RESUMEN
The interplay between the topological organization of the genome and the regulation of gene expression remains unclear. Depletion of molecular factors (e.g. CTCF) underlying topologically associating domains (TADs) leads to modest alterations in gene expression, whereas genomic rearrangements involving TAD boundaries disrupt normal gene expression and can lead to pathological phenotypes. Here, we targeted the TAD neighboring that of the noncoding transcript Xist, which controls X-chromosome inactivation. Inverting 245â kb within the TAD led to expected rearrangement of CTCF-based contacts but revealed heterogeneity in the 'contact' potential of different CTCF sites. Expression of most genes therein remained unaffected in mouse embryonic stem cells and during differentiation. Interestingly, expression of Xist was ectopically upregulated. The same inversion in mouse embryos led to biased Xist expression. Smaller inversions and deletions of CTCF clusters led to similar results: rearrangement of contacts and limited changes in local gene expression, but significant changes in Xist expression in embryos. Our study suggests that the wiring of regulatory interactions within a TAD can influence the expression of genes in neighboring TADs, highlighting the existence of mechanisms of inter-TAD communication.
Asunto(s)
ARN Largo no Codificante , Inactivación del Cromosoma X , Animales , Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Cromatina , Comunicación , Expresión Génica , Genoma , Ratones , ARN Largo no Codificante/genética , Inactivación del Cromosoma X/genéticaRESUMEN
Following fertilization in mammals, the chromatin landscape inherited from the two parental genomes and the nuclear organization are extensively reprogrammed. A tight regulation of nuclear organization is important for developmental success. One main nuclear feature is the organization of the chromosomes in discrete and individual nuclear spaces known as chromosome territories (CTs). In culture cells, their arrangements can be constrained depending on their genomic content (e.g., gene density or repeats) or by specific nuclear constrains such as the periphery or the nucleolus. However, during the early steps of mouse embryonic development, much less is known, specifically regarding how and when the two parental genomes intermingle. Here, we describe a three-dimensional fluorescence in situ hybridization (3D-FISH) for chromosome painting (3D-ChromoPaint) optimized to gain understanding in nuclear organization of specific CTs following fertilization. Our approach preserves the nuclear structure, and the acquired images allow full spatial analysis of interphase chromosome positioning and morphology across the cell cycle and during early development. This method will be useful in understanding the dynamics of chromosome repositioning during development as well as the alteration of chromosome territories upon changes in transcriptional status during key developmental steps. This protocol can be adapted to any other species or organoids in culture.
Asunto(s)
Blastocisto/citología , Pintura Cromosómica/métodos , Cromosomas/genética , Hibridación Fluorescente in Situ/métodos , Ratones/embriología , Animales , Blastocisto/metabolismo , Blastocisto/ultraestructura , ADN/genética , Desarrollo Embrionario , Imagenología Tridimensional/métodos , Ratones/genética , Microscopía/métodos , Imagen Óptica/métodosRESUMEN
Over the past two decades, the development of chromosome conformation capture technologies has allowed to intensively probe the properties of genome folding in various cell types. High-throughput versions of these C-based assays (named Hi-C) have released the mapping of 3D chromosome folding for the entire genomes. Applied to mammalian preimplantation embryos, it has revealed a unique chromosome organization after fertilization when a new individual is being formed. However, the questions of whether specific structures could arise depending on their parental origins or of their transcriptional status remain open. Our method chapter is dedicated to the technical description on how applying scHi-C to mouse embryos at different stages of preimplantation development. This approach capitalized with the limited amount of material available at these developmental stages. It also provides new research avenues, such as the study of mutant embryos for further functional studies.
Asunto(s)
Blastocisto/citología , Cromosomas/genética , Ratones/embriología , Análisis de la Célula Individual/métodos , Animales , Blastocisto/metabolismo , Cromosomas/química , Desarrollo Embrionario , Ratones/genética , Micromanipulación/métodos , Fijación del Tejido/métodosRESUMEN
The adaptation of Hi-C protocols to enable the investigation of chromosome organization in single cells opens new avenues to study the dynamics of this process during embryogenesis. However, the analysis of single-cell Hi-C data is not yet standardized and raises novel bioinformatic challenges. Here we describe a complete workflow for the analysis of single-cell Hi-C data, with a main focus on allele-specific analysis based on data obtained from hybrid embryos.
Asunto(s)
Blastocisto/citología , Biología Computacional/métodos , Ratones/embriología , Análisis de la Célula Individual/métodos , Alelos , Animales , Blastocisto/metabolismo , Ciclo Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Femenino , Masculino , Ratones/genética , Programas Informáticos , Flujo de TrabajoRESUMEN
Paternal and maternal epigenomes undergo marked changes after fertilization1. Recent epigenomic studies have revealed the unusual chromatin landscapes that are present in oocytes, sperm and early preimplantation embryos, including atypical patterns of histone modifications2-4 and differences in chromosome organization and accessibility, both in gametes5-8 and after fertilization5,8-10. However, these studies have led to very different conclusions: the global absence of local topological-associated domains (TADs) in gametes and their appearance in the embryo8,9 versus the pre-existence of TADs and loops in the zygote5,11. The questions of whether parental structures can be inherited in the newly formed embryo and how these structures might relate to allele-specific gene regulation remain open. Here we map genomic interactions for each parental genome (including the X chromosome), using an optimized single-cell high-throughput chromosome conformation capture (HiC) protocol12,13, during preimplantation in the mouse. We integrate chromosome organization with allelic expression states and chromatin marks, and reveal that higher-order chromatin structure after fertilization coincides with an allele-specific enrichment of methylation of histone H3 at lysine 27. These early parental-specific domains correlate with gene repression and participate in parentally biased gene expression-including in recently described, transiently imprinted loci14. We also find TADs that arise in a non-parental-specific manner during a second wave of genome assembly. These de novo domains are associated with active chromatin. Finally, we obtain insights into the relationship between TADs and gene expression by investigating structural changes to the paternal X chromosome before and during X chromosome inactivation in preimplantation female embryos15. We find that TADs are lost as genes become silenced on the paternal X chromosome but linger in regions that escape X chromosome inactivation. These findings demonstrate the complex dynamics of three-dimensional genome organization and gene expression during early development.
Asunto(s)
Blastocisto/citología , Blastocisto/metabolismo , Cromatina/metabolismo , Desarrollo Embrionario/genética , Fertilización/genética , Células Germinativas/citología , Padres , Alelos , Animales , Cromatina/química , Cromatina/genética , Posicionamiento de Cromosoma , Cromosomas de los Mamíferos/química , Cromosomas de los Mamíferos/genética , Cromosomas de los Mamíferos/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Genoma/genética , Impresión Genómica , Células Germinativas/metabolismo , Histonas/química , Histonas/metabolismo , Masculino , Metilación , Ratones , Proteínas del Grupo Polycomb/metabolismo , Análisis de la Célula Individual , Inactivación del Cromosoma X/genéticaRESUMEN
The Polycomb group of proteins is required for the proper orchestration of gene expression due to its role in maintaining transcriptional silencing. It is composed of several chromatin modifying complexes, including Polycomb Repressive Complex 2 (PRC2), which deposits H3K27me2/3. Here, we report the identification of a cofactor of PRC2, EZHIP (EZH1/2 Inhibitory Protein), expressed predominantly in the gonads. EZHIP limits the enzymatic activity of PRC2 and lessens the interaction between the core complex and its accessory subunits, but does not interfere with PRC2 recruitment to chromatin. Deletion of Ezhip in mice leads to a global increase in H3K27me2/3 deposition both during spermatogenesis and at late stages of oocyte maturation. This does not affect the initial number of follicles but is associated with a reduction of follicles in aging. Our results suggest that mature oocytes Ezhip-/- might not be fully functional and indicate that fertility is strongly impaired in Ezhip-/- females. Altogether, our study uncovers EZHIP as a regulator of chromatin landscape in gametes.
Asunto(s)
Proteínas Oncogénicas/metabolismo , Óvulo/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Espermatozoides/metabolismo , Adulto , Animales , Línea Celular Tumoral , Cromatina/metabolismo , Femenino , Células HEK293 , Histonas/metabolismo , Humanos , Masculino , Ratones , Ratones Noqueados , Mutación , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/aislamiento & purificación , Oogénesis , Ovario/citología , Ovario/patología , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Células Sf9 , Espermatogénesis , Testículo/citología , Testículo/patologíaRESUMEN
X-chromosome inactivation is established during early development. In mice, transcriptional repression of the paternal X-chromosome (Xp) and enrichment in epigenetic marks such as H3K27me3 is achieved by the early blastocyst stage. X-chromosome inactivation is then reversed in the inner cell mass. The mechanisms underlying Xp reactivation remain enigmatic. Using in vivo single-cell approaches (allele-specific RNAseq, nascent RNA-fluorescent in situ hybridization and immunofluorescence), we show here that different genes are reactivated at different stages, with more slowly reactivated genes tending to be enriched in H3meK27. We further show that in UTX H3K27 histone demethylase mutant embryos, these genes are even more slowly reactivated, suggesting that these genes carry an epigenetic memory that may be actively lost. On the other hand, expression of rapidly reactivated genes may be driven by transcription factors. Thus, some X-linked genes have minimal epigenetic memory in the inner cell mass, whereas others may require active erasure of chromatin marks.
Asunto(s)
Masa Celular Interna del Blastocisto/metabolismo , Epigénesis Genética , Factores de Transcripción/farmacocinética , Inactivación del Cromosoma X/genética , Animales , Femenino , Genes Ligados a X , Histonas/metabolismo , Hibridación Fluorescente in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Modelos Genéticos , Embarazo , ARN Largo no Codificante/genética , Análisis de Secuencia de ARN , Análisis de la Célula IndividualRESUMEN
Characterizing the maternal-to-zygotic transition (MZT) is a central question in embryogenesis, and is critical for our understanding of early post-fertilization events in mammals. High-throughput RNA sequencing (RNA Seq) of mouse oocytes and early embryos has recently revealed that elaborate transcription patterns of genes and repeats are established post-fertilization. This occurs in the context of the gradually depleted maternal pool of RNA provided by the oocyte, which can confound the accurate analysis of the zygotic genome activation when the mRNA population is sequenced. In this context, and given the limited amounts of material available from embryos, particularly when studying mutants, as well as the cost of sequencing, an alternative, complementary single cell approach is RNA FISH. This approach can assay the expression of specific genes or genetic elements during preimplantation development, in particular during the MZT. Here, we describe how RNA FISH can be applied to visualize nascent transcription at specific genomic loci in embryos at different stages of preimplantation development and also discuss possible analytical methods of RNA FISH data.
Asunto(s)
Embrión de Mamíferos/química , Hibridación Fluorescente in Situ/métodos , ARN/genética , Animales , Análisis Citogenético , Regulación del Desarrollo de la Expresión Génica , Ratones , Análisis de la Célula Individual , Activación TranscripcionalRESUMEN
MyoD is a master regulator of myogenesis. Chromatin modifications required to trigger MyoD expression are still poorly described. Here, we demonstrate that the histone demethylase LSD1/KDM1a is recruited on the MyoD core enhancer upon muscle differentiation. Depletion of Lsd1 in myoblasts precludes the removal of H3K9 methylation and the recruitment of RNA polymerase II on the core enhancer, thereby preventing transcription of the non-coding enhancer RNA required for MyoD expression (CEeRNA). Consistently, Lsd1 conditional inactivation in muscle progenitor cells during embryogenesis prevented transcription of the CEeRNA and delayed MyoD expression. Our results demonstrate that LSD1 is required for the timely expression of MyoD in limb buds and identify a new biological function for LSD1 by showing that it can activate RNA polymerase II-dependent transcription of enhancers.
Asunto(s)
Histona Demetilasas/metabolismo , Proteína MioD/metabolismo , Transcripción Genética/fisiología , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica/fisiología , Histonas/metabolismo , Esbozos de los Miembros/metabolismo , Ratones , Desarrollo de Músculos/fisiología , Mioblastos/metabolismo , Mioblastos/fisiología , ARN Polimerasa II/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/fisiologíaRESUMEN
The long noncoding RNA Xist is expressed from only the paternal X chromosome in mouse preimplantation female embryos and mediates transcriptional silencing of that chromosome. In females, absence of Xist leads to postimplantation lethality. Here, through single-cell RNA sequencing of early preimplantation mouse embryos, we found that the initiation of imprinted X-chromosome inactivation absolutely requires Xist. Lack of paternal Xist leads to genome-wide transcriptional misregulation in the early blastocyst and to failure to activate the extraembryonic pathway that is essential for postimplantation development. We also demonstrate that the expression dynamics of X-linked genes depends on the strain and parent of origin as well as on the location along the X chromosome, particularly at the first 'entry' sites of Xist. This study demonstrates that dosage-compensation failure has an effect as early as the blastocyst stage and reveals genetic and epigenetic contributions to orchestrating transcriptional silencing of the X chromosome during early embryogenesis.
Asunto(s)
Desarrollo Embrionario/genética , Impresión Genómica , ARN Largo no Codificante/genética , Inactivación del Cromosoma X/genética , Alelos , Animales , Blastocisto/citología , Blastocisto/metabolismo , Diferenciación Celular/genética , Compensación de Dosificación (Genética) , Implantación del Embrión/genética , Embrión de Mamíferos/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Silenciador del Gen , Genes Ligados a X , Cinética , Masculino , Ratones Endogámicos C57BL , Mutación/genética , ARN Largo no Codificante/metabolismo , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Factores de Tiempo , Cromosoma X/genéticaRESUMEN
The Polycomb repressive complexes PRC1 and PRC2 play a central role in developmental gene regulation in multicellular organisms. PRC1 and PRC2 modify chromatin by catalysing histone H2A lysine 119 ubiquitylation (H2AK119u1), and H3 lysine 27 methylation (H3K27me3), respectively. Reciprocal crosstalk between these modifications is critical for the formation of stable Polycomb domains at target gene loci. While the molecular mechanism for recognition of H3K27me3 by PRC1 is well defined, the interaction of PRC2 with H2AK119u1 is poorly understood. Here we demonstrate a critical role for the PRC2 cofactor Jarid2 in mediating the interaction of PRC2 with H2AK119u1. We identify a ubiquitin interaction motif at the amino-terminus of Jarid2, and demonstrate that this domain facilitates PRC2 localization to H2AK119u1 both in vivo and in vitro. Our findings ascribe a critical function to Jarid2 and define a key mechanism that links PRC1 and PRC2 in the establishment of Polycomb domains.
Asunto(s)
Histonas/metabolismo , Lisina/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Ubiquitinación , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Metilación de ADN , Metilación , Ratones , Nucleosomas/metabolismo , Complejo Represivo Polycomb 2/química , Unión Proteica , Dominios Proteicos , Inactivación del Cromosoma X/genéticaRESUMEN
Upon fertilization, the highly specialised sperm and oocyte genomes are remodelled to confer totipotency. The mechanisms of the dramatic reprogramming events that occur have remained unknown, and presumed roles of histone modifying enzymes are just starting to be elucidated. Here, we explore the function of the oocyte-inherited pool of a histone H3K4 and K9 demethylase, LSD1/KDM1A during early mouse development. KDM1A deficiency results in developmental arrest by the two-cell stage, accompanied by dramatic and stepwise alterations in H3K9 and H3K4 methylation patterns. At the transcriptional level, the switch of the maternal-to-zygotic transition fails to be induced properly and LINE-1 retrotransposons are not properly silenced. We propose that KDM1A plays critical roles in establishing the correct epigenetic landscape of the zygote upon fertilization, in preserving genome integrity and in initiating new patterns of genome expression that drive early mouse development.
Asunto(s)
Cromatina/metabolismo , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Histona Demetilasas/metabolismo , Transcripción Genética , Cigoto/enzimología , Cigoto/fisiología , Animales , Epigénesis Genética , Ratones , Oocitos/enzimología , Oocitos/fisiologíaRESUMEN
Polycomb Group (PcG) proteins maintain transcriptional repression throughout development, mostly by regulating chromatin structure. Polycomb Repressive Complex 2 (PRC2), a component of the Polycomb machinery, is responsible for the methylation of histone H3 lysine 27 (H3K27me2/3). Jarid2 was previously identified as a cofactor of PRC2, regulating PRC2 targeting to chromatin and its enzymatic activity. Deletion of Jarid2 leads to impaired orchestration of gene expression during cell lineage commitment. Here, we reveal an unexpected crosstalk between Jarid2 and PRC2, with Jarid2 being methylated by PRC2. This modification is recognized by the Eed core component of PRC2 and triggers an allosteric activation of PRC2's enzymatic activity. We show that Jarid2 methylation is important to promote PRC2 activity at a locus devoid of H3K27me3 and for the correct deposition of this mark during cell differentiation. Our results uncover a regulation loop where Jarid2 methylation fine-tunes PRC2 activity depending on the chromatin context.
Asunto(s)
Diferenciación Celular , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Animales , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Femenino , Células HEK293 , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Humanos , Lisina/genética , Lisina/metabolismo , Metilación , Ratones Noqueados , Modelos Genéticos , Mutación , Complejo Represivo Polycomb 2/genética , Interferencia de ARNRESUMEN
During X chromosome inactivation (XCI), the Polycomb Repressive Complex 2 (PRC2) is thought to participate in the early maintenance of the inactive state. Although Xist RNA is essential for the recruitment of PRC2 to the X chromosome, the precise mechanism remains unclear. Here, we demonstrate that the PRC2 cofactor Jarid2 is an important mediator of Xist-induced PRC2 targeting. The region containing the conserved B and F repeats of Xist is critical for Jarid2 recruitment via its unique N-terminal domain. Xist-induced Jarid2 recruitment occurs chromosome-wide independently of a functional PRC2 complex, unlike at other parts of the genome, such as CG-rich regions, where Jarid2 and PRC2 binding are interdependent. Conversely, we show that Jarid2 loss prevents efficient PRC2 and H3K27me3 enrichment to Xist-coated chromatin. Jarid2 thus represents an important intermediate between PRC2 and Xist RNA for the initial targeting of the PRC2 complex to the X chromosome during onset of XCI.
Asunto(s)
Complejo Represivo Polycomb 2/metabolismo , ARN Largo no Codificante/fisiología , Inactivación del Cromosoma X , Cromosoma X/metabolismo , Animales , Compensación de Dosificación (Genética) , Humanos , Ratones , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/fisiología , ARN Largo no Codificante/metabolismoRESUMEN
X chromosome inactivation (XCI) is an excellent model for studying how epigenetic marks are initiated during early embryogenesis. XCI is an essential process that takes place in females, leading to dosage compensation between males and females. In mouse, it occurs in two waves: the first one is paternally imprinted, during the preimplantation period and the second one occurs in a random fashion. We provide here an update of the main molecular steps and hypothesis underlining this complex process.
Asunto(s)
Ratones/genética , Inactivación del Cromosoma X/fisiología , Animales , Aberraciones Cromosómicas , Femenino , Impresión Genómica/genética , Impresión Genómica/fisiología , Masculino , Ratones/fisiología , Modelos Biológicos , Especificidad de la Especie , Espermatogénesis/genética , Espermatogénesis/fisiología , Inactivación del Cromosoma X/genéticaRESUMEN
A unique feature of the germ cell lineage is the generation of totipotency. A critical event in this context is DNA demethylation and the erasure of parental imprints in mouse primordial germ cells (PGCs) on embryonic day 11.5 (E11.5) after they enter into the developing gonads. Little is yet known about the mechanism involved, except that it is apparently an active process. We have examined the associated changes in the chromatin to gain further insights into this reprogramming event. Here we show that the chromatin changes occur in two steps. The first changes in nascent PGCs at E8.5 establish a distinctive chromatin signature that is reminiscent of pluripotency. Next, when PGCs are residing in the gonads, major changes occur in nuclear architecture accompanied by an extensive erasure of several histone modifications and exchange of histone variants. Furthermore, the histone chaperones HIRA and NAP-1 (NAP111), which are implicated in histone exchange, accumulate in PGC nuclei undergoing reprogramming. We therefore suggest that the mechanism of histone replacement is critical for these chromatin rearrangements to occur. The marked chromatin changes are intimately linked with genome-wide DNA demethylation. On the basis of the timing of the observed events, we propose that if DNA demethylation entails a DNA repair-based mechanism, the evident histone replacement would represent a repair-induced response event rather than being a prerequisite.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Epigénesis Genética , Células Germinativas/metabolismo , Animales , Metilación de ADN , Gónadas/citología , Gónadas/metabolismo , Histonas/metabolismo , Ratones , Células Madre/metabolismoRESUMEN
Blimp1, a transcriptional repressor, has a crucial role in the specification of primordial germ cells (PGCs) in mice at embryonic day 7.5 (E7.5). This SET-PR domain protein can form complexes with various chromatin modifiers in a context-dependent manner. Here, we show that Blimp1 has a novel interaction with Prmt5, an arginine-specific histone methyltransferase, which mediates symmetrical dimethylation of arginine 3 on histone H2A and/or H4 tails (H2A/H4R3me2s). Prmt5 has been shown to associate with Tudor, a component of germ plasm in Drosophila melanogaster. Blimp1-Prmt5 colocalization results in high levels of H2A/H4 R3 methylation in PGCs at E8.5. However, at E11.5, Blimp1-Prmt5 translocates from the nucleus to the cytoplasm, resulting in the loss of H2A/H4 R3 methylation at the time of extensive epigenetic reprogramming of germ cells. Subsequently, Dhx38, a putative target of the Blimp1-Prmt5 complex, is upregulated. Interestingly, expression of Dhx38 is also seen in pluripotent embryonic germ cells that are derived from PGCs when Blimp1 expression is lost. Our study demonstrates that Blimp1 is involved in a novel transcriptional regulatory complex in the mouse germ-cell lineage.
Asunto(s)
Arginina/metabolismo , Células Germinativas/metabolismo , Histonas/metabolismo , Proteínas Represoras/fisiología , Factores de Transcripción/fisiología , Transporte Activo de Núcleo Celular , Adenosina Trifosfatasas/genética , Factores de Edad , Animales , Embrión de Mamíferos , Regulación de la Expresión Génica , Metilación , Ratones , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Unión Proteica , Proteína Metiltransferasas/metabolismo , Proteína-Arginina N-Metiltransferasas , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Germ cell fate in mice is induced in pluripotent epiblast cells in response to signals from extraembryonic tissues. The specification of approximately 40 founder primordial germ cells and their segregation from somatic neighbours are important events in early development. We have proposed that a critical event during this specification includes repression of a somatic programme that is adopted by neighbouring cells. Here we show that Blimp1 (also known as Prdm1), a known transcriptional repressor, has a critical role in the foundation of the mouse germ cell lineage, as its disruption causes a block early in the process of primordial germ cell formation. Blimp1-deficient mutant embryos form a tight cluster of about 20 primordial germ cell-like cells, which fail to show the characteristic migration, proliferation and consistent repression of homeobox genes that normally accompany specification of primordial germ cells. Furthermore, our genetic lineage-tracing experiments indicate that the Blimp1-positive cells originating from the proximal posterior epiblast cells are indeed the lineage-restricted primordial germ cell precursors.
Asunto(s)
Linaje de la Célula , Células Germinativas/citología , Células Germinativas/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Gástrula/citología , Gástrula/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Ratones , Mutación/genética , Fenotipo , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Proteínas Represoras/genética , Células Madre/citología , Células Madre/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/genéticaRESUMEN
We investigated the control of telomere length by the human telomeric proteins TRF1 and TRF2. To this end, we established telomerase-positive cell lines in which the targeting of these telomeric proteins to specific telomeres could be induced. We demonstrate that their targeting leads to telomere shortening. This indicates that these proteins act in cis to repress telomere elongation. Inhibition of telomerase activity by a modified oligonucleotide did not further increase the pace of telomere erosion caused by TRF1 targeting, suggesting that telomerase itself is the target of TRF1 regulation. In contrast, TRF2 targeting and telomerase inhibition have additive effects. The possibility that TRF2 can activate a telomeric degradation pathway was directly tested in human primary cells that do not express telomerase. In these cells, overexpression of full-length TRF2 leads to an increased rate of telomere shortening.