Ian K. Greaves

Changes in 24-nt siRNA levels in Arabidopsis hybrids suggest an epigenetic contribution to hybrid vigor

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.

RNA interference demonstrates a novel role for H2A.Z in chromosome segregation

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 HP1α on the arms of chromosomes but not at centromeric regions. When H2A.Z is depleted, normal HP1α-chromatin interactions are disrupted on the chromosomal arms and, notably, also at pericentric regions. Therefore, H2A.Z controls the localization of HP1α. We conclude that H2A.Z is essential for the accurate transmission of chromosomes.

H2A.Z contributes to the unique 3D structure of the centromere.

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.

The replacement histone H2A.Z in a hyperacetylated form is a feature of active genes in the chicken

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.

Trans Chromosomal Methylation in Arabidopsis hybrids

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 mCG generally increased and mCHH 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.

The X and Y Chromosomes Assemble into H2A.Z, Containing Facultative Heterochromatin, following Meiosis

ABSTRACT 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 (HP1β 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.

The role of epigenetics in hybrid vigour

Hybrid vigour, or heterosis, refers to the increased yield and biomass of hybrid offspring relative to the parents. Although this has been exploited in plants for agriculture and horticulture, the molecular and cellular mechanisms underlying hybrid vigour are largely unknown. Genetic analyses show that there are a large number of quantitative trait loci (QTLs) that contribute to the heterotic phenotype, indicating that it is a complex phenomenon. Gene expression in hybrids is regulated by the interactions of the two parental epigenetic systems and the underlying genomes. Increasing understanding of the interplay of small RNA (sRNA) molecules, DNA methylation, and histone marks provides new opportunities to define the basis of hybrid vigour and to understand why F1 heterosis is not passed on to subsequent generations. We discuss recent findings that suggest the existence of several pathways that alter DNA methylation patterns, which may lead to transcriptional changes resulting in the heterotic phenotype.

Specific patterns of histone marks accompany X chromosome inactivation in a marsupial

The inactivation of one of the two X chromosomes in female placental mammals represents a remarkable example of epigenetic silencing. X inactivation occurs also in marsupial mammals, but is phenotypically different, being incomplete, tissue-specific and paternal. Paternal X inactivation occurs also in the extraembryonic cells of rodents, suggesting that imprinted X inactivation represents a simpler ancestral mechanism. This evolved into a complex and random process in placental mammals under the control of the XIST gene, involving notably variant and modified histones. Molecular mechanisms of X inactivation in marsupials are poorly known, but occur in the absence of an XIST homologue. We analysed the specific pattern of histone modifications using immunofluorescence on metaphasic chromosomes of a model kangaroo, the tammar wallaby. We found that all active marks are excluded from the inactive X in marsupials, as in placental mammals, so this represents a common feature of X inactivation throughout mammals. However, we were unable to demonstrate the accumulation of inactive histone marks, suggesting some fundamental differences in the molecular mechanism of X inactivation between marsupial and placental mammals. A better understanding of the epigenetic mechanisms underlying X inactivation in marsupials will provide important insights into the evolution of this complex process.

Conservation of chromosome arrangement and position of the X in mammalian sperm suggests functional significance

We used chromosome painting to show directly that chromosomes occupy fixed positions in the nuclei of mammal but not chicken sperm. We found that the positions of homologous chromosomes are conserved in sperm of two marsupial species that diverged 50–60 million years ago. We also discovered that the X chromosome lies in the region that makes first contact with the egg in marsupial and monotreme mammals, as well as eutherians, and suggest that this position may be related to its propensity for inactivation, and its high rate of loss from ICSI embryos. We propose that nuclear architecture in sperm is important for spatial chromatin differentiation and normal development of the fertilized egg, and evolved along with mammal-specific regulatory systems such as X inactivation and genomic imprinting.

Epigenetics in plants-vernalisation and hybrid vigour

In this review we have analysed two major biological systems involving epigenetic control of gene activity. In the first system we demonstrate the interplay between genetic and epigenetic controls over the transcriptional activity of FLC, a major repressor of flowering in Arabidopsis. FLC is down-regulated by low temperature treatment (vernalisation) releasing the repressor effect on flowering. We discuss the mechanisms of the reduced transcription and the memory of the vernalisation treatment through vegetative development. We also discuss the resetting of the repressed activity level of the FLC gene, following vernalisation, to the default high activity level and show it occurs during both male and female gametogenesis but with different timing in each. In the second part of the review discussed the complex multigenic system which is responsible for the patterns of gene activity which bring about hybrid vigour in crosses between genetically similar but epigenetically distinct parents. The epigenetic systems that we have identified as contributing to the heterotic phenotype are the 24nt siRNAs and their effects on RNA dependent DNA methylation (RdDM) at the target loci leading to changed expression levels. We conclude that it is likely that epigenetic controls are involved in expression systems in many aspects of plant development and plant function.