Differential acetylation of histone H4 lysine during development of in vitro fertilized,cloned and parthenogenetically activated bovine embryos

The oocyte is remarkable in its ability to remodel parental genomes following fertilization and to reprogram somatic nuclei after nuclear transfer (NT). To characterise the patterns of histone H4 acetylation and DNA methylation during development of bovine gametogenesis and embryogenesis, specific antibodies for histone H4 acetylated at lysine 5 (K5), K8, K12 and K16 residues and for methylated cytosine of CpG dinucleotides were used. Oocytes and sperm lacked the staining for histone acetylation, when DNA methylation staining was intense. In IVF zygotes, both pronuclei were transiently hyper-acetylated. However, the male pronucleus was faster in acquiring acetylated histones, and concurrently it was rapidly demethylated. Both pronuclei were equally acetylated during the S to G2-phase transition, while methylation staining was only still observed in the female pronucleus. In parthenogenetically activated oocytes, acetylation of the female pronucleus was enriched faster, while DNA remained methylated. A transient de-acetylation was observed in NT embryos reconstructed using a non-activated ooplast of a metaphase second arrested oocyte. Remarkably, the intensity of acetylation staining of most H4 lysine residues peaked at the 8-cell stage in IVF embryos, which coincided with zygotic genome activation and with lowest DNA methylation staining. At the blastocyst stage, trophectodermal cells of IVF and parthenogenetic embryos generally demonstrated more intense staining for most acetylated H4 lysine, whilst ICM cells stained very weakly. In contrast methylation of the DNA stained more intensely in ICM. NT blastocysts showed differential acetylation of blastomeres but not methylation. The inverse association of histone lysine acetylation and DNA methylation at different vital embryo stages suggests a mechanistically significant relationship. The complexities of these epigenetic interactions are discussed.     

Paternal H3K4 methylation is required for minor zygotic gene activation and early mouse embryonic development

Epigenetic modifications, such as DNA methylation and histone modifications, are dynamically altered predominantly in paternal pronuclei soon after fertilization. To identify which histone modifications are required for early embryonic development, we utilized histone K‐M mutants, which prevent endogenous histone methylation at the mutated site. We prepared four single K‐M mutants for histone H3.3, K4M, K9M, K27M, and K36M, and demonstrate that overexpression of H3.3 K4M in embryos before fertilization results in developmental arrest, whereas overexpression after fertilization does not affect the development. Furthermore, loss of H3K4 methylation decreases the level of minor zygotic gene activation (ZGA) predominantly in the paternal pronucleus, and we obtained similar results from knockdown of the H3K4 methyltransferase Mll3/4. We therefore conclude that H3K4 methylation, likely established by Mll3/4 at the early pronuclear stage, is essential for the onset of minor ZGA in the paternal pronucleus, which is necessary for subsequent preimplantation development in mice.     

Asymmetry in histone H3 variants and lysine methylation between paternal and maternal chromatin of the early mouse zygote

In mammalian fertilization, the paternal genome is delivered to the secondary oocyte by sperm with protamine compacted DNA, while the maternal genome is arrested in meiotic metaphase II. Thus, at the beginning of fertilization, the two gametic chromatin sets are strikingly different. We elaborate on this contrast by reporting asymmetry for histone H3 type in the pre-S-phase zygote when male chromatin is virtually devoid of histone H3.1/3.2. Localization of the histone H3.3/H4 assembly factor Hira with the paternal chromatin indicates the presence of histone H3.3. In conjunction with this, we performed a systematic immunofluorescence analysis of histone N-tail methylations at position H3K4, H3K9, H3K27 and H4K20 up to the young pronucleus stage and show that asymmetries reported earlier are systematic for virtually all di- and tri-methylations but not for mono-methylation of H3K4 and H4K20, the only marks studied present in the early male pronucleus. For H4K20 the expanding male chromatin is rapidly mono-methylated. This coincides with the formation of maternally derived nucleosomes, a process which is observed as early as sperm chromatin decondensation occurs. Absence of tri-methylated H3K9, tri-methylated H4K20 and presence of loosely anchored HP1-beta combined with the homogenous presence of mono-methylated H4K20 suggests the absence of a division of the paternal chromatin in eu- and heterochromatin. In summary the male, in contrast to female G1 chromatin, is uniform and contains predominantly histone H3.3 as histone H3 variant.     

Aging changes the chromatin configuration and histone methylation of mouse oocytes at germinal vesicle stage

Aging decreases the fertility of mammalian females. In old oocytes at metaphase II stage (MII) there are alterations of the chromatin configuration and chromatin modifications such as histone acetylation. Recent data indicate that alterations of histone acetylation at MII initially arise at germinal vesicle stage (GV). Therefore, we hypothesized that the chromatin configuration and histone methylation could also change in old GV oocytes. In agreement with our hypothesis, young GV oocytes had non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) chromatin configurations, while old GV oocytes also had chromatin configurations that could not be classified as NSN or SN. Regarding histone methylation, young GV and MII oocytes showed dimethylation of lysines 4, 9, 36 and 79 in histone 3 (H3K4me2, H3K9me2, H3K36me2, H3K79me2), lysine 20 in histone H4 (H4K20me2) and trimethylation of lysine 9 in histone 3 (H3K9me3) while a significant percentage of old GV and MII oocytes lacked H3K9me3, H3K36me2, H3K79me2 and H4K20me2. The percentage of old oocytes lacking histone methylation was similar at GV and MII suggesting that alterations of histone methylation in old MII oocytes initially arise at GV. Besides, the expression of the histone methylation-related factors Cbx1 and Sirt1 was also found to change in old GV oocytes. In conclusion, our study reports changes of chromatin configuration and histone methylation in old GV oocytes, which could be very useful for further understanding of human infertility caused by aging.     

Aging changes the chromatin configuration and histone methylation of mouse oocytes at germinal vesicle stage

Aging decreases the fertility of mammalian females. In old oocytes at metaphase II stage (MII) there are alterations of the chromatin configuration and chromatin modifications such as histone acetylation. Recent data indicate that alterations of histone acetylation at MII initially arise at germinal vesicle stage (GV). Therefore, we hypothesized that the chromatin configuration and histone methylation could also change in old GV oocytes. In agreement with our hypothesis, young GV oocytes had non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) chromatin configurations, while old GV oocytes also had chromatin configurations that could not be classified as NSN or SN. Regarding histone methylation, young GV and MII oocytes showed dimethylation of lysines 4, 9, 36 and 79 in histone 3 (H3K4me2, H3K9me2, H3K36me2, H3K79me2), lysine 20 in histone H4 (H4K20me2) and trimethylation of lysine 9 in histone 3 (H3K9me3) while a significant percentage of old GV and MII oocytes lacked H3K9me3, H3K36me2, H3K79me2 and H4K20me2. The percentage of old oocytes lacking histone methylation was similar at GV and MII suggesting that alterations of histone methylation in old MII oocytes initially arise at GV. Besides, the expression of the histone methylation-related factors Cbx1 and Sirt1 was also found to change in old GV oocytes. In conclusion, our study reports changes of chromatin configuration and histone methylation in old GV oocytes, which could be very useful for further understanding of human infertility caused by aging.     

Histone h3 lysine 4 methylation marks postreplicative human cytomegalovirus chromatin

In the nuclei of permissive cells, human cytomegalovirus genomes form nucleosomal structures initially resembling heterochromatin but gradually switching to a euchromatin-like state. This switch is characterized by a decrease in histone H3 K9 methylation and a marked increase in H3 tail acetylation and H3 K4 methylation across the viral genome. We used ganciclovir and a mutant virus encoding a reversibly destabilized DNA polymerase to examine the impact of DNA replication on histone modification dynamics at the viral chromatin. The changes in H3 tail acetylation and H3 K9 methylation proceeded in a DNA replication-independent fashion. In contrast, the increase in H3 K4 methylation proved to depend widely on viral DNA synthesis. Consistently, labeling of nascent DNA using "click chemistry" revealed preferential incorporation of methylated H3 K4 into viral (but not cellular) chromatin during or following DNA replication. This study demonstrates largely selective epigenetic tagging of postreplicative human cytomegalovirus chromatin.     

Dynamic chromatin modifications characterise the first cell cycle in mouse embryos

On fertilisation, gametes undergo epigenetic reorganisation and re-establish totipotency. Here, we investigate links between chromatin remodelling and asymmetric maintenance of DNA methylation in the early mouse embryo. Using antibodies for lysine specific H3 methylation reveals that the male pronucleus is negative for di- and trimethyl H3-K9 yet the female is positive for these residues. However, the male is positive for monomethyl H3-K9 and H3-K27 and these signals increase during pronuclear maturation. Non-histone chromatin proteins of the Polycomb group are found in the paternal compartment as early as sperm decondensation. However, trimethyl H3-K27 is not observed in the male until the completion of DNA replication. Heterochromatin protein 1 beta (HP1beta) is abundant in the male pronucleus, despite the absence of di- and trimethyl H3-K9, and co-localises with monomethyl H3-K9. Recent evidence identifies monomethyl H3-K9 as the preferred substrate of Suvar39h, the histone methyl transferase (HMT) responsible for heterochromatic H3-K9 trimethylation. The association of HP1beta with monomethyl H3-K9 may assist in preventing further modification of H3-K9. Association of dimethylation but not trimethylation of H3-K9 with DNA methylation, in the female pronucleus, suggests a mechanistically significant link. These differences begin to provide a chromatin based explanation for paternal-specific active DNA demethylation and maternal specific protection in the mouse.     

Holocarboxylase synthetase synergizes with methyl CpG binding protein 2 and DNA methyltransferase 1 in the transcriptional repression of long-terminal repeats

Holocarboxylase synthetase (HLCS) is a chromatin protein that facilitates the creation of histone H3 lysine 9-methylation (H3K9me) gene repression marks through physical interactions with the histone methyltransferase EHMT-1. HLCS knockdown causes a depletion of H3K9me marks in mammalian cell cultures and severe phenotypes such as short lifespan and low stress resistance in Drosophila melanogaster. HLCS displays a punctuate distribution pattern in chromatin despite lacking a strong DNA-binding domain. Previous studies suggest that the binding of HLCS to chromatin depends on DNA methylation. We tested the hypothesis that HLCS interacts physically with the DNA methyltransferase DNMT1 and the methyl CpG binding protein MeCP2 to facilitate the binding of HLCS to chromatin, and that these interactions contribute toward the repression of long-terminal repeats (LTRs) by H3K9me marks. Co-immunoprecipitation and limited proteolysis assays provided evidence suggesting that HLCS interacts physically with both DNMT1 and MeCP2. The abundance of H3K9me marks was 207% greater in the LTR15 locus in HLCS overexpression human embryonic kidney HEK293 cells compared with controls. This gain in H3K9me was inversely linked with a 87% decrease in mRNA coding for LTRs. Effects of HLCS abundance on LTR expression were abolished when DNA methylation marks were erased by treating cells with 5-azacytidine. We conclude that interactions between DNA methylation and HLCS are crucial for mediating gene repression by H3K9me, thereby providing evidence for epigenetic synergies between the protein biotin ligase HLCS and dietary methyl donors.     

Lineage-specific transition of histone signatures in the killer cell Ig-like receptor locus from hematopoietic progenitor to NK cells

The clonal distribution and stable expression of killer cell Ig-like receptor (KIR) genes is epigenetically regulated. To assess the epigenetic changes that occur during hemopoietic development we examined DNA methylation and chromatin structure of the KIR locus in early hemopoietic progenitor cells and major lymphocyte lineages. In hemopoietic progenitor cells, KIR genes exhibited the major hallmarks of epigenetic repression, which are dense DNA methylation, inaccessibility of chromatin to Micrococcus nuclease digest, and a repressive histone signature, characterized by strong H3K9 dimethylation and reduced H4K8 acetylation. In contrast, KIR genes of NK cells showed active histone signatures characterized by absence of H3K9 dimethylation and presence of H4K8 acetylation. Histone modifications correlated well with the competence of different lymphocyte lineages to express KIR; whereas H4K8 acetylation was high in NK and CD8+ T cells, it was almost absent in CD4+ T cells and B cells and, in the latter case, replaced by H3K9 dimethylation. In KIR-competent lineages, active histone signatures were also observed in silent KIR genes and in this case found in combination with dense DNA methylation of the promoter and nearby regions. The study suggests a two-step model of epigenetic regulation in which lineage-specific acquisition of euchromatic histone marks is a prerequisite for subsequent gene-specific DNA demethylation and expression of KIR genes.     

Epigenetic marks for chromosome imprinting during spermatogenesis in coccids

The establishment of sex-specific epigenetic marks during gametogenesis is one of the key feature of genomic imprinting. By immunocytological analysis, we thoroughly characterized the chromatin remodeling events that take place during gametogenesis in the mealybug Planococcus citri, in which an entire haploid set of (imprinted) chromosomes undergoes facultative heterochromatinization in male embryos. Building on our previous work, we have investigated the interplay of several epigenetic marks in the regulation of this genome-wide phenomenon. We characterized the germline patterns of histone modifications, Me(3)K9H3, Me(2)K9H3, and Me(3)K20H4, and of heterochromatic proteins, PCHET2 (HP1-like) and HP2-like during male and female gametogenesis. We found that at all stages in oogenesis chromatin is devoid of any detectable epigenetic marks. On the other hand, spermatogenesis is accompanied by a complex pattern of redistribution of epigenetic marks from euchromatin to heterochromatin, and vice versa. At the end of spermatogenesis, sperm heads are decorated by all the molecules we tested, except for PCHET2. However, only Me(3)K9H3 and Me(2)K9H3 are still detectable in the male pronucleus. We suggest that the histone H3 lysine 9 methylation is the signal used to establish the male-specific imprinting on the paternal genome, thus allowing it to be distinguished from the maternal genome in the developing embryo. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tissue-Specific Epigenetic Modifications in Root Apical Meristem Cells of Hordeum vulgare

Epigenetic modifications of chromatin structure are essential for many biological processes, including growth and reproduction. Patterns of DNA and histone modifications have recently been widely studied in many plant species, although there is virtually no data on the spatial and temporal distribution of epigenetic markers during plant development. Accordingly, we have used immunostaining techniques to investigate epigenetic modifications in the root apical meristem of Hordeum vulgare. Histone H4 acetylation (H4K5ac), histone H3 dimethylation (H3K4me2, H3K9me2) and DNA methylation (5mC) patterns were established for various root meristem tissues. Distinct levels of those modifications were visualised in the root cap, epidermis, cortex and vascular tissues. The lateral root cap cells seem to display the highest level of H3K9me2 and 5mC. In the epidermis, the highest level of 5mC and H3K9me2 was detected in the nuclei from the boundary of the proximal meristem and the elongation zone, while the vascular tissues were characterized by the highest level of H4K5ac. Some of the modified histones were also detectable in the cytoplasm in a highly tissue-specific manner. Immunolocalisation of epigenetic modifications of chromatin carried out in this way, on longitudinal or transverse sections, provides a unique topographic context within the organ, and will provide some answers to the significant biological question of tissue differentiation processes during root development in a monocotyledon plant species.