A Kinesin-14 Motor Activates Neocentromeres to Promote Meiotic Drive in Maize.

Maize abnormal chromosome 10 (Ab10) encodes a classic example of true meiotic drive that converts heterochromatic regions called knobs into motile neocentromeres that are preferentially transmitted to egg cells. Here, we identify a cluster of eight genes on Ab10, called the Kinesin driver (Kindr) complex, that are required for both neocentromere motility and preferential transmission. Two meiotic drive mutants that lack neocentromere activity proved to be kindr epimutants with increased DNA methylation across the entire gene cluster. RNAi of Kindr induced a third epimutant and corresponding loss of meiotic drive. Kinesin gliding assays and immunolocalization revealed that KINDR is a functional minus-end-directed kinesin that localizes specifically to knobs containing 180 bp repeats. Sequence comparisons suggest that Kindr diverged from a Kinesin-14A ancestor ∼12 mya and has driven the accumulation of > 500 Mb of knob repeats and affected the segregation of thousands of genes linked to knobs on all 10 chromosomes.

Metastable epialleles in humans.

First identified in isogenic mice, metastable epialleles (MEs) are loci where the extent of DNA methylation (DNAm) is variable between individuals but correlates across tissues derived from different germ layers within a given individual. This property, termed systemic interindividual variation (SIV), is attributed to stochastic methylation establishment before germ layer differentiation. Evidence suggests that some putative human MEs are sensitive to environmental exposures in early development. In this review we introduce key concepts pertaining to human MEs, describe methods used to identify MEs in humans, and review their genomic features. We also highlight studies linking DNAm at putative human MEs to early environmental exposures and postnatal (including disease) phenotypes.

Plant Transgenerational Epigenetics.

Transgenerational epigenetics is defined in opposition to developmental epigenetics and implies an absence of resetting of epigenetic states between generations. Unlike mammals, plants appear to be particularly prone to this type of inheritance. In this review, we summarize our knowledge about transgenerational epigenetics in plants, which entails heritable changes in DNA methylation. We emphasize the role of transposable elements and other repeat sequences in the creation of epimutable alleles. We also argue that because reprogramming of DNA methylation across generations seems limited in plants, the inheritance of DNA methylation defects results from the failure to reinforce rather than reset this modification during sexual reproduction. We compare genome-wide assessments of heritable DNA methylation variation and its phenotypic impact in natural populations to those made using near-isogenic populations derived from crosses between parents with experimentally induced DNA methylation differences. Finally, we question the role of the environment in inducing transgenerational epigenetic variation and briefly present theoretical models under which epimutability is expected to be selected for.

Genetic and epigenetic control of the plant metabolome.

Plant metabolites are mainly produced through chemical reactions catalysed by enzymes encoded in the genome. Mutations in enzyme-encoding or transcription factor-encoding genes can alter the metabolome by changing the enzyme's catalytic activity or abundance, respectively. Insertion of transposable elements into non-coding regions has also been reported to affect transcription and ultimately metabolite content. In addition to genetic mutations, transgenerational epigenetic variations have also been found to affect metabolic content by controlling the transcription of metabolism-related genes. However, the majority of cases reported so far, in which epigenetic mechanisms are associated with metabolism, are non-transgenerational, and are triggered by developmental signals or environmental stress. Although, accumulating research has provided evidence of strong genetic control of the metabolome, epigenetic control has been largely untouched. Here, we provide a review of the genetic and epigenetic control of metabolism with a focus on epigenetics. We discuss both transgenerational and non-transgenerational epigenetic marks regulating metabolism as well as prospects of the field of metabolic control where intricate interactions between genetics and epigenetics are involved.

Natural variation in DNA methylation homeostasis and the emergence of epialleles.

In plants and mammals, DNA methylation plays a critical role in transcriptional silencing by delineating heterochromatin from transcriptionally active euchromatin. A homeostatic balance between heterochromatin and euchromatin is essential to genomic stability. This is evident in many diseases and mutants for heterochromatin maintenance, which are characterized by global losses of DNA methylation coupled with localized ectopic gains of DNA methylation that alter transcription. Furthermore, we have shown that genome-wide methylation patterns in Arabidopsis thaliana are highly stable over generations, with the exception of rare epialleles. However, the extent to which natural variation in the robustness of targeting DNA methylation to heterochromatin exists, and the phenotypic consequences of such variation, remain to be fully explored. Here we describe the finding that heterochromatin and genic DNA methylation are highly variable among 725 A. thaliana accessions. We found that genic DNA methylation is inversely correlated with that in heterochromatin, suggesting that certain methylation pathway(s) may be redirected to genes upon the loss of heterochromatin. This redistribution likely involves a feedback loop involving the DNA methyltransferase, CHROMOMETHYLASE 3 (CMT3), H3K9me2, and histone turnover, as highly expressed, long genes with a high density of CMT3-preferred CWG sites are more likely to be methylated. Importantly, although the presence of CG methylation in genes alone may not affect transcription, genes containing CG methylation are more likely to become methylated at non-CG sites and silenced. These findings are consistent with the hypothesis that natural variation in DNA methylation homeostasis may underlie the evolution of epialleles that alter phenotypes.

KRAB zinc finger protein diversification drives mammalian interindividual methylation variability.

Most transposable elements (TEs) in the mouse genome are heavily modified by DNA methylation and repressive histone modifications. However, a subset of TEs exhibit variable methylation levels in genetically identical individuals, and this is associated with epigenetically conferred phenotypic differences, environmental adaptability, and transgenerational epigenetic inheritance. The evolutionary origins and molecular mechanisms underlying interindividual epigenetic variability remain unknown. Using a repertoire of murine variably methylated intracisternal A-particle (VM-IAP) epialleles as a model, we demonstrate that variable DNA methylation states at TEs are highly susceptible to genetic background effects. Taking a classical genetics approach coupled with genome-wide analysis, we harness these effects and identify a cluster of KRAB zinc finger protein (KZFP) genes that modifies VM-IAPs in trans in a sequence-specific manner. Deletion of the cluster results in decreased DNA methylation levels and altered histone modifications at the targeted VM-IAPs. In some cases, these effects are accompanied by dysregulation of neighboring genes. We find that VM-IAPs cluster together phylogenetically and that this is linked to differential KZFP binding, suggestive of an ongoing evolutionary arms race between TEs and this large family of epigenetic regulators. These findings indicate that KZFP divergence and concomitant evolution of DNA binding capabilities are mechanistically linked to methylation variability in mammals, with implications for phenotypic variation and putative paradigms of mammalian epigenetic inheritance.

Genomic and functional variation of human centromeres.

Centromeres are central to chromosome segregation and genome stability, and thus their molecular foundations are important for understanding their function and the ways in which they go awry. Human centromeres typically form at large megabase-sized arrays of alpha satellite DNA for which there is little genomic understanding due to its repetitive nature. Consequently, it has been difficult to achieve genome assemblies at centromeres using traditional next generation sequencing approaches, so that centromeres represent gaps in the current human genome assembly. The role of alpha satellite DNA has been debated since centromeres can form, albeit rarely, on non-alpha satellite DNA. Conversely, the simple presence of alpha satellite DNA is not sufficient for centromere function since chromosomes with multiple alpha satellite arrays only exhibit a single location of centromere assembly. Here, we discuss the organization of human centromeres as well as genomic and functional variation in human centromere location, and current understanding of the genomic and epigenetic mechanisms that underlie centromere flexibility in humans.

Application of 5-Methylcytosine DNA Glycosylase to the Quantitative Analysis of DNA Methylation.

In higher eukaryotes DNA methylation is a prominent epigenetic mark important for chromatin structure and gene expression. Thus, profiling DNA methylation is important for predicting gene expressions associated with specific traits or diseases. DNA methylation is achieved by DNA methyltransferases and can be actively removed by specific enzymes in a replication-independent manner. DEMETER (DME) is a bifunctional 5-methylcytosine (5mC) DNA glycosylase responsible for active DNA demethylation that excises 5mC from DNA and cleaves a sugar-phosphate bond generating a single strand break (SSB). In this study, DME was used to analyze DNA methylation levels at specific epialleles accompanied with gain or loss of DNA methylation. DME treatment on genomic DNA generates SSBs in a nonsequence-specific fashion proportional to 5mC density, and thus DNA methylation levels can be easily measured when combined with the quantitative PCR (qPCR) method. The DME-qPCR analysis was applied to measure DNA methylation levels at the FWA gene in late-flowering Arabidopsis mutants and the CNR gene during fruit ripening in tomato. Differentially methylated epialleles were successfully distinguished corresponding to their expression levels and phenotypes. DME-qPCR is proven a simple yet effective method for quantitative DNA methylation analysis, providing advantages over current techniques based on methylation-sensitive restriction digestion.

The Underlying Nature of Epigenetic Variation: Origin, Establishment, and Regulatory Function of Plant Epialleles.

In plants, the gene expression and associated phenotypes can be modulated by dynamic changes in DNA methylation, occasionally being fixed in certain genomic loci and inherited stably as epialleles. Epiallelic variations in a population can occur as methylation changes at an individual cytosine position, methylation changes within a stretch of genomic regions, and chromatin changes in certain loci. Here, we focus on methylated regions, since it is unclear whether variations at individual methylated cytosines can serve any regulatory function, and the evidence for heritable chromatin changes independent of genetic changes is limited. While DNA methylation is known to affect and regulate wide arrays of plant phenotypes, most epialleles in the form of methylated regions have not been assigned any biological function. Here, we review how epialleles can be established in plants, serve a regulatory function, and are involved in adaptive processes. Recent studies suggest that most epialleles occur as byproducts of genetic variations, mainly from structural variants and Transposable Element (TE) activation. Nevertheless, epialleles that occur spontaneously independent of any genetic variations have also been described across different plant species. Here, we discuss how epialleles that are dependent and independent of genetic architecture are stabilized in the plant genome and how methylation can regulate a transcription relative to its genomic location.

Fruit development and epigenetic modifications.

Fruit development is a complex process that is regulated not only by plant hormones and transcription factors, but also requires epigenetic modifications. Epigenetic modifications include DNA methylation, histone post-translational modifications, chromatin remodeling and noncoding RNAs. Together, these epigenetic modifications, which are controlled during development and in response to the environment, determine the chromatin state of genes and contribute to the transcriptomes of an organism. Recent studies have demonstrated that epigenetic regulation plays an important role in fleshy fruit ripening. Dysfunction of a DNA demethylase delayed ripening in tomato, and the application of a DNA methylation inhibitor altered ripening process in the fruits of several species. These studies indicated that manipulating the epigenome of fruit crops could open new ways for breeding in the future. In this review, we highlight recent progress and address remaining questions and challenges concerning the epigenetic regulation of fruit development and ripening.

Multi-omics association analysis revealed the role and mechanism of epialleles in environmental adaptive evolution of Arabidopsis thaliana.

Epialleles, generally referring to alleles whose expression is altered due to differential DNA methylation levels, have important roles in plant morphology, development, and various physiological processes. However, the influence of environmental factors on the plant epialleles under natural conditions is unclear. Meanwhile, the role and mechanism of epialleles in the environmental adaptive evolution of plants remain elusive. In this study, we collected the transcriptome, methylome and climate data from 623 Arabidopsis accessions, derived from worldwide distributions. Then the data were subject to multi-omics association analysis combined with protein interaction network and gene enrichment analysis to identify epialleles related to specific environmental factors and to explore their possible mechanisms involved in the environmental adaptive evolution of Arabidopsis thaliana. We focused on spring and summer precipitation and identified five potential epialleles with differential DNA methylation levels located in specific regions of the genes: AGL36, AT2G34100, AT4G09360, LSU4 and AT5G56910. Interestingly, the imprinted gene AGL36 related to seed development was discovered as an epiallele involved in the environmental adaptive evolution of Arabidopsis thaliana, and the other four genes are related to the response to biotic stress. By protein interaction, GO enrichment, and KEGG pathway analysis, we also found that LSU4 may participate in the sulfur metabolism network like other members of the LSU gene family, and be involved in the biotic stress response by affecting glucosinolate metabolism. In natural conditions low precipitation may affect the severity of local pests and diseases. Therefore, we speculate that DNA methylation associates with the expression of the four genes to regulate the resistance of Arabidopsis thaliana to local pests and diseases, and ultimately participates in the adaptation to local environments.

Alpha satellite DNA biology: finding function in the recesses of the genome.

Repetitive DNA, formerly referred to by the misnomer "junk DNA," comprises a majority of the human genome. One class of this DNA, alpha satellite, comprises up to 10% of the genome. Alpha satellite is enriched at all human centromere regions and is competent for de novo centromere assembly. Because of the highly repetitive nature of alpha satellite, it has been difficult to achieve genome assemblies at centromeres using traditional next-generation sequencing approaches, and thus, centromeres represent gaps in the current human genome assembly. Moreover, alpha satellite DNA is transcribed into repetitive noncoding RNA and contributes to a large portion of the transcriptome. Recent efforts to characterize these transcripts and their function have uncovered pivotal roles for satellite RNA in genome stability, including silencing "selfish" DNA elements and recruiting centromere and kinetochore proteins. This review will describe the genomic and epigenetic features of alpha satellite DNA, discuss recent findings of noncoding transcripts produced from distinct alpha satellite arrays, and address current progress in the functional understanding of this oft-neglected repetitive sequence. We will discuss unique challenges of studying human satellite DNAs and RNAs and point toward new technologies that will continue to advance our understanding of this largely untapped portion of the genome.

Transposable element insertions shape gene regulation and melanin production in a fungal pathogen of wheat.

BACKGROUND: Fungal plant pathogens pose major threats to crop yield and sustainable food production if they are highly adapted to their host and the local environment. Variation in gene expression contributes to phenotypic diversity within fungal species and affects adaptation. However, very few cases of adaptive regulatory changes have been reported in fungi and the underlying mechanisms remain largely unexplored. Fungal pathogen genomes are highly plastic and harbor numerous insertions of transposable elements, which can potentially contribute to gene expression regulation. In this work, we elucidated how transposable elements contribute to variation in melanin accumulation, a quantitative trait in fungi that affects survival under stressful conditions. RESULTS: We demonstrated that differential transcriptional regulation of the gene encoding the transcription factor Zmr1, which controls expression of the genes in the melanin biosynthetic gene cluster, is responsible for variation in melanin accumulation in the fungal plant pathogen Zymoseptoria tritici. We show that differences in melanin levels between two strains of Z. tritici are due to two levels of transcriptional regulation: (1) variation in the promoter sequence of Zmr1 and (2) an insertion of transposable elements upstream of the Zmr1 promoter. Remarkably, independent insertions of transposable elements upstream of Zmr1 occurred in 9% of Z. tritici strains from around the world and negatively regulated Zmr1 expression, contributing to variation in melanin accumulation. CONCLUSIONS: Our studies identified two levels of transcriptional control that regulate the synthesis of melanin. We propose that these regulatory mechanisms evolved to balance the fitness costs associated with melanin production against its positive contribution to survival in stressful environments.

Epiallelic changes in known stress-responsive genes under extreme drought conditions in Brassica juncea (L.) Czern.

KEY MESSAGE: Under severe drought conditions, Brassica juncea shows differential methylation and demethylation events, such that certain epialleles are silenced and some are activated. The plant employed avoidance strategy by delaying apoptosis through the activation of several genes. Harsh environmental conditions pose serious threat to normal growth and development of crops, sometimes leading to their death. However, plants have developed an essential mechanism of modulation of gene activities by epigenetic modifications. Brassica juncea is an important oilseed crop contributing effectively to the economy of India. In the present investigation, we studied the changes in the methylation level of various stress-responsive genes of B. juncea variety RH30 by methylation-dependent immune-precipitation-chip in response to severe drought. On the basis of changes in the number of differential methylation regions in response to drought, the promoter regions were designated as hypermethylated and hypomethylated. Gene body methylation increased in all the genes, whereas promoter methylation was dependent on the function of the gene. Overall, the genes responsible for delaying apoptosis were hypomethylated and many genes responsible for normal routine activities were hypermethylated at promoter regions, thereby suggesting that these may be suspending the activities under harsh conditions.

Hemizygosity for SMCHD1 in Facioscapulohumeral Muscular Dystrophy Type 2: Consequences for 18p Deletion Syndrome.

Facioscapulohumeral muscular dystrophy (FSHD) is most often associated with variegated expression in somatic cells of the normally repressed DUX4 gene within the D4Z4-repeat array. The most common form, FSHD1, is caused by a D4Z4-repeat array contraction to a size of 1-10 units (normal range 10-100 units). The less common form, FSHD2, is characterized by D4Z4 CpG hypomethylation and is most often caused by loss-of-function mutations in the structural maintenance of chromosomes hinge domain 1 (SMCHD1) gene on chromosome 18p. The chromatin modifier SMCHD1 is necessary to maintain a repressed D4Z4 chromatin state. Here, we describe two FSHD2 families with a 1.2-Mb deletion encompassing the SMCHD1 gene. Numerical aberrations of chromosome 18 are relatively common and the majority of 18p deletion syndrome (18p-) cases have, such as these FSHD2 families, only one copy of SMCHD1. Our finding therefore raises the possibility that 18p- cases are at risk of developing FSHD. To address this possibility, we combined genome-wide array analysis data with D4Z4 CpG methylation and repeat array sizes in individuals with 18p- and conclude that approximately 1:8 18p- cases might be at risk of developing FSHD.

Epigenetic mechanism causes Wnt9b deficiency and nonsyndromic cleft lip and palate in the A/WySn mouse strain.

BACKGROUND: The heritable multifactorial etiology of human nonsyndromic cleft lip with or without cleft palate (CL ± P) is not understood. CL ± P occurs in 15% of neonates in the homozygous A/WySn mouse strain, with a multifactorial genetic etiology, the clf1 and clf2 variant genes. Clf1 acts as a mutant allele of Wnt9b but its coding sequence is normal. An IAP (intracisternal A particle) retrotransposon inserted near the Wnt9b gene is associated with clf1. METHODS: Transcription of noncoding sequence between the IAP and the Wnt9b gene was examined in A/WySn embryos. The levels of Wnt9b transcript and of an "IAP antisense" transcript initiated in the IAP and extending into the noncoding interval were assayed in A/WySn and C57BL/6J whole embryos or heads across embryonic days 8 to 12. Methylation of the 5' LTR of the IAP was examined in E12 A/WySn embryo heads. RESULTS: Mean Wnt9b transcript levels were lower in A/WySn than in C57BL/6J at all ages examined and lower in CL ± P embryos than in their normal littermates. The "IAP antisense" transcript was found in all A/WySn embryos and was highest in CL ± P embryos. The IAP at Wnt9b was generally unmethylated in CL ± P embryos and approximately 50% methylated in normal littermates. CONCLUSION: The clf1 mutation in A/WySn is a "metastable epiallele", in which stochastic deficiency in some individuals of DNA methylation of a retrotransposon uniquely inserted near the Wnt9b gene allows transcriptional activity of the retrotransposon and interference with transcription from Wnt9b. Methylation of metastable epialleles should be investigated in human nonsyndromic CL ± P.

Establishing epigenetic variation during genome reprogramming.

Transgenerational reprogramming of DNA methylation is important for transposon silencing and epigenetic inheritance. A stochastic regulation of methylation states in the germline may lead to epigenetic variation and the formation of epialleles that contribute to phenotypic variation. In Arabidopsis thaliana inbred lines, the frequency of single base variation of DNA methylation is much higher than genetic mutation and, interestingly, variable epialleles are pre-methylated in the male germline. However, these same alleles are targeted for demethylation in the pollen vegetative nucleus, by a mechanism that seems to contribute to the accumulation of small RNAs that reinforce transcriptional gene silencing in the gametes. These observations are paving the way toward understanding the extent of epigenetic reprogramming in higher plants, and the mechanisms regulating the stability of acquired epigenetic states across generations.

An epi-allele of SMS causes Sanming dominant genic male sterility in rice.

Male sterility is an important trait in rice for hybrid rice (Oryza sativa) breeding. However, the factors involved in dominant male sterility are largely unknown. Here, we identified a gene from Sanming dominant genic male sterile rice, named Sanming dominant male sterility (SMS), and reported that an epi-allele of this locus contributes to male sterility. Segregation analysis attributed dominant male sterility to a single locus, SMS, which we characterized using a male-sterile near isogenic line (NIL) of rice cultivar 93-11. The SMS locus was heterozygous in the male-sterile 93-11 NIL, containing an epi-allele identical to that in 93-11, and an epi-allele identical to that in rice cultivar Nipponbare, which we refer to as SMS9 and SMSN, respectively. SMS9 is silent and hyper-methylated, whereas SMSN is expressed and hypo-methylated in the 93-11 NIL. Overexpressing SMSN led to male sterility. Mutations in SMS rescued the male sterility of the 93-11 NIL. Interestingly, we observed the duplication of SMSN in Nipponbare, but did not observe the duplication of SMS9 in 93-11. Together, these findings suggest that the reduced methylation and enhanced expression of the SMSN epi-allele in the 93-11 NIL is responsible for its role in conferring dominant male sterility.

Corrigendum: Will epigenetics be a key player in crop breeding?

[This corrects the article DOI: 10.3389/fpls.2022.958350.].

Epigenome editing: targeted manipulation of epigenetic modifications in plants.

BACKGROUND: Epigenetic modifications play important roles in diverse cellular processes such as X chromosome inactivation, cell differentiation, development and senescence. DNA methylation and histone modifications are major epigenetic modifications that regulate chromatin structure and gene expression without DNA sequence changes. Epigenetic alterations may induce phenotypic changes stable enough for mitotic or meiotic inheritance. Moreover, the reversibility of epigenetic marks makes the manipulation of chromatin and epigenetic signature an attractive strategy for therapeutic and breeding purposes. Targeted epigenetic manipulation, or epigenome editing, at the gene of interest commonly utilizes specific epigenetic modifiers fused with a targeting module of the conventional genome editing system. OBJECTIVE: This review aims to summarize essential epigenetic components and introduce currently available epigenetic mutants and the corresponding epialleles in plants. Furthermore, advances in epigenome editing technology are discussed while proposing its potential application to plant breeding. CONCLUSIONS: Epimutations associated with useful traits may provide a valuable resource for crop development. It is important to explore epimutations in a variety of crop species while understanding the fundamental aspects of epigenetic regulation of agronomically important traits such as yield, quality, disease resistance and stress tolerance. In the end, plant breeding programs through epigenome editing may help not only to expand the use of limited genetic resources but also to alleviate consumers' concerns about genetically manipulated crops.