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Changes in chromatin state may drive changes in gene expression, and it is of growing interest to understand the population genetic forces that drive differences in chromatin state. Here, we use the phenomenon of position effect variegation (PEV), a well-studied proxy for chromatin state, to survey variation in PEV among a naturally derived population. Further, we explore the genetic architecture of natural variation in factors that modify PEV. While previous mutation screens have identified over 150 suppressors and enhancers of PEV, it remains unknown to what extent allelic variation in these modifiers mediate interindividual variation in PEV. Is natural variation in PEV mediated by segregating genetic variation in known Su(var) and E(var) genes, or is the trait polygenic, with many variants mapping elsewhere in the genome? We designed a dominant mapping study that directly answers this question and suggests that the bulk of the variance in PEV does not map to genes with prior annotated impact to PEV. Instead, we find enrichment of top P-value ranked associations that suggest impact to active promoter and transcription start site proximal regions. This work highlights extensive variation in PEV within a population, and provides a quantitative view of the role naturally segregating autosomal variants play in modifying PEV—a phenomenon that continues to shape our understanding of chromatin state and epigenetics.

The establishment of modern graphics spatial concept is based on the disobeying of traditional perspective space, and it is beyond the limits of space where human beings live. Through the description and analysis of the historic evolution of graphics space as well as all kinds of forms that modern graphics space inheres, this article points out that modern graphics space boasts the characteristics of disobeying, variegation and creativity. Meanwhile, through the comparison and analysis between the logic regularity of reverse thinking and the essence of modern graphic creation, this article indicates that the creativity of modern graphics is based on reverse thinking.

Heterochromatin is characterized by dimethylated or trimethylated histone H3 Lys9 (H3K9me2 or H3K9me3, respectively) and is found at transposable elements, satellite repeats and genes, where it ensures their transcriptional silencing. The histone methyltransferases (HMTs) that methylate H3K9 — in mammals Suppressor of variegation 3–9 homologue 1 (SUV39H1), SUV39H2, SET domain bifurcated 1 (SETDB1), SETDB2, G9A and G9A-like protein (GLP) — and the ‘readers’ of H3K9me2 or H3K9me3 are highly conserved and show considerable redundancy. Despite their redundancy, genetic ablation or mistargeting of an individual H3K9 methyltransferase can correlate with impaired cell differentiation, loss of tissue identity, premature aging and/or cancer. In this Review, we discuss recent advances in understanding the roles of the known H3K9-specific HMTs in ensuring transcriptional homeostasis during tissue differentiation in mammals. We examine the effects of H3K9-methylation-dependent gene repression in haematopoiesis, muscle differentiation and neurogenesis in mammals, and compare them with mechanistic insights obtained from the study of model organisms, notably Caenorhabditis elegans and Drosophila melanogaster. In all these organisms, H3K9-specific HMTs have both unique and redundant roles that ensure the maintenance of tissue integrity by restricting the binding of transcription factors to lineage-specific promoters and enhancer elements.Histone H3 Lys9 (H3K9)-methylated heterochromatin ensures transcriptional silencing of repetitive elements and genes, and its deregulation leads to impaired cell and tissue identity, premature aging and cancer. Recent studies in mammals clarified the roles H3K9-specific histone methyltransferases in ensuring transcriptional homeostasis during tissue differentiation.

Position effect variegation (PEV) in Drosophila results from new juxtapositions of euchromatic and heterochromatic chromosomal regions, and manifests as striking bimodal patterns of gene expression. The semirandom patterns of PEV, reflecting clonal relationships between cells, have been interpreted as gene-expression states that are set in development and thereafter maintained without change through subsequent cell divisions. The rate of instability of PEV is almost entirely unexplored beyond the final expression of the modified gene; thus the origin of the expressivity and patterns of PEV remain unexplained. Many properties of PEV are not predicted from currently accepted biochemical and theoretical models. In this work we investigate the time at which expressivity of silencing is set, and find that it is determined before heterochromatin exists. We employ a mathematical simulation and a corroborating experimental approach to monitor switching (i.e., gains and losses of silencing) through development. In contrast to current views, we find that gene silencing is incompletely set early in embryogenesis, but nevertheless is repeatedly lost and gained in individual cells throughout development. Our data support an alternative to locus-specific “epigenetic” silencing at variegating gene promoters that more fully accounts for the final patterns of PEV.

Pseudouridine (Ψ) is widely distributed in mRNA and various non-coding RNAs in yeast and mammals, and the specificity of its distribution has been determined. However, knowledge about Ψs in the RNAs of plants, particularly in mRNA, is lacking. In this study, we performed genome-wide pseudouridine-sequencing in Arabidopsis and for the first time identified hundreds of Ψ sites in mRNA and multiple Ψ sites in non-coding RNAs. Many predicted and novel Ψ sites in rRNA and tRNA were detected. mRNA was extensively pseudouridylated, but with Ψs being under-represented in 3′-untranslated regions and enriched at position 1 of triple codons. The phenylalanine codon UUC was the most frequently pseudouridylated site. Some Ψs present in chloroplast 23S, 16S, and 4.5S rRNAs in wild-type Col-0 were absent in plants with a mutation of SVR1 (Suppressor of variegation 1), a chloroplast pseudouridine synthase gene. Many plastid ribosomal proteins and photosynthesis-related proteins were significantly reduced in svr1 relative to the wild-type, indicating the roles of SVR1 in chloroplast protein biosynthesis in Arabidopsis. Our results provide new insights into the occurrence of pseudouridine in Arabidopsis RNAs and the biological functions of SVR1, and will pave the way for further exploiting the mechanisms underlying Ψ modifications in controlling gene expression and protein biosynthesis in plants.

Air space-type variegation is the most diverse among the species of known variegated leaf plants and is caused by conspicuous intercellular spaces between the epidermal and palisade cells and among the palisade cells at non-green areas. Trifolium pratense, a species in Fabaceae with V-shaped air space-type variegation, was selected to explore the application potential of variegated leaf plants and accumulate basic data on the molecular regulatory mechanism and evolutionary history of leaf variegation. We performed comparative transcriptome analysis on young and adult leaflets of variegated and green plants and identified 43 candidate genes related to air space-type variegation formation. Most of the genes were related to cell-wall structure modification (CESA, CSL, EXP, FLA, PG, PGIP, PLL, PME, RGP, SKS, and XTH family genes), followed by photosynthesis (LHCB subfamily, RBCS, GOX, and AGT family genes), redox (2OG and GSH family genes), and nitrogen metabolism (NodGS family genes). Other genes were related to photooxidation, protein interaction, and protease degradation systems. The downregulated expression of light-responsive LHCB subfamily genes and the upregulated expression of the genes involved in cell-wall structure modification were important conditions for air space-type variegation formation in T. pratense. The upregulated expression of the ubiquitin-protein ligase enzyme (E3)-related genes in the protease degradation systems were conducive to air space-type variegation formation. Because these family genes are necessary for plant growth and development, the mechanism of the leaf variegation formation in T. pratense might be a widely existing regulation in air space-type variegation in nature.

Reporter genes inserted via P-element integration into different locations of the Drosophila melanogaster genome have been routinely used to monitor the functional state of chromatin domains. It is commonly thought that P-element-derived reporter genes are subjected to position effect variegation (PEV) when transposed into constitutive heterochromatin because they acquire heterochromatin-like epigenetic modifications that promote silencing. However, sequencing and annotation of the D. melanogaster genome have shown that constitutive heterochromatin is a genetically and molecularly heterogeneous compartment. In fact, in addition to repetitive DNAs, it harbors hundreds of functional genes, together accounting for a significant fraction of its entire genomic territory. Notably, most of these genes are actively transcribed in different developmental stages and tissues, irrespective of their location in heterochromatin. An open question in the genetic and molecular studies on PEV in D. melanogaster is whether functional heterochromatin domains, i.e., heterochromatin harboring active genes, are able to silence reporter genes therein transposed or, on the contrary, can drive their expression. In this work, we provide experimental evidence showing that strong silencing of the Pw+ reporters is induced even when they are integrated within or near actively transcribed loci in the pericentric regions of chromosome 2. Interestingly, some Pw+ reporters were found insensitive to the action of a known PEV suppressor. Two of them are inserted within Yeti, a gene expressed in the deep heterochromatin of chromosome 2 which carries active chromatin marks. The difference sensitivity to suppressors-exhibited Pw+ reporters supports the view that different epigenetic regulators or mechanisms control different regions of heterochromatin. Together, our results suggest that there may be more complexity regarding the molecular mechanisms underlying PEV.

In nature, foliar variegation has varied origins, and can be ascribed to two major mechanisms: pigment-related variegation and structural variegation caused by the optical properties of leaf structure. However, understanding of these mechanisms is still lacking, and structural mechanisms are often misinterpreted. In this study, six variegated plants native to Taiwan and two ornamental plants, with novel and unusual foliar variegation patterns, are studied to reveal their mechanisms of variegation. Two newly understood variegation patterns, the silvery white leaf surface of Begonia aptera and the varnish on basal leaflets of Oxalis corymbosa, are reported here. White to light green patches on leaf surfaces characterize the foliar variegation in the other six study species, Nervilia nipponica, O. acetosella subsp. griffithii var. formosana, Paphiopedilum concolor, Selaginella picta, Smilax bracteata subsp. verruculosa and Valeriana hsuii. All six Taiwan native plants exhibit structural variegation, five of which have air space type variegation. Surprisingly the silvery white leaf surface of B. aptera results from numerous sand-like white spots caused by intercellular space. The varnish on leaves of O. corymbosa is the epidermis type of variegation comprised of two newly identified subtypes, larger epidermal cells and thicker outer cell walls. The two ornamental plants have variegation caused by pigments: the chloroplast type from fewer chloroplasts in P. concolor and the chlorophyll type, from absence of chloroplasts in S. picta. This study extends understanding of the mechanisms of natural foliar variegation, illustrating the diversity of mechanisms by which plants may change the appearance of their leaves.

Position-effect variegation (PEV) results from the juxtaposition of euchromatic and heterochromatic components of eukaryotic genomes, silencing genes near the new euchromatin/heterochromatin junctions. Silencing is itself heritable through S phase, giving rise to distinctive random patterns of cell clones expressing the genes intermixed with clones in which the genes are silenced. Much of what we know about epigenetic inheritance in the soma stems from work on PEV aimed at identifying the components of the silencing machinery and its mechanism of inheritance. The roles of two central gene activities—the Su(var)3-9–encoded histone H3–lysine-9 methyltransferase and the Su(var)205-encoded methyl-H3–lysine-9 binding protein heterochromatin protein 1 (HP1a)—have been inferred from terminal phenotypes, leaving considerable gaps in understanding of how PEV behaves through development. Here, we investigate the PEV phenotypes of Su(var)3-9 and Su(var)205 mutations in live developing tissues. We discovered that mutation in Su(var)205 compromises the initial establishment of PEV in early embryogenesis. Later gains of heterochromatin-induced gene silencing are possible but are unstable and lost rapidly. In contrast, a strain with mutation in Su(var)3-9 exhibits robust silencing early in development but fails to maintain it through subsequent cell divisions. Our analyses show that, while the terminal phenotypes of these mutations may appear identical, they have arrived at them through different developmental trajectories. We discuss how our findings expand and clarify existing models for epigenetic inheritance of heterochromatin-induced gene silencing.

Haspin, a highly conserved kinase in eukaryotes, has been shown to be responsible for phosphorylation of histone H3 at threonine 3 (H3T3ph) during mitosis, in mammals and yeast. Here we report that haspin is the kinase that phosphorylates H3T3 in Drosophila melanogaster and it is involved in sister chromatid cohesion during mitosis. Our data reveal that haspin also phosphorylates H3T3 in interphase. H3T3ph localizes in broad silenced domains at heterochromatin and lamin-enriched euchromatic regions. Loss of haspin compromises insulator activity in enhancer-blocking assays and triggers a decrease in nuclear size that is accompanied by changes in nuclear envelope morphology. We show that haspin is a suppressor of position-effect variegation involved in heterochromatin organization. Our results also demonstrate that haspin is necessary for pairing-sensitive silencing and it is required for robust Polycomb-dependent homeotic gene silencing. Haspin associates with the cohesin complex in interphase, mediates Pds5 binding to chromatin and cooperates with Pds5-cohesin to modify Polycomb-dependent homeotic transformations. Therefore, this study uncovers an unanticipated role for haspin kinase in genome organization of interphase cells and demonstrates that haspin is required for homeotic gene regulation.