RESUMO
Skeletal muscle tissue is severely affected in myotonic dystrophy type 1 (DM1) patients, characterised by muscle weakness, myotonia and muscle immaturity in the most severe congenital form of the disease. Previously, it was not known at what stage during myogenesis the DM1 phenotype appears. In this study we differentiated healthy and DM1 human embryonic stem cells to myoblasts and myotubes and compared their differentiation potential using a comprehensive multi-omics approach. We found myogenesis in DM1 cells to be abnormal with altered myotube generation compared to healthy cells. We did not find differentially expressed genes between DM1 and non-DM1 cell lines within the same developmental stage. However, during differentiation we observed an aberrant inflammatory response and increased CpG methylation upstream of the CTG repeat at the myoblast level and RNA mis-splicing at the myotube stage. We show that early myogenesis modelled in hESC reiterates the early developmental manifestation of DM1.
Assuntos
Distrofia Miotônica , Células-Tronco Embrionárias/metabolismo , Humanos , Metilação , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Miotonina Proteína Quinase/genética , Miotonina Proteína Quinase/metabolismo , RNA/metabolismoRESUMO
Myotonic dystrophy type 1 (DM1) is caused by expansion of a CTG repeat in the DMPK gene, where expansion size and somatic mosaicism correlates with disease severity and age of onset. While it is known that the mismatch repair protein MSH2 contributes to the unstable nature of the repeat, its role on other disease-related features, such as CpG methylation upstream of the repeat, is unknown. In this study, we investigated the effect of an MSH2 knock-down (MSH2KD) on both CTG repeat dynamics and CpG methylation pattern in human embryonic stem cells (hESC) carrying the DM1 mutation. Repeat size in MSH2 wild-type (MSH2WT) and MSH2KD DM1 hESC was determined by PacBio sequencing and CpG methylation by bisulfite massive parallel sequencing. We found stabilization of the CTG repeat concurrent with a gradual loss of methylation upstream of the repeat in MSH2KD cells, while the repeat continued to expand and upstream methylation remained unchanged in MSH2WT control lines. Repeat instability was re-established and biased towards expansions upon MSH2 transgenic re-expression in MSH2KD lines while upstream methylation was not consistently re-established. We hypothesize that the hypermethylation at the mutant DM1 locus is promoted by the MMR machinery and sustained by a constant DNA repair response, establishing a potential mechanistic link between CTG repeat instability and upstream CpG methylation. Our work represents a first step towards understanding how epigenetic alterations and repair pathways connect and contribute to the DM1 pathology.
Assuntos
Desmetilação , Instabilidade Genômica , Células-Tronco Embrionárias Humanas/patologia , Proteína 2 Homóloga a MutS/antagonistas & inibidores , Distrofia Miotônica/patologia , Miotonina Proteína Quinase/genética , Expansão das Repetições de Trinucleotídeos , Sistemas CRISPR-Cas , Metilação de DNA , Reparo do DNA , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Distrofia Miotônica/genéticaRESUMO
Huntington's disease (HD) and myotonic dystrophy (DM1) are caused by trinucleotide repeat expansions. The repeats show different instability patterns according to the disorder, cell type and developmental stage. Here we studied the behavior of these repeats in DM1- and HD-derived human embryonic stem cells (hESCs) before and after differentiation, and its relationship to the DNA mismatch repair (MMR). The relatively small (CAG)44 HD expansion was stable in undifferentiated and differentiated HD hESCs. In contrast, the DM1 repeat showed instability from the earliest passages onwards in DM1 hESCs with (CTG)250 or (CTG)1800. Upon differentiation the DM1 repeat was stabilized. MMR genes, including hMSH2, hMSH3 and hMSH6 were assessed at the transcript and protein levels in differentiated cells. The coincidence of differentiation-induced down-regulated MMR expression with reduced instability of the long expanded repeats in hESCs is consistent with a known requirement of MMR proteins for repeat instability in transgenic mice. This is the first demonstration of a correlation between altered repeat instability of an endogenous DM1 locus and natural MMR down-regulation, in contrast to the commonly used murine knock-down systems.
Assuntos
Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/patologia , Doença de Huntington/patologia , Distrofia Miotônica/patologia , Expansão das Repetições de Trinucleotídeos/genética , Animais , Diferenciação Celular , Reparo de Erro de Pareamento de DNA/genética , Regulação para Baixo , Instabilidade Genômica , Humanos , Doença de Huntington/genética , Camundongos , Camundongos Transgênicos , Proteína 2 Homóloga a MutS/genética , Proteína 3 Homóloga a MutS , Distrofia Miotônica/genéticaRESUMO
Imprinting is a non-Mendelian form of inheritance where epigenetic modifications control mono-allelic expression depending on the parental origin. Methylation of CpG-dinucleotides at differentially methylated regions (DMRs) is one of the best-studied mechanisms directing expression to one specific parental allele. We studied the methylation patterns of the intergenic (IG)-DMR of DLK1 and GTL2. The methylation marks of the IG-DMR were analysed in human gametes, preimplantation embryos, amniocytes and blood of babies born after intracytoplasmic sperm injection (ICSI) and blood from adults using a bisulphite sequencing technique. In oocytes, the IG-DMR was mainly unmethylated while in sperm cells a generally methylated pattern was detected. This germ-line specific methylation mark was maintained in the preimplantation embryos until the second cleavage stage. Afterwards in the preimplantation embryos, intermediate methylation patterns (26-74% methylation) occurred, which may point to relaxation of the imprints. Intermediate patterns were also present in amniocytes, blood from ICSI babies and adults. We hypothesise that in the early cleavage stage embryo a strict differential methylation pattern is needed for the correct imprint establishment of surrounding imprinted genes. Once correct imprinting of the involved gene(s) is acquired, a more relaxed state of the IG-region is allowed.
Assuntos
Metilação de DNA , DNA Intergênico/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genética , Proteínas/genética , Proteínas de Ligação ao Cálcio , Impressão Genômica/fisiologia , Humanos , RNA Longo não Codificante , Sequências Repetitivas de Ácido NucleicoRESUMO
Recently, several reports have been published that showed a higher incidence of assisted reproductive technologies (ART) in patients with Beckwith-Wiedemann syndrome compared with the general population, and in most of these patients, aberrant methylation imprints of KvDMR1 have been found. This has led to the concern that ART might increase the incidence of imprinting syndromes such as Beckwith-Wiedemann syndrome. Not much is known on environmental or genetic factors that may interfere with the processes of imprint maintenance or resetting. A methylation analysis of KvDMR1 was performed in human oocytes at different stages of nuclear maturity and in sperm cells. The results indicate that the maternal methylation imprints were already established at the germinal vesicle stage, whereas all sperm cells were unmethylated, thereby showing that the KvDMR1 carries a germline methylation imprint. For one of the oocytes analysed, an unmethylated pattern was found, which highlights the need for further molecular studies that consider the safety of ART.
Assuntos
Proteínas de Membrana/genética , Oócitos/fisiologia , Sequência de Bases , Síndrome de Beckwith-Wiedemann/genética , DNA/sangue , DNA/genética , DNA/isolamento & purificação , Metilação de DNA , Feminino , Humanos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Injeções de Esperma IntracitoplásmicasRESUMO
The CTG repeat at the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene shows marked intergenerational and somatic instability in patients with myotonic dystrophy (DM1), when the repeat is expanded to more than approximately 55 repeats. Intensive research has yielded some insights into the timing and mechanism of these intergenerational changes: (1) increases in expansion sizes occur during gametogenesis but probably not during meiosis, (2) the marked somatic mosaicism becomes apparent from the 2nd trimester of development onward and increases during adult life, and (3) DNA repair mechanisms are involved. We have performed preimplantation genetic diagnosis for DM1 since 1995, which has given us the unique opportunity to study the expanded CTG repeat in affected embryos and in gametes from affected patients. We were able to demonstrate significant increases in the number of repeats in embryos from female patients with DM1 and in their immature and mature oocytes, whereas, in spermatozoa and embryos from male patients with DM1, smaller increases were detected. These data are in concordance with data on other tissues from adults and fetuses and fill a gap in our knowledge of the behavior of CTG triplet expansions in DM1.
Assuntos
Blastocisto/enzimologia , Células Germinativas/enzimologia , Distrofia Miotônica/genética , Proteínas Quinases/genética , Expansão das Repetições de Trinucleotídeos , Feminino , Instabilidade Genômica , Humanos , Masculino , Distrofia Miotônica/enzimologiaRESUMO
In Saccharomyces cerevisiae, which uses the nonlinear pathway of arginine biosynthesis, the first two enzymes, N-acetylglutamate synthase (NAGS) and N-acetylglutamate kinase (NAGK), are controlled by feedback inhibition. We have previously shown that NAGS and NAGK associate in a complex, essential to synthase activity and protein level [Abadjieva, A., Pauwels, K., Hilven, P. & Crabeel, M. (2001) J. Biol. Chem.276, 42869-42880]. The NAGKs of ascomycetes possess, in addition to the catalytic domain that is shared by all other NAGKs and whose structure has been determined, a C-terminal domain of unknown function and structure. Exploring the role of these two domains in the synthase/kinase interaction, we demonstrate that the ascomycete-specific domain is required to maintain synthase activity and protein level. Previous results had suggested a participation of the third enzyme of the pathway, N-acetylglutamylphosphate reductase, in the metabolon. Here, genetic analyses conducted in yeast at physiological level, or in a heterologous background, clearly demonstrate that the reductase is dispensable for synthase activity and protein level. Most importantly, we show that the arginine feedback regulation of the NAGS and NAGK enzymes is mutually interdependent. First, the kinase becomes less sensitive to arginine feedback inhibition in the absence of the synthase. Second, and as in Neurospora crassa, in a yeast kinase mutant resistant to arginine feedback inhibition, the synthase becomes feedback resistant concomitantly. We conclude that the NAGS/NAGK metabolon promotes the co-ordination of the catalytic activities and feedback regulation of the first two, flux controlling, enzymes of the arginine pathway.