Intraspecific hybrids between the Arabidopsis thaliana accessions C24 and Landsberg erecta have strong heterosis. The reciprocal hybrids show a decreased level of 24-nt small RNA (sRNA) relative to the parents with the decrease greatest for those loci where the parents had markedly different 24-nt sRNA levels. The genomic regions with reduced 24-nt sRNA levels were largely associated with genes and their flanking regions indicating a potential effect on gene expression. We identified several examples of genes with altered 24-nt sRNA levels that showed correlated changes in DNA methylation and expression levels. We suggest that such epigenetically generated differences in gene activity may contribute to hybrid vigor and that the epigenetic diversity between ecotypes provides increased allelic (epi-allelic) variability that could contribute to heterosis.yield increase | epigenome | transposons | transacting siRNA | transmethylation

The heterotic hybrid offspring of Arabidopsis accessions C24 and Landsberg erecta have altered methylomes. Changes occur most frequently at loci where parental methylation levels are different. There are context-specific biases in the nonadditive methylation patterns with m CG generally increased and m CHH decreased relative to the parents. These changes are a result of two main mechanisms, Trans Chromosomal Methylation and Trans Chromosomal deMethylation, where the methylation level of one parental allele alters to resemble that of the other parent. Regions of altered methylation are enriched around genic regions and are often correlated with changes in siRNA levels. We identified examples of genes with altered expression likely to be due to methylation changes and suggest that in crosses between the C24 and Ler accessions, epigenetic controls can be important in the generation of altered transcription levels that may contribute to the increased biomass of the hybrids.I n the formation of a hybrid, the genome and epigenome of each of the parents are brought together within the one nucleus. The interactions of these two sets of genetic instructions result in the unique characteristics of the hybrid, including superior performance. Both the level and pattern of expression of many genes are altered in hybrids (1-3). Altered transcription levels have mostly been explained by the interaction between the alleles of a gene delivered by the parents involving a range of interactions such as dominance, overdominance, and epistatic interactions between loci (1). Despite these genetic analyses, there is a lack of understanding of the molecular mechanisms underpinning heterosis.It has been suggested that the magnitude of hybrid vigor is positively correlated with the genetic distance or amount of sequence variation between the parental genomes (4, 5). However, crosses between genetically similar parents such as Arabidopsis accessions or subspecies of rice can produce hybrids displaying significant heterosis, apparently breaking down the relationship between genetic distance and extent of hybrid vigor (6, 7). It has been reported that the epigenome evolves at a significantly faster rate than the genetic sequence (8-10), consistent with genetically similar parents having markedly different epigenomes (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). These epigenomic systems, such as DNA methylation and small RNAs, play a vital role in genomic stability, development, and the regulation of genes within a plant. The epigenome may contribute the allelic variability needed to generate heterosis in crosses between genetically similar parents. We previously reported that the Arabidopsis C24 and Ler accessions have different siRNA distributions and that the reciprocal heterotic hybrids show a 27% reduction in the levels of 24-nt siRNAs (18). The major reduction in these 24-nt siRNA sequences corresponded to those segments of the genome, primarily the gene bodies and their flanking sequences, where the two parents had unequal levels of 24-nt ...

The histone variant H2A.Z plays an essential role in metazoans but its function remains to be determined. Here, we developed a new inducible RNAi strategy to elucidate the role of H2A.Z in mammalian cell lines. We show that in the absence of H2A.Z, the genome becomes highly unstable and that this instability is caused by defects in the chromosome segregation process. Analysis of H2A.Z localization reveals that in these cells it is enriched at heterochromatic foci with HP1alpha on the arms of chromosomes but not at centromeric regions. When H2A.Z is depleted, normal HP1alpha-chromatin interactions are disrupted on the chromosomal arms and, notably, also at pericentric regions. Therefore, H2A.Z controls the localization of HP1alpha. We conclude that H2A.Z is essential for the accurate transmission of chromosomes.

The replacement histone H2A.Z is variously reported as being linked to gene expression and preventing the spread of heterochromatin in yeast, or concentrated at heterochromatin in mammals. To resolve this apparent dichotomy, affinity-purified antibodies against the N-terminal region of H2A.Z, in both a triacetylated and non-acetylated state, are used in native chromatin immmuno-precipitation experiments with mononucleosomes from three chicken cell types. The hyperacetylated species concentrates at the 5′ end of active genes, both tissue specific and housekeeping but is absent from inactive genes, while the unacetylated form is absent from both active and inactive genes. A concentration of H2A.Z is also found at insulators under circumstances implying a link to barrier activity but not to enhancer blocking. Although acetylated H2A.Z is widespread throughout the interphase genome, at mitosis its acetylation is erased, the unmodified form remaining. Thus, although H2A.Z may operate as an epigenetic marker for active genes, its N-terminal acetylation does not.

Mammalian centromere function depends upon a specialized chromatin organization where distinct domains of CENP-A and dimethyl K4 histone H3, forming centric chromatin, are uniquely positioned on or near the surface of the chromosome. These distinct domains are embedded in pericentric heterochromatin (characterized by H3 methylated at K9). The mechanisms that underpin this complex spatial organization are unknown. Here, we identify the essential histone variant H2A.Z as a new structural component of the centromere. Along linear chromatin fibers H2A.Z is distributed nonuniformly throughout heterochromatin, and centric chromatin where regions of nucleosomes containing H2A.Z and dimethylated K4 H3 are interspersed between subdomains of CENP-A. At metaphase, using the inactive X chromosome centromere as a model, complex folding of this fiber produces spatially positioned domains where H2A.Z/dimethylated K4 H3 chromatin juxtaposes one side of CENP-A chromatin, whereas a region of H2A/trimethyl K9 H3 borders the other side. A second region of H2A.Z is found, with trimethyl K9 H3 at the inner centromere. We therefore propose that H2A.Z plays an integral role in organizing centromere structure.centromere organization ͉ chromosome structure ͉ histone variants A ll active centromeres contain an evolutionary conserved variant of histone H3 (CENP-A in mammals). Although poorly understood, fundamental to centromere function is the 3D organization of CENP-A containing chromatin, so that it is presented on the poleward face of a chromosome while being embedded in pericentric heterochromatin. This heterochromatin has a role in the cohesion of sister chromatids (1). The complex nature of this organization was demonstrated by showing that, whereas blocks of human CENP-A (and the Drosophila counterpart CID) and H3 nucleosomes (Ϸ15-40 kb) are interspersed along an extended chromatin fiber, the folding of this fiber in mitotic chromosomes yields separate CENP-A-and H3-containing domains, with the H3 domain located toward the inner chromatid region (together, these domains have been referred to as centric chromatin; ref. 2). Most interestingly, this H3-containing domain is enriched with a euchromatic mark, H3 dimethylated at K4 and not K9 H3 methylation associated with the flanking heterochromatin (3). Therefore, it was postulated that this distinct histone modification pattern might contribute to centromere structure and/or identity (3).Our recent studies have demonstrated that another histone variant, H2A.Z, has a role in mitosis (4). When H2A.Z is depleted in mammalian cells, major defects in chromosome segregation (and cytokinesis) are observed with a corresponding loss of HP1␣ from pericentric heterochromatin, indicating this domain is severely disrupted. The mechanism responsible for this defect remains unresolved. Although others (5) and we (6) have shown that the concentration of H2A.Z can dynamically increase at pericentric heterochromatin under different physiological contexts, whether H2A.Z is a core component of this domai...

Plant hybrids are extensively used in agriculture to deliver increases in yields, yet the molecular basis of their superior performance (heterosis) is not well understood. Our transcriptome analysis of a number of Arabidopsis F1 hybrids identified changes to defense and stress response gene expression consistent with a reduction in basal defense levels. Given the reported antagonism between plant immunity and growth, we suggest that these altered patterns of expression contribute to the greater growth of the hybrids. The altered patterns of expression in the hybrids indicate decreases to the salicylic acid (SA) biosynthesis pathway and increases in the auxin [indole-3-acetic acid (IAA)] biosynthesis pathway. SA and IAA are hormones known to control stress and defense responses as well as plant growth. We found that IAA-targeted gene activity is frequently increased in hybrids, correlating with a common heterotic phenotype of greater leaf cell numbers. Reduced SA concentration and target gene responses occur in the larger hybrids and promote increased leaf cell size. We demonstrated the importance of SA action to the hybrid phenotype by manipulating endogenous SA concentrations. Increasing SA diminished heterosis in SA-reduced hybrids, whereas decreasing SA promoted growth in some hybrids and phenocopied aspects of hybrid vigor in parental lines. Pseudomonas syringae infection of hybrids demonstrated that the reductions in basal defense gene activity in these hybrids does not necessarily compromise their ability to mount a defense response comparable to the parents.

Spermatogenesis is a complex sequential process that converts mitotically dividing spermatogonia stem cells into differentiated haploid spermatozoa. Not surprisingly, this process involves dramatic nuclear and chromatin restructuring events, but the nature of these changes are poorly understood. Here, we linked the appearance and nuclear localization of the essential histone variant H2A.Z with key steps during mouse spermatogenesis. H2A.Z cannot be detected during the early stages of spermatogenesis, when the bulk of X-linked genes are transcribed, but its expression begins to increase at pachytene, when meiotic sex chromosome inactivation (MSCI) occurs, peaking at the round spermatid stage. Strikingly, when H2A.Z is present, there is a dynamic nuclear relocalization of heterochromatic marks (HP1␤ and H3 di-and tri-methyl K9), which become concentrated at chromocenters and the inactive XY body, implying that H2A.Z may substitute for the function of these marks in euchromatin. We also show that the X and the Y chromosome are assembled into facultative heterochromatic structures postmeiotically that are enriched with H2A.Z, thereby replacing macroH2A. This indicates that XY silencing continues following MSCI. These results provide new insights into the large-scale changes in the composition and organization of chromatin associated with spermatogenesis and argue that H2A.Z has a unique role in maintaining sex chromosomes in a repressed state.

Heterosis is important for agriculture; however, little is known about the mechanisms driving hybrid vigor. Ultimately, heterosis depends on the interactions of specific alleles and epialleles provided by the parents, which is why hybrids can exhibit different levels of heterosis, even within the same species. We characterize the development of several intraspecific Arabidopsis (Arabidopsis thaliana) F1 hybrids that show different levels of heterosis at maturity. We identify several phases of heterosis beginning during embryogenesis and culminating in a final phase of vegetative maturity and seed production. During each phase, the hybrids show different levels and patterns of growth, despite the close relatedness of the parents. For instance, during the vegetative phases, the hybrids develop larger leaves than the parents to varied extents, and they do so by exploiting increases in cell size and cell numbers in different ratios. Consistent with this finding, we observed changes in the expression of genes known to regulate leaf size in developing rosettes of the hybrids, with the patterns of altered expression differing between combinations. The data show that heterosis is dependent on changes in development throughout the growth cycle of the hybrid, with the traits of mature vegetative biomass and reproductive yield as cumulative outcomes of heterosis at different levels, tissues, and times of development.