patologia , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Fator de Crescimento Insulin-Like II , Camundongos , Camundongos Mutantes , Família Multigênica , RNA Longo não Codificante , RNA não Traduzido/classificação , Teratoma/patologia
20.
PLoS One ; 3(4): e1972, 2008 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-18414667

RESUMO

BACKGROUND: Progress in oncology could improve survival rate in children, but would probably lead to impaired fertility and puberty. In pre-pubertal girls, the only therapeutic option is the cryopreservation of one ovary. Three births have been reported after reimplantation of cryopreserved mature ovary. Conversely, reimplantation of ovary preserved before puberty (defined as immature ovary) has never been performed in humans. METHODOLOGY/PRINCIPAL FINDINGS: In order to analyze ovarian function, we performed transplantation using fresh or cryopreserved immature grafts in pre-pubertal or adult mice. Puberty as well as cyclic hormonal activity was restored. All follicle populations were present although a significant reduction in follicle density was observed with or without cryopreservation. Although fertility was restored, the graft is of limited life span. Because ex vivo ovary manipulation and cryopreservation procedure, the status of genomic imprinting was investigated. Methylation status of the H19 and Lit1 Imprinting Control Regions in kidney, muscle and tongue of offsprings from grafted mice does not show significant alteration when compared to those of unoperated mice. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that immature ovarian grafting can restore spontaneous puberty and fertility. However, these data suggest that follicle depletion leads to premature ovarian failure. This study addresses the very important epigenetics issue, and provides valuable information to the study of ovarian transplantation suggesting that these procedures do not perturb normal epigenetics marks. These results are highly relevant to the reimplantation question of immature cortex in women.


Assuntos
Criopreservação/métodos , Epigênese Genética , Ovário/patologia , Ovário/fisiologia , Animais , Feminino , Fertilidade , Glândulas Mamárias Animais/patologia , Metilação , Camundongos , Folículo Ovariano/patologia , Ovário/transplante , Técnicas Reprodutivas , Maturidade Sexual , Fatores de Tempo , Útero/patologia
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