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1.  Histone H3.3 Variant Dynamics in the Germline of Caenorhabditis elegans 
PLoS Genetics  2006;2(6):e97.
Germline chromatin undergoes dramatic remodeling events involving histone variants during the life cycle of an organism. A universal histone variant, H3.3, is incorporated at sites of active transcription throughout the cell cycle. The presence of H3.3 in chromatin indicates histone turnover, which is the energy-dependent removal of preexisting histones and replacement with new histones. H3.3 is also incorporated during decondensation of the Drosophila sperm pronucleus, indicating a direct role in chromatin remodeling upon fertilization. Here we present a system to monitor histone turnover and chromatin remodeling during Caenorhabditis elegans development by following the developmental dynamics of H3.3. We generated worm strains expressing green fluorescent protein– or yellow fluorescent protein–fused histone H3.3 proteins, HIS-71 and HIS-72. We found that H3.3 is retained in mature sperm chromatin, raising the possibility that it transmits epigenetic information via the male germline. Upon fertilization, maternal H3.3 enters both male and female pronuclei and is incorporated into paternal chromatin, apparently before the onset of embryonic transcription, suggesting that H3.3 can be incorporated independent of transcription. In early embryos, H3.3 becomes specifically depleted from primordial germ cells. Strikingly, the X chromosome becomes deficient in H3.3 during gametogenesis, indicating a low level of histone turnover. These results raise the possibility that the asymmetry in histone turnover between the X chromosome and autosomes is established during gametogenesis. H3.3 patterns are similar to patterns of H3K4 methylation in the primordial germ cells and on the X chromosome during gametogenesis, suggesting that histone turnover and modification are coupled processes. Our demonstration of dynamic H3.3 incorporation in nondividing cells provides a mechanistic basis for chromatin changes during germ cell development.
Germ cells carry genetic information from one generation to the next. They are converted to gametes during meiosis, which are then reprogrammed for development in the fertilized egg. Gamete production and developmental reprogramming involve dramatic changes in DNA packaging, but little is understood about how these changes are involved in resetting the developmental program for the whole organism. In spermatogenesis, DNA is stripped and repackaged into highly condensed chromatin. After fertilization, sperm DNA is again repackaged as it dramatically decondenses to fuse with the egg nucleus. These repackaging processes involve the four core histone proteins, which tightly wrap DNA into nucleosome particles. A universal variant form of histone 3, H3.3, is abundant in the germ cells of all plants and animals studied and has been shown to turn over at sites of active transcription in various somatic cells. The authors show that H3.3 displays dynamic turnover throughout germ cell development of the roundworm Caenorhabditis elegans. H3.3 incorporates during the first germline stem cell division, continues through meiosis, and ends up in sperm and eggs. Strikingly, H3.3 becomes depleted from primordial germ cells, and the meiotically silenced X chromosome is deficient in H3.3, which suggests that H3.3 dynamics during meiosis and reprogramming transmit epigenetic information.
PMCID: PMC1484599  PMID: 16846252
2.  The Specification and Global Reprogramming of Histone Epigenetic Marks during Gamete Formation and Early Embryo Development in C. elegans 
PLoS Genetics  2014;10(10):e1004588.
In addition to the DNA contributed by sperm and oocytes, embryos receive parent-specific epigenetic information that can include histone variants, histone post-translational modifications (PTMs), and DNA methylation. However, a global view of how such marks are erased or retained during gamete formation and reprogrammed after fertilization is lacking. To focus on features conveyed by histones, we conducted a large-scale proteomic identification of histone variants and PTMs in sperm and mixed-stage embryo chromatin from C. elegans, a species that lacks conserved DNA methylation pathways. The fate of these histone marks was then tracked using immunostaining. Proteomic analysis found that sperm harbor ∼2.4 fold lower levels of histone PTMs than embryos and revealed differences in classes of PTMs between sperm and embryos. Sperm chromatin repackaging involves the incorporation of the sperm-specific histone H2A variant HTAS-1, a widespread erasure of histone acetylation, and the retention of histone methylation at sites that mark the transcriptional history of chromatin domains during spermatogenesis. After fertilization, we show HTAS-1 and 6 histone PTM marks distinguish sperm and oocyte chromatin in the new embryo and characterize distinct paternal and maternal histone remodeling events during the oocyte-to-embryo transition. These include the exchange of histone H2A that is marked by ubiquitination, retention of HTAS-1, removal of the H2A variant HTZ-1, and differential reprogramming of histone PTMs. This work identifies novel and conserved features of paternal chromatin that are specified during spermatogenesis and processed in the embryo. Furthermore, our results show that different species, even those with diverged DNA packaging and imprinting strategies, use conserved histone modification and removal mechanisms to reprogram epigenetic information.
Author Summary
Successful reproduction depends upon the receipt and processing of distinct chromatin packages from sperm and oocytes. This includes not only a unique complement of DNA, but information in the form of proteins, such as histones, that differentially package the DNA in each cell type. For example, histone variants and post-translationally modified histones can carry epigenetic information across generations. Such information is then reprogrammed in the new embryo to ensure proper development. However, it is unclear how many of these marks are established during gamete formation and reprogrammed after fertilization. We define a signature histone variant and post-translational modification profile of sperm chromatin from C. elegans. This profile is established during sperm formation in part by exchanging canonical histones with sperm-specific histone proteins. Histone variants and modifications passed from sperm and oocytes are differentially removed or retained, suggesting that the embryo can reprogram epigenetic information from each parent distinctly. These C. elegans studies reveal that both conserved and novel histone modification and exchange mechanisms are used across diverse species to establish and reprogram epigenetic information.
PMCID: PMC4191889  PMID: 25299455
3.  Dynamic Replacement of Histone H3 Variants Reprograms Epigenetic Marks in Early Mouse Embryos 
PLoS Genetics  2011;7(10):e1002279.
Upon fertilization, reprogramming of gene expression is required for embryo development. This step is marked by DNA demethylation and changes in histone variant composition. However, little is known about the molecular mechanisms causing these changes and their impact on histone modifications. We examined the global deposition of the DNA replication-dependent histone H3.1 and H3.2 variants and the DNA replication-independent H3.3 variant after fertilization in mice. We showed that H3.3, a euchromatic marker of gene activity, transiently disappears from the maternal genome, suggesting erasure of the oocyte-specific modifications carried by H3.3. After fertilization, H3.2 is incorporated into the transcriptionally silent heterochromatin, whereas H3.1 and H3.3 occupy unusual heterochromatic and euchromatin locations, respectively. After the two-cell stage, H3.1 and H3.3 variants resume their usual respective locations on heterochromatin and euchromatin. Preventing the incorporation of H3.1 and H3.2 by knockdown of the histone chaperone CAF-1 induces a reciprocal increase in H3.3 deposition and impairs heterochromatin formation. We propose that the deposition of different H3 variants influences the functional organization of chromatin. Taken together, these findings suggest that dynamic changes in the deposition of H3 variants are critical for chromatin reorganization during epigenetic reprogramming.
Author Summary
Histones play essential roles in the regulation of chromatin structure and gene expression. The functions of histone H3 non-centromeric variants (H3.1, H3.2, and H3.3) have not yet been fully understood. Little is known about the mammalian-specific H3.1 in terms of deposition pattern and role. We show here that the nuclear deposition of these three variants is dynamically changed after fertilization and in the process of embryo preimplantation development, during which genome reprogramming occurs. H3.3, an active gene marker, is removed from the female pronucleus soon after fertilization, suggesting that the epigenetic marks carried by H3.3 in differentiated oocytes are erased. This process might participate in generating totipotency in early embryos. During the late preimplantation stage, H3.1 appears to prevent H3.3 deposition in inactive chromatin domains. The interplay among deposition of H3 variants likely participates in the functional organization of chromatin. Together, our results suggest that dynamic replacement of histone variants plays important roles in genome remodeling.
PMCID: PMC3188537  PMID: 21998593
4.  Paternal heterochromatin formation in human embryos is H3K9/HP1 directed and primed by sperm-derived histone modifications 
Nature Communications  2014;5:5868.
The different configurations of maternal and paternal chromatin, acquired during oogenesis and spermatogenesis, have to be rearranged after fertilization to form a functional embryonic genome. In the paternal genome, nucleosomal chromatin domains are re-established after the protamine-to-histone exchange. We investigated the formation of constitutive heterochromatin (cHC) in human preimplantation embryos. Our results show that histones carrying canonical cHC modifications are retained in cHC regions of sperm chromatin. These modified histones are transmitted to the oocyte and contribute to the formation of paternal embryonic cHC. Subsequently, the modifications are recognized by the H3K9/HP1 pathway maternal chromatin modifiers and propagated over the embryonic cleavage divisions. These results are in contrast to what has been described for mouse embryos, in which paternal cHC lacks canonical modifications and is initially established by Polycomb group proteins. Our results show intergenerational epigenetic inheritance of the cHC structure in human embryos.
Following fertilization, the oocyte and sperm lose their distinct chromatin signature to form a functional embryonic genome. Here the authors find that, in human embryos, the paternal constitutive heterochromatin is inherited in the canonical configuration from the sperm and is propagated by the H3K9/HP1 pathway.
PMCID: PMC4284653  PMID: 25519718
5.  CAF-1 Is Essential for Heterochromatin Organization in Pluripotent Embryonic Cells 
PLoS Genetics  2006;2(11):e181.
During mammalian development, chromatin dynamics and epigenetic marking are important for genome reprogramming. Recent data suggest an important role for the chromatin assembly machinery in this process. To analyze the role of chromatin assembly factor 1 (CAF-1) during pre-implantation development, we generated a mouse line carrying a targeted mutation in the gene encoding its large subunit, p150CAF-1. Loss of p150CAF-1 in homozygous mutants leads to developmental arrest at the 16-cell stage. Absence of p150CAF-1 in these embryos results in severe alterations in the nuclear organization of constitutive heterochromatin. We provide evidence that in wild-type embryos, heterochromatin domains are extensively reorganized between the two-cell and blastocyst stages. In p150CAF-1 mutant 16-cell stage embryos, the altered organization of heterochromatin displays similarities to the structure of heterochromatin in two- to four-cell stage wild-type embryos, suggesting that CAF-1 is required for the maturation of heterochromatin during preimplantation development. In embryonic stem cells, depletion of p150CAF-1 using RNA interference results in the mislocalization, loss of clustering, and decondensation of pericentric heterochromatin domains. Furthermore, loss of CAF-1 in these cells results in the alteration of epigenetic histone methylation marks at the level of pericentric heterochromatin. These alterations of heterochromatin are not found in p150CAF-1-depleted mouse embryonic fibroblasts, which are cells that are already lineage committed, suggesting that CAF-1 is specifically required for heterochromatin organization in pluripotent embryonic cells. Our findings underline the role of the chromatin assembly machinery in controlling the spatial organization and epigenetic marking of the genome in early embryos and embryonic stem cells.
Chromatin is the support of our genetic information. It is composed of numerous repeated units called nucleosomes, in which DNA wraps around a core of histone proteins. Modifications in the composition and biochemical properties of nucleosomes play major roles in the regulation of genome function. Such modifications are termed “epigenetic” when they are inherited across cell divisions and confer new information to chromatin, in addition to the genetic information provided by DNA. It is usually believed that during genome replication, the basic chromatin assembly machinery builds up “naïve” nucleosomes, and, in a subsequent step, nucleosomes are selectively modified by a series of enzymes to acquire epigenetic information. Here, the authors studied the role of a basic chromatin assembly factor (CAF-1) in mouse embryonic stem cells and early embryos. Surprisingly, they show that CAF-1 confers epigenetic information to specific genomic regions. In addition, this study revealed that CAF-1 is required for the proper spatial organization of chromosomes in the nucleus. This new knowledge may contribute to better understanding the role of chromatin in the maintenance of embryonic stem cell identity and plasticity.
PMCID: PMC1630711  PMID: 17083276
6.  Dynamic alterations in the paternal epigenetic landscape following fertilization 
Frontiers in Genetics  2012;3:143.
Embryonic development is a complex and dynamic process with frequent changes in gene expression, ultimately leading to cellular differentiation and commitment of various cell lines. These changes are likely preceded by changes to signaling cascades and/or alterations to the epigenetic program in specific cells. The process of epigenetic remodeling begins early in development. In fact, soon after the union of sperm and egg massive epigenetic changes occur across the paternal and maternal epigenetic landscape. The epigenome of these cells includes modifications to the DNA itself, in the form of DNA methylation, as well as nuclear protein content and modification, such as modifications to histones. Sperm chromatin is predominantly packaged by protamines, but following fertilization the sperm pronucleus undergoes remodeling in which maternally derived histones replace protamines, resulting in the relaxation of chromatin and ultimately decondensation of the paternal pronucleus. In addition, active DNA demethylation occurs across the paternal genome prior to the first cell division, effectively erasing many spermatogenesis derived methylation marks. This complex interplay begins the dynamic process by which two haploid cells unite to form a diploid organism. The biology of these events is central to the understanding of sexual reproduction, yet our knowledge regarding the mechanisms involved is extremely limited. This review will explore what is known regarding the post-fertilization epigenetic alterations of the paternal chromatin and the implications suggested by the available literature.
PMCID: PMC3442791  PMID: 23024648
fertilization; epigenetics; chromatin; embryogenesis; DNA methylation
7.  GSE Is a Maternal Factor Involved in Active DNA Demethylation in Zygotes 
PLoS ONE  2013;8(4):e60205.
After fertilization, the sperm and oocyte genomes undergo extensive epigenetic reprogramming to form a totipotent zygote. The dynamic epigenetic changes during early embryo development primarily involve DNA methylation and demethylation. We have previously identified Gse (gonad-specific expression gene) to be expressed specifically in germ cells and early embryos. Its encoded protein GSE is predominantly localized in the nuclei of cells from the zygote to blastocyst stages, suggesting possible roles in the epigenetic changes occurring during early embryo development. Here, we report the involvement of GSE in epigenetic reprogramming of the paternal genome during mouse zygote development. Preferential binding of GSE to the paternal chromatin was observed from pronuclear stage 2 (PN2) onward. A knockdown of GSE by antisense RNA in oocytes produced no apparent effect on the first and second cell cycles in preimplantation embryos, but caused a significant reduction in the loss of 5-methylcytosine (5mC) and the accumulation of 5-hydroxymethylcytosine (5hmC) in the paternal pronucleus. Furthermore, DNA methylation levels in CpG sites of LINE1 transposable elements, Lemd1, Nanog and the upstream regulatory region of the Oct4 (also known as Pou5f1) gene were clearly increased in GSE-knockdown zygotes at mid-pronuclear stages (PN3-4), but the imprinted H19-differential methylated region was not affected. Importantly, DNA immunoprecipitation of 5mC and 5hmC also indicates that knockdown of GSE in zygotes resulted in a significant reduction of the conversion of 5mC to 5hmC on LINE1. Therefore, our results suggest an important role of maternal GSE for mediating active DNA demethylation in the zygote.
PMCID: PMC3613368  PMID: 23560077
8.  Epigenetic Patterns Maintained in Early Caenorhabditis elegans Embryos Can Be Established by Gene Activity in the Parental Germ Cells 
PLoS Genetics  2011;7(6):e1001391.
Epigenetic information, such as parental imprints, can be transmitted with genetic information from parent to offspring through the germ line. Recent reports show that histone modifications can be transmitted through sperm as a component of this information transfer. How the information that is transferred is established in the parent and maintained in the offspring is poorly understood. We previously described a form of imprinted X inactivation in Caenorhabditis elegans where dimethylation on histone 3 at lysine 4 (H3K4me2), a mark of active chromatin, is excluded from the paternal X chromosome (Xp) during spermatogenesis and persists through early cell divisions in the embryo. Based on the observation that the Xp (unlike the maternal X or any autosome) is largely transcriptionally inactive in the paternal germ line, we hypothesized that transcriptional activity in the parent germ line may influence epigenetic information inherited by and maintained in the embryo. We report that chromatin modifications and histone variant patterns assembled in the germ line can be retained in mature gametes. Furthermore, despite extensive chromatin remodeling events at fertilization, the modification patterns arriving with the gametes are largely retained in the early embryo. Using transgenes, we observe that expression in the parental germline correlates with differential chromatin assembly that is replicated and maintained in the early embryo. Expression in the adult germ cells also correlates with more robust expression in the somatic lineages of the offspring. These results suggest that differential expression in the parental germ lines may provide a potential mechanism for the establishment of parent-of-origin epigenomic content. This content can be maintained and may heritably affect gene expression in the offspring.
Author Summary
Epigenetic information such as parental imprints can be transmitted along with genetic information through the germ line from parent to offspring. Recent reports show that histone modifications marking developmentally regulated loci can be transmitted through sperm as a component of this information transfer. How the information that is transferred is established in the parent and maintained in the offspring is poorly understood. Here we show that expression in the parental germ line can influence the establishment of information that is then replicated and maintained in the early embryo, suggesting a potential mechanism for the establishment of parent-of-origin epigenomic content.
PMCID: PMC3111476  PMID: 21695223
9.  Should I stay or should I go 
Epigenetics  2012;7(9):969-975.
Recent findings shed light on the coordination of two fundamental, yet mechanistically opposing, processes in the early mammalian embryo. During the oocyte-to-embryo transition and early preimplantation development nuclear reprogramming occurs. This resetting of the epigenome in maternal and paternal pronuclei to a ground state is the essential step ensuring totipotency in the zygote, the first embryonic stage. Radical, global DNA demethylation, which occurs actively in the paternal and passively in the maternal genome, is a prominent feature of nuclear reprogramming; yet, this process poses a danger to a subset of methylated sequences that must be preserved for their germline to soma inheritance. Genomic imprinting and its importance were demonstrated three decades ago by a series of experiments generating non-viable mammalian uniparental embryos. Indeed, imprinted loci, gene clusters with parent-of-origin specific gene expression patterns, must retain their differential methylation status acquired during gametogenesis throughout embryogenesis and in adult tissues. It is just recently that the molecular players that protect/maintain imprinting marks during reprogramming in preimplantation embryos have been identified, in particular, an epigenetic modifier complex formed by ZFP57 and TRIM28/KAP1. The interaction of these and other molecules with the newly formed embryonic chromatin and imprinted genes is discussed and highlighted herein.
PMCID: PMC3515016  PMID: 22869105
Kap1; Trim28; Zfp57; 5-methyl-cytosine; embryogenesis; epigenetics; imprinting; reprogramming
10.  Mouse Zygotes Respond to Severe Sperm DNA Damage by Delaying Paternal DNA Replication and Embryonic Development 
PLoS ONE  2013;8(2):e56385.
Mouse zygotes do not activate apoptosis in response to DNA damage. We previously reported a unique form of inducible sperm DNA damage termed sperm chromatin fragmentation (SCF). SCF mirrors some aspects of somatic cell apoptosis in that the DNA degradation is mediated by reversible double strand breaks caused by topoisomerase 2B (TOP2B) followed by irreversible DNA degradation by a nuclease(s). Here, we created zygotes using spermatozoa induced to undergo SCF (SCF zygotes) and tested how they responded to moderate and severe paternal DNA damage during the first cell cycle. We found that the TUNEL assay was not sensitive enough to identify the breaks caused by SCF in zygotes in either case. However, paternal pronuclei in both groups stained positively for γH2AX, a marker for DNA damage, at 5 hrs after fertilization, just before DNA synthesis, while the maternal pronuclei were negative. We also found that both pronuclei in SCF zygotes with moderate DNA damage replicated normally, but paternal pronuclei in the SCF zygotes with severe DNA damage delayed the initiation of DNA replication by up to 12 hrs even though the maternal pronuclei had no discernable delay. Chromosomal analysis of both groups confirmed that the paternal DNA was degraded after S-phase while the maternal pronuclei formed normal chromosomes. The DNA replication delay caused a marked retardation in progression to the 2-cell stage, and a large portion of the embryos arrested at the G2/M border, suggesting that this is an important checkpoint in zygotic development. Those embryos that progressed through the G2/M border died at later stages and none developed to the blastocyst stage. Our data demonstrate that the zygote responds to sperm DNA damage through a non-apoptotic mechanism that acts by slowing paternal DNA replication and ultimately leads to arrest in embryonic development.
PMCID: PMC3576397  PMID: 23431372
11.  Drosophila Yemanuclein and HIRA Cooperate for De Novo Assembly of H3.3-Containing Nucleosomes in the Male Pronucleus 
PLoS Genetics  2013;9(2):e1003285.
The differentiation of post-meiotic spermatids in animals is characterized by a unique reorganization of their nuclear architecture and chromatin composition. In many species, the formation of sperm nuclei involves the massive replacement of nucleosomes with protamines, followed by a phase of extreme nuclear compaction. At fertilization, the reconstitution of a nucleosome-based paternal chromatin after the removal of protamines requires the deposition of maternally provided histones before the first round of DNA replication. This process exclusively uses the histone H3 variant H3.3 and constitutes a unique case of genome-wide replication-independent (RI) de novo chromatin assembly. We had previously shown that the histone H3.3 chaperone HIRA plays a central role for paternal chromatin assembly in Drosophila. Although several conserved HIRA-interacting proteins have been identified from yeast to human, their conservation in Drosophila, as well as their actual implication in this highly peculiar RI nucleosome assembly process, is an open question. Here, we show that Yemanuclein (YEM), the Drosophila member of the Hpc2/Ubinuclein family, is essential for histone deposition in the male pronucleus. yem loss of function alleles affect male pronucleus formation in a way remarkably similar to Hira mutants and abolish RI paternal chromatin assembly. In addition, we demonstrate that HIRA and YEM proteins interact and are mutually dependent for their targeting to the decondensing male pronucleus. Finally, we show that the alternative ATRX/XNP-dependent H3.3 deposition pathway is not involved in paternal chromatin assembly, thus underlining the specific implication of the HIRA/YEM complex for this essential step of zygote formation.
Author Summary
Chromosome organization relies on a basic functional unit called the nucleosome, in which DNA is wrapped around a core of histone proteins. However, during male gamete formation, the majority of histones are replaced by sperm-specific proteins that are adapted to sexual reproduction but incompatible with the formation of the first zygotic nucleus. These proteins must therefore be replaced by histones upon fertilization, in a replication-independent chromatin assembly process that requires the histone deposition factor HIRA. In this study, we identified the protein Yemanuclein (YEM) as a new partner of HIRA at fertilization. We show that, in eggs laid by yem mutant females, the male pronucleus fails to assemble its nucleosomes, resulting in the loss of paternal chromosomes at the first zygotic division. In addition, we found that YEM and HIRA are mutually dependent to perform chromatin assembly at fertilization, demonstrating that they tightly cooperate in vivo. Finally, we demonstrate that the replication-independent chromatin assembly factor ATRX/XNP is not involved in the assembly of paternal nucleosomes. In conclusion, our results shed new light into critical mechanisms controlling paternal chromosome formation at fertilization.
PMCID: PMC3567178  PMID: 23408912
12.  Maternal Depletion of Piwi, a Component of the RNAi System, Impacts Heterochromatin Formation in Drosophila 
PLoS Genetics  2013;9(9):e1003780.
A persistent question in epigenetics is how heterochromatin is targeted for assembly at specific domains, and how that chromatin state is faithfully transmitted. Stable heterochromatin is necessary to silence transposable elements (TEs) and maintain genome integrity. Both the RNAi system and heterochromatin components HP1 (Swi6) and H3K9me2/3 are required for initial establishment of heterochromatin structures in S. pombe. Here we utilize both loss of function alleles and the newly developed Drosophila melanogaster transgenic shRNA lines to deplete proteins of interest at specific development stages to dissect their roles in heterochromatin assembly in early zygotes and in maintenance of the silencing chromatin state during development. Using reporters subject to Position Effect Variegation (PEV), we find that depletion of key proteins in the early embryo can lead to loss of silencing assayed at adult stages. The piRNA component Piwi is required in the early embryo for reporter silencing in non-gonadal somatic cells, but knock-down during larval stages has no impact. This implies that Piwi is involved in targeting HP1a when heterochromatin is established at the late blastoderm stage and possibly also during embryogenesis, but that the silent chromatin state created is transmitted through cell division independent of the piRNA system. In contrast, heterochromatin structural protein HP1a is required for both initial heterochromatin assembly and the following mitotic inheritance. HP1a profiles in piwi mutant animals confirm that Piwi depletion leads to decreased HP1a levels in pericentric heterochromatin, particularly in TEs. The results suggest that the major role of the piRNA system in assembly of heterochromatin in non-gonadal somatic cells occurs in the early embryo during heterochromatin formation, and further demonstrate that failure of heterochromatin formation in the early embryo impacts the phenotype of the adult.
Author Summary
Most eukaryotes harbor a high proportion of transposable elements (TEs) in their genomes. Heterochromatin, a condensed chromatin state found at domains enriched for TEs and other repetitious elements, is important for silencing TEs and maintaining the integrity of the genome. The RNAi system has been shown to be important for the establishment and maintenance of heterochromatin in both fungi and plants. To investigate whether this mechanism is also utilized in animals, we selectively depleted the proteins of interest in different developmental stages of Drosophila melanogaster, the fruit fly. Observing reporters subject to Position Effect Variegation (silencing due to heterochromatin formation), and using chromatin immunoprecipitation to establish heterochromatin protein levels at these sites, we find that the piRNA component Piwi is important for heterochromatin establishment during embryogenesis, but not for maintenance of the chromatin state in somatic cells. In contrast, the heterochromatin structural protein HP1a is essential for both establishment and maintenance. This suggests that RNAi components (specifically Piwi) play a guiding role in heterochromatin structure formation during embryogenesis, but have a minor role in maintenance. The findings emphasize the importance of maternal materials for the development of the Drosophila offspring, and demonstrate that failure of heterochromatin formation in the embryo impacts the adult phenotype.
PMCID: PMC3777992  PMID: 24068954
13.  The Essential Role of Drosophila HIRA for De Novo Assembly of Paternal Chromatin at Fertilization 
PLoS Genetics  2007;3(10):e182.
In many animal species, the sperm DNA is packaged with male germ line–specific chromosomal proteins, including protamines. At fertilization, these non-histone proteins are removed from the decondensing sperm nucleus and replaced with maternally provided histones to form the DNA replication competent male pronucleus. By studying a point mutant allele of the Drosophila Hira gene, we previously showed that HIRA, a conserved replication-independent chromatin assembly factor, was essential for the assembly of paternal chromatin at fertilization. HIRA permits the specific assembly of nucleosomes containing the histone H3.3 variant on the decondensing male pronucleus. We report here the analysis of a new mutant allele of Drosophila Hira that was generated by homologous recombination. Surprisingly, phenotypic analysis of this loss of function allele revealed that the only essential function of HIRA is the assembly of paternal chromatin during male pronucleus formation. This HIRA-dependent assembly of H3.3 nucleosomes on paternal DNA does not require the histone chaperone ASF1. Moreover, analysis of this mutant established that protamines are correctly removed at fertilization in the absence of HIRA, thus demonstrating that protamine removal and histone deposition are two functionally distinct processes. Finally, we showed that H3.3 deposition is apparently not affected in Hira mutant embryos and adults, suggesting that different chromatin assembly machineries could deposit this histone variant.
Author Summary
Chromatin is composed of basic units called nucleosomes, in which DNA wraps around a core of histone proteins. HIRA is a histone chaperone that is specifically involved in the assembly of nucleosomes containing H3.3, a universally conserved type of histone 3. To understand the function of HIRA in vivo, the authors generated mutant fruit flies with a non-functional Hira gene. Surprisingly, mutant flies were viable, but females were completely sterile. By analysing the female fruit flies' eggs, the authors found that in the absence of HIRA protein, the sperm nucleus was unable to participate in the formation of the zygote. In Drosophila, as in many animals, the condensed sperm chromatin contains protamines instead of histones. The authors found that the only crucial role of HIRA in flies was to assemble nucleosomes containing H3.3 in the male pronucleus, after the removal of protamines. This fundamental process, which is presumably also controlled by HIRA in vertebrates, allows the paternal DNA to reconstitute its chromatin and participate in the development of the embryo.
PMCID: PMC2041997  PMID: 17967064
14.  Sperm Chromatin-Induced Ectopic Polar Body Extrusion in Mouse Eggs after ICSI and Delayed Egg Activation 
PLoS ONE  2009;4(9):e7171.
Meiotic chromosomes in an oocyte are not only a maternal genome carrier but also provide a positional signal to induce cortical polarization and define asymmetric meiotic division of the oocyte, resulting in polar body extrusion and haploidization of the maternal genome. The meiotic chromosomes play dual function in determination of meiosis: 1) organizing a bipolar spindle formation and 2) inducing cortical polarization and assembly of a distinct cortical cytoskeleton structure in the overlying cortex for polar body extrusion. At fertilization, a sperm brings exogenous paternal chromatin into the egg, which induces ectopic cortical polarization at the sperm entry site and leads to a cone formation, known as fertilization cone. Here we show that the sperm chromatin-induced fertilization cone formation is an abortive polar body extrusion due to lack of spindle induction by the sperm chromatin during fertilization. If experimentally manipulating the fertilization process to allow sperm chromatin to induce both cortical polarization and spindle formation, the fertilization cone can be converted into polar body extrusion. This suggests that sperm chromatin is also able to induce polar body extrusion, like its maternal counterpart. The usually observed cone formation instead of ectopic polar body extrusion induced by sperm chromatin during fertilization is due to special sperm chromatin compaction which restrains it from rapid spindle induction and therefore provides a protective mechanism to prevent a possible paternal genome loss during ectopic polar body extrusion.
PMCID: PMC2746308  PMID: 19787051
15.  Transgenerational Propagation and Quantitative Maintenance of Paternal Centromeres Depends on Cid/Cenp-A Presence in Drosophila Sperm 
PLoS Biology  2012;10(12):e1001434.
Analysis of centromeres in progeny of Drosophila sperm with experimentally altered centromere-specific histone CenH3 levels reveals quantitative inheritance of this epigenetic mark.
In Drosophila melanogaster, as in many animal and plant species, centromere identity is specified epigenetically. In proliferating cells, a centromere-specific histone H3 variant (CenH3), named Cid in Drosophila and Cenp-A in humans, is a crucial component of the epigenetic centromere mark. Hence, maintenance of the amount and chromosomal location of CenH3 during mitotic proliferation is important. Interestingly, CenH3 may have different roles during meiosis and the onset of embryogenesis. In gametes of Caenorhabditis elegans, and possibly in plants, centromere marking is independent of CenH3. Moreover, male gamete differentiation in animals often includes global nucleosome for protamine exchange that potentially could remove CenH3 nucleosomes. Here we demonstrate that the control of Cid loading during male meiosis is distinct from the regulation observed during the mitotic cycles of early embryogenesis. But Cid is present in mature sperm. After strong Cid depletion in sperm, paternal centromeres fail to integrate into the gonomeric spindle of the first mitosis, resulting in gynogenetic haploid embryos. Furthermore, after moderate depletion, paternal centromeres are unable to re-acquire normal Cid levels in the next generation. We conclude that Cid in sperm is an essential component of the epigenetic centromere mark on paternal chromosomes and it exerts quantitative control over centromeric Cid levels throughout development. Hence, the amount of Cid that is loaded during each cell cycle appears to be determined primarily by the preexisting centromeric Cid, with little flexibility for compensation of accidental losses.
Author Summary
Genetic information in eukaryotic cells is parceled into chromosomes. These information strings are precisely transmitted to daughter cells during mitotic and meiotic cell divisions, but only if the centromere, a specialized chromosomal region, is functional. The centromere region within chromosomes of many species—including humans and the fly Drosophila melanogaster—is thought to be specified epigenetically by incorporation of a centromere-specific histone H3 variant (CenH3). After chromosome replication, the centromeres in the resulting two sister chromatids might be expected to be composed of a mixture of pre-existing CenH3 evenly distributed onto the two copies during replication and new CenH3 recruited by the partitioned pool in a stoichiometric manner. Here, we have addressed whether centromeres are indeed replicated in this manner by experimentally altering the levels of centromeric CenH3 in Drosophila sperm. We show that centromeres on paternal chromosomes cannot recruit new CenH3 in embryos fertilized with sperm lacking CenH3. By using sperm with increased or reduced amounts of centromeric CenH3, we demonstrate that altered CenH3 levels are at least partially propagated on paternal centromeres throughout development of the offspring. We conclude that pre-existing CenH3 in Drosophila sperm is therefore not only required for transgenerational centromere maintenance, but that it also exerts quantitative control of this process.
PMCID: PMC3531477  PMID: 23300376
16.  Rapid Elimination of the Histone Variant MacroH2A from Somatic Cell Heterochromatin after Nuclear Transfer 
Cellular Reprogramming  2010;12(1):43-53.
Oocytes contain a maternal store of the histone variant MacroH2A, which is eliminated from zygotes shortly after fertilization. Preimplantation embryos then execute three cell divisions without MacroH2A before the onset of embryonic MacroH2A expression at the 16-cell stage. During subsequent development, MacroH2A is expressed in most cells, where it is assembled into facultative heterochromatin. Because differentiated cells contain heterochromatin rich in MacroH2A, we investigated the fate of MacroH2A during somatic cell nuclear transfer (SCNT). The results show that MacroH2A is rapidly eliminated from the chromosomes of transplanted somatic cell nuclei by a process in which MacroH2A is first stripped from chromosomes, and then degraded. Furthermore, MacroH2A is eliminated from transplanted nuclei by a mechanism requiring intact microtubules and nuclear envelope break down. Preimplantation SCNT embryos express endogenous MacroH2A once they reach the morula stage, similar to the timing observed in embryos produced by natural fertilization. We also show that the ability to reprogram somatic cell heterochromatin by SCNT is tied to the developmental stage of recipient cell cytoplasm because enucleated zygotes fail to support depletion of MacroH2A from transplanted somatic nuclei. Together, the results indicate that nuclear reprogramming by SCNT utilizes the same chromatin remodeling mechanisms that act upon the genome immediately after fertilization.
PMCID: PMC2998967  PMID: 20132012
17.  Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term 
Genome reprogramming in early mouse embryos is associated with nuclear reorganization and particular features such as the peculiar distribution of centromeric and pericentric heterochromatin during the first developmental stage. This zygote-specific heterochromatin organization could be observed both in maternal and paternal pronuclei after natural fertilization as well as in embryonic stem (ES) cell nuclei after nuclear transfer suggesting that this particular type of nuclear organization was essential for embryonic reprogramming and subsequent development.
Here, we show that remodeling into a zygotic-like organization also occurs after somatic cell nuclear transfer (SCNT), supporting the hypothesis that reorganization of constitutive heterochromatin occurs regardless of the source and differentiation state of the starting material. However, abnormal nuclear remodeling was frequently observed after SCNT, in association with low developmental efficiency. When transient treatment with the histone deacetylase inhibitor trichostatin A (TSA) was tested, we observed improved nuclear remodeling in 1-cell SCNT embryos that correlated with improved rates of embryonic development at subsequent stages.
Together, the results suggest that proper organization of constitutive heterochromatin in early embryos is involved in the initial developmental steps and might have long term consequences, especially in cloning procedures.
PMCID: PMC2656487  PMID: 19210795
18.  Chronological Reorganization of Microtubules, Actin Microfilaments, and Chromatin during the First Cell Cycle in Swamp Buffalo (Bubalus bubalis) Embryos 
This paper aimed to study the dynamics of early embryonic development, in terms of redistribution of cytoskeleton (microtubules, actin microfilaments) and chromatin configurations during the first cell cycle in swamp buffalo embryos. Oocytes were matured and fertilized in vitro, and they were fixed at various time points after IVF. At 6 h after IVF, 44.4% matured oocytes were penetrated by spermatozoa. Partial ZP digestion, however, did not improve fertilization rate compared to control (P > .05). At 12 h after IVF, the fertilized oocytes progressed to the second meiotic division and formed the female pronucleus simultaneously with the paternal chromatin continued to decondense. A sperm aster was observed radiating from the base of the decondensing sperm head. At 18 h after IVF, most presumptive zygotes had reached the pronuclear stage. The sperm aster was concurrently enlarged to assist the migration and apposition of pronuclei. Cell cleavage was facilitated by microfilaments and firstly observed by 30 h after IVF. In conclusion, the cytoskeleton actively involves with the process of fertilization and cleavage in swamp buffalo oocytes. The centrosomal material is paternally inherited. Fertilization failure is predominantly caused by poor sperm penetration. However, partial digestion of ZP did not improve fertilization rate.
PMCID: PMC3014712  PMID: 21234419
19.  Histone Methyltransferases MES-4 and MET-1 Promote Meiotic Checkpoint Activation in Caenorhabditis elegans 
PLoS Genetics  2012;8(11):e1003089.
Chromosomes that fail to synapse during meiosis become enriched for chromatin marks associated with heterochromatin assembly. This response, called meiotic silencing of unsynapsed or unpaired chromatin (MSUC), is conserved from fungi to mammals. In Caenorhabditis elegans, unsynapsed chromosomes also activate a meiotic checkpoint that monitors synapsis. The synapsis checkpoint signal is dependent on cis-acting loci called Pairing Centers (PCs). How PCs signal to activate the synapsis checkpoint is currently unknown. We show that a chromosomal duplication with PC activity is sufficient to activate the synapsis checkpoint and that it undergoes heterochromatin assembly less readily than a duplication of a non-PC region, suggesting that the chromatin state of these loci is important for checkpoint function. Consistent with this hypothesis, MES-4 and MET-1, chromatin-modifying enzymes associated with transcriptional activity, are required for the synapsis checkpoint. In addition, a duplication with PC activity undergoes heterochromatin assembly when mes-4 activity is reduced. MES-4 function is required specifically for the X chromosome, while MES-4 and MET-1 act redundantly to monitor autosomal synapsis. We propose that MES-4 and MET-1 antagonize heterochromatin assembly at PCs of unsynapsed chromosomes by promoting a transcriptionally permissive chromatin environment that is required for meiotic checkpoint function. Moreover, we suggest that different genetic requirements to monitor the behavior of sex chromosomes and autosomes allow for the lone unsynapsed X present in male germlines to be shielded from inappropriate checkpoint activation.
Author Summary
Sexual reproduction relies on meiosis. This specialized cell division generates gametes, such as sperm and eggs, with a single copy of the genome so that fertilization restores diploidy. During meiosis, homologous chromosomes undergo synapsis, in which they assemble a proteinaceous structure called the synaptonemal complex to promote proper chromosome segregation. In C. elegans, a checkpoint monitors synapsis and removes nuclei that have unsynapsed chromosomes by activating programmed cell death or apoptosis. Activation of this checkpoint coincides with, but is not dependent on, the assembly of heterochromatin on unsynapsed chromosomes. The acquisition of heterochromatic marks is a conserved response to asynapsis. A specific portion of each chromosome, called a Pairing Center (PC), is required for this checkpoint; but how PCs send a signal when chromosomes are unsynapsed is unknown. We present evidence that suggests that unsynapsed PCs need to maintain a transcriptionally open chromatin environment, even as the rest of the chromosome undergoes heterochromatin assembly, to activate the checkpoint. Furthermore, we demonstrate that sex chromosomes have different genetic requirements than autosomes to monitor synapsis, providing a potential explanation for observed sexual dimorphism in synapsis checkpoint activation.
PMCID: PMC3499413  PMID: 23166523
20.  Symposium: Nuclear reprogramming and the control of differentiation in mammalian embryos 
Though spermatozoon and egg contribute an equal share of nuclear DNA content to the newly formed embryo, there are inherent epigenetic differences between the paternal and maternal pronuclei in early cleavage stage embryos. Information about how to decipher sperm DNA in the embryo is established via sperm-specific DNA packaging that occurs during spermatogenesis. In addition to protamines, paternal factors that package sperm DNA distinctly from oocyte or somatic DNA include histones and their modifications, histone variants, chromatin-binding proteins, and non-coding RNAs. These evolutionarily conserved factors play interconnected roles in heterochromatin formation, gene regulation, and maintenance of genome integrity, which influence key processes after fertilization. This review focuses on recent developments from genomic and proteomic studies in model organisms showing that components closely associated with sperm DNA contribute to embryonic survival. These advances may reveal important insights into the treatment of infertility and use of assisted reproductive technologies.
PMCID: PMC2875117  PMID: 18252043
chromatin; embryogenesis; epigenetics; fertility; paternal; spermatogenesis
21.  Spatial Separation of Parental Genomes in Preimplantation Mouse Embryos 
The Journal of Cell Biology  2000;148(4):629-634.
We have used two different experimental approaches to demonstrate topological separation of parental genomes in preimplantation mouse embryos: mouse eggs fertilized with 5-bromodeoxyuridine (BrdU)-labeled sperm followed by detection of BrdU in early diploid embryos, and differential heterochromatin staining in mouse interspecific hybrid embryos. Separation of chromatin according to parental origin was preserved up to the four-cell embryo stage and then gradually disappeared. In F1 hybrid animals, genome separation was also observed in a proportion of somatic cells. Separate nuclear compartments during preimplantation development, when extreme chromatin remodelling occurs, and possibly in some differentiated cell types, may be associated with epigenetic reprogramming.
PMCID: PMC2169371  PMID: 10684246
5-bromodeoxyuridine; fluorescence in situ hybridization; mouse interspecific hybrids; nuclear architecture; preimplantation embryo
22.  Reprogramming the Maternal Zebrafish Genome after Fertilization to Match the Paternal Methylation Pattern 
Cell  2013;153(4):759-772.
Early vertebrate embryos must achieve totipotency and prepare for zygotic genome activation (ZGA). To understand this process, we determined the DNA methylation (DNAme) profiles of zebrafish gametes, embryos at different stages, and somatic muscle and compared them to gene activity and histone modifications. Sperm chromatin patterns are virtually identical to those at ZGA. Unexpectedly, the DNA of many oocyte genes important for germ-line functions (i.e., piwil1) or early development (i.e., hox genes) is methylated, but the loci are demethylated during zygotic cleavage stages to precisely the state observed in sperm, even in parthenogenetic embryos lacking a replicating paternal genome. Furthermore, this cohort constitutes the genes and loci that acquire DNAme during development (i.e., ZGA to muscle). Finally, DNA methyltransferase inhibition experiments suggest that DNAme silences particular gene and chromatin cohorts at ZGA, preventing their precocious expression. Thus, zebrafish achieve a totipotent chromatin state at ZGA through paternal genome competency and maternal genome DNAme reprogramming.
PMCID: PMC4030421  PMID: 23663776
23.  Heterochromatin formation in the mouse embryo requires critical residues of the histone variant H3.3 
Nature cell biology  2010;12(9):853-862.
In mammals, oocyte fertilisation by sperm initiates development. This is followed by epigenetic reprogramming of both parental genomes, which involves de-novo establishment of chromatin domains. In the mouse embryo, methylation of histone H31 establishes an epigenetic asymmetry and is predominant in the maternal pronucleus2-5. However, the role of (i) differential incorporation of histone H3 variants in the parental chromatin and of (ii) modified residues within specific histone variants has 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 localises to paternal pericentromeric chromatin during S-phase at the time of transcription of pericentromeric repeats. Mutation of H3.3K27, but not H3.1K27, results in aberrant accumulation of pericentromeric transcripts, HP1 mislocalisation, dysfunctional chromosome segregation and developmental arrest. This phenotype is rescued by injection of dsRNA derived from pericentromeric transcripts, indicating a functional link between H3.3K27 and silencing of such regions via an 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.
PMCID: PMC3701880  PMID: 20676102
24.  Differential dynamics of histone H3 methylation at positions K4 and K9 in the mouse zygote 
In the mouse zygote the paternal genome undergoes dramatic structural and epigenetic changes. Chromosomes are decondensed, protamines replaced by histones and DNA is rapidly and actively demethylated. The epigenetic asymmetry between parental genomes remains at least until the 2-cell stage suggesting functional differences between paternal and maternal genomes during early cleavage stages.
Here we analyzed the timing of histone deposition on the paternal pronucleus and the dynamics of histone H3 methylation (H3/K4mono-, H3/K4tri- and H3/K9di-methylation) immediately after fertilization. Whereas maternal chromatin maintains all types of histone H3 methylation throughout the zygotic development, paternal chromosomes acquire new and unmodified histones shortly after fertilization. In the following hours we observe a gradual increase in H3/K4mono-methylation whereas H3/K4tri-methylation is not present before latest pronuclear stages. Histone H3/K9di-methylation is completely absent from the paternal pronucleus, including metaphase chromosomes of the first mitotic stage.
Parallel to the epigenetic asymmetry in DNA methylation, chromatin modifications are also different between both parental genomes in the very first hours post fertilization. Whereas methylation at H3/K4 gradually becomes similar between both genomes, H3/K9 methylation remains asymmetric.
PMCID: PMC521682  PMID: 15383155
25.  Genome-wide reprogramming in the mouse germ line entails the base excision repair pathway 
Science (New York, N.Y.)  2010;329(5987):10.1126/science.1187945.
The mouse germ-line presents a unique opportunity to study epigenetic reprogramming in vivo1. Recently we showed that genome-wide active DNA demethylation in primordial germ cells (PGCs) is linked to changes in nuclear architecture and extensive loss of histone modifications brought about by widespread histone replacement2. Notably, this chromatin remodelling follows the onset of genome-wide DNA demethylation, which raises a possibility that DNA demethylation may be linked to a DNA repair process2. Here we show the activation of components of the base excision repair (BER) pathway and the presence of single-strand DNA (ssDNA) breaks at the time of genome-wide DNA demethylation in PGCs. We found high levels of expression of critical BER components as well as chromatin-bound XRCC1 together with nuclear poly-ADP-ribosylation (PAR), specifically at the time when PGCs are undergoing DNA demethylation. A similar wave of genome-wide DNA demethylation occurs in the zygote affecting only the paternal genome3-5 where we observed a strikingly similar activation of BER components. Notably, maternally inherited Stella promotes this epigenetic asymmetry, since in zygotes lacking this protein, DNA demethylation is detected in both pronuclei. Crucially, zygotes lacking Stella exhibit aberrant targeting of active BER to both pronuclei. Finally we demonstrate that small molecule inhibitors of diverse BER components administered during in vitro fertilisation interfere with the progress of DNA demethylation.
Our combined observations demonstrate that DNA repair through BER represents a core component of genome-wide DNA demethylation and provides a vital mechanistic link to the extensive chromatin remodelling in developing PGCs2.
PMCID: PMC3863715  PMID: 20595612

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