DNA methylation is a conserved epigenetic mark in plants and many animals. How parental alleles interact in progeny to influence the epigenome is poorly understood. We analyzed the DNA methylomes of Arabidopsis Col and C24 ecotypes, and their hybrid progeny. Hybrids displayed nonadditive DNA methylation levels, termed methylation interactions, throughout the genome. Approximately 2,500 methylation interactions occurred at regions where parental DNA methylation levels are similar, whereas almost 1,000 were at differentially methylated regions in parents. Methylation interactions were characterized by an abundance of 24-nt small interfering RNAs. Furthermore, dysfunction of the RNA-directed DNA methylation pathway abolished methylation interactions but did not affect the increased biomass observed in hybrid progeny. Methylation interactions correlated with altered genetic variation within the genome, suggesting that they may play a role in genome evolution.D NA methylation is a conserved epigenetic mark in many eukaryotes (1-6). In plants and mammals, DNA methylation plays important roles in genome stability, genomic imprinting, paramutation, and gene regulation during development and diseases (1-6). Parental genetic alleles interact in the filial 1 (F1) progeny in a Mendelian manner. DNA methylation may affect this interaction such that methylated epialleles may show nonadditive interactions. This notion is supported by recent observations that hybridization results in nonadditive changes in the F1 plant DNA methylome (7,8). It has been proposed that genome-wide interactions between parental epialleles in F1 hybrids are critical for hybrid vigor, the superior performance of hybrids compared with their parents (8-13). Epigenome interactions may confer nonadditive transcriptional and epigenetic activities in hybrid offspring compared with the parental lines. In contrast to additive regulation, which leads to midparent value (MPV) levels equal to the average of the two parental lines, nonadditive regulation results in a deviation from the MPV, such that the F1 hybrid resembles the high-or low-expression parent. In addition, nonadditive regulation can be transgressive, which is beyond the range of the parental levels (14).Studies in rice and Arabidopsis have identified nonadditive changes in DNA methylation levels at loci where parental methylation levels are different [differentially methylated regions (DMRs)] (7,8,11,12,15,16). These "methylation interactions" were attributed to two mechanisms, transchromosomal methylation (TCM) and transchromosomal demethylation (TCdM), whereby the methylation level of one parental allele is altered to resemble the methylation level of the other parental allele (7,8,17). At some genomic loci, the transmethylation events in F1 were shown to be inherited to the next generation, as observed for paramutation (17). Nonetheless, a thorough profile of DNA methylation interactions across the whole Arabidopsis genome has yet to be determined. In particular, it remains unclear whether DNA m...

Recent research suggests that epigenetic alterations involving DNA methylation can be causative for neurodevelopmental, growth and metabolic disorders. Although lymphoblastoid cell lines have been an invaluable resource for the study of both genetic and epigenetic disorders, the impact of EBV transformation, cell culturing and freezing on epigenetic patterns is unknown. We compared genome-wide DNA methylation patterns of four white blood cell samples, four low-passage lymphoblastoid cell lines pre and post freezing and four high-passage lymphobastoid cell lines, using two microarray platforms: Illumina HumanMethylation27 platform containing 27,578 CpG sites and Agilent Human CpG island Array containing 27,800 CpG islands. Comparison of genome-wide methylation profiles between white blood cells and lymphoblastoid cell lines demonstrated methylation alterations in lymphoblastoid cell lines occurring at random genomic locations. These changes were more profound in high-passage cells. Freezing at low-passages did not have a significant effect on DNA methylation. Methylation changes were observed in several imprinted differentially methylated regions, including DIRAS3, NNAT, H19, MEG3, NDN and MKRN3, but not in known imprinting centers. Our results suggest that lymphoblastoid cell lines should be used with caution for the identification of disease-associated DNA methylation changes or for discovery of new imprinted genes, as the methylation patterns seen in these cell lines may not always be representative of DNA methylation present in the original B-lymphocytes of the patient.

BackgroundA number of neurodevelopmental syndromes are caused by mutations in genes encoding proteins that normally function in epigenetic regulation. Identification of epigenetic alterations occurring in these disorders could shed light on molecular pathways relevant to neurodevelopment.ResultsUsing a genome-wide approach, we identified genes with significant loss of DNA methylation in blood of males with intellectual disability and mutations in the X-linked KDM5C gene, encoding a histone H3 lysine 4 demethylase, in comparison to age/sex matched controls. Loss of DNA methylation in such individuals is consistent with known interactions between DNA methylation and H3 lysine 4 methylation. Further, loss of DNA methylation at the promoters of the three top candidate genes FBXL5, SCMH1, CACYBP was not observed in more than 900 population controls. We also found that DNA methylation at these three genes in blood correlated with dosage of KDM5C and its Y-linked homologue KDM5D. In addition, parallel sex-specific DNA methylation profiles in brain samples from control males and females were observed at FBXL5 and CACYBP.ConclusionsWe have, for the first time, identified epigenetic alterations in patient samples carrying a mutation in a gene involved in the regulation of histone modifications. These data support the concept that DNA methylation and H3 lysine 4 methylation are functionally interdependent. The data provide new insights into the molecular pathogenesis of intellectual disability. Further, our data suggest that some DNA methylation marks identified in blood can serve as biomarkers of epigenetic status in the brain.

ORCID IDs: 0000-0002-4656-3189 (H.S.); 0000-0003-0518-5924 (C.Z.); 0000-0001-9320-9628 (W.J.).Anther cuticle and pollen exine are protective barriers for pollen development and fertilization. Despite that several regulators have been identified for anther cuticle and pollen exine development in rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), few genes have been characterized in maize (Zea mays) and the underlying regulatory mechanism remains elusive. Here, we report a novel male-sterile mutant in maize, irregular pollen exine1 (ipe1), which exhibited a glossy outer anther surface, abnormal Ubisch bodies, and defective pollen exine. Using map-based cloning, the IPE1 gene was isolated as a putative glucose-methanolcholine oxidoreductase targeted to the endoplasmic reticulum. Transcripts of IPE1 were preferentially accumulated in the tapetum during the tetrad and early uninucleate microspore stage. A biochemical assay indicated that ipe1 anthers had altered constituents of wax and a significant reduction of cutin monomers and fatty acids. RNA sequencing data revealed that genes implicated in wax and flavonoid metabolism, fatty acid synthesis, and elongation were differentially expressed in ipe1 mutant anthers. In addition, the analysis of transfer DNA insertional lines of the orthologous gene in Arabidopsis suggested that IPE1 and their orthologs have a partially conserved function in male organ development. Our results showed that IPE1 participates in the putative oxidative pathway of C16/C18 v-hydroxy fatty acids and controls anther cuticle and pollen exine development together with MALE STERILITY26 and MALE STERILITY45 in maize.Male sterility is a common biological phenomenon in plants and widely used in the production of hybrid seeds, which can reduce costs and enhance seed purity (Tester and Langridge, 2010). According to inheritance or origin, male sterility includes three types: genic male sterility, cytoplasmic male sterility, and cytoplasmicgenic male sterility (Rhee et al., 2015). The generation of mature pollen grains relies on anther development. The start of anther formation occurs in differentiated flower tissues (floral meristem), which consist of three histogenic layers: L1, L2, and L3. After continuous cell division and differentiation, L1 forms the epidermis and the L3 layer develops into the stomium and vascular bundles. L2 is the most important layer; it undergoes a series of periclinal and anticlinal divisions and eventually grows into the endothecium, the middle layer, the tapetum, and the pollen mother cells. When anther morphogenesis is completed, the anther has centrally localized pollen mother cells enclosed by four somatic layers, which are, from the surface to the interior, the epidermis, endothecium, middle layer, and tapetum. Then, the pollen mother cells undergo meiosis and mitosis, resulting in trinucleate pollen grains, and the endothecium, middle layer, and tapetum are gradually degraded (Goldberg et al., 1993(Goldberg et al., , 1995Ma, 2005).The anther cuticle and poll...

A contribution of DNA methylation to defense against invading nucleic acids and maintenance of genome integrity is uncontested; however, our understanding of the extent of involvement of this epigenetic mark in genome-wide gene regulation and plant developmental control is incomplete. Here, we knock out all five known DNA methyltransferases in Arabidopsis, generating DNA methylation-free plants. This quintuple mutant exhibits a suite of developmental defects, unequivocally demonstrating that DNA methylation is essential for multiple aspects of plant development. We show that CG methylation and non-CG methylation are required for a plethora of biological processes, including pavement cell shape, endoreduplication, cell death, flowering, trichome morphology, vasculature and meristem development, and root cell fate determination. Moreover, we find that DNA methylation has a strong dose-dependent effect on gene expression and repression of transposable elements. Taken together, our results demonstrate that DNA methylation is dispensable for Arabidopsis survival but essential for the proper regulation of multiple biological processes.

Although much progress has been made towards understanding the ripening of non-climacteric fruit using the strawberry as a model plant, the defined molecular mechanisms remain unclear. Here, RNA-sequencing was performed using four cDNA libraries around the onset of ripening, and a total of 31,793 unigenes and 335 pathways were annotated including the top five pathways, which were involved in ribosome, spliceosome, protein processing, plant-pathogen interaction and plant hormone signaling, and the important DEGs related to ripening were annotated to be mainly involved in protein translation and processing, sugar metabolism, energy metabolism, phytohormones, antioxidation, pigment and softening, especially finding a decreased trend of oxidative phosphorylation during red-coloring. VIGS-mediated downregulation of the pyruvate dehydrogenase gene PDHE1α, a key gene for glycolysis-derived oxidative phosphorylation, could inhibit respiration and ATP biosynthesis, whilst promote the accumulation of sugar, ABA, ETH, and PA, ultimately accelerating the ripening. In conclusion, our results demonstrate that a set of metabolism transition occurred during green-to-white-to-red stages that are coupled with more-to-less DEGs, and the oxidative phosphorylation plays an important role in the regulation of ripening. On the basis of our results, we discuss an oxidative phosphorylation-based model underlying strawberry fruit ripening.

In plants, hybrid vigor is influenced by genetic and epigenetic mechanisms; however, the molecular pathways are poorly understood. We investigated the potential contributions of epigenetic regulators to heterosis in Arabidposis and found that the chromatin remodeler DECREASED DNA METHYLATION 1 (DDM1) affects early seedling growth heterosis in Col/C24 hybrids. ddm1 mutants showed impaired heterosis and increased expression of non-additively expressed genes related to salicylic acid metabolism. Interestingly, our data suggest that salicylic acid is a hormetic regulator of seedling growth heterosis, and that hybrid vigor arises from crosses that produce optimal salicylic acid levels. Although DNA methylation failed to correlate with differential non-additively expressed gene expression, we uncovered DDM1 as an epigenetic link between salicylic acid metabolism and heterosis, and propose that the endogenous salicylic acid levels of parental plants can be used to predict the heterotic outcome. Salicylic acid protects plants from pathogens and abiotic stress. Thus, our findings suggest that stress-induced hormesis, which has been associated with increased longevity in other organisms, may underlie specific hybrid vigor traits.

Two recent papers in Nature show that human blastocyst-like structures (or blastoids) can be generated from human pluripotent stem cells (Yu et al 2021) or through reprogramming of fibroblasts (Liu et al 2021), respectively. Both papers perform extensive single cell transcriptional analysis and compare blastoid cells with the cells in preimplantation human embryos, leading to a conclusion that the blastoids contain cell lineages corresponding to the epiblast, primitive endoderm and trophectoderm in preimplantation human embryos. Transcriptional analysis is, however, critically dependent on having relevant reference samples, not only of targeted cell types but also of potential alternative cell lineages. For this reason, we have reevaluated the blastoid data with a more comprehensive cellular reference, including extended cultures of blastocysts, several stem cell-based embryo models and a gastrulation stage human specimen. From this reanalysis we resolve that reprogrammed blastoids by Liu et al. fail to generate cells with trophectoderm profiles. Instead, cells identified as trophectoderm lineages in reprogrammed blastoids possess a transcriptional profile more representative of amniotic cells in post-implantation human embryos. Our reanalysis also shows that stem cell-derived blastoids did contain trophectoderm-like cells, highlighting the potential of human blastoids to model blastocyst development.