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DNA methylation plays crucial roles in responses to environmental stimuli. Modification of DNA methylation during development and abiotic stress responses has been confirmed in increasingly numbers of plants, mainly annual plants. However, the epigenetic regulation mechanism underlying the immune response to pathogens remains largely unknown in plants, especially trees. To investigate whether DNA methylation is involved in the response to infection process or is related to the resistance differences among poplars, we performed comprehensive whole-genome bisulfite sequencing of the infected stem of the susceptible type Populus × euramerican ‘74/76’ and resistant type Populus tomentosa ‘henan’ upon Lonsdalea populi infection. The results revealed that DNA methylation changed dynamically in poplars during the infection process with a remarkable decrease seen in the DNA methylation ratio. Intriguingly, the resistant Populus tomentosa ‘henan’ had a much lower basal DNA methylation ratio than the susceptible Populus × euramerican ‘74/76’. Compared to mock-inoculation, both poplar types underwent post-inoculation CHH hypomethylation, however, significant decreases in mC and mCHH proportions were found in resistant poplar. In addition, most differentially CHH-hypomethylated regions were distributed in repeat and promoter regions. Based on comparison of DNA methylation modification with the expression profiles of genes, DNA methylation occurred in resistance genes, pathogenesis-related genes, and phytohormone genes in poplars during pathogen infection. Additionally, transcript levels of genes encoding methylation-related enzymes changed during pathogen infection. Interestingly, small-regulator miRNAs were subject to DNA methylation in poplars experiencing pathogen infection. This investigation highlights the critical role of DNA methylation in the poplar immune response to pathogen infection and provides new insights into epigenetic regulation in perennial plants in response to biotic stress.

DNA methylation plays crucial roles in responses to environmental stimuli. Modification of DNA methylation during development and abiotic stress responses has been confirmed in increasingly numbers of plants, mainly annual plants. However, the epigenetic regulation mechanism underlying the immune response to pathogens remains largely unknown in plants, especially trees. To investigate whether DNA methylation is involved in the response to infection process or is related to the resistance differences among poplars, we performed comprehensive whole-genome bisulfite sequencing of the infected stem of the susceptible type Populus × euramerican ‘74/76’ and resistant type Populus tomentosa ‘henan’ upon Lonsdalea populi infection. The results revealed that DNA methylation changed dynamically in poplars during the infection process with a remarkable decrease seen in the DNA methylation ratio. Intriguingly, the resistant Populus tomentosa ‘henan’ had a much lower basal DNA methylation ratio than the susceptible Populus × euramerican ‘74/76’. Compared to mock-inoculation, both poplar types underwent post-inoculation CHH hypomethylation, however, significant decreases in mC and mCHH proportions were found in resistant poplar. In addition, most differentially CHH-hypomethylated regions were distributed in repeat and promoter regions. Based on comparison of DNA methylation modification with the expression profiles of genes, DNA methylation occurred in resistance genes, pathogenesis-related genes, and phytohormone genes in poplars during pathogen infection. Additionally, transcript levels of genes encoding methylation-related enzymes changed during pathogen infection. Interestingly, small-regulator miRNAs were subject to DNA methylation in poplars experiencing pathogen infection. This investigation highlights the critical role of DNA methylation in the poplar immune response to pathogen infection and provides new insights into epigenetic regulation in perennial plants in response to biotic stress.

DNA methylation plays crucial roles in responses to environmental stimuli. Modification of DNA methylation during development and abiotic stress responses has been confirmed in increasingly numbers of plants, mainly annual plants. However, the epigenetic regulation mechanism underlying the immune response to pathogens remains largely unknown in plants, especially trees. To investigate whether DNA methylation is involved in the response to infection process or is related to the resistance differences among poplars, we performed comprehensive whole-genome bisulfite sequencing of the infected stem of the susceptible type Populus × euramerican ‘74/76’ and resistant type Populus tomentosa ‘henan’ upon Lonsdalea populi infection. The results revealed that DNA methylation changed dynamically in poplars during the infection process with a remarkable decrease seen in the DNA methylation ratio. Intriguingly, the resistant Populus tomentosa ‘henan’ had a much lower basal DNA methylation ratio than the susceptible Populus × euramerican ‘74/76’. Compared to mock-inoculation, both poplar types underwent post-inoculation CHH hypomethylation, however, significant decreases in mC and mCHH proportions were found in resistant poplar. In addition, most differentially CHH-hypomethylated regions were distributed in repeat and promoter regions. Based on comparison of DNA methylation modification with the expression profiles of genes, DNA methylation occurred in resistance genes, pathogenesis-related genes, and phytohormone genes in poplars during pathogen infection. Additionally, transcript levels of genes encoding methylation-related enzymes changed during pathogen infection. Interestingly, small-regulator miRNAs were subject to DNA methylation in poplars experiencing pathogen infection. This investigation highlights the critical role of DNA methylation in the poplar immune response to pathogen infection and provides new insights into epigenetic regulation in perennial plants in response to biotic stress.

<p>DNA methylation plays crucial roles in responses to environmental stimuli. Modification of DNA methylation during development and abiotic stress responses has been confirmed in increasing numbers of plants, mainly annual plants. However, the epigenetic regulation mechanism underlying the immune response to pathogens remains largely unknown in plants, especially trees. To investigate whether DNA methylation is involved in the response to infection process or is related to the resistance differences among poplars, we performed comprehensive whole-genome bisulfite sequencing of the infected stem of the susceptible type Populus × euramerican ‘74/76’ and resistant type Populus tomentosa ‘henan’ upon Lonsdalea populi infection. The results revealed that DNA methylation changed dynamically in poplars during the infection process with a remarkable decrease seen in the DNA methylation ratio. Intriguingly, the resistant P. tomentosa ‘henan’ had a much lower basal DNA methylation ratio than the susceptible P. × euramerican ‘74/76’. Compared to mock-inoculation, both poplar types underwent post-inoculation CHH hypomethylation; however, significant decreases in mC and mCHH proportions were found in resistant poplar. In addition, most differentially CHH-hypomethylated regions were distributed in repeat and promoter regions. Based on comparison of DNA methylation modification with the expression profiles of genes, DNA methylation occurred in resistance genes, pathogenesis-related genes, and phytohormone genes in poplars during pathogen infection. Additionally, transcript levels of genes encoding methylation-related enzymes changed during pathogen infection. Interestingly, small-regulator miRNAs were subject to DNA methylation in poplars experiencing pathogen infection. This investigation highlights the critical role of DNA methylation in the poplar immune response to pathogen infection and provides new insights into epigenetic regulation in perennial plants in response to biotic stress.</p>

Update on epigenetics in allergic disease

Alternative splicing (AS) increases transcript and proteomic diversity of intron containing genes and has emerged as a pervasive property of eukaryotic genes. DNA methylation is a regulator of gene expression which is present in many eukaryotes. Recently, chromatin structure and histone modifications have been shown to regulate AS in humans (1), but it is unknown if this also occurs in plants. Here, I investigated the regulatory role that DNA and histone methylation have on AS in Arabidopsis thaliana. I utilized the SR protein gene family as a model system to examine changes in isoform abundance in response to chemical inhibition of DNA and histone methylation using the histone deacetylase inhibitor Tricostatin A (TSA) and the DNA demethylating agent 5-Azadeoxycytidine (Azad). RT-PCR revealed that 12 SR genes had isoforms that had altered abundance in response to TSA and Azad-C. Antagonistic effects were found when both drugs were applied since changes in splicing were only seen for 10 of the SR genes. AS patterns of the SR genes are known to change according to organ and developmental stage (2) and DNA methylation is also known to change over the lifespan of the plant (3). I investigated if organ type and developmental patterns of AS are disrupted in a DNA hypermethylation mutant. Splicing patterns of the SR genes displayed tissue and developmental specific changes in the DNA hypermethylation mutant. Computational analysis of three different DNA methylation mutants was performed using a publically available Illumina dataset (4). Widespread changes in AS was detected in each of the mutants across all types of AS. Changes in methylcytosine content within the coding region of the gene did not account for a large proportion of the novel AS events detected. Splice site sequence analysis of introns uniquely retained in each of the mutant genotypes uncovered sequence changes around the functionally important sequence elements. The results of my thesis indicate for the first time that AS in plants is regulated in part by changes in DNA methylation and histone modifications.

BackgroundSomatostatin receptor type 2 (SST2) expression is critical for the diagnosis and treatment of neuroendocrine tumors and is associated with improved patient survival. Recent data suggest that epigenetic changes such as DNA methylation and histone modifications play an important role in regulating SST2 expression and tumorigenesis of NETs. However, there are limited data on the association between epigenetic marks and SST2 expression in small intestinal neuroendocrine tumors (SI-NETs).MethodsTissue samples from 16 patients diagnosed with SI-NETs and undergoing surgical resection of the primary tumor at Erasmus MC Rotterdam were analysed for SST2 expression levels and epigenetic marks surrounding the SST2 promoter region, i.e. DNA methylation and histone modifications H3K27me3 and H3K9ac. As a control, 13 normal SI-tissue samples were included.ResultsThe SI-NET samples had high SST2 protein and mRNA expression levels; a median (IQR) of 80% (70-95) SST2-positive cells and 8.2 times elevated SST2 mRNA expression level compared to normal SI-tissue (p=0.0042). In comparison to normal SI-tissue, DNA methylation levels and H3K27me3 levels were significantly lower at five out of the eight targeted CpG positions and at two out of the three examined locations within the SST2 gene promoter region of the SI-NET samples, respectively. No differences in the level of activating histone mark H3K9ac were observed between matched samples. While no correlation was found between histone modification marks and SST2 expression, SST2 mRNA expression levels correlated negatively with DNA methylation within the SST2 promoter region in both normal SI-tissue and SI-NETs (p=0.006 and p=0.04, respectively).ConclusionSI-NETs have lower SST2 promoter methylation levels and lower H3K27me3 methylation levels compared to normal SI-tissue. Moreover, in contrast to the absence of a correlation with SST2 protein expression levels, significant negative correlations were found between SST2 mRNA expression level and the mean level of DNA methylation within the SST2 promoter region in both normal SI-tissue and SI-NET tissue. These results indicate that DNA methylation might be involved in regulating SST2 expression. However, the role of histone modifications in SI-NETs remains elusive.

Epigenetic regulation of cellular identity and function is at least partly achieved through changes in covalent modifications on DNA and histones. Much progress has been made in recent years to understand how these covalent modifications affect cell identity and function. Despite the advances, whether and how epigenetic factors contribute to memory formation is still poorly understood. In this review, we discuss recent progress in elucidating epigenetic mechanisms of learning and memory, primarily at the DNA level, and look ahead to discuss their potential implications in reward memory and development of drug addiction.

DNA methylation and histone modifications critically regulate the expression of many genes and repeat regions during spermatogenesis. However, the molecular details of these processes in male germ cells remain to be addressed. Here, using isolated murine sperm cells, ultra-low-input native ChIP-Seq (ULI-NChIP-Seq), and whole genome bisulfite sequencing (WGBS), we investigated genome-wide DNA methylation patterns and histone 3 Lys-9 trimethylation (H3K9me3) modifications during mouse spermatogenesis. We found that DNA methylation and H3K9me3 have distinct sequence preferences and dynamics in promoters and repeat elements during spermatogenesis. H3K9me3 modifications in histones at gene promoters were highly enriched in round spermatids. H3K9me3 modification on long terminal repeats (LTRs) and long interspersed nuclear elements (LINEs) was involved in silencing active transcription from these regions in conjunction with reestablishment of DNA methylation. Furthermore, H3K9me3 remodeling on the X chromosome was involved in meiotic sex chromosome inactivation and in partial transcriptional reactivation of sex chromosomes in spermatids. Our findings also revealed the DNA methylation patterns and H3K9me3 modification profiles of paternal and maternal germline imprinting control regions (gICRs) during spermatogenesis. Taken together, our results provide a genome-wide map of H3K9me3 modifications during mouse spermatogenesis that may be helpful for understanding male reproductive disorders.

Background: The neonatal mammalian heart has a transient capacity for regeneration, which is lost shortly after birth. A series of critical developmental transitions including a switch from hyperplastic to hypertrophic growth occur during this postnatal regenerative window, preparing the heart for the increased contractile demands of postnatal life. Postnatal cardiomyocyte maturation and loss of regenerative capacity are associated with expression alterations of thousands of genes embedded within tightly controlled transcriptional networks, which remain poorly understood. Interestingly, although mitogenic stimulation of neonatal cardiomyocytes results in proliferation, the same stimuli induce hypertrophy in adult cardiomyocytes by activating different transcriptional pathways, indicating that cardiomyocyte maturation may result from epigenetic modifications during development. Notably, DNA methylation and chromatin compaction are both important epigenetic modifications associated with a decrease in transcription factor (TF) accessibility to DNA. However, the role of both DNA methylation and chromatin compaction during postnatal cardiac maturation remain largely unknown.Hypothesis: Postnatal changes in DNA methylation and chromatin compaction silence transcriptional networks required for cardiomyocyte proliferation during postnatal heart development.Aims: This PhD Thesis focuses on characterising the role of DNA methylation and chromatin dynamics during postnatal cardiac development and maturation using in vitro and in vivo model systems. Three major Aims are addressed:• Aim 1: To characterize transcription and DNA methylation dynamics during postnatal rodent cardiac development (Chapter 3).• Aim 2: To determine the role of DNA methylation in regulating the expression of cell cycle related genes in mouse and human cardiomyocytes (Chapter 4).• Aim 3: To understand the relationship between chromatin accessibility and transcription during mouse and human cardiomyocyte development (Chapter 5).Results: In Aim 1, global DNA methylation changes were characterized during postnatal development. Marked changes in the expression levels of key DNA methylation enzymes were observed during the first month of postnatal heart development. Postnatal inhibition of DNA methylation using 5-aza-2’-deoxycytidine (5aza-dC; 1mg/kg/day) reduced global DNA methylation levels in the heart in vivo and was associated with a marked increase in cardiomyocyte proliferation (~3-fold) and ~50% reduction in binucleated cardiomyocytes compared to saline-treated controls, suggesting DNA methylation is required for postnatal cardiomyocyte cell cycle arrest. Nextgeneration mRNA sequencing (RNA-seq) and genome-wide sequencing of methylated DNA (methyl-CpG binding domain enrichment and sequencing (MBD-seq)) identified dynamic changes in the cardiac methylome during postnatal development (2545 differentially methylated regions (DMRs) from postnatal day (P) 1 to P14 in the mouse). ~80% of DMRs were hypermethylated and these DMRs were associated with transcriptional shut down of important developmental signalling pathways.In Aim 2, a specific subset of cell cycle genes displaying differentially regulated gene expression and DNA methylation patterns were profiled to understand how DNA methylation is involved in regulating cardiomyocyte proliferation. Gene expression profiling of these cell cycle genes during development and following 5aza-dC treatment in mouse hearts, as well as 3D human cardiac organoids (hCOs), verified that DNA methylation was required for transcriptional regulation of a small group of cell cycle genes. Interestingly, 5aza-dC (10µM) also induced ~2-fold increase in cardiomyocyte proliferation in hCOs. These findings suggest that DNA methylation has a direct role in the regulation of a subset of genes required for cardiomyocyte proliferation in both mice and humans.Chromatin accessibility results from the integrated action of multiple epigenetic marks. In Aim 3, cardiomyocyte nuclei were isolated from both human and mouse developmental heart samples for RNA-seq and for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) to profile changes in transcription and chromatin accessibility during cardiomyocyte development in mice (P1 to P56) and humans (foetal (14-19 weeks (wks)), 0-10 years (yrs), 10-30 yrs and 30+ yrs). RNA-seq confirmed that cardiomyocyte cell cycle arrest occurs between P1 and P14 in the mouse. Moreover, ATAC-seq identified open chromatin signatures at various genomic features, including CG islands, transcription start sites (TSSs), enhancers, TF binding sites and protein coding regions, while intergenic regions were associated with compact chromatin. Integration of RNA-seq and ATAC-seq data demonstrated a strong correlation between transcription and chromatin accessibility at the different stages of cardiomyocyte development. TF motif analysis identified E2F transcription factor 4 (E2f4) and Forkhead box M1 (Foxm1) sites as being transcriptionally repressed and undergoing chromatin compaction from P1 to P56. E2f4 and Foxm1 are well known TFs that regulate cell cycle, thus suggesting that cell cycle genes are epigenetically silenced during cardiomyocyte maturation.Conclusion: This PhD Thesis provides novel evidence for widespread alterations in DNA methylation during postnatal heart maturation and suggests that cardiomyocyte cell cycle arrest during the neonatal period is subject to regulation by DNA methylation and chromatin compaction. Together, this PhD Thesis provides new insights into the role of DNA methylation and chromatin dynamics during cardiac development. This Thesis provides a new framework for the epigenetic regulation of transcription during postnatal cardiomyocyte maturation and points towards an epigenetic mechanism for cardiomyocyte terminal differentiation during the neonatal period.

Coming from a single zygote, monozygotic (MZ) twins share nearly all of their genetic materials and are almost strikingly similar in appearance. Yet they are often discordant for important phenotypes including complex diseases. The study of discordant MZ twins allows us to understand the role of epigenetics in complex traits by controlling many potential confounders, such as genetic factors, maternal effects, age and sex. As we know, DNA methylation is the most stable epigenetic modification. In human, many causal genes, regulating disease status by DNA methylation modification in imprinting disorders, psychological disorders, autoimmune diseases and cancers, have been discovered, which provides a foundation for studying epigenetic regulation of diseases and application of epigenetic drugs. This article reviews the latest findings on DNA methylation status, estimates of DNA methylation heritability as well as studies of DNA methylation regulation in complex traits using MZ twins, in order to provide reference for research on epigenetic modification in complex traits.

The nucleosome remodeling and deacetylation (NuRD) complex is required for modulating the transcription of essential pluripotency genes in ESC self-renewal. MBD3 is considered a key player in the formation of a functional NuRD complex. The recruitment of MBD3 to methylated promoters may require other prerequisite factors. We show that cyclin-dependent kinase 2-associated protein 1 (CDK2AP1), an essential gene for early embryonic development, plays a role in pluripotency of ESC by engaging MBD3 to the promoter region of Wnt signaling genes. The occupancy of MBD3 on several promoters of Wnt genes was significantly lost in the absence of CDK2AP1, resulting in hyperactivation of Wnt. We propose that the transcriptional modulation of the Wnt pathway mediated by NuRD requires the presence of essential auxiliary components such as CDK2AP1, which may aid the association of the complex with specific focal regions of the target promoters.

ROR1 Protein Expression Is Regulated By UHRF1 in t(1;19) Pre B-Cell ALL

BackgroundSmall RNAs generated by RNA polymerase IV (Pol IV) are the most abundant class of small RNAs in flowering plants. In Arabidopsis thaliana Pol IV-dependent short interfering (p4-si)RNAs are imprinted and accumulate specifically from maternal chromosomes in the developing seeds. Imprinted expression of protein-coding genes is controlled by differential DNA or histone methylation placed in gametes. To identify epigenetic factors required for maternal-specific expression of p4-siRNAs we analyzed the effect of a series of candidate mutations, including those required for genomic imprinting of protein-coding genes, on uniparental expression of a representative p4-siRNA locus.ResultsPaternal alleles of imprinted genes are marked by DNA or histone methylation placed by DNA METHYLTRANSFERASE 1 or the Polycomb Repressive Complex 2. Here we demonstrate that repression of paternal p4-siRNA expression at locus 08002 is not controlled by either of these mechanisms. Similarly, loss of several chromatin modification enzymes, including a histone acetyltransferase, a histone methyltransferase, and two nucleosome remodeling proteins, does not affect maternal expression of locus 08002. Maternal alleles of imprinted genes are hypomethylated by DEMETER DNA glycosylase, yet expression of p4-siRNAs occurs irrespective of demethylation by DEMETER or related glycosylases.ConclusionsDifferential DNA methylation and other chromatin modifications associated with epigenetic silencing are not required for maternal-specific expression of p4-siRNAs at locus 08002. These data indicate that there is an as yet unknown epigenetic mechanism causing maternal-specific p4-siRNA expression that is distinct from the well-characterized mechanisms associated with DNA methylation or the Polycomb Repressive Complex 2.

Background and rationale:Obesity is a metabolic condition brought on by the interplay of hereditary and environmental factors, making it one of the most common diseases in the world. Insulin resistance (IR) and obesity have a close connection and can both be advantageous. One of the main methods of epigenetic regulation is DNA methylation modification. Studies have demonstrated over the past few years that DNA methylation is crucial to the emergence of obesity and DNA methylation can lead to IR. Adipose tissue participates in the physiopathological processes of obesity and IR and functions as an endocrine organ controlling the body’s balanced metabolism, thus, adipose tissue-associated gene DNA methylation affects the development of obesity and IR by influencing the function of adipose tissue. Hence, an explanation of current research on DNA methylation, IR, and obesity, following the most recent developments, exploring changes in DNA methylation in different types of adipose tissue in insulin-resistant patients and obese patients may enable the identification of novel targets in clinical obesity prevention and treatment.Method and analysis:The following electronic bibliographic databases will be searched from inception for peer-reviewed original research published: MEDLINE (through PubMed), Scopus, and EMBASE. Cochrane Library, Cochrane Clinical Trials Registry, the National Institutes for Health Clinical Trials Registry, and the WHO International Clinical Trials Registry Platform from inception to December 31, 2021 will be conducted. Systematic reviews will adhere to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses reporting guidelines. The development of search strategies will make use of medical issue phrases and keywords associated with DNA methylation, Adipose tissue DNA methylation, obesity, and IR. Identified citations will be independently reviewed by two authors to determine eligibility at the title and abstract level, and then at the full text and data extraction phases. Disagreements and conflicts will be resolved through discussion with a third author. Two authors will extract the necessary data from the included studies independently, and The Cochrane Risk of Bias Assessment Tool will be used to assess the bias of randomized controlled studies, and the Newcastle–Ottawa scale for nonrandomized controlled studies. If the interventions and outcomes evaluated are sufficiently homogeneous, results from subgroups of studies will be pooled together in a meta-analysis.