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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 heterochrotininduced 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 heterochrotininduced gene silencing. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

FoxP3+ regulatory T cells (Tregs) are a central element of immunological tolerance. FoxP3 is the key determining transcription factor of the Treg lineage, interacting with numerous cofactors and transcriptional targets to determine the many facets of Treg function. Its absence leads to devastating lymphoproliferation and autoimmunity in scurfy mutant mice and immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) patients. To finely map transcriptionally active regions of the protein, with respect to disease-causing variation, we performed a systematic alanine-scan mutagenesis of FoxP3, assessing mutational impacts on DNA binding and transcriptional activation or repression. The mutations affected transcriptional activation and repression in a variegated manner involving multiple regions of the protein and varying between different transcriptional targets of FoxP3. There appeared to be different modalities for target genes related to classic immunosuppressive function vs. those related to atypical or tissue-Treg functions. Relevance to in vivo Treg biology was established by introducing some of the subtle Foxp3 mutations into the mouse germline by CRISPR-based genome editing. The resulting mice showed Treg populations in normal numbers and exhibited no overt autoimmune manifestations. However, Treg functional defects were revealed upon competition or by system stress, manifest as a strikingly heightened susceptibility to provoked colitis, and conversely by greater resistance to tumors. These observations suggest that some of the missense mutations that segregate in human populations, but do not induce IPEX manifestations, may have unappreciated consequences in other diseases. [ABSTRACT FROM AUTHOR]

Maintenance of genome stability is essential for the accurate propagation of genetic information and cell growth and survival. Organisms have therefore developed efficient strategies to prevent DNA lesions and rearrangements. Much of the information concerning these strategies has been obtained through the study of bacterial and nuclear genomes. Comparatively, little is known about how organelle genomes maintain a stable structure. Here, we report that the plastid-localized Whirly ssDNA-binding proteins are required for plastid genome stability in Arabidopsis. We show that a double KO of the genes AtWhy1 and AtWhy3 leads to the appearance of plants with variegated green/white/yellow leaves, symptomatic of nonfunctional chloroplasts. This variegation is maternally inherited, indicating defects in the plastid genome. Indeed, in all variegated lines examined, reorganized regions of plastid DNA are amplified as circular and/or head-tail concatemers. All amplified regions are delimited by short direct repeats of 10-18 bp, strongly suggesting that these regions result from illegitimate recombination between repeated sequences. This type of recombination occurs frequently in plants lacking both Whirlies, to a lesser extent in single KO plants and rarely in WT individuals. Maize mutants for the ZmWhy1 Whirly protein also show an increase in the frequency of illegitimate recombination. We propose a model where Whirlies contribute to plastid genome stability by protecting against illegitimate repeat-mediated recombination. [ABSTRACT FROM AUTHOR]

The CTLA4 gene is important for T lymphocyte-mediated immunoregulation and has been associated with several autoimmune diseases, in particular, type 1 diabetes. To model the impact of natural genetic variants of CTLA4, we constructed RNA interference (RNAi) ‘knockdown’ mice through lentiviral transgenesis. Variegation of expression was observed in founders but proved surmountable because it reflected parental imprinting, with derepression by transmission from male lentigenics. Unlike the indiscriminate multiorgan autoimmune phenotype of the corresponding knockout mice, Ctla4 knockdown animals had a disease primarily focused on the pancreas, with rapid progression to diabetes. As with the human disease, the knockdown phenotype was tempered by genetic-modifier loci. RNAi should be more pertinent than gene ablation in modeling disease pathogenesis linked to a gene-dosage variation. [ABSTRACT FROM AUTHOR]

The Arabidopsis var2 variegation mutant defines a nuclear gene for a chloroplast FtsH metalloprotease. Leaf variegation is expressed only in homozygous recessive plants. The cells in the green leaf sectors of this mutant contain morphologically normal chloroplasts, whereas cells in the white sectors contain abnormal plastids lacking organized lamellar structures. var2 mutants are hypersusceptible to photoinhibition, and VAR2 degrades unassembled polypeptides and is involved in the D1 repair cycle of photosystem II, likely by affecting turnover of the photodamaged D1 polypeptide. A second-site suppressor screen of var2 yielded a normal-appearing, nonvariegated line. Map-based cloning revealed that the suppression of variegation in this line is due to a splice site mutation in ClpC2, a chloroplast Hsp100 chaperone, that results in sharply reduced ClpC2 protein accumulation. Isolation of clpC2 single mutants showed that clpC2 is epistatic to var2, and that a lack of ClpC2 does not markedly alter the composition of the thylakoid membrane. Suppression by clpC2 is not allele-specific. Our results suggest that clpC2 is a suppressor of thylakoid biogenesis and maintenance and that ClpC2 might act by accelerating photooxidative stress. Arabidopsis has two ClpC genes (ClpC1 and ClpC2), and mutants with down-regulated expression of both genes have a phenotype different from clpC2, suggesting that ClpC1 and ClpC2 act synergistically and/or that they are only partially redundant. The isolation of a clpC2 mutant represents an important advance in the generation of tools to understand Hsp100 function and insight into the mechanisms of protein quality control in plants. [ABSTRACT FROM AUTHOR]

Methylation of lysine-79 (K79) within the globular domain of histone H3 by Dot1 methylase is important for transcriptional silencing and for association of the 5Jr silencing proteins in yeast. Here, we show that the level of H3-K79 methylation is low at all Sir-dependent silenced loci but not at other transcriptionally repressed regions. Hypomethylation of H3-K79 at the telomeric and silent mating-type loci, but not the ribosomal DNA, requires the Sir proteins. Overexpression of Sir3 concomitantly extends the domain of Sir protein association and H3-K79 hypomethylation at telomeres. In mammalian cells, H3-K79 methylation is found at loci that are active for V(D)J recombination, but not at recombinationally inactive loci that are heterochromatic. These results suggest that H3-K79 methylation is an evolutionarily conserved marker of active chromatin regions, and that silencing proteins block the ability of Dot1 to methylate histone H3. Further, they suggest that Sir proteins preferentially bind chromatin with hypomethylated H3-K79 and then block H3-K79 methylation. This positive feedback loop, and the reverse loop in which H3-K79 methylation weakens Sir protein association and leads to further methylation, suggests a model for position-effect variegation. [ABSTRACT FROM AUTHOR]