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1.
Cell ; 176(5): 952-965, 2019 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-30794780

RESUMEN

Complex multicellular organisms, such as mammals, express two complete sets of chromosomes per nucleus, combining the genetic material of both parents. However, epigenetic studies have demonstrated violations to this rule that are necessary for mammalian physiology; the most notable parental allele expression phenomenon is genomic imprinting. With the identification of endogenous imprinted genes, genomic imprinting became well-established as an epigenetic mechanism in which the expression pattern of a parental allele influences phenotypic expression. The expanding study of genomic imprinting is revealing a significant impact on brain functions and associated diseases. Here, we review key milestones in the field of imprinting and discuss mechanisms and systems in which imprinted genes exert a significant role.


Asunto(s)
Impresión Genómica/genética , Impresión Genómica/fisiología , Mamíferos/genética , Alelos , Animales , Evolución Biológica , Cromosomas , Metilación de ADN , Epigénesis Genética/genética , Epigénesis Genética/fisiología , Mamíferos/metabolismo , Fenómenos Fisiológicos
2.
Genes Dev ; 38(3-4): 131-150, 2024 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-38453481

RESUMEN

Maternal inactivation of genes encoding components of the subcortical maternal complex (SCMC) and its associated member, PADI6, generally results in early embryo lethality. In humans, SCMC gene variants were found in the healthy mothers of children affected by multilocus imprinting disturbances (MLID). However, how the SCMC controls the DNA methylation required to regulate imprinting remains poorly defined. We generated a mouse line carrying a Padi6 missense variant that was identified in a family with Beckwith-Wiedemann syndrome and MLID. If homozygous in female mice, this variant resulted in interruption of embryo development at the two-cell stage. Single-cell multiomic analyses demonstrated defective maturation of Padi6 mutant oocytes and incomplete DNA demethylation, down-regulation of zygotic genome activation (ZGA) genes, up-regulation of maternal decay genes, and developmental delay in two-cell embryos developing from Padi6 mutant oocytes but little effect on genomic imprinting. Western blotting and immunofluorescence analyses showed reduced levels of UHRF1 in oocytes and abnormal localization of DNMT1 and UHRF1 in both oocytes and zygotes. Treatment with 5-azacytidine reverted DNA hypermethylation but did not rescue the developmental arrest of mutant embryos. Taken together, this study demonstrates that PADI6 controls both nuclear and cytoplasmic oocyte processes that are necessary for preimplantation epigenetic reprogramming and ZGA.


Asunto(s)
Oocitos , Cigoto , Animales , Niño , Femenino , Humanos , Ratones , Proteínas Potenciadoras de Unión a CCAAT/genética , Citoplasma/genética , Citoplasma/metabolismo , Metilación de ADN/genética , Desarrollo Embrionario/genética , Impresión Genómica/genética , Ubiquitina-Proteína Ligasas/metabolismo
3.
Genes Dev ; 35(11-12): 821-834, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-34074696

RESUMEN

Genomic imprinting is the monoallelic expression of a gene based on parent of origin and is a consequence of differential epigenetic marking between the male and female germlines. Canonically, genomic imprinting is mediated by allelic DNA methylation. However, recently it has been shown that maternal H3K27me3 can result in DNA methylation-independent imprinting, termed "noncanonical imprinting." In this review, we compare and contrast what is currently known about the underlying mechanisms, the role of endogenous retroviral elements, and the conservation of canonical and noncanonical genomic imprinting.


Asunto(s)
Impresión Genómica/fisiología , Metilación de ADN , Epigenómica , Humanos , Retroelementos/genética
4.
Nat Rev Mol Cell Biol ; 21(10): 566-567, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32839539
5.
Nature ; 600(7890): 737-742, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34880491

RESUMEN

Stability of the epigenetic landscape underpins maintenance of the cell-type-specific transcriptional profile. As one of the main repressive epigenetic systems, DNA methylation has been shown to be important for long-term gene silencing; its loss leads to ectopic and aberrant transcription in differentiated cells and cancer1. The developing mouse germ line endures global changes in DNA methylation in the absence of widespread transcriptional activation. Here, using an ultra-low-input native chromatin immunoprecipitation approach, we show that following DNA demethylation the gonadal primordial germ cells undergo remodelling of repressive histone modifications, resulting in a sex-specific signature in mice. We further demonstrate that Polycomb has a central role in transcriptional control in the newly hypomethylated germline genome as the genetic loss of Ezh2 leads to aberrant transcriptional activation, retrotransposon derepression and dramatic loss of developing female germ cells. This sex-specific effect of Ezh2 deletion is explained by the distinct landscape of repressive modifications observed in male and female germ cells. Overall, our study provides insight into the dynamic interplay between repressive chromatin modifications in the context of a developmental reprogramming system.


Asunto(s)
Ensamble y Desensamble de Cromatina , Células Germinativas , Animales , Cromatina/genética , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Metilación de ADN , Epigénesis Genética , Femenino , Células Germinativas/metabolismo , Masculino , Ratones , Proteínas del Grupo Polycomb/metabolismo
6.
Genome Res ; 33(1): 18-31, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36690445

RESUMEN

EHMT1 (also known as GLP) is a multifunctional protein, best known for its role as an H3K9me1 and H3K9me2 methyltransferase through its reportedly obligatory dimerization with EHMT2 (also known as G9A). Here, we investigated the role of EHMT1 in the oocyte in comparison to EHMT2 using oocyte-specific conditional knockout mouse models (Ehmt2 cKO, Ehmt1 cKO, Ehmt1/2 cDKO), with ablation from the early phase of oocyte growth. Loss of EHMT1 in Ehmt1 cKO and Ehmt1/2 cDKO oocytes recapitulated meiotic defects observed in the Ehmt2 cKO; however, there was a significant impairment in oocyte maturation and developmental competence in Ehmt1 cKO and Ehmt1/2 cDKO oocytes beyond that observed in the Ehmt2 cKO. Consequently, loss of EHMT1 in oogenesis results, upon fertilization, in mid-gestation embryonic lethality. To identify H3K9 methylation and other meaningful biological changes in each mutant to explore the molecular functions of EHMT1 and EHMT2, we performed immunofluorescence imaging, multi-omics sequencing, and mass spectrometry (MS)-based proteome analyses in cKO oocytes. Although H3K9me1 was depleted only upon loss of EHMT1, H3K9me2 was decreased, and H3K9me2-enriched domains were eliminated equally upon loss of EHMT1 or EHMT2. Furthermore, there were more significant changes in the transcriptome, DNA methylome, and proteome in Ehmt1/2 cDKO than Ehmt2 cKO oocytes, with transcriptional derepression leading to increased protein abundance and local changes in genic DNA methylation in Ehmt1/2 cDKO oocytes. Together, our findings suggest that EHMT1 contributes to local transcriptional repression in the oocyte, partially independent of EHMT2, and is critical for oogenesis and oocyte developmental competence.


Asunto(s)
Multiómica , Proteoma , Animales , Ratones , Proteoma/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Oogénesis/genética , Oocitos/metabolismo
7.
Nature ; 576(7787): 487-491, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31827285

RESUMEN

Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes1-5. Global epigenetic reprogramming accompanies these changes6-8, but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.


Asunto(s)
Metilación de ADN , Epigénesis Genética , Gástrula/citología , Gástrula/metabolismo , Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica , ARN/genética , Análisis de la Célula Individual , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Cromatina/genética , Cromatina/metabolismo , Desmetilación , Cuerpos Embrioides/citología , Endodermo/citología , Endodermo/embriología , Endodermo/metabolismo , Elementos de Facilitación Genéticos/genética , Epigenoma/genética , Eritropoyesis , Análisis Factorial , Gástrula/embriología , Gastrulación/fisiología , Mesodermo/citología , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , ARN/análisis , Factores de Tiempo , Dedos de Zinc
8.
BMC Genomics ; 25(1): 335, 2024 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-38580918

RESUMEN

BACKGROUND: Mammalian follicle development is characterized by extensive changes in morphology, endocrine responsiveness, and function, providing the optimum environment for oocyte growth, development, and resumption of meiosis. In cattle, the first signs of transcription activation in the oocyte are observed in the secondary follicle, later than during mouse and human oogenesis. While many studies have generated extensive datasets characterizing gene expression in bovine oocytes, they are mostly limited to the analysis of fully grown and matured oocytes. The aim of the present study was to apply single-cell RNA sequencing to interrogate the transcriptome of the growing bovine oocyte from the secondary follicle stage through to the mid-antral follicle stage. RESULTS: Single-cell RNA-seq libraries were generated from oocytes of known diameters (< 60 to > 120 µm), and datasets were binned into non-overlapping size groups for downstream analysis. Combining the results of weighted gene co-expression network and Trendy analyses, and differently expressed genes (DEGs) between size groups, we identified a decrease in oxidative phosphorylation and an increase in maternal -genes and transcription regulators across the bovine oocyte growth phase. In addition, around 5,000 genes did not change in expression, revealing a cohort of stable genes. An interesting switch in gene expression profile was noted in oocytes greater than 100 µm in diameter, when the expression of genes related to cytoplasmic activities was replaced by genes related to nuclear activities (e.g., chromosome segregation). The highest number of DEGs were detected in the comparison of oocytes 100-109 versus 110-119 µm in diameter, revealing a profound change in the molecular profile of oocytes at the end of their growth phase. CONCLUSIONS: The current study provides a unique dataset of the key genes and pathways characteristic of each stage of oocyte development, contributing an important resource for a greater understanding of bovine oogenesis.


Asunto(s)
Oogénesis , Transcriptoma , Femenino , Bovinos , Animales , Humanos , Ratones , Oogénesis/genética , Oocitos/metabolismo , Folículo Ovárico/metabolismo , Proliferación Celular , Mamíferos/genética
9.
Nucleic Acids Res ; 50(4): 1993-2004, 2022 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-35137160

RESUMEN

Histone 3 lysine 4 trimethylation (H3K4me3) is an epigenetic mark found at gene promoters and CpG islands. H3K4me3 is essential for mammalian development, yet mechanisms underlying its genomic targeting are poorly understood. H3K4me3 methyltransferases SETD1B and MLL2 (KMT2B) are essential for oogenesis. We investigated changes in H3K4me3 in Setd1b conditional knockout (cKO) oocytes using ultra-low input ChIP-seq, with comparisons to DNA methylation and gene expression analyses. H3K4me3 was redistributed in Setd1b cKO oocytes showing losses at active gene promoters associated with downregulated gene expression. Remarkably, many regions also gained H3K4me3, in particular those that were DNA hypomethylated, transcriptionally inactive and CpG-rich, which are hallmarks of MLL2 targets. Consequently, loss of SETD1B disrupts the balance between MLL2 and de novo DNA methyltransferases in determining the epigenetic landscape during oogenesis. Our work reveals two distinct, complementary mechanisms of genomic targeting of H3K4me3 in oogenesis, with SETD1B linked to gene expression and MLL2 to CpG content.


Asunto(s)
Histonas , Lisina , Animales , Islas de CpG/genética , Metilación de ADN , Histona Metiltransferasas/genética , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Mamíferos/genética , Oogénesis/genética
10.
Mol Cell ; 60(4): 611-25, 2015 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-26549683

RESUMEN

The integrity of chromatin, which provides a dynamic template for all DNA-related processes in eukaryotes, is maintained through replication-dependent and -independent assembly pathways. To address the role of histone deposition in the absence of DNA replication, we deleted the H3.3 chaperone Hira in developing mouse oocytes. We show that chromatin of non-replicative developing oocytes is dynamic and that lack of continuous H3.3/H4 deposition alters chromatin structure, resulting in increased DNase I sensitivity, the accumulation of DNA damage, and a severe fertility phenotype. On the molecular level, abnormal chromatin structure leads to a dramatic decrease in the dynamic range of gene expression, the appearance of spurious transcripts, and inefficient de novo DNA methylation. Our study thus unequivocally shows the importance of continuous histone replacement and chromatin homeostasis for transcriptional regulation and normal developmental progression in a non-replicative system in vivo.


Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Oogénesis , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Metilación de ADN , Femenino , Fertilización , Regulación de la Expresión Génica , Ratones , Oocitos/metabolismo , Transcripción Genética
11.
Genes Dev ; 29(23): 2449-62, 2015 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-26584620

RESUMEN

Erasure and subsequent reinstatement of DNA methylation in the germline, especially at imprinted CpG islands (CGIs), is crucial to embryogenesis in mammals. The mechanisms underlying DNA methylation establishment remain poorly understood, but a number of post-translational modifications of histones are implicated in antagonizing or recruiting the de novo DNA methylation complex. In mouse oogenesis, DNA methylation establishment occurs on a largely unmethylated genome and in nondividing cells, making it a highly informative model for examining how histone modifications can shape the DNA methylome. Using a chromatin immunoprecipitation (ChIP) and genome-wide sequencing (ChIP-seq) protocol optimized for low cell numbers and novel techniques for isolating primary and growing oocytes, profiles were generated for histone modifications implicated in promoting or inhibiting DNA methylation. CGIs destined for DNA methylation show reduced protective H3K4 dimethylation (H3K4me2) and trimethylation (H3K4me3) in both primary and growing oocytes, while permissive H3K36me3 increases specifically at these CGIs in growing oocytes. Methylome profiling of oocytes deficient in H3K4 demethylase KDM1A or KDM1B indicated that removal of H3K4 methylation is necessary for proper methylation establishment at CGIs. This work represents the first systematic study performing ChIP-seq in oocytes and shows that histone remodeling in the mammalian oocyte helps direct de novo DNA methylation events.


Asunto(s)
Metilación de ADN , Código de Histonas , Oocitos/enzimología , Oogénesis/fisiología , Animales , Inmunoprecipitación de Cromatina , Islas de CpG , Citometría de Flujo , Histona Demetilasas/genética , Histonas/metabolismo , Ratones , Oxidorreductasas N-Desmetilantes/genética , Análisis de Secuencia de ADN
12.
Nucleic Acids Res ; 48(20): 11394-11407, 2020 11 18.
Artículo en Inglés | MEDLINE | ID: mdl-33053156

RESUMEN

Genomic imprinting is an epigenetic process regulated by germline-derived DNA methylation that is resistant to embryonic reprogramming, resulting in parental origin-specific monoallelic gene expression. A subset of individuals affected by imprinting disorders (IDs) displays multi-locus imprinting disturbances (MLID), which may result from aberrant establishment of imprinted differentially methylated regions (DMRs) in gametes or their maintenance in early embryogenesis. Here we investigated the extent of MLID in a family harbouring a ZFP57 truncating variant and characterize the interactions between human ZFP57 and the KAP1 co-repressor complex. By ectopically targeting ZFP57 to reprogrammed loci in mouse embryos using a dCas9 approach, we confirm that ZFP57 recruitment is sufficient to protect oocyte-derived methylation from reprogramming. Expression profiling in human pre-implantation embryos and oocytes reveals that unlike in mice, ZFP57 is only expressed following embryonic-genome activation, implying that other KRAB-zinc finger proteins (KZNFs) recruit KAP1 prior to blastocyst formation. Furthermore, we uncover ZNF202 and ZNF445 as additional KZNFs likely to recruit KAP1 to imprinted loci during reprogramming in the absence of ZFP57. Together, these data confirm the perplexing link between KZFPs and imprint maintenance and highlight the differences between mouse and humans in this respect.


Asunto(s)
Metilación de ADN , Embrión de Mamíferos/metabolismo , Impresión Genómica , Células Germinativas/metabolismo , Oocitos/metabolismo , Proteínas Represoras/metabolismo , Síndrome de Beckwith-Wiedemann/metabolismo , Estudios de Cohortes , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Humanos , Análisis por Micromatrices , Mutación , Linaje , Seudohipoparatiroidismo/metabolismo , RNA-Seq , Proteínas Represoras/genética , Hermanos , Transcriptoma , Proteína 28 que Contiene Motivos Tripartito
13.
Int J Mol Sci ; 23(24)2022 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-36555801

RESUMEN

Controlled ovarian stimulation is a necessary step in some assisted reproductive procedures allowing a higher collection of female gametes. However, consequences of this stimulation for the gamete or the offspring have been shown in several mammals. Most studies used comparisons between oocytes from different donors, which may contribute to different responses. In this work, we use the bovine model in which each animal serves as its own control. DNA methylation profiles were obtained by single-cell whole-genome bisulfite sequencing of oocytes from pre-ovulatory unstimulated follicles compared to oocytes from stimulated follicles. Results show that the global percentage of methylation was similar between groups, but the percentage of methylation was lower for non-stimulated oocytes in the imprinted genes APEG3, MEG3, and MEG9 and higher in TSSC4 when compared to stimulated oocytes. Differences were also found in CGI of imprinted genes: higher methylation was found among non-stimulated oocytes in MEST (PEG1), IGF2R, GNAS (SCG6), KvDMR1 ICR UMD, and IGF2. In another region around IGF2, the methylation percentage was lower for non-stimulated oocytes when compared to stimulated oocytes. Data drawn from this study might help to understand the molecular reasons for the appearance of certain syndromes in assisted reproductive technologies-derived offspring.


Asunto(s)
Metilación de ADN , Superovulación , Animales , Bovinos , Femenino , Superovulación/fisiología , Impresión Genómica , Oocitos/metabolismo , Técnicas Reproductivas Asistidas , Mamíferos
14.
Int J Mol Sci ; 22(8)2021 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-33924072

RESUMEN

The complex nature of folliculogenesis regulation accounts for its susceptibility to maternal physiological fitness. In obese mothers, progressive expansion of adipose tissue culminates with severe hyperestrogenism and hyperleptinemia with detrimental effects for ovarian performance. Indeed, maternal obesity is associated with the establishment of ovarian leptin resistance. This review summarizes current knowledge on potential effects of impaired leptin signaling throughout folliculogenesis and oocyte developmental competence in mice and women.


Asunto(s)
Diferenciación Celular , Leptina/metabolismo , Obesidad/metabolismo , Oocitos/metabolismo , Oogénesis , Folículo Ovárico/metabolismo , Transducción de Señal , Adipoquinas/metabolismo , Animales , Biomarcadores , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Modelos Biológicos , Madres , Obesidad/etiología , Oocitos/citología , Folículo Ovárico/citología , Ovulación , Embarazo
15.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-34204008

RESUMEN

Assisted reproductive technologies impact transcriptome and epigenome of embryos and can result in long-term phenotypic consequences. Whole-genome DNA methylation profiles from individual bovine blastocysts in vivo- and in vitro-derived (using three sources of protein: reproductive fluids, blood serum and bovine serum albumin) were generated. The impact of in vitro culture on DNA methylation was analyzed, and sex-specific methylation differences at blastocyst stage were uncovered. In vivo embryos showed the highest levels of methylation (29.5%), close to those produced in vitro with serum, whilst embryos produced in vitro with reproductive fluids or albumin showed less global methylation (25-25.4%). During repetitive element analysis, the serum group was the most affected. DNA methylation differences between in vivo and in vitro groups were more frequent in the first intron than in CpGi in promoters. Moreover, hierarchical cluster analysis showed that sex produced a stronger bias in the results than embryo origin. For each group, distance between male and female embryos varied, with in vivo blastocyst showing a lesser distance. Between the sexually dimorphic methylated tiles, which were biased to X-chromosome, critical factors for reproduction, developmental process, cell proliferation and DNA methylation machinery were included. These results support the idea that blastocysts show sexually-dimorphic DNA methylation patterns, and the known picture about the blastocyst methylome should be reconsidered.


Asunto(s)
Blastocisto/metabolismo , Reprogramación Celular/genética , Medios de Cultivo/farmacología , Epigénesis Genética/efectos de los fármacos , Caracteres Sexuales , Animales , Blastocisto/efectos de los fármacos , Bovinos , Cromosomas de los Mamíferos/genética , Islas de CpG/genética , Metilación de ADN/efectos de los fármacos , Metilación de ADN/genética , Femenino , Fertilización In Vitro , Ontología de Genes , Modelos Logísticos , Masculino , Anotación de Secuencia Molecular , Análisis de Componente Principal
16.
BMC Genomics ; 21(1): 385, 2020 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-32493210

RESUMEN

BACKGROUND: Lipopolysaccharide (LPS) endotoxin stimulates pro-inflammatory pathways and is a key player in the pathological mechanisms involved in the development of endometritis. This study aimed to investigate LPS-induced DNA methylation changes in bovine endometrial epithelial cells (bEECs), which may affect endometrial function. Following in vitro culture, bEECs from three cows were either untreated (0) or exposed to 2 and 8 µg/mL LPS for 24 h. RESULTS: DNA samples extracted at 0 h and 24 h were sequenced using reduced representation bisulfite sequencing (RRBS). When comparing DNA methylation results at 24 h to time 0 h, a larger proportion of hypomethylated regions were identified in the LPS-treated groups, whereas the trend was opposite in controls. When comparing LPS groups to controls at 24 h, a total of 1291 differentially methylated regions (DMRs) were identified (55% hypomethylated and 45% hypermethylated). Integration of DNA methylation data obtained here with our previously published gene expression data obtained from the same samples showed a negative correlation (r = - 0.41 for gene promoter, r = - 0.22 for gene body regions, p < 0.05). Differential methylation analysis revealed that effects of LPS treatment were associated with methylation changes for genes involved in regulation of immune and inflammatory responses, cell adhesion, and external stimuli. Gene ontology and pathway analyses showed that most of the differentially methylated genes (DMGs) were associated with cell proliferation and apoptotic processes; and pathways such as calcium-, oxytocin- and MAPK-signaling pathways with recognized roles in innate immunity. Several DMGs were related to systemic inflammation and tissue re-modelling including HDAC4, IRAK1, AKT1, MAP3K6, Wnt7A and ADAMTS17. CONCLUSIONS: The present results show that LPS altered the DNA methylation patterns of bovine endometrial epithelial cells. This information, combined with our previously reported changes in gene expression related to endometrial function, confirm that LPS activates pro-inflammatory mechanisms leading to perturbed immune balance and cell adhesion processes in the endometrium.


Asunto(s)
Metilación de ADN/efectos de los fármacos , Endometrio/citología , Redes Reguladoras de Genes/efectos de los fármacos , Lipopolisacáridos/efectos adversos , Análisis de Secuencia de ADN/veterinaria , Animales , Bovinos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Endometrio/química , Endometrio/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Ontología de Genes , Lipopolisacáridos/farmacología , Regiones Promotoras Genéticas
17.
Cell Physiol Biochem ; 54(3): 417-437, 2020 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-32348667

RESUMEN

BACKGROUND/AIMS: Obesity is associated with infertility, decreased ovarian performance and lipotoxicity. However, little is known about the aetiology of these reproductive impairments. Here, we hypothesise that the majority of changes in ovarian physiology in diet-induced obesity (DIO) are a consequence of transcriptional changes downstream of altered leptin signalling. Therefore, we investigated the extent to which leptin signalling is altered in the ovary upon obesity with particular emphasis on effects on cumulus cells (CCs), the intimate functional companions of the oocyte. Furthermore, we used the pharmacological hyperleptinemic (LEPT) mouse model to compare transcriptional profiles to DIO. METHODS: Mice were subjected to DIO for 4 and 16 weeks (wk) and leptin treatment for 16 days, to study effects in the ovary in components of leptin signalling at the transcript and protein levels, using Western blot, Real-time PCR and immunostaining. Furthermore, we used low-cell RNA sequencing to characterise changes in the transcriptome of CCs in these models. RESULTS: In the DIO model, obesity led to establishment of ovarian leptin resistance after 16 wk high fat diet (HFD), as evidenced by increases in the feedback regulator suppressor of cytokine signalling 3 (SOCS3) and decreases in the positive effectors phosphorylation of tyrosine 985 of leptin receptor (ObRb-pTyr985) and Janus kinase 2 (pJAK2). Transcriptome analysis of the CCs revealed a complex response to DIO, with large numbers and distinct sets of genes deregulated at early and late stages of obesity; in addition, there was a striking correlation between body weight and global transcriptome profile of CCs. Further analysis indicated that the transcriptome profile in 4 wk HFD CCs resembled that of LEPT CCs, in the upregulation of cellular trafficking and impairment in cytoskeleton organisation. Conversely, after 16 wk HFD CCs showed expression changes indicative of augmented inflammatory responses, cell morphogenesis, and decreased metabolism and transport, mainly as a consequence of the physiological changes of obesity. CONCLUSION: Obesity leads to ovarian leptin resistance and major time-dependent changes in gene expression in CCs, which in early obesity may be caused by increased leptin signalling in the ovary, whereas in late obesity are likely to be a consequence of metabolic changes taking place in the obese mother.


Asunto(s)
Células del Cúmulo/metabolismo , Leptina/farmacología , Obesidad/metabolismo , Oocitos/metabolismo , Ovario/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Inflamación/metabolismo , Janus Quinasa 2/metabolismo , Leptina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Obesidad/genética , Ovario/fisiología , Fosforilación , RNA-Seq , Receptores de Leptina/metabolismo , Transducción de Señal/fisiología , Proteína 3 Supresora de la Señalización de Citocinas/genética
18.
Development ; 144(14): 2606-2617, 2017 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-28619824

RESUMEN

Germ cell development involves major reprogramming of the epigenome to prime the zygote for totipotency. Histone 3 lysine 4 (H3K4) methylations are universal epigenetic marks mediated in mammals by six H3K4 methyltransferases related to fly Trithorax, including two yeast Set1 orthologs: Setd1a and Setd1b. Whereas Setd1a plays no role in oogenesis, we report that Setd1b deficiency causes female sterility in mice. Oocyte-specific Gdf9-iCre conditional knockout (Setd1bGdf9 cKO) ovaries develop through all stages; however, follicular loss accumulated with age and unfertilized metaphase II (MII) oocytes exhibited irregularities of the zona pellucida and meiotic spindle. Most Setd1bGdf9 cKO zygotes remained in the pronuclear stage and displayed polyspermy in the perivitelline space. Expression profiling of Setd1bGdf9 cKO MII oocytes revealed (1) that Setd1b promotes the expression of the major oocyte transcription factors including Obox1, 2, 5, 7, Meis2 and Sall4; and (2) twice as many mRNAs were upregulated than downregulated, suggesting that Setd1b also promotes the expression of negative regulators of oocyte development with multiple Zfp-KRAB factors implicated. Together, these findings indicate that Setd1b serves as maternal effect gene through regulation of the oocyte gene expression program.


Asunto(s)
N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Oogénesis/genética , Oogénesis/fisiología , Animales , Blastocisto/citología , Blastocisto/metabolismo , Epigénesis Genética , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Factor 9 de Diferenciación de Crecimiento/deficiencia , Factor 9 de Diferenciación de Crecimiento/genética , Factor 9 de Diferenciación de Crecimiento/metabolismo , N-Metiltransferasa de Histona-Lisina/deficiencia , Masculino , Herencia Materna , Ratones , Ratones Noqueados , Ratones Transgénicos , Oocitos/citología , Oocitos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de la Célula Individual , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Zona Pelúcida/metabolismo , Zona Pelúcida/patología , Cigoto/citología , Cigoto/metabolismo
19.
Int J Mol Sci ; 21(17)2020 Aug 25.
Artículo en Inglés | MEDLINE | ID: mdl-32854421

RESUMEN

TNFα is the main proinflammatory cytokine implicated in the pathogenesis of neurodegenerative disorders, but it also modulates physiological functions in both the developing and adult brain. In this study, we investigated a potential direct role of TNFα in determining phenotypic changes of a recently established cellular model of human basal forebrain cholinergic neuroblasts isolated from the nucleus basalis of Meynert (hfNBMs). Exposing hfNBMs to TNFα reduced the expression of immature markers, such as nestin and ß-tubulin III, and inhibited primary cilium formation. On the contrary, TNFα increased the expression of TNFα receptor TNFR2 and the mature neuron marker MAP2, also promoting neurite elongation. Moreover, TNFα affected nerve growth factor receptor expression. We also found that TNFα induced the expression of DNA-methylation enzymes and, accordingly, downregulated genes involved in neuronal development through epigenetic mechanisms, as demonstrated by methylome analysis. In summary, TNFα showed a dual role on hfNBMs phenotypic plasticity, exerting a negative influence on neurogenesis despite a positive effect on differentiation, through mechanisms that remain to be elucidated. Our results help to clarify the complexity of TNFα effects in human neurons and suggest that manipulation of TNFα signaling could provide a potential therapeutic approach against neurodegenerative disorders.


Asunto(s)
Prosencéfalo Basal/citología , Núcleo Basal de Meynert/citología , Metilación de ADN , Factor de Necrosis Tumoral alfa/metabolismo , Prosencéfalo Basal/efectos de los fármacos , Prosencéfalo Basal/metabolismo , Núcleo Basal de Meynert/efectos de los fármacos , Núcleo Basal de Meynert/metabolismo , Línea Celular , Neuronas Colinérgicas/citología , Neuronas Colinérgicas/metabolismo , Metilación de ADN/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas del Tejido Nervioso/genética , Plasticidad Neuronal/efectos de los fármacos , Receptores de Factor de Crecimiento Nervioso/genética , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Factor de Necrosis Tumoral alfa/farmacología , Secuenciación Completa del Genoma
20.
Genome Res ; 26(6): 756-67, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26769960

RESUMEN

The maternal and paternal copies of the genome are both required for mammalian development, and this is primarily due to imprinted genes, those that are monoallelically expressed based on parent-of-origin. Typically, this pattern of expression is regulated by differentially methylated regions (DMRs) that are established in the germline and maintained after fertilization. There are a large number of germline DMRs that have not yet been associated with imprinting, and their function in development is unknown. In this study, we developed a genome-wide approach to identify novel imprinted DMRs in the human placenta and investigated the dynamics of these imprinted DMRs during development in somatic and extraembryonic tissues. DNA methylation was evaluated using the Illumina HumanMethylation450 array in 134 human tissue samples, publicly available reduced representation bisulfite sequencing in the human embryo and germ cells, and targeted bisulfite sequencing in term placentas. Forty-three known and 101 novel imprinted DMRs were identified in the human placenta by comparing methylation between diandric and digynic triploid conceptions in addition to female and male gametes. Seventy-two novel DMRs showed a pattern consistent with placental-specific imprinting, and this monoallelic methylation was entirely maternal in origin. Strikingly, these DMRs exhibited polymorphic imprinted methylation between placental samples. These data suggest that imprinting in human development is far more extensive and dynamic than previously reported and that the placenta preferentially maintains maternal germline-derived DNA methylation.


Asunto(s)
Metilación de ADN , Impresión Genómica , Placenta/fisiología , Femenino , Humanos , Masculino , Polimorfismo Genético , Embarazo , Análisis de Secuencia de ADN
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