DNA Methylation In Gene Regulation Research Articles

A combination of 5-azacytidine and nivolumab is a potentially effective rescue therapy in relapsed/refractory AITL.

Angioimmunoblastic T-cell lymphoma (AITL) is a peripheral T-cell lymphoma characterized by a T follicular helper cell phenotype expressing PD-1 (programmed cell death-1). AITL exhibits a poor response to conventional chemotherapy, with a median 5-year overall survival of 44% and a progression-free survival of 32%. Relapse is common, resulting in a median overall survival of 6 months. Recurrent mutations are detected in genes regulating DNA methylation, including TET2, DNMT3A, and IDH2 variants, along with the prevalent RHOA G17V mutation. In this context, patients treated with the hypomethylating agent 5-azacytidine achieved overall response and complete response rates of 75% and 41%, respectively. We hypothesized that targeted therapies combining anti-PD-1 checkpoint blockers with hypomethylating agents could be efficient in AITL patients and less toxic than standard chemotherapy. Here, we report the efficacy of a regimen combining 5-azacytidine and nivolumab in nine relapsed or refractory AITL patients. This regimen was well-tolerated, especially in elderly patients. The overall response rate was 78%, including four partial responses (44%) and three complete responses (33%). Allogeneic hematopoietic stem cell transplantation was performed in two patients who reached complete response. These preliminary favorable results may serve as a basis for further investigation in prospective studies.

Identification and experimental validation of druggable epigenetic targets in hepatoblastoma

Hepatoblastoma (HB) is the most frequent childhood liver cancer. Patients with aggressive tumors have limited therapeutic options; therefore, a better understanding of HB pathogenesis is needed to improve treatment. HBs have a very low mutational burden; however, epigenetic alterations are increasingly recognized. We aimed to identify epigenetic regulators consistently dysregulated in HB and to evaluate the therapeutic efficacy of their targeting in clinically relevant models. We performed a comprehensive transcriptomic analysis of 180 epigenetic genes. Data from fetal, pediatric, adult, peritumoral (n= 72) and tumoral (n= 91) tissues were integrated. Selected epigenetic drugs were tested in HB cells. The most relevant epigenetic target identified was validated in primary HB cells, HB organoids, a patient-derived xenograft model, and a genetic mouse model. Transcriptomic, proteomic and metabolomic mechanistic analyses were performed. Altered expression of genes regulating DNA methylation and histone modifications was consistently observed in association with molecular and clinical features of poor prognosis. The histone methyltransferase G9a was markedly upregulated in tumors with epigenetic and transcriptomic traits of increased malignancy. Pharmacological targeting of G9a significantly inhibited growth of HB cells, organoids and patient-derived xenografts. Development of HB induced by oncogenic forms of β-catenin and YAP1 was ablated in mice with hepatocyte-specific deletion of G9a. We observed that HBs undergo significant transcriptional rewiring in genes involved in amino acid metabolism and ribosomal biogenesis. G9a inhibition counteracted these pro-tumorigenic adaptations. Mechanistically, G9a targeting potently repressed the expression of c-MYC and ATF4, master regulators of HB metabolic reprogramming. HBs display a profound dysregulation of the epigenetic machinery. Pharmacological targeting of key epigenetic effectors exposes metabolic vulnerabilities that can be leveraged to improve the treatment of these patients. In spite of recent advances in the management of hepatoblastoma (HB), treatment resistance and drug toxicity are still major concerns. This systematic study reveals the remarkable dysregulation in the expression of epigenetic genes in HB tissues. Through pharmacological and genetic experimental approaches, we demonstrate that the histone-lysine-methyltransferase G9a is an excellent drug target in HB, which can also be harnessed to enhance the efficacy of chemotherapy. Furthermore, our study highlights the profound pro-tumorigenic metabolic rewiring of HB cells orchestrated by G9a in coordination with the c-MYC oncogene. From a broader perspective, our findings suggest that anti-G9a therapies may also be effective in other c-MYC-dependent tumors.

Fragile sites, chromosomal lesions, tandem repeats, and disease.

Expanded tandem repeat DNAs are associated with various unusual chromosomal lesions, despiralizations, multi-branched inter-chromosomal associations, and fragile sites. Fragile sites cytogenetically manifest as localized gaps or discontinuities in chromosome structure and are an important genetic, biological, and health-related phenomena. Common fragile sites (∼230), present in most individuals, are induced by aphidicolin and can be associated with cancer; of the 27 molecularly-mapped common sites, none are associated with a particular DNA sequence motif. Rare fragile sites ( 40 known), 5% of the population (may be as few as a single individual), can be associated with neurodevelopmental disease. All 10 molecularly-mapped folate-sensitive fragile sites, the largest category of rare fragile sites, are caused by gene-specific CGG/CCG tandem repeat expansions that are aberrantly CpG methylated and include FRAXA, FRAXE, FRAXF, FRA2A, FRA7A, FRA10A, FRA11A, FRA11B, FRA12A, and FRA16A. The minisatellite-associated rare fragile sites, FRA10B, FRA16B, can be induced by AT-rich DNA-ligands or nucleotide analogs. Despiralized lesions and multi-branched inter-chromosomal associations at the heterochromatic satellite repeats of chromosomes 1, 9, 16 are inducible by de-methylating agents like 5-azadeoxycytidine and can spontaneously arise in patients with ICF syndrome (Immunodeficiency Centromeric instability and Facial anomalies) with mutations in genes regulating DNA methylation. ICF individuals have hypomethylated satellites I-III, alpha-satellites, and subtelomeric repeats. Ribosomal repeats and subtelomeric D4Z4 megasatellites/macrosatellites, are associated with chromosome location, fragility, and disease. Telomere repeats can also assume fragile sites. Dietary deficiencies of folate or vitamin B12, or drug insults are associated with megaloblastic and/or pernicious anemia, that display chromosomes with fragile sites. The recent discovery of many new tandem repeat expansion loci, with varied repeat motifs, where motif lengths can range from mono-nucleotides to megabase units, could be the molecular cause of new fragile sites, or other chromosomal lesions. This review focuses on repeat-associated fragility, covering their induction, cytogenetics, epigenetics, cell type specificity, genetic instability (repeat instability, micronuclei, deletions/rearrangements, and sister chromatid exchange), unusual heritability, disease association, and penetrance. Understanding tandem repeat-associated chromosomal fragile sites provides insight to chromosome structure, genome packaging, genetic instability, and disease.

Oral Azacytidine in Patients with Relapsed/Refractory Angioimmunoblastic T-Cell Lymphoma: Final Analysis of the Oracle Phase III Study

Background: Angioimmunoblastic T-cell Lymphoma (AITL) and nodal lymphomas of follicular helper T-cell origin have in common frequent mutations affecting genes regulating DNA methylation and hydroxymethylation such as TET2, DNMT3A and IDH2. Preliminary studies suggested efficacy of the hypomethylating agent 5-azacytidine (Blood. 2018; 132(21):2305-09) in relapsed/refractory AITL patients. We present here the final results of the ORACLE study (NCT03593018), a phase III trial comparing CC-486, an oral form of 5-azacytidine, to single agent treatment chosen by the investigator. Patients & methods: Eighty-six patients with relapsed/refractory AITL or nodal follicular helper T-cell lymphoma were randomized between CC-486 (n=42) and investigator's choice (gemcitabine, n=24, bendamustine n=16, romidepsin n=4) between November 2018 and February 2021. CC-486 was given at a daily dose of 300 mg/day (200 mg/day in Asian patients, based on previous phase I pharmacokinetics results) every day for 14 days out of 28 day-cycles, until progression or unacceptable toxicity. Gemcitabine and bendamustine were both administered for 6 cycles, while romidepsin was administered until progression. Patients were evaluated for response using 2014 international working group CT-based criteria. The primary endpoint was progression-free survival (PFS) based on local assessment of progressive disease, and overall survival was a key secondary endpoint in this study. Main secondary endpoints were PFS based on central review, overall response rate (ORR), complete response (CR) rate and safety. Statistical hypotheses were built on a PFS of 12 months in the experimental arm vs 5 months in the standard arm (HR=0.417), with a one-sided alpha risk of 0.025 and 90% power. Results: Median age was 69 years (IQR 62-76), and the male/female ratio was 1.4. Patients had received a median of 2 (IQR 1-2) previous lines of treatment, 90.6% of them had advanced stage (III-IV), 57% had elevated LDH, 23.3% had an ECOG performance status >1, 31.4% had an IPI score 4-5 and 35.8% had a bone marrow involvement. Patients received a median number of 5.5 (IQR 2;14) cycles of CC486, and respectively 2 (1-4), 3.5 (2-6) and 13.5 (5-25) cycles of gemcitabine, bendamustine and romidepsin. The primary endpoint was analysed after a follow-up of 14.4 months. Median PFS in the CC-486 arm was 5.6 (95%CI, 2.66-8.11) months vs 2.8 (95%CI, 1.87-4.83) months in the standard arm (stratified log-rank test p=0.0421), with a hazard ratio of 0.634 (95%CI, 0.38; 1.07), which did not reach the required significance of p<0.025. Median overall survival was 18.4 months (95%CI, 12.9-31.5) in the CC-486 arm vs 10.3 (95%CI, 4.2-13.5) in the standard arm, with a hazard ratio of 0.557 (95%CI, 0.323-0.961). Best overall response and complete response rates at 3 months in the CC-486 arm were 33.3% (95%CI, 19.6%-49.5%) and 11.9% (95%CI, 4%-25.6%) respectively, vs 43.2% (95%CI, 28.3%-59.0%) and 22.7% (95%CI, 11.5%-37.8%) in the standard arm. The most frequent (>40%) treatment emergent adverse events (TEAEs) for the CC-486 versus (vs) standard arm respectively were: blood and lymphatic system disorders (76.2% vs 93%) [neutropenia (42.9% vs 58.1%) and thrombocytopenia (23.8% vs 48.8%)], infections (35.7% vs 67.4%), gastrointestinal disorders (71.4% vs 55.8%). At least one grade 3/4 TEAE occurred in 76.2% patients in CC-486 arm vs 97.7% in the standard arm, and at least one serious TEAE occurred in 26.2% patients in CC-486 arm vs 44.2% patients in the standard arm. Central pathological review confirmed the diagnosis of AITL and TFH PTCL in 69 and 9 cases. We detected TET2, RHOA, DNMT3A and IDH2 mutations in 68/77 (88%), 51/75 (68%), 24/75 (32%) and 25/75 (33%) samples. None of these mutations were associated with PFS or OS of the whole population or the 5-azacytidine treated patients. Conclusion: The trial did not meet its primary endpoint, most likely due to an optimistic hypothesis of PFS improvement, resulting in a study including 86 patients which could be underpowered to detect a clinically meaningful difference. 5-azacytidine had a favourable safety profile compared to standard of care and was associated with prolonged overall survival, suggesting that this drug might have a role in the treatment of TFH PTCL and could be investigated in combination with other agents. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Decoding the sorghum methylome: understanding epigenetic contributions to agronomic traits.

DNA methylation is a chromatin modification that plays an essential role in regulating gene expression and genome stability and it is typically associated with gene silencing and heterochromatin. Owing to its heritability, alterations in the patterns of DNA methylation have the potential to provide for epigenetic inheritance of traits. Contemporary epigenomic technologies provide information beyond sequence variation and could supply alternative sources of trait variation for improvement in crops such as sorghum. Yet, compared with other species such as maize and rice, the sorghum DNA methylome is far less well understood. The distribution of CG, CHG, and CHH methylation in the genome is different compared with other species. CG and CHG methylation levels peak around centromeric segments in the sorghum genome and are far more depleted in the gene dense chromosome arms. The genes regulating DNA methylation in sorghum are also yet to be functionally characterised; better understanding of their identity and functional analysis of DNA methylation machinery mutants in diverse genotypes will be important to better characterise the sorghum methylome. Here, we catalogue homologous genes encoding methylation regulatory enzymes in sorghum based on genes in Arabidopsis, maize, and rice. Discovering variation in the methylome may uncover epialleles that provide extra information to explain trait variation and has the potential to be applied in epigenome-wide association studies or genomic prediction. DNA methylation can also improve genome annotations and discover regulatory elements underlying traits. Thus, improving our knowledge of the sorghum methylome can enhance our understanding of the molecular basis of traits and may be useful to improve sorghum performance.

Large-scale DNA demethylation occurs in proliferating ovarian granulosa cells during mouse follicular development

During ovarian follicular development, granulosa cells proliferate and progressively differentiate to support oocyte maturation and ovulation. To determine the underlying links between proliferation and differentiation in granulosa cells, we determined changes in 1) the expression of genes regulating DNA methylation and 2) DNA methylation patterns, histone acetylation levels and genomic DNA structure. In response to equine chorionic gonadotropin (eCG), granulosa cell proliferation increased, DNA methyltransferase (DNMT1) significantly decreased and Tet methylcytosine dioxygenase 2 (TET2) significantly increased in S-phase granulosa cells. Comprehensive MeDIP-seq analyses documented that eCG treatment decreased methylation of promoter regions in approximately 40% of the genes in granulosa cells. The expression of specific demethylated genes was significantly increased in association with specific histone modifications and changes in DNA structure. These epigenetic processes were suppressed by a cell cycle inhibitor. Based on these results, we propose that the timing of sequential epigenetic events is essential for progressive, stepwise changes in granulosa cell differentiation.

Genome-wide identification of genes regulating DNA methylation using genetic anchors for causal inference

BackgroundDNA methylation is a key epigenetic modification in human development and disease, yet there is limited understanding of its highly coordinated regulation. Here, we identify 818 genes that affect DNA methylation patterns in blood using large-scale population genomics data.ResultsBy employing genetic instruments as causal anchors, we establish directed associations between gene expression and distant DNA methylation levels, while ensuring specificity of the associations by correcting for linkage disequilibrium and pleiotropy among neighboring genes. The identified genes are enriched for transcription factors, of which many consistently increased or decreased DNA methylation levels at multiple CpG sites. In addition, we show that a substantial number of transcription factors affected DNA methylation at their experimentally determined binding sites. We also observe genes encoding proteins with heterogenous functions that have widespread effects on DNA methylation, e.g., NFKBIE, CDCA7(L), and NLRC5, and for several examples, we suggest plausible mechanisms underlying their effect on DNA methylation.ConclusionWe report hundreds of genes that affect DNA methylation and provide key insights in the principles underlying epigenetic regulation.

MethGET: web-based bioinformatics software for correlating genome-wide DNA methylation and gene expression

BackgroundDNA methylation is a major epigenetic modification involved in regulating gene expression. The effects of DNA methylation on gene expression differ by genomic location and vary across kingdoms, species and environmental conditions. To identify the functional regulatory roles of DNA methylation, the correlation between DNA methylation changes and alterations in gene expression is crucial. With the advance of next-generation sequencing, genome-wide methylation and gene expression profiling have become feasible. Current bioinformatics tools for investigating such correlation are designed to the assessment of DNA methylation at CG sites. The correlation of non-CG methylation and gene expression is very limited. Some bioinformatics databases allow correlation analysis, but they are limited to specific genomes such as that of humans and do not allow user-provided data.ResultsHere, we developed a bioinformatics web tool, MethGET (Methylation and Gene Expression Teller), that is specialized to analyse the association between genome-wide DNA methylation and gene expression. MethGET is the first web tool to which users can supply their own data from any genome. It is also the tool that correlates gene expression with CG, CHG, and CHH methylation based on whole-genome bisulfite sequencing data. MethGET not only reveals the correlation within an individual sample (single-methylome) but also performs comparisons between two groups of samples (multiple-methylomes). For single-methylome analyses, MethGET provides Pearson correlations and ordinal associations to investigate the relationship between DNA methylation and gene expression. It also groups genes with different gene expression levels to view the methylation distribution at specific genomic regions. Multiple-methylome analyses include comparative analyses and heatmap representations between two groups. These functions enable the detailed investigation of the role of DNA methylation in gene regulation. Additionally, we applied MethGET to rice regeneration data and discovered that CHH methylation in the gene body region may play a role in the tissue culture process, which demonstrates the capability of MethGET for use in epigenomic research.ConclusionsMethGET is a Python software that correlates DNA methylation and gene expression. Its web interface is publicly available at https://paoyang.ipmb.sinica.edu.tw/Software.html. The stand-alone version and source codes are available on GitHub at https://github.com/Jason-Teng/MethGET.

Differential CpG DNA methylation in peripheral na\xefve CD4+ T-cells in early rheumatoid arthritis patients

BackgroundThe genetic risk associated with rheumatoid arthritis (RA) includes genes regulating DNA methylation, one of the hallmarks of epigenetic re-programing, as well as many T-cell genes, with a strong MHC association, pointing to immunogenetic mechanisms as disease triggers leading to chronicity. The aim of our study was to explore DNA methylation in early, drug-naïve RA patients, towards a better understanding of early events in pathogenesis.ResultMonocytes, naïve and memory CD4+ T-cells were sorted from 6 healthy controls and 10 RA patients. DNA methylation was assessed using a genome-wide Illumina 450K CpG promoter array. Differential methylation was confirmed using bisulfite sequencing for a specific gene promoter, ELISA for several cytokines and flow cytometry for cell surface markers. Differentially methylated (DM) CpGs were observed in 1047 genes in naïve CD4+ T-cells, 913 in memory cells and was minimal in monocytes with only 177 genes. Naive CD4+ T-cells were further investigated as presenting differential methylation in the promoter of > 500 genes associated with several disease-relevant pathways, including many cytokines and their receptors. We confirmed hypomethylation of a region of the TNF-alpha gene in early RA and differential expression of 3 cytokines (IL21, IL34 and RANKL). Using a bioinformatics package (DMRcate) and an in-house analysis based on differences in β values, we established lists of DM genes between health and RA. Publicly available gene expression data were interrogated to confirm differential expression of over 70 DM genes. The lists of DM genes were further investigated based on a functional relationship database analysis, which pointed to an IL6/JAK1/STAT3 node, related to TNF-signalling and engagement in Th17 cell differentiation amongst many pathways. Five DM genes for cell surface markers (CD4, IL6R, IL2RA/CD25, CD62L, CXCR4) were investigated towards identifying subpopulations of CD4+ T-cells undergoing these modifications and pointed to a subset of naïve T-cells, with high levels of CD4, IL2R, and CXCR4, but reduction and loss of IL6R and CD62L, respectively.ConclusionOur data provided novel conceptual advances in the understanding of early RA pathogenesis, with implications for early treatment and prevention.

ATAC-Me Captures Prolonged DNA Methylation of Dynamic Chromatin Accessibility Loci during Cell Fate Transitions.

DNA methylation of enhancers is dynamic, cell-type specific, and vital for cell fate progression. However, current models inadequately define its role within the hierarchy of gene regulation. Analysis of independent datasets shows an unanticipated overlap between DNA methylation and chromatin accessibility at enhancers of steady-state stem cells, suggesting that these two opposing features might exist concurrently. To define their temporal relationship, we developed ATAC-Me, which probes accessibility and methylation from single DNA library preparations. We identified waves of accessibility occurring rapidly across thousands of myeloid enhancers in amonocyte-to-macrophage cell fate model. Prolonged methylation states were observed at a majority of these sites, while transcription of nearby genes tracked closely with accessibility. ATAC-Me uncovers a significant disconnect between chromatin accessibility, DNA methylation status, and gene activity. This unexpected observation highlights the value of ATAC-Me in constructing precise molecular timelines for understanding the role of DNA methylation in gene regulation.

S843 THE PROLIFERATIVE HISTORY SHAPES THE DNA METHYLOME OF B‐CELL TUMORS AND PREDICTS CLINICAL OUTCOME

Background:Beyond the known role of DNA methylation in gene regulation, recent studies indicate that most DNA methylation changes in cancer accumulate in silent regions without affecting gene expression. To obtain new insights into the biological and clinical implications of DNA methylation, we systematically analyzed genetic, epigenetic, transcriptional, and clinical data of five categories of B‐cell tumors including 1553 samples. This analysis unraveled that mitotic activity shapes the overall genetic and epigenetic landscapes of B cell tumors and affects the clinical behavior of patients.Aims:To provide a holistic view on the biological and clinical implications of DNA methylation variability in B‐cell tumors, with particular emphasis in non‐regulatory regions.Methods:We used 450k methylation arrays from normal B cell subpopulations as well as ALL, MCL DLBCL, CLL and MM patients (n = 67, 830, 74, 27, 490 and 65, respectively). We integrated ChIP‐seq data of 6 histone marks from normal B cells, MCL, CLL and MM (n = 15, 5, 7 and 5, respectively). This allowed us to construct the epigenetic Cumulative Mitosis (epiCMIT) score. We validated the epiCMIT using whole‐genome sequencing and gene expression data of the same CLL patients (n = 135). We additionally used 477 CLL patients with whole exome sequencing to find genetic driver alterations associated with high epiCMIT. From the clinical perspective, we used a machine‐learning approach to build an accurate pan B‐cell cancer diagnostic tool and related the epiCMIT to clinical outcome of ALL, MCL and CLL patients.Results:We dissected the DNA methylome of B‐cell tumors at different levels, including entity‐specific, subentity‐specific and patient‐specific variability. We showed that virtually the entire methylome (87%) is modulated in normal and/or neoplastic conditions, and subsequently dissected the sources of such variability. We initially identified the presence of differential methylation patterns among B‐cell tumors and their subentities. This analysis allowed us to construct an accurate pan B‐cell cancer diagnostic tool for 14 subentities of tumors requiring different clinical management. Furthermore, of particular interest were the hypomethylation patterns in ALL, MCL and CLL, which we could relate to differential binding of transcription factors important for the pathogenesis of each disease, such as ETS1 and MEIS1A in ALL, TCF/ZEB in MCL and NFAT in CLL. Despite the importance of DNA methylation changes in these regulatory regions, we identified that the majority of the changes were actually located in inactive non‐regulatory regions. By means of genetic and transcriptomic associations, we demonstrated that those changes reflect the proliferative history of cancer specimens. Interestingly, we found that cell mitosis leaves different imprints onto the DNA methylome depending on the maturation stage of cancer cells. Subsequently, we built a mitotic clock called epiCMIT (representing the proliferative history of the tumor) and applied it to 1,553 samples. Putting the epiCMIT in the context of the clinical behavior of the ALL, MCL and CLL patients, we found that it represents a strong and independent prognostic factor. Finally, we identified driver genetic alterations related to higher proliferative capacity, a finding that could explain their well‐established clinical impact.Summary/Conclusion:Our analytic approach reveals DNA methylation as a holistic tracker of B cell tumor developmental history, with implications in the differential diagnosis and prediction of prognosis of the patients

Genetic drivers of oncogenic pathways in molecular subgroups of peripheral T-cell lymphoma

AbstractPeripheral T-cell lymphoma (PTCL) is a group of complex clinicopathological entities, often associated with an aggressive clinical course. Angioimmunoblastic T-cell lymphoma (AITL) and PTCL-not otherwise specified (PTCL-NOS) are the 2 most frequent categories, accounting for >50% of PTCLs. Gene expression profiling (GEP) defined molecular signatures for AITL and delineated biological and prognostic subgroups within PTCL-NOS (PTCL-GATA3 and PTCL-TBX21). Genomic copy number (CN) analysis and targeted sequencing of these molecular subgroups revealed unique CN abnormalities (CNAs) and oncogenic pathways, indicating distinct oncogenic evolution. PTCL-GATA3 exhibited greater genomic complexity that was characterized by frequent loss or mutation of tumor suppressor genes targeting the CDKN2A/B-TP53 axis and PTEN-PI3K pathways. Co-occurring gains/amplifications of STAT3 and MYC occurred in PTCL-GATA3. Several CNAs, in particular loss of CDKN2A, exhibited prognostic significance in PTCL-NOS as a single entity and in the PTCL-GATA3 subgroup. The PTCL-TBX21 subgroup had fewer CNAs, primarily targeting cytotoxic effector genes, and was enriched in mutations of genes regulating DNA methylation. CNAs affecting metabolic processes regulating RNA/protein degradation and T-cell receptor signaling were common in both subgroups. AITL showed lower genomic complexity compared with other PTCL entities, with frequent co-occurring gains of chromosome 5 (chr5) and chr21 that were significantly associated with IDH2R172 mutation. CN losses were enriched in genes regulating PI3K–AKT–mTOR signaling in cases without IDH2 mutation. Overall, we demonstrated that novel GEP-defined PTCL subgroups likely evolve by distinct genetic pathways and provided biological rationale for therapies that may be investigated in future clinical trials.

DNA Methylation Patterns in the Social Spider, Stegodyphus dumicola.

Variation in DNA methylation patterns among genes, individuals, and populations appears to be highly variable among taxa, but our understanding of the functional significance of this variation is still incomplete. We here present the first whole genome bisulfite sequencing of a chelicerate species, the social spider Stegodyphus dumicola. We show that DNA methylation occurs mainly in CpG context and is concentrated in genes. This is a pattern also documented in other invertebrates. We present RNA sequence data to investigate the role of DNA methylation in gene regulation and show that, within individuals, methylated genes are more expressed than genes that are not methylated and that methylated genes are more stably expressed across individuals than unmethylated genes. Although no causal association is shown, this lends support for the implication of DNA CpG methylation in regulating gene expression in invertebrates. Differential DNA methylation between populations showed a small but significant correlation with differential gene expression. This is consistent with a possible role of DNA methylation in local adaptation. Based on indirect inference of the presence and pattern of DNA methylation in chelicerate species whose genomes have been sequenced, we performed a comparative phylogenetic analysis. We found strong evidence for exon DNA methylation in the horseshoe crab Limulus polyphemus and in all spider and scorpion species, while most Parasitiformes and Acariformes species seem to have lost DNA methylation.

Sequencing in High Definition Drives a Changing Worldview of the Epigenome.

Single-molecule sequencing approaches have transformed the study of the human epigenome, accelerating efforts to describe genome function beyond the sequences that encode proteins. The post-genome era has ignited strong interest in the noncoding genome and profiling epigenetic signatures genome-wide have been critical for the identification and characterization of noncoding gene-regulatory sequences in various cellular and developmental contexts. These technologies enable quantification of epigenetic marks through digital assessment of DNA fragments. With the capacity to probe both the DNA sequence and count DNA molecules at once with unparalleled throughput and sensitivity, deep sequencing has been especially transformative to the study of DNA methylation. This review will discuss advances in epigenome profiling with a particular focus on DNA methylation, highlighting how deep sequencing has generated new insights into the role of DNA methylation in gene regulation. Technical aspects of profiling DNA methylation, remaining challenges, and the future of DNA methylation sequencing are also described.

The complete methylome of an entomopathogenic bacterium reveals the existence of loci with unmethylated Adenines

DNA methylation can serve to control diverse phenomena in eukaryotes and prokaryotes, including gene regulation leading to cell differentiation. In bacteria, DNA methylomes (i.e., methylation state of each base of the whole genome) have been described for several species, but methylome profile variation during the lifecycle has rarely been studied, and only in a few model organisms. Moreover, major phenotypic changes have been reported in several bacterial strains with a deregulated methyltransferase, but the corresponding methylome has rarely been described. Here we report the first methylome description of an entomopathogenic bacterium, Photorhabdus luminescens. Eight motifs displaying a high rate of methylation (>94%) were identified. The methylome was strikingly stable over course of growth, but also in a subpopulation responsible for a critical step in the bacterium’s lifecycle: successful survival and proliferation in insects. The rare unmethylated GATC motifs were preferentially located in putative promoter regions, and most of them were methylated after Dam methyltransferase overexpression, suggesting that DNA methylation is involved in gene regulation. Our findings bring key insight into bacterial methylomes and encourage further research to decipher the role of loci protected from DNA methylation in gene regulation.

Extensive genetic and DNA methylation variation contribute to heterosis in triploid loquat hybrids.

We aim to overcome the unclear origin of the loquat and elucidate the heterosis mechanism of the triploid loquat. Here we investigated the genetic and epigenetic variations between the triploid plant and its parental lines using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified fragment length polymorphism (MSAP) analyses. We show that in addition to genetic variations, extensive DNA methylation variation occurred during the formation process of triploid loquat, with the triploid hybrid having increased DNA methylation compared to the parents. Furthermore, a correlation existed between genetic variation and DNA methylation remodeling, suggesting that genome instability may lead to DNA methylation variation or vice versa. Sequence analysis of the MSAP bands revealed that over 53% of them overlap with protein-coding genes, which may indicate a functional role of the differential DNA methylation in gene regulation and hence heterosis phenotypes. Consistent with this, the genetic and epigenetic alterations were associated closely to the heterosis phenotypes of triploid loquat, and this association varied for different traits. Our results suggested that the formation of triploid is accompanied by extensive genetic and DNA methylation variation, and these changes contribute to the heterosis phenotypes of the triploid loquats from the two cross lines.

The origin and purpose of layers of subcutaneous adipose tissue in pigs and man.

Adipose tissue exists in many locations or depots that differ from one another based on numerous and various characteristics. The unique "layered" anatomical feature of subcutaneous adipose tissue (SAT) in man and the pig is reviewed and discussed. The origin of fetal pig adipose tissue subcutaneous layers is reviewed before the onset of adipogenesis and after the overt adipogenesis. Furthermore, the distinguishing characteristics of developing outer SAT layer (OSQ) and middle SAT layer (MSQ) in pigs are reviewed. These characteristics include adipocyte hypertrophy, metabolism and genetic regulation. The MSQ layer is the major layer in the pig and expands to the greatest degree in obesity and growth. Abdominal SAT in man is composed of deep SAT (dSAT) and superficial SAT (sSAT) layers. Clearly, dSAT expands disproportionally more than sSAT with increasing obesity in Caucasian males which precipitates a number of human pathologies associated with increased adiposity. We reviewed the considerable evidence that demonstrates the distinction between sSAT and dSAT which includes higher levels of saturated fatty acids (FAs) and greater levels of lipolysis in dSAT. Furthermore, dSAT expresses more metabolic and inflammatory genes. Studies comparing visceral adipose tissue (VAT) and dSAT indicate that both depots are implicated in insulin resistance (IR) and other human pathologies. Epigenetic studies of MSQ and dSAT have begun to indicate a role for DNA methylation in gene regulation of these depots. Further studies of dSAT and MSQ are warranted as they are clearly a major manifestation of obesity.

A distinct immunophenotype identifies a subset of NPM1-mutated AML with TET2 or IDH1/2 mutations and improved outcome.

Recent work has identified distinct molecular subgroups of acute myeloid leukemia (AML) with implications for disease classification and prognosis. NPM1 is one of the most common recurrently mutated genes in AML. NPM1 mutations often co-occur with FLT3-ITDs and mutations in genes regulating DNA methylation, such as DNMT3A, TET2, and IDH1/2. It remains unclear whether these genetic alterations are associated with distinct immunophenotypic findings or affect prognosis. We identified 133 cases of NPM1-mutated AML and correlated sequencing data with immunophenotypic and clinical findings. Of 84 cases (63%) that lacked monocytic differentiation ("myeloid AML"), 40 (48%) demonstrated an acute promyelocytic leukemia-like (APL-like) immunophenotype by flow cytometry, with absence of CD34 and HLA-DR and strong myeloperoxidase expression, in the absence of a PML-RARA translocation. Pathologic variants in TET2, IDH1, or IDH2 were identified in 39/40 APL-like cases. This subset of NPM1-mutated AML was associated with longer relapse-free and overall survival, when compared with cases that were positive for CD34 and/or HLA-DR. The combination of NPM1 and TET2 or IDH1/2 mutations along with an APL-like immunophenotype identifies a distinct subtype of AML. Further studies addressing its biology and clinical significance may be especially relevant in the era of IDH inhibitors and recent work showing efficacy of ATRA therapy in NPM1 and IDH1-mutated AML.

Readers of DNA methylation, the MBD family as potential therapeutic targets.

DNA methylation represents a fundamental epigenetic modification that regulates chromatin architecture and gene transcription. Many diseases, including cancer, show aberrant methylation patterns that contribute to the disease phenotype. DNA methylation inhibitors have been used to block methylation dependent gene silencing to treat hematopoietic neoplasms and to restore expression of developmentally silenced genes. However, these inhibitors disrupt methylation globally and show significant off-target toxicities. As an alternative approach, we have been studying readers of DNA methylation, the 5-methylcytosine binding domain family of proteins, as potential therapeutic targets to restore expression of aberrantly and developmentally methylated and silenced genes. In this review, we discuss the role of DNA methylation in gene regulation and cancer development, the structure and function of the 5-methylcytosine binding domain family of proteins, and the possibility of targeting the complexes these proteins form to treat human disease.

DNMT3A moderates cognitive decline in subjects with mild cognitive impairment: replicated evidence from two mild cognitive impairment cohorts.

Epigenetic dysregulation has been associated with cognitive decline and Alzheimer's disease. The present study investigated associations between common SNPs in genes regulating DNA methylation and age-related changes in cognitive decline in two independent prospective cohorts of patients suffering from mild cognitive impairment. An association between the rs1187120 SNP in DNMT3A and annual decline in cognitive functioning was discovered and replicated, suggesting that DNMT3A moderates cognitive decline in subjects with mild cognitive impairment.