47 results found
AZIM SURANI, ULRIKE LANGE, PETRA HAJKOVA, et al., Epigenetic regulatory complex for control of gene expression
An epigenetic regulatory polypeptide complex comprises at least a first domain having site-specific DNA binding activity and at least a second domain having an arginine methyltransferase activity, wherein the second domain is capable of methylating an arginine residue located in the tail region of a histone H2A. The complex is able to regulate gene expression in cells, particularly in mammalian stem cells by controlling the methylation of R3 in the tail regions of histones H2A and H4. The complex is exemplified by a polypeptide complex comprising the DNA binding activity of Blimpi and the arginine methyltransferase activity of Prmt5.
Benesova M, Trejbalova K, Kucerova D, et al., Overexpression of TET dioxygenases in seminomas associates with low levels of DNA methylation and hydroxymethylation, Molecular Carcinogenesis, ISSN: 1098-2744
Germ cell tumors and particularly seminomas reflect the epigenomic features of their parental primordial germ cells, including the genomic DNA hypomethylation and expression of pluripotent cell markers. Because the DNA hypomethylation might be a result of TET dioxygenase activity, we examined expression of TET1-3 enzymes and the level of their product, 5-hydroxymethylcytosine, in a panel of histologically characterized seminomas and non-seminomatous germ cell tumors. Expression of TET dioxygenase mRNAs was quantified by real-time PCR. TET1 expression and the level of 5-hydroxymethylcytosine were examined immunohistochemically. Quantitative assessment of 5-methylcytosine and 5-hydroxymethylcytosine levels was done by liquid chromatography-mass spectroscopy technique. We found highly increased expression of TET1 dioxygenase in most seminomas and a strong TET1 staining in seminoma cells. Is ocitrate dehydrogenase 1 and 2 mutations were not detected suggest ing the enzymatic activity of TET1. The levels of 5-methylcytosine and 5-hydroxymethylcytosine in seminomas were found decreased in comparison to non-seminoma to us germ cell tumors and healthy testicular tissue. We propose TET1 expression as a marker of seminoma and mixed germ cell tumor and we suggest that high levels of TET1 expression are associated with the maintenance of low DNA methylation levels in seminomas. This “anti-methylator” phenotype of seminomas is in contrast to the CpG island methylator phenotype observed in a fraction of tumors of various types.
Benesova M, Trejbalova K, Kucerova D, et al., 2017, Overexpression of TET dioxygenases in seminomas associates with low levels of DNA methylation and hydroxymethylation, MOLECULAR CARCINOGENESIS, Vol: 56, Pages: 1837-1850, ISSN: 0899-1987
Germ cell tumors and particularly seminomas reflect the epigenomic features of their parental primordial germ cells (PGCs), including genomic DNA hypomethylation and expression of pluripotent cell markers. Because the DNA hypomethylation might be a result of TET dioxygenase activity, we examined expression of TET1-3 enzymes and the level of their product, 5-hydroxymethylcytosine (5hmC), in a panel of histologically characterized seminomas and non-seminomatous germ cell tumors. Expression of TET dioxygenase mRNAs was quantified by real-time PCR. TET1 expression and the level of 5hmC were examined immunohistochemically. Quantitative assessment of 5-methylcytosine (5mC) and 5hmC levels was done by the liquid chromatography-mass spectroscopy technique. We found highly increased expression of TET1 dioxygenase in most seminomas and strong TET1 staining in seminoma cells. Isocitrate dehydrogenase 1 and 2 mutations were not detected, suggesting the enzymatic activity of TET1. The levels of 5mC and 5hmC in seminomas were found decreased in comparison to non-seminomatous germ cell tumors and healthy testicular tissue. We propose that TET1 expression should be studied as a potential marker of seminomas and mixed germ cell tumors and we suggest that elevated expression of TET dioxygenase enzymes is associated with the maintenance of low DNA methylation levels in seminomas. This "anti-methylator" phenotype of seminomas is in contrast to the CpG island methylator phenotype (CIMP) observed in a fraction of tumors of various types.
Izzo A, Ziegler-Birling C, Hill PWS, et al., 2017, Dynamic changes in H1 subtype composition during epigenetic reprogramming., J Cell Biol, ISSN: 0021-9525
In mammals, histone H1 consists of a family of related proteins, including five replication-dependent (H1.1-H1.5) and two replication-independent (H1.10 and H1.0) subtypes, all expressed in somatic cells. To systematically study the expression and function of H1 subtypes, we generated knockin mouse lines in which endogenous H1 subtypes are tagged. We focused on key developmental periods when epigenetic reprogramming occurs: early mouse embryos and primordial germ cell development. We found that dynamic changes in H1 subtype expression and localization are tightly linked with chromatin remodeling and might be crucial for transitions in chromatin structure during reprogramming. Although all somatic H1 subtypes are present in the blastocyst, each stage of preimplantation development is characterized by a different combination of H1 subtypes. Similarly, the relative abundance of somatic H1 subtypes can distinguish male and female chromatin upon sex differentiation in developing germ cells. Overall, our data provide new insights into the chromatin changes underlying epigenetic reprogramming. We suggest that distinct H1 subtypes may mediate the extensive chromatin remodeling occurring during epigenetic reprogramming and that they may be key players in the acquisition of cellular totipotency and the establishment of specific cellular states.
Amouroux R, Nashun B, Shirane K, et al., 2016, De novo DNA methylation drives 5hmC accumulation in mouse zygotes, NATURE CELL BIOLOGY, Vol: 18, Pages: 225-+, ISSN: 1465-7392
Zygotic epigenetic reprogramming entails genome-wide DNA demethylation that is accompanied by Tet methylcytosine dioxygenase 3 (Tet3)-driven oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC; refs 1-4). Here we demonstrate using detailed immunofluorescence analysis and ultrasensitive LC-MS-based quantitative measurements that the initial loss of paternal 5mC does not require 5hmC formation. Small-molecule inhibition of Tet3 activity, as well as genetic ablation, impedes 5hmC accumulation in zygotes without affecting the early loss of paternal 5mC. Instead, 5hmC accumulation is dependent on the activity of zygotic Dnmt3a and Dnmt1, documenting a role for Tet3-driven hydroxylation in targeting de novo methylation activities present in the early embryo. Our data thus provide further insights into the dynamics of zygotic reprogramming, revealing an intricate interplay between DNA demethylation, de novo methylation and Tet3-driven hydroxylation.
Eguizabal C, Herrera L, De Onate L, et al., 2016, Characterization of the Epigenetic Changes During Human Gonadal Primordial Germ Cells Reprogramming, STEM CELLS, Vol: 34, Pages: 2418-2428, ISSN: 1066-5099
Epigenetic reprogramming is a central process during mammalian germline development. Genome-wide DNA demethylation in primordial germ cells (PGCs) is a prerequisite for the erasure of epigenetic memory, preventing the transmission of epimutations to the next generation. Apart from DNA demethylation, germline reprogramming has been shown to entail reprogramming of histone marks and chromatin remodelling. Contrary to other animal models, there is limited information about the epigenetic dynamics during early germ cell development in humans. Here, we provide further characterization of the epigenetic configuration of the early human gonadal PGCs. We show that early gonadal human PGCs are DNA hypomethylated and their chromatin is characterized by low H3K9me2 and high H3K27me3 marks. Similarly to previous observations in mice, human gonadal PGCs undergo dynamic chromatin changes concomitant with the erasure of genomic imprints. Interestingly, and contrary to mouse early germ cells, expression of BLIMP1/PRDM1 persists in through all gestational stages in human gonadal PGCs and is associated with nuclear lysine-specific demethylase-1. Our work provides important additional information regarding the chromatin changes associated with human PGCs development between 6 and 13 weeks of gestation in male and female gonads. Stem Cells 2016;34:2418-2428.
Leitch HG, Surani MA, Hajkova P, et al., 2016, DNA (De)Methylation: The Passive Route to Naivety?, TRENDS IN GENETICS, Vol: 32, Pages: 592-595, ISSN: 0168-9525
Mouse pluripotent embryonic stem (ES) cells can exist in distinct yet interchangeable epigenetic states dictated by their culture environment. Previous reports have shown that naïve pluripotent cells grown in the presence of 2i are characterised by global DNA hypomethylation and changes in the abundance and distribution of histone modifications. New research provides insights regarding how this might be achieved.
Nashun B, Hill PW, Hajkova P, et al., 2015, Reprogramming of cell fate: epigenetic memory and the erasure of memories past., EMBO Journal, Vol: 34, Pages: 1296-1308, ISSN: 0261-4189
Cell identity is a reflection of a cell type-specific gene expression profile, and consequently, cell type-specific transcription factor networks are considered to be at the heart of a given cellular phenotype. Although generally stable, cell identity can be reprogrammed in vitro by forced changes to the transcriptional network, the most dramatic example of which was shown by the induction of pluripotency in somatic cells by the ectopic expression of defined transcription factors alone. Although changes to cell fate can be achieved in this way, the efficiency of such conversion remains very low, in large part due to specific chromatin signatures constituting an epigenetic barrier to the transcription factor-mediated reprogramming processes. Here we discuss the two-way relationship between transcription factor binding and chromatin structure during cell fate reprogramming. We additionally explore the potential roles and mechanisms by which histone variants, chromatin remodelling enzymes, and histone and DNA modifications contribute to the stability of cell identity and/or provide a permissive environment for cell fate change during cellular reprogramming.
Nashun B, Hill PWS, Smallwood SA, et al., 2015, Continuous Histone Replacement by Hira Is Essential for Normal Transcriptional Regulation and De Novo DNA Methylation during Mouse Oogenesis, MOLECULAR CELL, Vol: 60, Pages: 611-625, ISSN: 1097-2765
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.
Amouroux R, McEwen KR, Hajkova P, et al., 2014, Current technological advances in mapping new DNA modifications, Drug Discovery Today: Disease Models, Vol: 12, Pages: 15-26, ISSN: 1740-6757
Hill PS, Amouroux R, Hajkova P, et al., 2014, DNA demethylation, Tet proteins and 5-hydroxymethylcytosine in epigenetic reprogramming: An emerging complex story, GENOMICS, Vol: 104, Pages: 324-333, ISSN: 0888-7543
Epigenetic reprogramming involves processes that lead to the erasure of epigenetic information, reverting the chromatin template to a less differentiated state. Extensive epigenetic reprogramming occurs both naturally during mammalian development in the early embryo and the developing germ line, and artificially in various in vitro reprogramming systems. Global DNA demethylation appears to be a shared attribute of reprogramming events, and understanding DNA methylation dynamics is thus of considerable interest. Recently, the Tet enzymes, which catalyse the iterative oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine, have emerged as potential drivers of epigenetic reprogramming. Although some of the recent studies point towards the direct role of Tet proteins in the removal of DNA methylation, the accumulating evidence suggests that the processes underlying DNA methylation dynamics might be more complex. Here, we review the current evidence, highlighting the agreements and the discrepancies between the suggested models and the experimental evidence.
Supek F, Lehner B, Hajkova P, et al., 2014, Hydroxymethylated Cytosines Are Associated with Elevated C to G Transversion Rates, PLOS GENETICS, Vol: 10, Pages: e1004585-e1004585, ISSN: 1553-7390
It has long been known that methylated cytosines deaminate at higher rates than unmodified cytosines and constitute mutational hotspots in mammalian genomes. The repertoire of naturally occurring cytosine modifications, however, extends beyond 5-methylcytosine to include its oxidation derivatives, notably 5-hydroxymethylcytosine. The effects of these modifications on sequence evolution are unknown. Here, we combine base-resolution maps of methyl- and hydroxymethylcytosine in human and mouse with population genomic, divergence and somatic mutation data to show that hydroxymethylated and methylated cytosines show distinct patterns of variation and evolution. Surprisingly, hydroxymethylated sites are consistently associated with elevated C to G transversion rates at the level of segregating polymorphisms, fixed substitutions, and somatic mutations in tumors. Controlling for multiple potential confounders, we find derived C to G SNPs to be 1.43-fold (1.22-fold) more common at hydroxymethylated sites compared to methylated sites in human (mouse). Increased C to G rates are evident across diverse functional and sequence contexts and, in cancer genomes, correlate with the expression of Tet enzymes and specific components of the mismatch repair pathway (MSH2, MSH6, and MBD4). Based on these and other observations we suggest that hydroxymethylation is associated with a distinct mutational burden and that the mismatch repair pathway is implicated in causing elevated transversion rates at hydroxymethylated cytosines.
Leitch HG, McEwen KR, Turp A, et al., 2013, Naive pluripotency is associated with global DNA hypomethylation, NATURE STRUCTURAL & MOLECULAR BIOLOGY, Vol: 20, Pages: 311-316, ISSN: 1545-9993
Naive pluripotent embryonic stem cells (ESCs) and embryonic germ cells (EGCs) are derived from the preimplantation epiblast and primordial germ cells (PGCs), respectively. We investigated whether differences exist between ESCs and EGCs, in view of their distinct developmental origins. PGCs are programmed to undergo global DNA demethylation; however, we find that EGCs and ESCs exhibit equivalent global DNA methylation levels. Inhibition of MEK and Gsk3b by 2i conditions leads to pronounced reduction in DNA methylation in both cell types. This is driven by Prdm14 and is associated with downregulation of Dnmt3a and Dnmt3b. However, genomic imprints are maintained in 2i, and we report derivation of EGCs with intact genomic imprints. Collectively, our findings establish that culture in 2i instills a naive pluripotent state with a distinctive epigenetic configuration that parallels molecular features observed in both the preimplantation epiblast and nascent PGCs.
McEwen KR, Leitch HG, Amouroux R, et al., 2013, The impact of culture on epigenetic properties of pluripotent stem cells and pre-implantation embryos, BIOCHEMICAL SOCIETY TRANSACTIONS, Vol: 41, Pages: 711-719, ISSN: 0300-5127
Cultured pluripotent stem cells hold great promise for regenerative medicine. Considerable efforts have been invested into the refinement and definition of improved culture systems that sustain self-renewal and avoid differentiation of pluripotent cells in vitro. Recent studies have, however, found that the choice of culture condition has a significant impact on epigenetic profiles of cultured pluripotent cells. Mouse and human ESCs (embryonic stem cells) show substantial epigenetic differences that are dependent on the culture condition, including global changes to DNA methylation and histone modifications and, in female human ESCs, to the epigenetic process of X chromosome inactivation. Epigenetic perturbations have also been detected during culture of pre-implantation embryos; limited research undertaken in mouse suggests a direct effect of the in vitro environment on epigenetic processes in this system. Widespread epigenetic changes induced by the culture condition in stem cells thus emphasize the necessity for extensive research into both immediate and long-term epigenetic effects of embryo culture during assisted reproductive technologies.
Piccolo FM, Bagci H, Brown KE, et al., 2013, Different Roles for Tet1 and Tet2 Proteins in Reprogramming-Mediated Erasure of Imprints Induced by EGC Fusion., Molecular cell, Vol: 49, Pages: 1176-1176, ISSN: 1097-2765
Piccolo FM, Bagci H, Brown KE, et al., 2013, Different Roles for Tet1 and Tet2 Proteins in Reprogramming-Mediated Erasure of Imprints Induced by EGC Fusion, MOLECULAR CELL, Vol: 49, Pages: 1023-1033, ISSN: 1097-2765
Genomic imprinting directs the allele-specific marking and expression of loci according to their parental origin. Differential DNA methylation at imprinted control regions (ICRs) is established in gametes and, although largely preserved through development, can be experimentally reset by fusing somatic cells with embryonic germ cell (EGC) lines. Here, we show that the Ten-Eleven Translocation proteins Tet1 and Tet2 participate in the efficient erasure of imprints in this model system. The fusion of B cells with EGCs initiates pluripotent reprogramming, in which rapid re-expression of Oct4 is accompanied by an accumulation of 5-hydroxymethylcytosine (5hmC) at several ICRs. Tet2 was required for the efficient reprogramming capacity of EGCs, whereas Tet1 was necessary to induce 5-methylcytosine oxidation specifically at ICRs. These data show that the Tet1 and Tet2 proteins have discrete roles in cell-fusion-mediated pluripotent reprogramming and imprint erasure in somatic cells.
Hajkova P, Hajkova P, Hajkova P, et al., 2011, Epigenetic reprogramming in the germline: towards the ground state of the epigenome, PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES, Vol: 366, Pages: 2266-2273, ISSN: 0962-8436
Epigenetic reprogramming in the germline provides a developmental model to study the erasure of epigenetic memory as it occurs naturally in vivo in the course of normal embryonic development. Our data show that germline reprogramming comprises both active DNA demethylation and extensive chromatin remodelling that are mechanistically linked through the activation of the base excision DNA repair pathway involved in the DNA demethylation process. The observed molecular hallmarks of the germline reprogramming exhibit intriguing similarities to other dedifferentiation or regeneration systems, pointing towards the existence of unifying molecular pathways underlying cell fate reversal. Elucidation of molecular processes involved in the resetting of epigenetic information in vivo will thus add to our ability to manipulate cell fate and to restore pluripotency in in vitro settings.
Hajkova P, Hajkova P, Hajkova P, et al., 2010, Epigenetic reprogramming - taking a lesson from the embryo, CURRENT OPINION IN CELL BIOLOGY, Vol: 22, Pages: 342-350, ISSN: 0955-0674
Epigenetic reprogramming involves processes that lead to the erasure of epigenetic information. Such instances are typically connected with the reversal of differentiation and can potentially lead to the re-establishment of the pluripotent (embryonic stem (ES)-like) phenotype. Genome-wide epigenetic reprogramming occurs naturally in vivo in the course of normal mammalian development. Although in vitro reprogramming systems that can restore pluripotency in somatic cell have been designed, they are still very inefficient and the process requires considerably more time than the reprogramming processes that occur in vivo. Careful analysis of the developmental reprogramming events can give us mechanistic clues and enable us to design better in vitro experimental strategies.
Hajkova P, Jeffries SJ, Lee C, et al., 2010, Genome-Wide Reprogramming in the Mouse Germ Line Entails the Base Excision Repair Pathway, SCIENCE, Vol: 329, Pages: 78-82, ISSN: 0036-8075
Genome-wide active DNA demethylation in primordial germ cells (PGCs), which reprograms the epigenome for totipotency, is linked to changes in nuclear architecture, loss of histone modifications, and widespread histone replacement. Here, we show that DNA demethylation in the mouse PGCs is mechanistically linked to the appearance of single-stranded DNA (ssDNA) breaks and the activation of the base excision repair (BER) pathway, as is the case in the zygote where the paternal pronucleus undergoes active DNA demethylation shortly after fertilization. Whereas BER might be triggered by deamination of a methylcytosine (5mC), cumulative evidence indicates other mechanisms in germ cells. We demonstrate that DNA repair through BER represents a core component of genome-wide DNA demethylation in vivo and provides a mechanistic link to the extensive chromatin remodeling in developing PGCs.
Surani MA, Hajkova P, Surani MA, et al., 2010, Epigenetic reprogramming of mouse germ cells toward totipotency., Cold Spring Harb Symp Quant Biol, Vol: 75, Pages: 211-218, ISSN: 0091-7451
Primordial germ cells (PGCs), the precursors of sperm and eggs, are the route to totipotency and require establishment of a unique epigenome in this lineage. The genetic program for PGC specification in the mouse also initiates epigenetic reprogramming that continues when PGCs migrate into the developing gonads. Among these later events is active and genome-wide DNA demethylation, which is linked to extensive chromatin remodeling. These extensive epigenetic changes erase most, if not all, of the existing epigenetic information, which resets the epigenome for totipotency. Recent evidence suggests that active DNA demethylation involves a base excision repair (BER) pathway. BER is mechanistically linked to DNA demethylation, but what triggers BER is currently under investigation. The methylated cytosine (5mC) could be modified by deamination or to 5hmC, which could induce BER. Detection of Tet1 expression specifically and coincidentally, at the time of BER in PGCs, suggests that conversion of 5mC to 5hmC might be involved, at least in part, during epigenetic reprogramming and DNA demethylation in germ cells.
Tee W-W, Pardo M, Theunissen TW, et al., 2010, Prmt5 is essential for early mouse development and acts in the cytoplasm to maintain ES cell pluripotency, GENES & DEVELOPMENT, Vol: 24, Pages: 2772-2777, ISSN: 0890-9369
Prmt5, an arginine methyltransferase, has multiple roles in germ cells, and possibly in pluripotency. Here we show that loss of Prmt5 function is early embryonic-lethal due to the abrogation of pluripotent cells in blastocysts. Prmt5 is also up-regulated in the cytoplasm during the derivation of embryonic stem (ES) cells together with Stat3, where they persist to maintain pluripotency. Prmt5 in association with Mep50 methylates cytosolic histone H2A (H2AR3me2s) to repress differentiation genes in ES cells. Loss of Prmt5 or Mep50 results in derepression of differentiation genes, indicating the significance of the Prmt5/Mep50 complex for pluripotency, which may occur in conjunction with the leukemia inhibitory factor (LIF)/Stat3 pathway.
Gebert C, Wrenzycki C, Herrmann D, et al., 2009, DNA methylation in the IGF2 intragenic DMR is re-established in a sex-specific manner in bovine blastocysts after somatic cloning, GENOMICS, Vol: 94, Pages: 63-69, ISSN: 0888-7543
The recent identification of an intragenic differentially methylated region (DMR) within the last exon of the bovine Insulin-like growth factor 2 (IGF2) gene provides a diagnostic tool for in-depth investigation of bovine imprinting and regulatory mechanisms which are active during embryo development. Here, we used bisulfite sequencing to compare sex-specific DNA methylation patterns within this DMR in bovine blastocysts produced in vivo, by in vitro fertilization and culture, SCNT, androgenesis or parthenogenesis. In in vivo derived embryos, DNA methylation was removed from this intragenic DMR after fertilization, but partially replaced by the time the embryo reached the blastocyst stage. Among embryos developing in vivo, the level of DNA methylation was significantly lower in female than in male blastocysts. This sexual dimorphism was also found between parthenogenetic and androgenetic embryos, and followed the donor cell sex in SCNT derived blastocysts and is evidence for correct methylation reprogramming in SCNT embryos.
Hajkova P, Ancelin K, Waldmann T, et al., 2008, Chromatin dynamics during epigenetic reprogramming in the mouse germ line, NATURE, Vol: 452, Pages: 877-U6, ISSN: 0028-0836
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.
Hayashi K, Lopes SMCDS, Kaneda M, et al., 2008, MicroRNA Biogenesis Is Required for Mouse Primordial Germ Cell Development and Spermatogenesis, PLOS ONE, Vol: 3, Pages: e1738-e1738, ISSN: 1932-6203
BACKGROUND: MicroRNAs (miRNAs) are critical regulators of transcriptional and post-transcriptional gene silencing, which are involved in multiple developmental processes in many organisms. Apart from miRNAs, mouse germ cells express another type of small RNA, piwi-interacting RNAs (piRNAs). Although it has been clear that piRNAs play a role in repression of retrotransposons during spermatogenesis, the function of miRNA in mouse germ cells has been unclear. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we first revealed the expression pattern of miRNAs by using a real-time PCR-based 220-plex miRNA expression profiling method. During development of germ cells, miR-17-92 cluster, which is thought to promote cell cycling, and the ES cell-specific cluster encoding miR-290 to -295 (miR-290-295 cluster) were highly expressed in primordial germ cells (PGCs) and spermatogonia. A set of miRNAs was developmentally regulated. We next analysed function of miRNA biogenesis in germ cell development by using conditional Dicer-knockout mice in which Dicer gene was deleted specifically in the germ cells. Dicer-deleted PGCs and spermatogonia exhibited poor proliferation. Retrotransposon activity was unexpectedly suppressed in Dicer-deleted PGCs, but not affected in the spermatogonia. In Dicer-deleted testis, spermatogenesis was retarded at an early stage when proliferation and/or early differentiation. Additionally, we analysed spermatogenesis in conditional Argonaute2-deficient mice. In contrast to Dicer-deficient testis, spermatogenesis in Argonaute2-deficient testis was indistinguishable from that in wild type. CONCLUSION/SIGNIFICANCE: These results illustrate that miRNAs are important for the proliferation of PGCs and spermatogonia, but dispensable for the repression of retrotransposons in developing germ cells. Consistently, miRNAs promoting cell cycling are highly expressed in PGCs and spermatogonia. Furthermore, based on normal spermatogenesis in Argonaute2-deficient testis, t
Surani MA, Durcova-Hills G, Hajkova P, et al., 2008, Germ Line, Stem Cells, and Epigenetic Reprogramming, CONTROL AND REGULATION OF STEM CELLS, Vol: 73, Pages: 9-15, ISSN: 0091-7451
The germ cell lineage has the unique attribute of generating the totipotent state. Development of blastocysts from the totipotent zygote results in the establishment of pluripotent primitive ectoderm cells in the inner cell mass of blastocysts, which subsequently develop into epiblast cells in postimplantation embryos. The germ cell lineage in mice originates from these pluripotent epiblast cells of postimplantation embryos in response to specific signals. Pluripotent stem cells and unipotent germ cells share some fundamental properties despite significant phenotypic differences between them. Additionally, early primordial germ cells can be induced to undergo dedifferentiation into pluripotent embryonic germ cells. Investigations on the relationship between germ cells and pluripotent stem cells may further elucidate the nature of the pluripotent state. Furthermore, comprehensive epigenetic reprogramming of the genome in early germ cells, including extensive erasure of epigenetic modifications, is a critical step toward establishment of totipotency. The mechanisms involved may be relevant for gaining insight into events that lead to reprogramming of somatic cells into pluripotent stem cells.
Tang F, Hajkova P, O'Carroll D, et al., 2008, MicroRNAs are tightly associated with RNA-induced gene silencing complexes in vivo, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol: 372, Pages: 24-29, ISSN: 0006-291X
Previous work has shown that synthesized siRNA/miRNA is tightly associated with RNA-induced Gene Silencing Complexes (RISCs) in vitro. However, it is unknown if the endogenous miRNAs are also stably bound to RISC complexes in vivo in cells under physiological conditions. Here we describe the use of the looped real-time PCR-based method to trace the location of endogenous miRNAs in intact cells. We found that most of the endogenous miRNAs are tightly bound to RISC complexes, and only a very small proportion of them are free in cells. Furthermore, synthesized single-stranded mature miRNA or hairpin miRNA precursor cannot replace endogenous miRNAs already present in RISC complexes. However, we found that modified 2-O-Methyl-ribonucleotides were able to dissociate the target miRNA specifically from the RISC complex. These findings have important implications for understanding the basis for the stability and metabolism of miRNAs in living cells.
Genetic and epigenetic mechanisms regulate the transition from the totipotent zygote to pluripotent primitive ectoderm cells in the inner cell mass of mouse blastocysts. These pluripotent cells can be propagated indefinitely in vitro, underpinned by a unique epigenetic state. Following implantation of the blastocyst, diverse epigenetic modifiers control differentiation of pluripotent epiblast cells into somatic cells, while specification of germ cells requires repression of the somatic program. Regenerating totipotency during development of germ cells entails re-expression of pluripotency-specific genes and extensive erasure of epigenetic modifications. Increasing knowledge of key underlying mechanisms heightens prospects for creating pluripotent cells directly from adult somatic cells.
Tang F, Kaneda M, O'Carroll D, et al., 2007, Maternal microRNAs are essential for mouse zygotic development, GENES & DEVELOPMENT, Vol: 21, Pages: 644-648, ISSN: 0890-9369
MicroRNAs (miRNAs) have important roles in diverse cellular processes, but little is known about their identity and functions during early mammalian development. Here, we show the effects of the loss of maternal inheritance of miRNAs following specific deletion of Dicer from growing oocytes. The mutant mature oocytes were almost entirely depleted of all miRNAs, and they failed to progress through the first cell division, probably because of disorganized spindle formation. By comparing single-cell cDNA microarray profiles of control and mutant oocytes, our data are compatible with the notion that a large proportion of the maternal genes are directly or indirectly under the control of miRNAs, which demonstrates that the maternal miRNAs are essential for the earliest stages of mouse embryonic development.
Ancelin K, Lange UC, Hajkova P, et al., 2006, Blimp1 associates with Prmt5 and directs histone arginine methylation in mouse germ cells, NATURE CELL BIOLOGY, Vol: 8, Pages: 623-630, ISSN: 1465-7392
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.
Durcova-Hills G, Hajkova P, Sullivan S, et al., 2006, Influence of sex chromosome constitution on the genomic imprinting of germ cells, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 103, Pages: 11184-11188, ISSN: 0027-8424
Germ cells in XY male mice establish site-specific methylation on imprinted genes during spermatogenesis, whereas germ cells in XX females establish their imprints in growing oocytes. We showed previously that in vitro, sex-specific methylation patterns of pluripotent stem cell lines derived from germ cells were influenced more by the sex chromosome constitution of the cells themselves than by the gender of the embryo from which they had been derived. To see whether the same situation would prevail in vivo, we have now determined the methylation status of H19 expressed from the maternal allele, and the expression and methylation status of a paternally expressed gene Peg3, in germ cells from sex-reversed and control embryos. For these imprinted genes, we conclude that the female imprint is a response of the germ cells to undergoing oogenesis, rather than to their XX chromosome constitution. Similarly, both our XY and our sex-reversed XX male germ cells clearly showed a male rather than a female pattern of DNA methylation; here, however, the sex chromosome constitution had a significant effect, with XX male germ cells less methylated than the XY controls.
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