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1.
Nat Commun ; 11(1): 2267, 2020 05 08.
Article in English | MEDLINE | ID: mdl-32385287

ABSTRACT

To faithfully transmit genetic information, cells must replicate their entire genome before division. This is thought to be ensured by the temporal separation of replication and chromosome segregation. Here we show that in 20-40% of unperturbed yeast cells, DNA synthesis continues during anaphase, late in mitosis. High cyclin-Cdk activity inhibits DNA synthesis in metaphase, and the decrease in cyclin-Cdk activity during mitotic exit allows DNA synthesis to finish at subtelomeric and some difficult-to-replicate regions. DNA synthesis during late mitosis correlates with elevated mutation rates at subtelomeric regions, including copy number variation. Thus, yeast cells temporally overlap DNA synthesis and chromosome segregation during normal growth, possibly allowing cells to maximize population-level growth rate while simultaneously exploring greater genetic space.


Subject(s)
Chromosome Segregation , Chromosomes, Fungal/metabolism , DNA, Fungal/metabolism , Saccharomycetales/metabolism , Anaphase/genetics , Cell Nucleus/metabolism , Chromatin/metabolism , Cyclin-Dependent Kinases/metabolism , DNA Replication , Genes, Fungal , Metaphase , Mitosis , Mutation Rate , Saccharomyces cerevisiae Proteins/metabolism , Saccharomycetales/genetics , Telomere/metabolism
2.
Nat Struct Mol Biol ; 25(8): 743, 2018 Aug.
Article in English | MEDLINE | ID: mdl-29995840

ABSTRACT

In this article, the Ponceau staining presented in Fig. 1b (right, bottom) does not follow best practices for figure preparation since itinadvertently included duplications from the Ponceau staining presented in Supplementary Fig. 1b (for which the same preparation ofnucleosomes from HeLa cells had been used). A new Fig. 1b is provided in the Author Correction.

3.
J Cell Biol ; 216(10): 3017-3028, 2017 10 02.
Article in English | MEDLINE | ID: mdl-28794128

ABSTRACT

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.

4.
Epigenetics ; 11(8): 553-62, 2016 08 02.
Article in English | MEDLINE | ID: mdl-26479850

ABSTRACT

In mammals, the time period that follows fertilization is characterized by extensive chromatin remodeling, which enables epigenetic reprogramming of the gametes. Major changes in chromatin structure persist until the time of implantation, when the embryo develops into a blastocyst, which comprises the inner cell mass and the trophectoderm. Changes in DNA methylation, histone variant incorporation, and covalent modifications of the histones tails have been intensively studied during pre-implantation development. However, modifications within the core of the nucleosomes have not been systematically analyzed. Here, we report the first characterization and temporal analysis of 3 key acetylated residues in the core of the histone H3: H3K64ac, H3K122ac, and H3K56ac, all located at structurally important positions close to the DNA. We found that all 3 acetylations occur during pre-implantation development, but with different temporal kinetics. Globally, H3K64ac and H3K56ac were detected throughout cleavage stages, while H3K122ac was only weakly detectable during this time. Our work contributes to the understanding of the contribution of histone modifications in the core of the nucleosome to the "marking" of the newly established embryonic chromatin and unveils new modification pathways potentially involved in epigenetic reprogramming.


Subject(s)
Gene Expression Regulation, Developmental , Histones/metabolism , Nucleosomes/metabolism , Acetylation , Animals , Chromatin Assembly and Disassembly , Embryo Implantation , Epigenesis, Genetic , Female , Histone Code , Histones/genetics , Mice , Nucleosomes/genetics , Protein Processing, Post-Translational
5.
Nat Struct Mol Biol ; 22(9): 662-71, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26237512

ABSTRACT

Cellular plasticity is essential for early embryonic cells. Unlike pluripotent cells, which form embryonic tissues, totipotent cells can generate a complete organism including embryonic and extraembryonic tissues. Cells resembling 2-cell-stage embryos (2C-like cells) arise at very low frequency in embryonic stem (ES) cell cultures. Although induced reprogramming to pluripotency is well established, totipotent cells remain poorly characterized, and whether reprogramming to totipotency is possible is unknown. We show that mouse 2C-like cells can be induced in vitro through downregulation of the chromatin-assembly activity of CAF-1. Endogenous retroviruses and genes specific to 2-cell embryos are the highest-upregulated genes upon CAF-1 knockdown. Emerging 2C-like cells exhibit molecular characteristics of 2-cell embryos and higher reprogrammability than ES cells upon nuclear transfer. Our results suggest that early embryonic-like cells can be induced by modulating chromatin assembly and that atypical histone deposition may trigger the emergence of totipotent cells.


Subject(s)
Cell Differentiation , Chromatin Assembly and Disassembly , Proteins/antagonists & inhibitors , Totipotent Stem Cells/physiology , Animals , Exoribonucleases , Mice , Repressor Proteins , Ribonucleases
6.
Mol Cell ; 56(4): 580-94, 2014 Nov 20.
Article in English | MEDLINE | ID: mdl-25457167

ABSTRACT

Constitutive heterochromatin is typically defined by high levels of DNA methylation and H3 lysine 9 trimethylation (H3K9Me3), whereas facultative heterochromatin displays DNA hypomethylation and high H3 lysine 27 trimethylation (H3K27Me3). The two chromatin types generally do not coexist at the same loci, suggesting mutual exclusivity. During development or in cancer, pericentromeric regions can adopt either epigenetic state, but the switching mechanism is unknown. We used a quantitative locus purification method to characterize changes in pericentromeric chromatin-associated proteins in mouse embryonic stem cells deficient for either the methyltransferases required for DNA methylation or H3K9Me3. DNA methylation controls heterochromatin architecture and inhibits Polycomb recruitment. BEND3, a protein enriched on pericentromeric chromatin in the absence of DNA methylation or H3K9Me3, allows Polycomb recruitment and H3K27Me3, resulting in a redundant pathway to generate repressive chromatin. This suggests that BEND3 is a key factor in mediating a switch from constitutive to facultative heterochromatin.


Subject(s)
DNA Methylation , DNA-Binding Proteins/physiology , Gene Silencing , Heterochromatin/genetics , Animals , CCAAT-Enhancer-Binding Proteins , Cell Nucleus/metabolism , Cells, Cultured , Centromere/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , Embryonic Stem Cells/physiology , Genetic Loci , Histones/metabolism , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Microsatellite Repeats , Nuclear Proteins/metabolism , Proteome/metabolism , Repressor Proteins , Ubiquitin-Protein Ligases , DNA Methyltransferase 3B
7.
Nat Struct Mol Biol ; 20(11): 1321-4, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24096363

ABSTRACT

The spatiotemporal organization of genomes in the nucleus is an emerging key player to regulate genome function. Live imaging of nuclear organization dynamics would be a breakthrough toward uncovering the functional relevance and mechanisms regulating genome architecture. Here, we used transcription activator-like effector (TALE) technology to visualize endogenous repetitive genomic sequences. We established TALE-mediated genome visualization (TGV) to label genomic sequences and follow nuclear positioning and chromatin dynamics in cultured mouse cells and in the living organism. TGV is highly specific, thus allowing differential labeling of parental chromosomes by distinguishing between single-nucleotide polymorphisms (SNPs). Our findings provide a framework to address the function of genome architecture through visualization of nuclear dynamics in vivo.


Subject(s)
Cell Nucleus/chemistry , Cell Nucleus/metabolism , Chromatin/metabolism , Microscopy, Fluorescence/methods , Staining and Labeling/methods , Animals , Cells, Cultured , Mice
8.
Nat Commun ; 4: 2233, 2013.
Article in English | MEDLINE | ID: mdl-23903902

ABSTRACT

To ensure genome stability, pericentromeric regions are compacted in a dense heterochromatic structure through a combination of specific 'epigenetic' factors and modifications. A cascadal pathway is responsible for establishing pericentromeric chromatin involving chromatin modifiers and 'readers', such as H3K9 histone methyltransferases (Suv)39h and heterochromatin protein 1. Here we define how H3K64me3 on the lateral surface of the histone octamer integrates within the heterochromatinization cascade. Our data suggest that enrichment of H3K64me3 at pericentromeric chromatin foci is dependent on H3K9me3 but independent of a number of central factors such as heterochromatin protein 1, DNA methyltransferases and Suv4-20h histone methyltransferases. Our results support a model in which pericentromeric heterochromatin foci are formed along distinct pathways upon H3K9 trimethylation, involving H3K64me3 to potentially stabilize DNA-histone interactions, as well as sequential recruitment of repressive histone tail and DNA modifications. We hence suggest that multiple mechanisms ensure heterochromatin integrity at pericentromeres, with H3K64me3 as an important factor.


Subject(s)
Centromere/metabolism , Heterochromatin/metabolism , Histones/metabolism , Lysine/metabolism , Animals , Chromobox Protein Homolog 5 , Chromosomal Proteins, Non-Histone/metabolism , DNA Methylation/genetics , Fluorescent Antibody Technique , Histone-Lysine N-Methyltransferase/metabolism , Humans , Mice , NIH 3T3 Cells , Zygote/metabolism
9.
Nat Struct Mol Biol ; 20(3): 332-8, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23353788

ABSTRACT

How a more plastic chromatin state is maintained and reversed during development is unknown. Heterochromatin-mediated silencing of repetitive elements occurs in differentiated cells. Here, we used repetitive elements, including retrotransposons, as model loci to address how and when heterochromatin forms during development. RNA sequencing throughout early mouse embryogenesis revealed that repetitive-element expression is dynamic and stage specific, with most repetitive elements becoming repressed before implantation. We show that LINE-1 and IAP retrotransposons become reactivated from both parental genomes after fertilization. Chromatin immunoprecipitation for H3K4me3 and H3K9me3 in 2- and 8-cell embryos indicates that their developmental silencing follows loss of activating marks rather than acquisition of conventional heterochromatic marks. Furthermore, short LINE-1 RNAs regulate LINE-1 transcription in vivo. Our data indicate that reprogramming after mammalian fertilization comprises a robust transcriptional activation of retrotransposons and that repetitive elements are initially regulated through RNA.


Subject(s)
Blastocyst/physiology , DNA Methylation , Gene Expression Regulation, Developmental , Heterochromatin/genetics , Long Interspersed Nucleotide Elements , Animals , Chromatin Immunoprecipitation , Embryonic Development/genetics , Female , Histones/genetics , Histones/metabolism , Male , Mice , Mice, Inbred C57BL , Nucleic Acid Conformation , RNA Caps , Retroelements , Transcription, Genetic
10.
Genes Dev ; 26(23): 2580-9, 2012 Dec 01.
Article in English | MEDLINE | ID: mdl-23152447

ABSTRACT

PR-Set7 is the sole monomethyltransferase responsible for H4K20 monomethylation (H4K20me1) that is the substrate for further methylation by Suv4-20h1/h2. PR-Set7 is required for proper cell cycle progression and is subject to degradation by the CRL4(Cdt2) ubiquitin ligase complex as a function of the cell cycle and DNA damage. This report demonstrates that PR-Set7 is an important downstream effector of CRL4(Cdt2) function during origin of DNA replication licensing, dependent on Suv4-20h1/2 activity. Aberrant rereplication correlates with decreased levels of H4K20me1 and increased levels of H4K20 trimethylation (H4K20me3). Expression of a degradation-resistant PR-Set7 mutant in the mouse embryo that is normally devoid of Suv4-20 does not compromise development or cell cycle progression unless Suv4-20h is coexpressed. PR-Set7 targeting to an artificial locus results in recruitment of the origin recognition complex (ORC) in a manner dependent on Suv4-20h and H4K20me3. Consistent with this, H4K20 methylation status plays a direct role in recruiting ORC through the binding properties of ORC1 and ORCA/LRWD1. Thus, coordinating the status of H4K20 methylation is pivotal for the proper selection of DNA replication origins in higher eukaryotes.


Subject(s)
Cell Cycle/physiology , DNA Replication/physiology , Histone-Lysine N-Methyltransferase/metabolism , Animals , Cell Cycle/genetics , Cell Differentiation , Cell Line, Tumor , Cell Proliferation , DNA Damage , DNA Replication/genetics , Embryo, Mammalian , Fibroblasts/cytology , HeLa Cells , Histone-Lysine N-Methyltransferase/genetics , Humans , Mice , Origin Recognition Complex/metabolism , Protein Binding , Ubiquitin-Protein Ligases/metabolism
11.
Epigenetics ; 7(7): 747-57, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22647320

ABSTRACT

Early embryonic development is characterized by dramatic changes in cell potency and chromatin organization. The role of histone variants in the context of chromatin remodeling during embryogenesis remains under investigated. In particular, the nuclear distribution of the histone variant H2A.Z and its modifications have not been examined. Here we investigated the dynamics of acetylation of H2A.Z and two other active chromatin marks, H3K9ac and H3K36me3, throughout murine and bovine pre-implantation development. We show that H2A.Z distribution is dynamic during the earliest stages of mouse development, with protein levels significantly varying across stages and lowest at the 2-cell stage. When present, H2A.Z localizes preferentially to euchromatin at all stages analyzed. H2A.Z is acetylated in pre-implantation blastomeres and is preferentially localized to euchromatin, in line with the known role of H2A.Zac in transcriptional activation. Interestingly, however, H2A.Zac is undetectable in mouse embryos at the 2-cell stage, the time of major embryonic genome activation (EGA). Similarly, H3K36me3 is present exclusively in the maternal chromatin immediately after fertilization but becomes undetectable in interphase nuclei at the 2-cell stage, suggesting uncoupling of these active marks with global embryonic transcription activation. In bovine embryos, which undergo EGA at the 8-cell stage, H2A.Zac can be detected in zygotes, 4-, 8- and 16-cell stage embryos as well as in blastocysts, indicating that the dynamics of H2A.Zac is not conserved in mammals. In contrast, H3K36me3 displays mostly undetectable and heterogeneous localization pattern throughout bovine pre-implantation development. Thus, our results suggest that 'canonical' active chromatin marks exhibit a dynamic behavior in embryonic nuclei, which is both stage- and species-specific. We hypothesize that chromatin of early embryonic nuclei is subject to fine-tuning through differential acquisition of histone marks, allowing for proper chromatin remodeling and developmental progression in a species-specific fashion.


Subject(s)
Embryo, Mammalian/metabolism , Embryonic Development/genetics , Epigenesis, Genetic , Histones/metabolism , Acetylation , Animals , Blastocyst/metabolism , Cattle , Chromatin/metabolism , Gene Expression Regulation, Developmental , Methylation , Mice , Transcriptional Activation
12.
Nat Cell Biol ; 12(9): 853-62, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20676102

ABSTRACT

In mammals, oocyte fertilization by sperm initiates development. This is followed by epigenetic reprogramming of both parental genomes, which involves the de novo establishment of chromatin domains. In the mouse embryo, methylation of histone H3 establishes an epigenetic asymmetry and is predominant in the maternal pronucleus. However, the roles of differential incorporation of histone H3 variants in the parental chromatin, and of modified residues within specific histone variants, have not been addressed. Here we show that the histone variant H3.3, and in particular lysine 27, is required for the establishment of heterochromatin in the mouse embryo. H3.3 localizes to paternal pericentromeric chromatin during S phase at the time of transcription of pericentromeric repeats. Mutation of H3.3 K27, but not of H3.1 K27, results in aberrant accumulation of pericentromeric transcripts, HP1 mislocalization, dysfunctional chromosome segregation and developmental arrest. This phenotype is rescued by injection of double-stranded RNA (dsRNA) derived from pericentromeric transcripts, indicating a functional link between H3.3K27 and the silencing of such regions by means of an RNA-interference (RNAi) pathway. Our work demonstrates a role for a modifiable residue within a histone-variant-specific context during reprogramming and identifies a novel function for mammalian H3.3 in the initial formation of dsRNA-dependent heterochromatin.


Subject(s)
Embryo, Mammalian/metabolism , Genetic Variation , Heterochromatin/metabolism , Histones/genetics , Histones/metabolism , Amino Acid Substitution/genetics , Animals , Blastocyst/cytology , Blastocyst/metabolism , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Segregation/genetics , DNA, Satellite/genetics , Embryo, Mammalian/cytology , Embryonic Development/genetics , Epigenesis, Genetic/genetics , Female , Heterochromatin/genetics , Lysine/genetics , Male , Methylation , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , RNA, Double-Stranded/administration & dosage , RNA, Double-Stranded/genetics , RNA, Messenger/administration & dosage , RNA, Messenger/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Time Factors , Zygote/cytology , Zygote/metabolism
13.
Genes Dev ; 23(18): 2210-23, 2009 Sep 15.
Article in English | MEDLINE | ID: mdl-19759265

ABSTRACT

Development of the germline requires consecutive differentiation events. Regulation of these has been associated with germ cell-specific and pluripotency-associated transcription factors, but the role of general transcription factors (GTFs) remains elusive. TATA-binding protein (TBP) is a GTF involved in transcription by all RNA polymerases. During ovarian folliculogenesis in mice the vertebrate-specific member of the TBP family, TBP2/TRF3, is expressed exclusively in oocytes. To determine TBP2 function in vivo, we generated TBP2-deficient mice. We found that Tbp2(-/-) mice are viable with no apparent phenotype. However, females lacking TBP2 are sterile due to defective folliculogenesis, altered chromatin organization, and transcriptional misregulation of key oocyte-specific genes. TBP2 binds to promoters of misregulated genes, suggesting that TBP2 directly regulates their expression. In contrast, TBP ablation in the female germline results in normal ovulation and fertilization, indicating that in these cells TBP is dispensable. We demonstrate that TBP2 is essential for the differentiation of female germ cells, and show the mutually exclusive functions of these key core promoter-binding factors, TBP and TBP2, in the mouse.


Subject(s)
Cell Differentiation , Chromatin/metabolism , Gene Expression Regulation, Developmental , Oocytes/growth & development , Oocytes/metabolism , TATA-Box Binding Protein/metabolism , Animals , Cell Proliferation , Female , Gene Expression Profiling , Genome/genetics , Mice , Mice, Knockout , Oocytes/cytology , Promoter Regions, Genetic , Protein Binding , Zygote/metabolism
14.
Int J Dev Biol ; 53(7): 1003-11, 2009.
Article in English | MEDLINE | ID: mdl-19598117

ABSTRACT

The cells in the preimplantation mammalian embryo undergo several rounds of fast cell division. Whether the known DNA repair pathways are active during these early stages of development where cell division is of primary importance, has not been fully established. Because of the important role of phosphorylated H2A.X (gammaH2A.X) in the DNA damage response as well as its putative role in assembly of embryonic chromatin, we analysed its distribution in the preimplantation mouse embryo. We found that H2A.X is highly phosphorylated throughout preimplantation development in the absence of any induced DNA damage. Moreover, gammaH2A.X levels vary significantly throughout the cell cycle. Interestingly, after the 4-cell stage, we detected high levels of H2A.X phosphorylation in mitosis, where telomeres appeared focally enriched with gammaH2A.X. In contrast, 53BP1, which is known to be recruited to DNA damage sites, is undetectable at mitotic chromosomes at these stages and its localisation changes upon blastocyst formation from mainly nuclear to cytoplasmic. We also show that 53BP1 and gammaH2A.X rarely colocalise, suggesting that the high levels of phosphorylation of H2A.X in the embryo might not be directly linked to the DNA damage response in the embryo. Our data suggest that phosphorylation of H2A.X is an important event in the fast dividing cells of the early embryo in the absence of any induced DNA damage. We discuss the possible consequences of these findings on the genome-wide chromatin remodelling that ocurs in the preimplantation mammalian embryo.


Subject(s)
DNA Damage , Embryonic Development/physiology , Histones/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Animals , Blastocyst/cytology , Blastocyst/metabolism , Blastomeres/cytology , Blastomeres/metabolism , Chromosomal Proteins, Non-Histone , Cleavage Stage, Ovum/cytology , Cleavage Stage, Ovum/metabolism , DNA-Binding Proteins , Female , Male , Mice , Mitosis , Morula/cytology , Morula/metabolism , Phosphorylation , Pregnancy , Tumor Suppressor p53-Binding Protein 1 , Zygote/metabolism
15.
Nat Struct Mol Biol ; 16(7): 777-81, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19561610

ABSTRACT

Histone modifications are central to the regulation of all DNA-dependent processes. Lys64 of histone H3 (H3K64) lies within the globular domain at a structurally important position. We identify trimethylation of H3K64 (H3K64me3) as a modification that is enriched at pericentric heterochromatin and associated with repeat sequences and transcriptionally inactive genomic regions. We show that this new mark is dynamic during the two main epigenetic reprogramming events in mammals. In primordial germ cells, H3K64me3 is present at the time of specification, but it disappears transiently during reprogramming. In early mouse embryos, it is inherited exclusively maternally; subsequently, the modification is rapidly removed, suggesting an important role for H3K64me3 turnover in development. Taken together, our findings establish H3K64me3 as a previously uncharacterized histone modification that is preferentially localized to repressive chromatin. We hypothesize that H3K64me3 helps to 'secure' nucleosomes, and perhaps the surrounding chromatin, in an appropriately repressed state during development.


Subject(s)
Epigenesis, Genetic , Heterochromatin/metabolism , Histones/metabolism , Lysine/metabolism , Animals , Cell Line , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA Methylation , Embryo, Mammalian/anatomy & histology , Embryo, Mammalian/metabolism , Heterochromatin/chemistry , Heterochromatin/genetics , Histones/genetics , Humans , Methyltransferases/genetics , Methyltransferases/metabolism , Mice , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Nucleosomes/chemistry , Nucleosomes/metabolism , Protein Conformation , Repressor Proteins/genetics , Repressor Proteins/metabolism , Xenopus laevis
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