Maintenance of CpG methylation is essential for epigenetic inheritance during plant gametogenesis

In mammals, the DNA methyltransferase 1 (Dnmt1) faithfully copies the pattern of cytosine methylation at CpG sites to the newly synthesized strand, and this is essential for epigenetic inheritance. In Arabidopsis thaliana, several DNA methyltransferases or chromatin modifiers coupled to methylation changes have been characterized, and mutations that cause loss of their function are recessive. This is surprising because plant gametogenesis includes postmeiotic DNA replication in haploid nuclei before fertilization. Therefore, the recessive character of the mutations excludes the affected components from a regulatory role in postmeiotic maintenance or modification of epigenetic states. Here we show, however, that depletion of A. thaliana MET1, a homolog of mammalian Dnmt1 (ref. 8), results in immense epigenetic diversification of gametes. This diversity seems to be a consequence of passive postmeiotic demethylation, leading to gametes with fully demethylated and hemidemethylated DNA, followed by remethylation of hemimethylated templates once MET1 is again supplied in a zygote.     

Targets and dynamics of promoter DNA methylation during early mouse development

DNA methylation is extensively reprogrammed during the early phases of mammalian development, yet genomic targets of this process are largely unknown. We optimized methylated DNA immunoprecipitation for low numbers of cells and profiled DNA methylation during early development of the mouse embryonic lineage in vivo. We observed a major epigenetic switch during implantation at the transition from the blastocyst to the postimplantation epiblast. During this period, DNA methylation is primarily targeted to repress the germline expression program. DNA methylation in the epiblast is also targeted to promoters of lineage-specific genes such as hematopoietic genes, which are subsequently demethylated during terminal differentiation. De novo methylation during early embryogenesis is catalyzed by Dnmt3b, and absence of DNA methylation leads to ectopic gene activation in the embryo. Finally, we identify nonimprinted genes that inherit promoter DNA methylation from parental gametes, suggesting that escape of post-fertilization DNA methylation reprogramming is prevalent in the mouse genome.     

Meiotic pairing and imprinted X chromatin assembly in Caenorhabditis elegans

The genetic imprinting of individual loci or whole chromosomes, as in imprinted X-chromosome inactivation in mammals, is established and reset during gametogenesis; defects in this process in the parent can result in disease in the offspring. We describe a sperm-specific chromatin-based imprinting of the X chromosome in the nematode Caenorhabditis elegans that is restricted to histone H3 modifications. The epigenetic imprint is established during spermatogenesis and its stability in the offspring is affected by the presence of a pairing partner during meiosis in the parental germ line. We observed that DNA lacking a pairing partner during meiosis, the normal situation for the X chromosome in males, is targeted for methylation of histone H3 at Lys9 (H3-Lys9) and can be silenced. Targeting unpaired DNA for silencing during meiosis, a potential hallmark of genome defense, could therefore have a conserved role in imprinted X-chromosome inactivation and, ultimately, in sex chromosome evolution.     

Genetic and epigenetic incompatibilities underlie hybrid dysgenesis in Peromyscus

Crosses between the two North American rodent species Peromyscus polionotus (PO) and Peromyscus maniculatus (BW) yield parent-of-origin effects on both embryonic and placental growth. The two species are approximately the same size, but a female BW crossed with a male PO produces offspring that are smaller than either parent. In the reciprocal cross, the offspring are oversized and typically die before birth. Rare survivors are exclusively female, consistent with Haldane's rule, which states that in instances of hybrid sterility or inviability, the heterogametic sex tends to be more severely affected. To understand these sex- and parent-of-origin-specific patterns of overgrowth, we analysed reciprocal backcrosses. Our studies reveal that hybrid inviability is partially due to a maternally expressed X-linked PO locus and an imprinted paternally expressed autosomal BW locus. In addition, the hybrids display skewing of X-chromosome inactivation in favour of the expression of the BW X chromosome. The most severe overgrowth is accompanied by widespread relaxation of imprinting of mostly paternally expressed genes. Both genetic and epigenetic mechanisms underlie hybrid inviability in Peromyscus and hence have a role in the establishment and maintenance of reproductive isolation barriers in mammals.     

Aberrant methylation of donor genome in cloned bovine embryos

Despite recent successes in cloning various animal species, the use of somatic cells as the source of donor nuclei has raised many practically relevant questions such as increased abortion rates, high birth weight and perinatal death. These anomalies may be caused by incomplete epigenetic reprogramming of donor DNA. Genome-wide demethylation occurs during early development, 'erasing' gamete-specific methylation patterns inherited from the parents. This process may be a prerequisite for the formation of pluripotent stem cells that are important for the later development. Here, we provide evidence that cloned bovine embryos may have impaired epigenetic reprogramming capabilities. We found highly aberrant methylation patterns in various genomic regions of cloned embryos. Cloned blastocysts closely resembled donor cells in their overall genomic methylation status, which was very different from that of normal blastocysts produced in vitro or in vivo. We found demethylation of the Bov-B long interspersed nuclear element sequence in normal embryos, but not in cloned embryos, in which the donor-type methylation was simply maintained during preimplantation development. There were also significant variations in the degree of methylation among individual cloned blastocysts. Our findings indicate that the developmental anomalies of cloned embryos could be due to incomplete epigenetic reprogramming of donor genomic DNA.     

Incomplete methylation reprogramming in SCNT embryos

The cloning of Dolly the sheep was a remarkable demonstration of the oocyte's ability to reprogram a specialized nucleus. However, embryos derived from such somatic cell nuclear transfer (SCNT) very rarely result in live births-a fate that may be linked to observed epigenetic defects. A new genome-wide study shows that epigenetic reprogramming in SCNT embryos does not fully recapitulate the natural DNA demethylation events occurring at fertilization, resulting in aberrant methylation at some promoters and repetitive elements that may contribute to developmental failure.     

Epigenetic asymmetry of imprinted genes in plant gametes

Plant imprinted genes show parent-of-origin expression in seed endosperm, but little is known about the nature of parental imprints in gametes before fertilization. We show here that single differentially methylated regions (DMRs) correlate with allele-specific expression of two maternally expressed genes in the seed and that one DMR is differentially methylated between gametes. Thus, plants seem to have developed similar strategies as mammals to epigenetically mark imprinted genes.     

Histone H3 lysine 9 methylation is an epigenetic imprint of facultative heterochromatin.

Post-translational modifications of histone amino termini are an important regulatory mechanism that induce transitions in chromatin structure, thereby contributing to epigenetic gene control and the assembly of specialized chromosomal subdomains. Methylation of histone H3 at lysine 9 (H3-Lys9) by site-specific histone methyltransferases (Suv39h HMTases) marks constitutive heterochromatin. Here, we show that H3-Lys9 methylation also occurs in facultative heterochromatin of the inactive X chromosome (Xi) in female mammals. H3-Lys9 methylation is retained through mitosis, indicating that it might provide an epigenetic imprint for the maintenance of the inactive state. Disruption of the two mouse Suv39h HMTases abolishes H3-Lys9 methylation of constitutive heterochromatin but not that of the Xi. In addition, HP1 proteins, which normally associate with heterochromatin, do not accumulate with the Xi. These observations suggest the existence of an Suv39h-HP1-independent pathway regulating H3-Lys9 methylation of facultative heterochromatin.     

Reverse breeding in Arabidopsis thaliana generates homozygous parental lines from a heterozygous plant

Traditionally, hybrid seeds are produced by crossing selected inbred lines. Here we provide a proof of concept for reverse breeding, a new approach that simplifies meiosis such that homozygous parental lines can be generated from a vigorous hybrid individual. We silenced DMC1, which encodes the meiotic recombination protein DISRUPTED MEIOTIC cDNA1, in hybrids of A. thaliana, so that non-recombined parental chromosomes segregate during meiosis. We then converted the resulting gametes into adult haploid plants, and subsequently into homozygous diploids, so that each contained half the genome of the original hybrid. From 36 homozygous lines, we selected 3 (out of 6) complementing parental pairs that allowed us to recreate the original hybrid by intercrossing. In addition, this approach resulted in a complete set of chromosome-substitution lines. Our method allows the selection of a single choice offspring from a segregating population and preservation of its heterozygous genotype by generating homozygous founder lines.     

Identification of genetic elements that autonomously determine DNA methylation states

Cytosine methylation is a repressive, epigenetically propagated DNA modification. Although patterns of DNA methylation seem tightly regulated in mammals, it is unclear how these are specified and to what extent this process entails genetic or epigenetic regulation. To dissect the role of the underlying DNA sequence, we sequentially inserted over 50 different DNA elements into the same genomic locus in mouse stem cells. Promoter sequences of approximately 1,000 bp autonomously recapitulated correct DNA methylation in pluripotent cells. Moreover, they supported proper de novo methylation during differentiation. Truncation analysis revealed that this regulatory potential is contained within small methylation-determining regions (MDRs). MDRs can mediate both hypomethylation and de novo methylation in cis, and their activity depends on developmental state, motifs for DNA-binding factors and a critical CpG density. These results demonstrate that proximal sequence elements are both necessary and sufficient for regulating DNA methylation and reveal basic constraints of this regulation.     

Evidence for an epigenetic mechanism by which Hsp90 acts as a capacitor for morphological evolution

Morphological alterations have been shown to occur in Drosophila melanogaster when function of Hsp90 (heat shock 90-kDa protein 1alpha, encoded by Hsp83) is compromised during development. Genetic selection maintains the altered phenotypes in subsequent generations. Recent experiments have shown, however, that phenotypic variation still occurs in nearly isogenic recombinant inbred strains of Arabidopsis thaliana. Using a sensitized isogenic D. melanogaster strain, iso-Kr(If-1), we confirm this finding and present evidence supporting an epigenetic mechanism for Hsp90's capacitor function, whereby reduced activity of Hsp90 induces a heritably altered chromatin state. The altered chromatin state is evidenced by ectopic expression of the morphogen wingless in eye imaginal discs and a corresponding abnormal eye phenotype, both of which are epigenetically heritable in subsequent generations, even when function of Hsp90 is restored. Mutations in nine different genes of the trithorax group that encode chromatin-remodeling proteins also induce the abnormal phenotype. These findings suggest that Hsp90 acts as a capacitor for morphological evolution through epigenetic and genetic mechanisms.