Relationship between DNA methylation, histone H4 acetylation and gene expression in the mouse imprinted Igf2-H19 domain

DNA methylation and histone H4 acetylation play a role in gene regulation by modulating the structure of the chromatin. Recently, these two epigenetic modifications have dynamically and physically been linked. Evidence suggests that both modifications are involved in regulating imprinted genes - a subset of genes whose expression depends on their parental origin. Using immunoprecipitation assays, we investigate the relationship between DNA methylation, histone H4 acetylation and gene expression in the well-characterised imprinted Igf2-H19 domain on mouse chromosome 7. A systematic regional analysis of the acetylation status of the domain shows that parental-specific differences in acetylation of the core histone H4 are present in the promoter regions of both Igf2 and H19 genes, with the expressed alleles being more acetylated than the silent alleles. A correlation between DNA methylation, histone hypoacetylation and gene repression is evident only at the promoter region of the H19 gene. Treatment with trichostatin A, a specific inhibitor of histone deacetylase, reduces the expression of the active maternal H19 allele and this can be correlated with regional changes in acetylation within the upstream regulatory domain. The data suggest that histone H4 acetylation and DNA methylation have distinct functions on the maternal and paternal Igf2-H19 domains.     

Inhibition of histone deacetylases alters allelic chromatin conformation at the imprinted U2af1-rs1 locus in mouse embryonic stem cells

Most loci that are regulated by genomic imprinting have differentially methylated regions (DMRs). Previously, we showed that the DMRs of the mouse Snrpn and U2af1-rs1 genes have paternal allele-specific patterns of acetylation on histones H3 and H4. To investigate the maintenance of acetylation at these DMRs, we performed chromatin immunoprecipitation on trichostatin-A (TSA)-treated and control cells. In embryonic stem (ES) cells and fibroblasts, brief (6-h) TSA treatment induces global hyperacetylation of H3 and H4. In ES cells only, TSA led to a selective increase in maternal acetylation at U2af1-rs1, at lysine 5 of H4 and at lysine 14 of H3. TSA treatment of ES cells did not affect DNA methylation or expression of U2af1-rs1, but was sufficient to increase DNase I sensitivity along the maternal allele to a level comparable with that of the paternal allele. In fibroblasts, TSA did not alter U2af1-rs1 acetylation, and the parental alleles retained their differential DNase I sensitivity. At Snrpn, no changes in acetylation were observed in the TSA-treated cells. Our data suggest that the mechanisms regulating histone acetylation at DMRs are locus and developmental stage-specific and are distinct from those effecting global levels of acetylation. Furthermore, it seems that the allelic U2af1-rs1 acetylation determines DNase I sensitivity/chromatin conformation.     

DNA Methylation Is Linked to Deacetylation of Histone H3, but Not H4, on the Imprinted Genes Snrpn and U2af1-rs1

The relationship between DNA methylation and histone acetylation at the imprinted mouse genes U2af1-rs1 and Snrpn is explored by chromatin immunoprecipitation (ChIP) and resolution of parental alleles using single-strand conformational polymorphisms. The U2af1-rs1 gene lies within a differentially methylated region (DMR), while Snrpn has a 5' DMR (DMR1) with sequences homologous to the imprinting control center of the Prader-Willi/Angelman region. For both DMR1 of Snrpn and the 5' untranslated region (5'-UTR) and 3'-UTR of U2af1-rs1, the methylated and nonexpressed maternal allele was underacetylated, relative to the paternal allele, at all H3 lysines tested (K14, K9, and K18). For H4, underacetylation of the maternal allele was exclusively (U2af1-rs1) or predominantly (Snrpn) at lysine 5. Essentially the same patterns of differential acetylation were found in embryonic stem (ES) cells, embryo fibroblasts, and adult liver from F1 mice and in ES cells from mice that were dipaternal or dimaternal for U2af1-rs1. In contrast, in a region within Snrpn that has biallelic methylation in the cells and tissues analyzed, the paternal (expressed) allele showed relatively increased acetylation of H4 but not of H3. The methyl-CpG-binding-domain (MBD) protein MeCP2 was found, by ChIP, to be associated exclusively with the maternal U2af1-rs1 allele. To ask whether DNA methylation is associated with histone deacetylation, we produced mice with transgene-induced methylation at the paternal allele of U2af1-rs1. In these mice, H3 was underacetylated across both the parental U2af1-rs1 alleles whereas H4 acetylation was unaltered. Collectively, these data are consistent with the hypothesis that CpG methylation leads to deacetylation of histone H3, but not H4, through a process that involves selective binding of MBD proteins.     

Differential histone modifications mark mouse imprinting control regions during spermatogenesis

Only some imprinting control regions (ICRs) acquire their DNA methylation in the male germ line. These imprints are protected against the global demethylation of the sperm genome following fertilisation, and are maintained throughout development. We find that in somatic cells and tissues, DNA methylation at these ICRs is associated with histone H4-lysine-20 and H3-lysine-9 trimethylation. The unmethylated allele, in contrast, has H3-lysine-4 dimethylation and H3 acetylation. These differential modifications are also detected at maternally methylated ICRs, and could be involved in the somatic maintenance of imprints. To explore whether the post-fertilisation protection of imprints relates to events during spermatogenesis, we assayed chromatin at stages preceding the global histone-to-protamine exchange. At these stages, H3-lysine-4 methylation and H3 acetylation are enriched at maternally methylated ICRs, but are absent at paternally methylated ICRs. H4 acetylation is enriched at all regions analysed. Thus, paternally and maternally methylated ICRs carry different histone modifications during the stages preceding the global histone-to-protamine exchange. These differences could influence the way ICRs are assembled into specific structures in late spermatogenesis, and may thus influence events after fertilisation.     

The relationship between DNA methylation and chromosome imprinting in the coccid Planococcus citri

The phenomenon of chromosome, or genomic, imprinting indicates the relevance of parental origin in determining functional differences between alleles, homologous chromosomes, or haploid sets. In mealybug males (Homoptera, Coccoidea), the haploid set of paternal origin undergoes heterochromatization at midcleavage and remains so in most of the tissues. This different behavior of the two haploid sets, which depends on their parental origin, represents one of the most striking examples of chromosome imprinting. In mammals, DNA methylation has been postulated as a possible molecular mechanism to differentially imprint DNA sequences during spermatogenesis or oogenesis. In the present article we addressed the role of DNA methylation in the imprinting of whole haploid sets as it occurs in Coccids. We investigated the DNA methylation patterns at both the molecular and chromosomal level in the mealybug Planococcus citri. We found that in both males and females the paternally derived haploid set is hypomethylated with respect to the maternally derived one. Therefore, in males, it is the paternally derived hypomethylated haploid set that is heterochromatized. Our data suggest that the two haploid sets are imprinted by parent-of-origin-specific DNA methylation with no correlation with the known gene-silencing properties of this base modification.     

Allele-specific histone modifications regulate expression of the Dlk1-Gtl2 imprinted domain

Dlk1 and Gtl2 are reciprocally expressed imprinted genes located on mouse chromosome 12. The Dlk1-Gtl2 locus carries three differentially methylated regions (DMRs), which are methylated only on the paternal allele. Of these, the intergenic (IG) DMR, located 12 kb upstream of Gtl2, is required for proper imprinting of linked genes on the maternal chromosome, while the Gtl2 DMR, located across the promoter of the Gtl2 gene, is implicated in imprinting on both parental chromosomes. In addition to DNA methylation, modification of histone proteins is also an important regulator of imprinted gene expression. Chromatin immunoprecipitation was therefore used to examine the pattern of histone modifications across the IG and Gtl2 DMRs. The data show maternal-specific histone acetylation at the Gtl2 DMR, but not at the IG DMR. In contrast, only low levels of histone methylation were observed throughout the region, and there was no difference between the two parental alleles. An existing mouse line carrying a deletion/insertion upstream of Gtl2 is unable to imprint the Dlk1-Gtl2 locus properly and demonstrates loss of allele-specific methylation at the Gtl2 DMR. Further analysis of these animals now shows that the loss of allele-specific methylation is accompanied by increased paternal histone acetylation at the Gtl2 DMR, with the activated paternal allele adopting a maternal acetylation pattern. These data indicate that interactions between DNA methylation and histone acetylation are involved in regulating the imprinting of the Dlk1-Gtl2 locus.     

Histone acetylation and gene expression analysis of sex lethal mutants in Drosophila

The evolution of sex determination mechanisms is often accompanied by reduction in dosage of genes on a whole chromosome. Under these circumstances, negatively acting regulatory genes would tend to double the expression of the genome, which produces compensation of the single-sex chromosome and increases autosomal gene expression. Previous work has suggested that to reduce the autosomal expression to the female level, these dosage effects are modified by a chromatin complex specific to males, which sequesters a histone acetylase to the X. The reduced autosomal histone 4 lysine 16 (H4Lys16) acetylation results in lowered autosomal expression, while the higher acetylation on the X is mitigated by the male-specific lethal complex, preventing overexpression. In this report, we examine how mutations in the principal sex determination gene, Sex lethal (Sxl), impact the H4 acetylation and gene expression on both the X and autosomes. When Sxl expression is missing in females, we find that the sequestration occurs concordantly with reductions in autosomal H4Lys16 acetylation and gene expression on the whole. When Sxl is ectopically expressed in Sxl(M) mutant males, the sequestration is disrupted, leading to an increase in autosomal H4Lys16 acetylation and overall gene expression. In both cases we find relatively little effect upon X chromosomal gene expression.     

Maternal and paternal alleles exhibit differential histone methylation and acetylation at maize imprinted genes

Imprinting is an epigenetically controlled form of gene regulation in which the expression of a gene is based on its parent of origin. This epigenetic regulation is likely to involve allele-specific DNA or histone modifications. The relative abundance of eight different histone modifications was tested at various regions in several imprinted maize (Zea mays) genes using a chromatin immunoprecipitation protocol coupled with quantitative allele-specific single nucleotide polymorphism assays. Histone H3 lysine-27 di- and tri-methylation are paternally enriched at the imprinted loci Mez1, ZmFie1 and Nrp1. In contrast, acetylation of histones H3 and H4 and H3K4 dimethylation are enriched at the maternal alleles of these genes. Di- and tri-methylation of H3 lysine-9, which is generally associated with constitutively silenced chromatin, was not enriched at either allele of imprinted loci. These patterns of enrichment were specific to tissues that exhibit imprinting. In addition, the enrichment of these modifications was dependent upon the parental origin of an allele and not sequence differences between the alleles, as demonstrated by reciprocal crosses. This study presents a detailed view of the chromatin modifications that are associated with the maternal and paternal alleles at imprinted loci and provides evidence for common histone modifications at multiple imprinted loci.     

Parent-of-origin specific histone acetylation and reactivation of a key imprinted gene locus in Prader-Willi syndrome

To examine the chromatin basis of imprinting in chromosome 15q11-q13, we have investigated the status of histone acetylation of the SNURF-SNRPN locus, which is a key imprinted gene locus in Prader-Willi syndrome (PWS). Chromatin immunoprecipitation (ChIP) studies revealed that the unmethylated CpG island of the active, paternally derived allele of SNURF-SNRPN was associated with acetylated histones, whereas the methylated maternally derived, inactive allele was specifically hypoacetylated. The body of the SNURF-SNRPN gene was associated with acetylated histones on both alleles. Furthermore, treatment of PWS cells with the DNA methyltransferase inhibitor 5-azadeoxycytidine (5-aza-dC) induced demethylation of the SNURF-SNRPN CpG island and restoration of gene expression on the maternal allele. The reactivation was associated with increased H4 acetylation but not with H3 acetylation at the SNURF-SNRPN CpG island. These findings indicate that (1) a significant role for histone deacetylation in gene silencing is associated with imprinting in 15q11-q13 and (2) silenced genes in PWS can be reactivated by drug treatment.     

Dynamic reprogramming of histone acetylation and methylation in the first cell cycle of cloned mouse embryos

Epigenetic reprogramming is thought to play an important role in the development of cloned embryos reconstructed by somatic cell nuclear transfer (SCNT). In the present study, dynamic reprogramming of histone acetylation and methylation modifications was investigated in the first cell cycle of cloned embryos. Our results demonstrated that part of somatic inherited lysine acetylation on core histones (H3K9, H3K14, H4K16) could be quickly deacetylated following SCNT, and reacetylation occurred following activation treatment. However, acetylation marks of the other lysine residues on core histones (H4K8, H4K12) persisted in the genome of cloned embryos with only mild deacetylation occurring in the process of SCNT and activation treatment. The somatic cloned embryos established histone acetylation modifications resembling those in normal embryos produced by intracytoplasmic sperm injection through these two different programs. Moreover, treatment of cloned embryos with a histone deacetylase inhibitor, Trichostatin A (TSA), improved the histone acetylation in a manner similar to that in normal embryos, and the improved histone acetylation in cloned embryos treated with TSA might contribute to improved development of TSA-treated clones. In contrast to the asymmetric histone H3K9 tri- and dimethylation present in the parental genomes of fertilized embryos, the tri- and dimethylations of H3K9 were gradually demethylated in the cloned embryos, and this histone H3K9 demethylation may be crucial for gene activation of cloned embryos. Together, our results indicate that dynamic reprogramming of histone acetylation and methylation modifications in cloned embryos is developmentally regulated.     

Epigenetic discrimination by mouse metaphase II oocytes mediates asymmetric chromatin remodeling independently of meiotic exit

In mammalian fertilization, paternal chromatin is exhaustively remodeled, yet the maternal contribution to this process is unknown. To address this, we prevented the induction of meiotic exit by spermatozoa and examined sperm chromatin remodeling in metaphase II (mII) oocytes. Methylation of paternal H3-K4 and H3-K9 remained low, unlike maternal H3, although paternal H3-K4 methylation increased in zygotes. Thus, mII cytoplasm can sustain epigenetic asymmetry in a cell-cycle dependent manner. Paternal genomic DNA underwent oocyte-mediated cytosine demethylation and acquired maternally-derived K12-acetylated H4 (AcH4-K12) independently of microtubule assembly and maternal chromatin. AcH4-K12 persisted without typical maturation-associated deacetylation, irrespective of paternal pan-genomic cytosine methylation. Contrastingly, somatic cell nuclei underwent rapid H4 deacetylation; sperm and somatic chromatin exhibited asymmetric AcH4-K12 dynamics simultaneously within the same mII oocyte. Inhibition of somatic histone deacetylation revealed endogenous histone acetyl transferase activity. Oocytes thus specify the histone acetylation status of given nuclei by differentially targeting histone deacetylase and acetyl transferase activities. Asymmetric H4 acetylation during and immediately after fertilization was dispensable for development when both parental chromatin sets were hyperacetylated. These studies delineate non-zygotic chromatin remodeling and suggest a powerful model with which to study de novo genomic reprogramming.     

Roles for Gcn5 in promoting nucleosome assembly and maintaining genome integrity

The coordinated process of DNA replication and nucleosome assembly, termed replication-coupled (RC) nucleosome assembly, is important for the maintenance of genome integrity. Loss of genome integrity is linked to aging and cancer. RC nucleosome assembly involves deposition of histone H3-H4 by the histone chaperones CAF-1, Rtt106 and Asf1 onto newly-replicated DNA. Coordinated actions of these three histone chaperones are regulated by modifications on the histone proteins. One such modification is histone H3 lysine 56 acetylation (H3K56Ac), a mark of newly-synthesized histone H3 that regulates the interaction between H3-H4 and the histone chaperones CAF-1 and Rtt106 following DNA replication and DNA repair. Recently, we have shown that the lysine acetyltransferase Gcn5 and H3 N-terminal tail lysine acetylation also regulates the interaction between H3-H4 and CAF-1 to promote the deposition of newly-synthesized histones. Genetic studies indicate that Gcn5 and Rtt109, the H3K56Ac lysine acetyltransferase, function in parallel to maintain genome stability. Utilizing synthetic genetic array analysis, we set out to identify additional genes that function in parallel with Gcn5 in response to DNA damage. We summarize here the role of Gcn5 in nucleosome assembly and suggest that Gcn5 impacts genome integrity via multiple mechanisms, including nucleosome assembly.     

Histone methylation defines epigenetic asymmetry in the mouse zygote

The oocyte cytoplasm regulates and enhances the epigenetic asymmetry between parental genomes and, consequently, functional differences observed between them during development in mammals. Here we demonstrate a preferential interaction of HP1beta with the maternal genome immediately after fertilisation in the mouse zygote, which also shows a high level of lysine 9-methylated histone H3. In contrast, the paternal genome has neither HP1beta binding nor methylated histone H3 at these early stages. Paternal binding of HP1beta is only detected at the pronuclear stage, prior to the appearance of lysine 9-methylated histone H3. The early recruitment of heterochromatic factors specifically to the maternal genome could explain the preferential DNA demethylation of the paternal genome in the zygote.     

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.