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Abstract Vitamin D deficiency is a common deficiency worldwide, particularly among women of reproductive age. During pregnancy, it increases the risk of immune-related diseases in offspring later in life. However, how the body remembers exposure to an adverse environment during development is poorly understood. Herein, we explore the effects of prenatal vitamin D deficiency on immune cell proportions in offspring using vitamin D deficient mice established by dietary manipulation. We found that prenatal vitamin D deficiency alters immune cell proportions in offspring by changing the transcriptional properties of genes downstream of vitamin D receptor signaling in hematopoietic stem and progenitor cells of both the fetus and adults. Moreover, further investigations of the associations between maternal vitamin D levels and cord blood immune cell profiles from 75 healthy pregnant women and their term offspring also confirm that maternal vitamin D levels in the second trimester significantly affect immune cell proportions in the offspring. These findings imply that the differentiation properties of hematopoiesis act as long-term memories of prenatal vitamin D deficiency exposure in later life.

Abstract The ten-eleven-translocation family of proteins (TET1/2/3) are epigenetic regulators of gene expression. They regulate genes by promoting DNA demethylation (i.e., catalytic activity) and by partnering with regulatory proteins (i.e., non-catalytic functions). Unlike Tet1 and Tet2, Tet3 is not expressed in mouse embryonic stem cells (ESCs) but is induced upon ESC differentiation. However, the significance of its dual roles in lineage specification is less defined. By generating TET3 catalytic-mutant (Tet3 m/m ) and knockout (Tet3 –/– ) mouse ESCs and differentiating them to neuroectoderm (NE), we identify distinct catalytic-dependent and independent roles of TET3 in NE specification. We find that the catalytic activity of TET3 is important for activation of neural genes while its non-catalytic functions are involved in suppressing mesodermal programs. Interestingly, the vast majority of differentially methylated regions (DMRs) in Tet3 m/m and Tet3 –/– NE cells are hypomethylated. The hypo-DMRs are associated to aberrantly upregulated genes while the hyper-DMRs are linked to downregulated neural genes. We find the maintenance methyltransferase Dnmt1 as a direct target of TET3, which is downregulated in TET3-deficient NE cells and may contribute to the increased DNA hypomethylation. Our findings establish that the catalytic-dependent and -independent roles of TET3 have distinct contributions to NE specification with potential implications in development.

Objective: We implemented an online visitation system named “telepresence” in the neonatal intensive care unit (NICU) for family members at home to communicate with their babies in real-time using video and audio. This study evaluated the impact of this system on families and medical staff. Methods: Nineteen families of babies admitted to the NICU between 2022 and 2023 and 65 medical staff participated. Each family experienced two weeks of virtual visits. Changes in parental depression and attachment were assessed. Result: Before and after telepresence, the median Edinburgh Postnatal Depression Scale score reduced from 6 to 4 (p = 0.026), and the Mother-to-Infant Bonding Scale score showed a decreasing trend, with both medians at 2 (p = 0.057). Eighty-nine percent of the parents and 97% of staff reported that telepresence did not increase parental stress, and 88% of parents felt positive changes in their baby's siblings. All parents wanted to visit their babies in person after seeing them on camera. Conclusion: Telepresence improved parental mental health, reduced family distress, and supported connection with their infants, making them eager to visit in person. Innovations: This technology potentially make parents want to visit more by helping them feel more connected to their infants.

Background/Objectives: Refeeding syndrome (RFS) is recognized as a potentially fatal metabolic disturbance, particularly concerning for non-critically ill patients who do not receive frequent electrolyte assessments. Assessing the risk of developing RFS and implementing preventive strategies is essential in these cases. We investigated the proportion of risk and its association with prognosis in a high-dependency unit (HDU). Method: This observational study was conducted in a tertiary care hospital’s HDU in Japan. We consecutively enrolled all patients who had been admitted urgently to the HDU and hospitalized for three days or more. We evaluated the National Institute for Health and Clinical Excellence (NICE) RFS risk factors at admission and classified patients into four groups based on the modified NICE criteria. The primary outcome was 30-day in-hospital mortality. The secondary outcome was a composite of 30-day in-hospital mortality and transfer to the intensive care unit, or discharge to locations other than home. Using logistic regression, we assessed the association between the four risk groups and outcomes, using the no-risk group as a reference. Results: A total of 955 patients were analyzed, of which 33.1%, 26.7%, 37.8%, and 2.4% were classified into the no-risk, low-risk, high-risk, and very high-risk groups, respectively. The 30-day in-hospital mortality was 4.4%, 5.5%, 5.0%, and 21.7%, respectively (Log-rank trend test: p = 0.047). In multivariable logistic regression, adjusting for sepsis, comorbidities, and age, only the very high-risk group was associated with 30-day in-hospital mortality (odds ratio: 5.54, 95% confidence interval: 1.73–17.79) A similar association was observed for the secondary outcomes. Conclusions: For patients admitted urgently to the HDU, there may be an opportunity to improve outcomes for very high-risk patients through preventive strategies.

Sporadic inclusion body myositis (sIBM) is a muscle disease in older people and is characterized by inflammatory cell invasion into intact muscle fibers and rimmed vacuoles. The pathomechanism of sIBM is not fully elucidated yet, and controversy exists as to whether sIBM is a primary autoimmune disease or a degenerative muscle disease with secondary inflammation. Previously, we established a method of collecting CD56-positive myoblasts from human skeletal muscle biopsy samples. We hypothesized that the myoblasts derived from these patients are useful to see the cell-autonomous pathomechanism of sIBM. With these resources, myoblasts were differentiated into myotubes, and the expression profiles of cell-autonomous pathology of sIBM were analyzed. Myoblasts from three sIBM cases and six controls were differentiated into myotubes. In the RNA-sequencing analysis of these "myotube" samples, 104 differentially expressed genes (DEGs) were found to be significantly upregulated by more than twofold in sIBM, and 13 DEGs were downregulated by less than twofold. For muscle biopsy samples, a comparative analysis was conducted to determine the extent to which "biopsy" and "myotube" samples differed. Fifty-three DEGs were extracted of which 32 (60%) had opposite directions of expression change (e.g., increased in biopsy vs decreased in myotube). Apolipoprotein E (apoE) and transmembrane protein 8C (TMEM8C or MYMK) were commonly upregulated in muscle biopsies and myotubes from sIBM. ApoE and myogenin protein levels were upregulated in sIBM. Given that enrichment analysis also captured changes in muscle contraction and development, the triggering of muscle atrophy signaling and abnormal muscle differentiation via MYMK or myogenin may be involved in the pathogenesis of sIBM. The presence of DEGs in sIBM suggests that the myotubes formed from sIBM-derived myoblasts revealed the existence of muscle cell-autonomous degeneration in sIBM. The catalog of DEGs will be an important resource for future studies on the pathogenesis of sIBM focusing on primary muscle degeneration.

Abstract Background Single-cell technologies to analyze transcription and chromatin structure have been widely used in many research areas to reveal the functions and molecular properties of cells at single-cell resolution. Sample multiplexing techniques are valuable when performing single-cell analysis, reducing technical variation and permitting cost efficiencies. Several commercially available methods have been used in many scRNA-seq studies. On the other hand, while several methods have been published, multiplexing techniques for single nuclear assay for transposase-accessible chromatin (snATAC)-seq assays remain under development. We developed a simple nucleus hashing method using oligonucleotide-conjugated antibodies recognizing nuclear pore complex proteins, NuHash, to perform snATAC-seq library preparations by multiplexing. Results We performed multiplexing snATAC-seq analyses on a mixture of human and mouse cell samples (two samples, 2-plex, and four samples, 4-plex) using NuHash. The analyses on nuclei with at least 10,000 read counts showed that the demultiplexing accuracy of NuHash was high, and only ten out of 9144 nuclei (2-plex) and 150 of 12,208 nuclei (4-plex) had discordant classifications between NuHash demultiplexing and discrimination using reference genome alignments. The differential open chromatin region (OCR) analysis between female and male samples revealed that male-specific OCRs were enriched in chromosome Y (four out of nine). We also found that five female-specific OCRs (20 OCRs) were on chromosome X. A comparative analysis between snATAC-seq and deeply sequenced bulk ATAC-seq on the same samples revealed that the bulk ATAC-seq signal intensity was positively correlated with the number of cell clusters detected in snATAC-seq. Moreover, when we categorized snATAC-seq peaks based on the number of cell clusters in which the peak was present, we observed different distributions over different genomic features between the groups. This result suggests that the peak intensities of bulk ATAC-seq can be used to identify different types of functional loci. Conclusions Our multiplexing method using oligo-conjugated anti-nuclear pore complex proteins, NuHash, permits high-accuracy demultiplexing of samples. The NuHash protocol is straightforward, works on frozen samples, and requires no modifications for snATAC-seq library preparation.

Summary: Early-life stress and ovarian hormones contribute to increased female vulnerability to cocaine addiction. Here, we reveal molecular substrates in the reward area, the nucleus accumbens, through which these female-specific factors affect immediate and conditioning responses to cocaine. We find shared involvement of X chromosome inactivation-related and estrogen signaling-related gene regulation in enhanced conditioning responses following early-life stress and during the low-estrogenic state in females. Low-estrogenic females respond to acute cocaine by opening neuronal chromatin enriched for the sites of ΔFosB, a transcription factor implicated in chronic cocaine response and addiction. Conversely, high-estrogenic females respond to cocaine by preferential chromatin closing, providing a mechanism for limiting cocaine-driven chromatin and synaptic plasticity. We find that physiological estrogen withdrawal, early-life stress, and absence of one X chromosome all nullify the protective effect of a high-estrogenic state on cocaine conditioning in females. Our findings offer a molecular framework to enable understanding of sex-specific neuronal mechanisms underlying cocaine use disorder.

Abstract Background Cellular differentiation is marked by temporally and spatially coordinated gene expression regulated at multiple levels. DNA methylation represents a universal mechanism to control chromatin organization and its accessibility. Cytosine methylation of CpG dinucleotides regulates binding of methylation-sensitive DNA-binding transcription factors within regulatory regions of transcription, including promoters and distal enhancers. Ocular lens differentiation represents an advantageous model system to examine these processes as lens comprises only two cell types, the proliferating lens epithelium and postmitotic lens fiber cells all originating from the epithelium. Results Using whole genome bisulfite sequencing (WGBS) and microdissected lenses, we investigated dynamics of DNA methylation and chromatin changes during mouse lens fiber and epithelium differentiation between embryos (E14.5) and newborns (P0.5). Histone H3.3 variant chromatin landscapes were also generated for both P0.5 lens epithelium and fibers by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Tissue-specific features of DNA methylation patterns are demonstrated via comparative studies with embryonic stem (ES) cells and neural progenitor cells (NPCs) at Nanog, Pou5f1, Sox2, Pax6 and Six3 loci. Comparisons with ATAC-seq and RNA-seq data demonstrate that reduced methylation is associated with increased expression of fiber cell abundant genes, including crystallins, intermediate filament (Bfsp1 and Bfsp2) and gap junction proteins (Gja3 and Gja8), marked by high levels of histone H3.3 within their transcribed regions. Interestingly, Pax6-binding sites exhibited predominantly DNA hypomethylation in lens chromatin. In vitro binding of Pax6 proteins showed Pax6’s ability to interact with sites containing one or two methylated CpG dinucleotides. Conclusions Our study has generated the first data on methylation changes between two different stages of mammalian lens development and linked these data with chromatin accessibility maps, presence of histone H3.3 and gene expression. Reduced DNA methylation correlates with expression of important genes involved in lens morphogenesis and lens fiber cell differentiation.

The Assay for Transposase Accessible Chromatin by sequencing (ATAC-seq) is becoming popular in the neuroscience field where chromatin regulation is thought to be involved in neurodevelopment, activity-dependent gene regulation, hormonal and environmental responses, and pathophysiology of neuropsychiatric disorders. The advantages of using ATAC-seq include a small amount of material needed, fast protocol, and the ability to capture a range of gene regulatory elements with a single assay. With increasing interest in chromatin research, it is an imperative to have feasible, reliable assays that are compatible with a range of neuroscience study designs. Here we tested three protocols for neuronal chromatin accessibility analysis, including a varying brain tissue freezing method followed by fluorescence-activated nuclei sorting (FANS) and ATAC-seq. Our study shows that the cryopreservation method impacts the number of open chromatin regions identified from frozen brain tissue using ATAC-seq. However, we show that all protocols generate consistent and robust data and enable the identification of functional regulatory elements in neuronal cells. Our study implies that the broad biological interpretation of chromatin accessibility data is not significantly affected by the freezing condition. We also reveal additional challenges of doing chromatin analysis on post-mortem human brain tissue. Overall, ATAC-seq coupled with FANS is a powerful method to capture cell-type-specific chromatin accessibility information in mouse and human brain. Our study provides alternative brain preservation methods that generate high-quality ATAC-seq data while fitting in different study designs, and further encourages the use of this method to uncover the role of epigenetic (dys)regulation in the brain.

Abstract Background Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify types of cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and cross-validated resource for epigenetics research from the FDA’s Epigenomics Quality Control Group. Results Each sample is processed in multiple replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), enzymatic deamination method (EMSeq), targeted methylation sequencing (Illumina Methyl Capture EPIC), single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies, and 850k Illumina methylation arrays. After rigorous quality assessment and comparison to Illumina EPIC methylation microarrays and testing on a range of algorithms (Bismark, BitmapperBS, bwa-meth, and BitMapperBS), we find overall high concordance between assays, but also differences in efficiency of read mapping, CpG capture, coverage, and platform performance, and variable performance across 26 microarray normalization algorithms. Conclusions The data provided herein can guide the use of these DNA reference materials in epigenomics research, as well as provide best practices for experimental design in future studies. By leveraging seven human cell lines that are designated as publicly available reference materials, these data can be used as a baseline to advance epigenomics research.

Abstract Preterm birth is a leading cause of neonatal morbidity. Survivors have a greater risk for kidney dysfunction and hypertension. Little is known about the molecular changes that occur in the kidney of individuals born preterm. Here, we demonstrate that mice delivered two days prior to full term gestation undergo premature cessation of nephrogenesis, resulting in a lower glomerular density. Kidneys from preterm and term groups exhibited differences in gene expression profiles at 20- and 27-days post-conception, including significant differences in the expression of fat-soluble vitamin-related genes. Kidneys of the preterm mice exhibited decreased proportions of endothelial cells and a lower expression of genes promoting angiogenesis compared to the term group. Kidneys from the preterm mice also had altered nephron progenitor subpopulations, early Six2 depletion, and altered Jag1 expression in the nephrogenic zone, consistent with premature differentiation of nephron progenitor cells. In conclusion, preterm birth alone was sufficient to shorten the duration of nephrogenesis and cause premature differentiation of nephron progenitor cells. These candidate genes and pathways may provide targets to improve kidney health in preterm infants.

Abstract Background In genomics, we often assume that continuous data, such as gene expression, follow a specific kind of distribution. However we rarely stop to question the validity of this assumption, or consider how broadly applicable it may be to all genes that are in the transcriptome. Our study investigated the prevalence of a range of gene expression distributions in three different tumor types from the Cancer Genome Atlas (TCGA). Results Surprisingly, the expression of less than 50% of all genes was Normally-distributed, with other distributions including Gamma, Bimodal, Cauchy, and Lognormal also represented. Most of the distribution categories contained genes that were significantly enriched for unique biological processes. Different assumptions based on the shape of the expression profile were used to identify genes that could discriminate between patients with good versus poor survival. The prognostic marker genes that were identified when the shape of the distribution was accounted for reflected functional insights into cancer biology that were not observed when standard assumptions were applied. We showed that when multiple types of distributions were permitted, i.e. the shape of the expression profile was used, the statistical classifiers had greater predictive accuracy for determining the prognosis of a patient versus those that assumed only one type of gene expression distribution. Conclusions Our results highlight the value of studying a gene’s distribution shape to model heterogeneity of transcriptomic data and the impact on using analyses that permit more than one type of gene expression distribution. These insights would have been overlooked when using standard approaches that assume all genes follow the same type of distribution in a patient cohort.

Vitamin D, a fat-soluble vitamin, plays a critical role in calcium homeostasis, the immune system, and normal development. Many epidemiological cohort studies globally have found high prevalence rates of vitamin D deficiency and insufficiency, recognized as an important health issue that needs to be solved. In particular, reproductive age and pregnant women low in vitamin D status may confer risks of diseases like obesity on their offspring. While observational studies have suggested associations between prenatal vitamin D deficiency and metabolic phenotypes in offspring, not yet determined is whether prenatal vitamin D deficiency permanently alters the development of the liver, a major metabolic organ. We tested the histopathology and the transcriptomic profiles of livers from male C57BL/6J mice exposed to prenatal vitamin D deficiency through a maternal dietary intervention model. We found that prenatal vitamin D deficiency increases the prevalence of histopathological changes in the liver, and alters its gene expression profile. Cell subtype proportion analysis showed that the liver of prenatal vitamin D deficiency alters non-parenchymal cells of the liver, specifically macrophages, a subset of endothelial cells, and dendritic cells. Our results indicate the long-term memory of prenatal vitamin D deficiency exposure in the adult liver, a potential contributor to offspring health risks.

Vitamin A, an essential fat-soluble micronutrient, plays a critical role in the body, by regulating vision, immune responses, and normal development, for instance. Vitamin A deficiency (VAD) is a major cause of xerophthalmia and increases the risk of death from infectious diseases. It is also emerging that prenatal exposure to VAD is associated with disease risks later in life. The overall prevalence of VAD has significantly declined over recent decades; however, the rate of VAD is still high in many low- and mid-income countries and even in high-income countries among specific ethnic/race groups. While VAD occurs when dietary intake is insufficient to meet demands, establishing a strong association between food insecurity and VAD, and vitamin A supplementation is the primary solution to treat VAD, genetic contributions have also been reported to effect serum vitamin A levels. In this review, we discuss genetic variations associated with vitamin A status and vitamin A bioactivity-associated genes, specifically those linked to uptake of the vitamin in the small intestine and its storage in the liver, as well as their potential contribution to vitamin A deficiency risks among different ethnic groups.

Abstract Background Suicide mortality is high in Japan and early interventional strategies to solve that problem are needed. An accurate evaluation of the regional status of current suicide mortality would be useful for community interventions. A few studies in Kanagawa prefecture, located next to Tokyo and with the second largest population in Japan, have identified spatial clusters of suicide mortality at regional levels. This study examined spatial clustering and clustering over time of such events using spatial data from regional statistics on suicide deaths. Methods Data were obtained from regional statistics (58 regions in Kanagawa prefecture) of the National Vital Statistics of Japan from 2011 to 2017. The standardized mortality ratio (SMR) and Empirical Bayes estimator for the SMR (EBSMR) were used as measures. Spatial clusters were examined by Kulldorff’s circular spatial scan statistic, Tango-Takahashi’s flexible spatial scan statistic and Tango’s test. Linear regression and conditional autoregressive (CAR) models were used not only to adjust for covariates but also to estimate regional effects. The analyses were conducted for each year, inclusive. Results Among male suicide deaths, being unemployed (50%) was most frequently related to suicide while among female health problem (50%) were frequent. Spatial clusters with significance detected by FlexScan, SatScan and Tango’s test were few and varied somewhat according to the method used. Spatial clusters were detected in some regions including Kawasaki ward after adjustment by covariates. By the linear regression models, selected variables with significance were different between the sexes. For males, unemployment, family size, and proportion of higher education were detected for several of the years studied while for females, family size and divorce rate were detected over this period. These variables were also observed by the CAR model with 5 covariates. Regional effects were much clearer by considering the spatial parameter for both males and females and especially, Kawasaki ward was detected as a high risk region in many years. Conclusion The present results detected some spatial clustering of suicide deaths within certain regions. Factors related to suicide deaths were also indicated. These results would provide important information in policy making for suicide prevention.

A decrease in nicotinamide adenine dinucleotide (NAD+) levels is a hallmark of aging in multiple organisms, including humans. We report a human clinical trial designed to assess the efficacy of single-dose supplementation of nicotinamide, a form of vitamin B3, on increasing NAD+ levels. Nicotinamide intake within the daily tolerable upper level (200 mg) led to the maximal whole blood concentration by a 30-fold increase at 0.5 h with a consistent decrease until 6 h in all 6 healthy male volunteers. The increase in blood nicotinamide amounted to 17%, whereas renal clearance accounted for 1.8% of the ingested dose. NAD+ levels reached a maximum concentration at 12 h, with all time points showing a trend of increase. Blood metabolome assay indicates that the metabolomic changes with NAM supplementation lasted 24 h and then returned to baseline by 48 h. The basal levels of whole blood NAD+ varied among individuals with a negative correlation with body height as well as nicotinamide mononucleotide (NMN) levels. These data warrant consideration for further clinical evaluation with repeated doses of nicotinamide within the safety dose range for the potency to increase NAD+ levels and potential accompanying benefits for healthy longevity.

To gain insights into the molecular interactions of an intracellular pathogen and its host cell, we studied the gene expression and chromatin states of human fibroblasts infected with the Apicomplexan parasite Toxoplasma gondii. We show a striking activation of host cell genes that regulate a number of cellular processes, some of which are protective of the host cell, others likely to be advantageous to the pathogen. The simultaneous capture of host and parasite genomic information allowed us to gain insights into the regulation of the T. gondii genome. We show how chromatin accessibility and transcriptional profiling together permit novel annotation of the parasite's genome, including more accurate mapping of known genes and the identification of new genes and cis-regulatory elements. Motif analysis reveals not only the known T. gondii AP2 transcription factor-binding site but also a previously-undiscovered candidate TATA box-containing motif at one-quarter of promoters. By inferring the transcription factor and upstream cell signaling responses involved in the host cell, we can use genomic information to gain insights into T. gondii's perturbation of host cell physiology. Our resulting model builds on previously-described human host cell signalling responses to T. gondii infection, linked to induction of specific transcription factors, some of which appear to be solely protective of the host cell, others of which appear to be co-opted by the pathogen to enhance its own survival.

Functional variants have been proposed to alter transcription factor binding. Here, the authors provide direct evidence that functional variants within the TBC1D4 gene, encoding an NFκB binding site, can alter transcription factor binding, and use CRISPR-Cas9 to reveal localization of the transcription factor to be the regulator of chromatin accessibility and p65 binding and ultimately TBC1D4 expression.

The molecular mechanisms underlying the dynamic nature of the female brain structure and function remain poorly understood. Here the authors characterise chromatin organization in the mouse female ventral hippocampus, finding it fluctuates with the oestrous cycle, and identify changes in chromatin organization associated with the transcription of genes important for neuronal function and behaviour.

Regulatory T (Treg) cells are developed in the thymus, and are essential for suppressing detrimental autoimmunity. Here the authors show, using mice with dampened interleukin 2 (IL-2) signaling, that IL-2 helps position the pioneer factor SATB1 to control genome-wide chromatin accessibility to facilitate Treg cell lineage commitment in the thymus.

Abstract: Erythroid differentiation is associated with global DNA demethylation, but a complete methylome was lacking in the erythroid lineage. We have generated allele-specific base resolution methylomes of primary basophilic erythroblasts (BasoEs) and compared these with 8 other cell types. We found that DNA demethylation during differentiation from hematopoietic stem/progenitor cells (HSPCs) to BasoEs occurred predominantly in intergenic sequences and in inactive gene bodies causing the formation of partially methylated domains (PMDs) in 74% of the BasoE methylome. Moreover, differentially methylated regions (DMRs) between HSPCs and BasoEs occurred mostly in putative enhancer regions and were most often associated with GATA, EKLF, and AP1 binding motifs. Surprisingly, promoters silent in both HSPCs and BasoEs exhibited much more dramatic chromatin changes during differentiation than activated promoters. Unmethylated silent promoters were often associated with active chromatin states in highly methylated domains (HMDs) but with polycomb-repression in PMDs, indicating that silent promoters are generally regulated differently in HMDs and PMDs. We show that long PMDs replicate late, but that short PMDs replicate early and therefore that the partial methylation of DNA after replication during erythroid expansion occurs throughout S phase of the cell cycle. We propose that baseline maintenance methylation following replication decreases during erythroid differentiation resulting in PMD formation and that the presence of HMDs in the BasoE methylome results from transcription-associated DNA methylation of gene bodies. We detected ∼700 large allele-specific DMRs that were enriched in single-nucleotide polymorphisms, suggesting that primary DNA sequence might be a determinant of DNA methylation levels within PMDs.

Abstract Background Cell-type-specific genes exhibit heterogeneity in genomic contexts and may be subject to different epigenetic regulations through different gene transcriptional processes depending on the cell type involved. The gene-body regions (GBRs) of some cardiomyocyte (CM)-specific genes are long and highly hypomethylated in CMs. To explore the cell-type specificities of epigenetic patterns and functions, multiple epigenetic modifications of GBRs were compared among CMs, liver cells and embryonic stem cells (ESCs). Results We found that most genes show a moderately negative correlation between transcript levels and gene lengths. As CM-specific genes are generally longer than other cell-type-specific genes, we hypothesized that the gene-body epigenetic features of CMs may support the transcriptional regulation of CM-specific genes. We found gene-body DNA hypomethylation in a CM-specific gene subset co-localized with rare gene-body marks, including RNA polymerase II (Pol II) and p300. Interestingly, 5-hydroxymethylcytosine (5hmC) within the gene body marked cell-type-specific genes at neonatal stages and active gene-body histone mark H3K36 trimethylation declined and overlapped with cell-type-specific gene-body DNA hypomethylation and selective Pol II/p300 accumulation in adulthood. Different combinations of gene-body epigenetic modifications were also observed with genome-wide scale cell-type specificity, revealing the occurrence of dynamic epigenetic rearrangements in GBRs across different cell types. Conclusions As 5hmC enrichment proceeded to hypomethylated GBRs, we considered that hypomethylation may not represent a static state but rather an equilibrium state of turnover due to the balance between local methylation linked to transcription and Tet oxidative modification causing demethylation. Accordingly, we conclude that demethylation in CMs can be a used to establish such cell-type-specific epigenetic domains in relation to liver cells. The establishment of cell-type-specific epigenetic control may also change genomic contexts of evolution and may contribute to the development of cell-type-specific transcriptional coordination.

Abstract Studies on genetic–epigenetic interactions, including the mapping of methylation quantitative trait loci (mQTLs) and haplotype-dependent allele-specific DNA methylation (hap-ASM), have become a major focus in the post-genome-wide-association-study (GWAS) era. Such maps can nominate regulatory sequence variants that underlie GWAS signals for common diseases, ranging from neuropsychiatric disorders to cancers. Conversely, mQTLs need to be filtered out when searching for non-genetic effects in epigenome-wide association studies (EWAS). Sequence variants in CCCTC-binding factor (CTCF) and transcription factor binding sites have been mechanistically linked to mQTLs and hap-ASM. Identifying these sites can point to disease-associated transcriptional pathways, with implications for targeted treatment and prevention.

Even though pancreatic ductal adenocarcinoma (PDAC) is associated with fibrotic stroma, the molecular pathways regulating the formation of cancer associated fibroblasts (CAFs) are not well elucidated. An epigenomic analysis of patient-derived and de-novo generated CAFs demonstrated widespread loss of cytosine methylation that was associated with overexpression of various inflammatory transcripts including CXCR4. Co-culture of neoplastic cells with CAFs led to increased invasiveness that was abrogated by inhibition of CXCR4. Metabolite tracing revealed that lactate produced by neoplastic cells leads to increased production of alpha-ketoglutarate (aKG) within mesenchymal stem cells (MSCs). In turn, aKG mediated activation of the demethylase TET enzyme led to decreased cytosine methylation and increased hydroxymethylation during de novo differentiation of MSCs to CAF. Co-injection of neoplastic cells with TET-deficient MSCs inhibited tumor growth in vivo. Thus, in PDAC, a tumor-mediated lactate flux is associated with widespread epigenomic reprogramming that is seen during CAF formation.

Cell subtype proportion variability between samples contributes significantly to the variation of functional genomic properties such as gene expression or DNA methylation. Although the impact of the variation of cell subtype composition on measured genomic quantities is recognized, and some innovative tools have been developed for the analysis of heterogeneous samples, most functional genomics studies using samples with mixed cell types still ignore the influence of cell subtype proportion variation, or just deal with it as a nuisance variable to be eliminated. Here we demonstrate how harvesting information about cell subtype proportions from functional genomics data can provide insights into cellular changes associated with phenotypes. We focused on two types of mixed cell populations, human blood and mouse kidney. Cell type prediction is well developed in the former, but not currently in the latter. Estimating the cellular repertoire is easier when a reference dataset from purified samples of all cell types in the tissue is available, as is the case for blood. However, reference datasets are not available for most other tissues, such as the kidney. In this study, we showed that the proportion of alterations attributable to changes in the cellular composition varies strikingly in the two disorders (asthma and systemic lupus erythematosus), suggesting that the contribution of cell subtype proportion changes to functional genomic properties can be disease-specific. We also showed that a reference dataset from a single-cell RNA-seq study successfully estimated the cell subtype proportions in mouse kidney and allowed us to distinguish altered cell subtype differences between two different knock-out mouse models, both of which had reported a reduced number of glomeruli compared to their wild-type counterparts. These findings demonstrate that testing for changes in cell subtype proportions between conditions can yield important insights in functional genomics studies.

Background: While the current national prevalence rate of vitamin A deficiency (VAD) is estimated to be less than 1%, it is suggested that it varies between different ethnic groups and races within the U.S. We assessed the prevalence of VAD in pregnant women of different ethnic groups and tested these prevalence rates for associations with the vitamin A-related single nucleotide polymorphism (SNP) allele frequencies in each ethnic group. Methods: We analyzed two independent datasets of serum retinol levels with self-reported ethnicities and the differences of allele frequencies of the SNPs associated with vitamin A metabolism between groups in publicly available datasets. Results: Non-Hispanic Black and Hispanic pregnant women showed high VAD prevalence in both datasets. Interestingly, the VAD prevalence for Hispanic pregnant women significantly differed between datasets (p = 1.973 × 10−10, 95%CI 0.04–0.22). Alleles known to confer the risk of low serum retinol (rs10882272 C and rs738409 G) showed higher frequencies in the race/ethnicity groups with more VAD. Moreover, minor allele frequencies of a set of 39 previously reported SNPs associated with vitamin A metabolism were significantly different between the populations of different ancestries than those of randomly selected SNPs (p = 0.030). Conclusions: Our analysis confirmed that VAD prevalence varies between different ethnic groups/races and may be causally associated with genetic variants conferring risk for low retinol levels. Assessing genetic variant information prior to performing an effective nutrient supplementation program will help us plan more effective food-based interventions.

Aberrant cytosine 5-methylation underlies many deregulated elements of cancer. Among paired non-small cell lung cancers (NSCLC), we sought to profile DNA 5-methyl-cytosine features which may underlie genome-wide deregulation. In one of the more dense interrogations of the methylome, we sampled 1.2 million CpG sites from twenty-four NSCLC tumor (T)-non-tumor (NT) pairs using a methylation-sensitive restriction enzyme- based HELP-microarray assay. We found 225,350 differentially methylated (DM) sites in adenocarcinomas versus adjacent non-tumor tissue that vary in frequency across genomic compartment, particularly notable in gene bodies (GB; p

DNA mutational events are increasingly being identified in autism spectrum disorder (ASD), but the potential additional role of dysregulation of the epigenome in the pathogenesis of the condition remains unclear. The epigenome is of interest as a possible mediator of environmental effects during development, encoding a cellular memory reflected by altered function of progeny cells. Advanced maternal age (AMA) is associated with an increased risk of having a child with ASD for reasons that are not understood. To explore whether AMA involves covert aneuploidy or epigenetic dysregulation leading to ASD in the offspring, we tested a homogeneous ectodermal cell type from 47 individuals with ASD compared with 48 typically developing (TD) controls born to mothers of ≥35 years, using a quantitative genome-wide DNA methylation assay. We show that DNA methylation patterns are dysregulated in ectodermal cells in these individuals, having accounted for confounding effects due to subject age, sex and ancestral haplotype. We did not find mosaic aneuploidy or copy number variability to occur at differentially-methylated regions in these subjects. Of note, the loci with distinctive DNA methylation were found at genes expressed in the brain and encoding protein products significantly enriched for interactions with those produced by known ASD-causing genes, representing a perturbation by epigenomic dysregulation of the same networks compromised by DNA mutational mechanisms. The results indicate the presence of a mosaic subpopulation of epigenetically-dysregulated, ectodermally-derived cells in subjects with ASD. The epigenetic dysregulation observed in these ASD subjects born to older mothers may be associated with aging parental gametes, environmental influences during embryogenesis or could be the consequence of mutations of the chromatin regulatory genes increasingly implicated in ASD. The results indicate that epigenetic dysregulatory mechanisms may complement and interact with DNA mutations in the pathogenesis of the disorder.

Disruption of circadian rhythm is believed to play a critical role in cancer development. Cryptochrome 1 (CRY1) is a core component of the mammalian circadian clock and we have previously shown its deregulated expression in a subgroup of patients with chronic lymphocytic leukemia (CLL). Using real-time RT-PCR in a cohort of 76 CLL patients and 35 normal blood donors we now demonstrate that differential CRY1 mRNA expression in high-risk (HR) CD38+/immunoglobulin variable heavy chain gene (IgVH) unmutated patients as compared to low-risk (LR) CD38-/IgVH mutated patients can be attributed to down-modulation of CRY1 in LR CLL cases. Analysis of the DNA methylation profile of the CRY1 promoter in a subgroup of 57 patients revealed that CRY1 expression in LR CLL cells is silenced by aberrant promoter CpG island hypermethylation. The methylation pattern of the CRY1 promoter proved to have high prognostic impact in CLL where aberrant promoter methylation predicted a favourable outcome. CRY1 mRNA transcript levels did not change over time in the majority of patients where sequential samples were available for analysis. We also compared the CRY1 expression in CLL with other lymphoid malignancies and observed epigenetic silencing of CRY1 in a patient with B cell acute lymphoblastic leukemia (B-ALL).

Although a combination of genomic and epigenetic alterations are implicated in the multistep transformation of normal squamous esophageal epithelium to Barrett esophagus, dysplasia, and adenocarcinoma, the combinatorial effect of these changes is unknown. By integrating genome-wide DNA methylation, copy number, and transcriptomic datasets obtained from endoscopic biopsies of neoplastic progression within the same individual, we are uniquely able to define the molecular events associated progression of Barrett esophagus. We find that the previously reported global hypomethylation phenomenon in cancer has its origins at the earliest stages of epithelial carcinogenesis. Promoter hypomethylation synergizes with gene amplification and leads to significant upregulation of a chr4q21 chemokine cluster and other transcripts during Barrett neoplasia. In contrast, gene-specific hypermethylation is observed at a restricted number of loci and, in combination with hemi-allelic deletions, leads to downregulatation of selected transcripts during multistep progression. We also observe that epigenetic regulation during epithelial carcinogenesis is not restricted to traditionally defined "CpG islands," but may also occur through a mechanism of differential methylation outside of these regions. Finally, validation of novel upregulated targets (CXCL1 and 3, GATA6, and DMBT1) in a larger independent panel of samples confirms the utility of integrative analysis in cancer biomarker discovery.

In this article, I focus on the difference between proverbs and idioms which derive from proverbs. Both proverbs and idioms are hard to defi ne, as it is also difficult to categorize a specific expression being either a proverb or an idiom. Fixed form is one feature of proverbs as well as that a proverb appears as a sentence, but today one can easily find variations and/or modifications of proverbs. To see how those idioms which derive from proverbs are used in actual speech, I have collected examples from a number of corpuses. Some of these idioms are truncated and, being quite conventional, used almost only in their reduced form. Some idioms lose the negations which the original proverbs show, but they still carry their original meaning implicated by the proverbs. A proverb, as a whole, is lexicalized and this is why one can use part of the proverb and/or the proverb modified. However, when a part or a component of a proverb becomes strongly lexicalized and no longer connected with its original meaning as a proverb, then such an expression can be regarded as an idiom.

This editorial, published in the journal Frontiers in Cell & Developmental Biology, focuses on the contributions of women in the field of developmental epigenetics. It acknowledges the gender disparity in academia and publishing, with fewer women in senior positions and fewer women credited as authors. The editorial highlights the pioneering work of women scientists in the field and includes reviews and research articles on topics related to genomic imprinting, epigenetic programming, and developmental exposures. It discusses the impact of epigenetic perturbations on prenatal and perinatal development and long-term health outcomes, as well as the effects of prenatal exposure to cadmium and environmental toxicants on epigenetic alterations. The conclusion emphasizes the importance of gender parity in publishing and the need for progress in this area. [Extracted from the article]