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Transgenic T-cell receptor (TCR) adoptive cell therapies recognizing tumor antigens are associated with robust initial response rates, but frequent disease relapse. This usually occurs in the setting of poor long-term persistence of cells expressing the transgenic TCR, generated using murine stem cell virus (MSCV) γ-retroviral vectors. Analysis of clinical transgenic adoptive cell therapy products in vivo revealed that despite strong persistence of the transgenic TCR DNA sequence over time, its expression was profoundly decreased over time at the RNA and protein levels. Patients with the greatest degrees of expression suppression displayed significant increases in DNA methylation over time within the MSCV promoter region, as well as progressive increases in DNA methylation within the entire MSCV vector over time. These increases in vector methylation occurred independently of its integration site within the host genomes. These results have significant implications for the design of future viral vector gene–engineered adoptive cell transfer therapies.Significance:Cellular immunotherapies' reliance on retroviral vectors encoding foreign genetic material can be vulnerable to progressive acquisition of DNA methylation and subsequent epigenetic suppression of the transgenic product in TCR adoptive cell therapy. This must be considered in the design of future generations of cellular immunotherapies for cancer.This article is highlighted in the In This Issue feature, p. 1611

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Oncogenic kinase activity and the resulting aberrant growth and survival signaling are a common driving force of cancer. Accordingly, many successful molecularly targeted anticancer therapeutics are directed at inhibiting kinase activity. To assess kinase activity in minute patient samples, we have developed an immunocapture-based in vitro kinase assay on an integrated polydimethylsiloxane microfluidics platform that can reproducibly measure kinase activity from as few as 3,000 cells. For this platform, we adopted the standard radiometric 32P-ATP–labeled phosphate transfer assay. Implementation on a microfluidic device required us to develop methods for repeated trapping and mixing of solid-phase affinity microbeads. We also developed a solid-state beta-particle camera imbedded directly below the microfluidic device for real-time quantitative detection of the signal from this and other microfluidic radiobioassays. We show that the resulting integrated device can measure ABL kinase activity from BCR-ABL–positive leukemia patient samples. The low sample input requirement of the device creates new potential for direct kinase activity experimentation and diagnostics on patient blood, bone marrow, and needle biopsy samples. Cancer Res; 70(21); 8299–308. ©2010 AACR.

Background Influenza virus infection alters the promoter DNA methylation of key immune response-related genes, including type-1 interferons and proinflammatory cytokines. However, less is known about the effect of the influenza vaccine on the epigenome. We utilized a targeted DNA methylation approach to study the longitudinal effects (day 0 pre-vaccination and day 28 post-vaccination) on influenza vaccination responses in peripheral blood mononuclear cells. Results We found that baseline, pre-vaccination methylation profiles are associated with pre-existing, protective serological immunity. Additionally, we identified 481 sites that were differentially methylated between baseline and day 28 post-vaccination. These were enriched for genes involved in the regulation of the RIG-I signaling pathway, an important regulator of viral responses. Conclusions Our results suggest that DNA methylation changes to components of the RIG-I pathway are associated with vaccine effectiveness. Therefore, immunization strategies that target this pathway may improve serological responses to influenza vaccination.

Unlike genomes, which are static throughout the lifespan of an organism, DNA methylomes are dynamic. To study these dynamics we developed quantitative models that measure the effect of multiple factors on DNA methylomes including, age, sex, weight and genetics. We conducted our study in canids, which prove to be an ideal species to assess epigenetic moderators due to their extreme variability in size and well-characterized genetic structure. We collected buccal swabs from 217 canids (207 domestic dogs and 10 gray wolves) and used targeted bisulfite sequencing to measure methylomes. We also measured genotypes at over one thousand single nucleotide polymorphisms (SNPs). We found that DNA methylomes are strongly associated with age, enabling the construction of epigenetic clocks. We also show that methylomes are strongly impacted by sex, weight and sterilization status, leading to accurate predators of these factors. Methylomes are also affected by genetics and we observe multiple associations between SNP loci and methylated CpGs. Finally, we show that several factors moderate the relationship between epigenetic ages and real ages, such as body weight, which increases epigenetic aging. In conclusion, we demonstrate that the plasticity of DNA methylomes is impacted by myriad genetic and physiological factors, and that DNA methylation biomarkers are accurate predictors of age, sex and sterilization status.

Cytosine methylation is one of the best studied epigenetic modifications. In mammals, DNA methylation patterns vary among cells and is mainly found in the CpG context. DNA methylation is involved in important processes during development and differentiation and its dysregulation can lead to or is associated with diseases, such as cancer, loss-of-imprinting syndromes and neurological disorders. It has been also shown that DNA methylation at the cellular, tissue and organism level varies with age. To overcome the costs of Whole-Genome Bisulfite Sequencing, the gold standard method to detect 5-methylcytosines at a single base resolution, DNA methylation arrays have been developed and extensively used. This method allows one to assess the status of a fraction of the CpG sites present in the genome of an organism. In order to combine the relatively low cost of Methylation Arrays and digital signals of bisulfite sequencing, we developed a Targeted Bisulfite Sequencing method that can be applied to biomarker discovery for virtually any phenotype. Here we describe a comprehensive step-by-step protocol to build a DNA methylation-based epigenetic clock.

Transgenic T-cell receptor (TCR) adoptive cell therapies recognizing tumor antigens are associated with robust initial response rates, but frequent disease relapse. This usually occurs in the setting of poor long-term persistence of cells expressing the transgenic TCR, generated using murine stem cell virus (MSCV) γ-retroviral vectors. Analysis of clinical transgenic adoptive cell therapy products in vivo revealed that despite strong persistence of the transgenic TCR DNA sequence over time, its expression was profoundly decreased over time at the RNA and protein levels. Patients with the greatest degrees of expression suppression displayed significant increases in DNA methylation over time within the MSCV promoter region, as well as progressive increases in DNA methylation within the entire MSCV vector over time. These increases in vector methylation occurred independently of its integration site within the host genomes. These results have significant implications for the design of future viral vector gene–engineered adoptive cell transfer therapies. Significance: Cellular immunotherapies' reliance on retroviral vectors encoding foreign genetic material can be vulnerable to progressive acquisition of DNA methylation and subsequent epigenetic suppression of the transgenic product in TCR adoptive cell therapy. This must be considered in the design of future generations of cellular immunotherapies for cancer. This article is highlighted in the In This Issue feature, p. 1611

Human DNA-methylation data have been used to develop highly accurate biomarkers of aging ("epigenetic clocks"). Recent studies demonstrate that similar epigenetic clocks for mice (Mus Musculus) can be slowed by gold standard anti-aging interventions such as calorie restriction and growth hormone receptor knock-outs. Using DNA methylation data from previous publications with data collected in house for a total 1189 samples spanning 193,651 CpG sites, we developed 4 novel epigenetic clocks by choosing different regression models (elastic net- versus ridge regression) and by considering different sets of CpGs (all CpGs vs highly conserved CpGs). We demonstrate that accurate age estimators can be built on the basis of highly conserved CpGs. However, the most accurate clock results from applying elastic net regression to all CpGs. While the anti-aging effect of calorie restriction could be detected with all types of epigenetic clocks, only ridge regression based clocks replicated the finding of slow epigenetic aging effects in dwarf mice. Overall, this study demonstrates that there are trade-offs when it comes to epigenetic clocks in mice. Highly accurate clocks might not be optimal for detecting the beneficial effects of anti-aging interventions.

Macrophages (Mϕs) of patients with Alzheimer’s disease and mild cognitive impairment (MCI) are defective in amyloid-β1-42 (Aβ) phagocytosis and have low resistance to apoptosis by Aβ. Omega-3 fatty acids (ω-3s) in vitro and in vivo and the ω-3 mediator, resolvin D1, in vitro increase Aβ phagocytosis by Mϕs of patients with MCI. We have investigated the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress by Mϕs in a longitudinal study of fish-derived, ω-3–supplemented patients with MCI. Patients in the apolipoprotein E (ApoE)e3/e3 subgroup over time exhibited an increase of protein kinase RNA-like ER kinase (PERK) expression, Aβ phagocytosis, intermediate M1-M2 Mϕ type, and a Mini-Mental State Examination (MMSE) rate of change of +1.8 points per year, whereas patients in the ApoEe3/e4 subgroup showed individually divergent results with an MMSE rate of change of −3.2 points per year. In vitro treatment of Mϕs by fish-derived ω-3 emulsion increased Aβ phagocytosis, PERK expression, and UPR RNA signature, and decreased ER stress signature. Augmented genes in the UPR signature included chaperones, lectins, foldases, and N-linked glycosylation enzymes. In summary, fish-derived ω-3s increase cytoprotective genes and decrease proapoptotic genes, improve immune clearance of Aβ, and are associated with an improved MMSE rate of change in ApoEe3/e3 vs. ApoEe3/e4 patients.—Olivera-Perez, H. M., Lam, L., Dang, J., Jiang, W., Rodriguez, F., Rigali, E., Weitzman, S., Porter, V., Rubbi, L., Morselli, M., Pellegrini, M., Fiala, M. Omega-3 fatty acids increase the unfolded protein response and improve amyloid-β phagocytosis by macrophages of patients with mild cognitive impairment.

Applications of embryonic stem cells (ESCs) require faithful chromatin changes during differentiation, but the fate of the X chromosome state in differentiating ESCs is unclear. Female human ESC lines either carry two active X chromosomes (XaXa), an Xa and inactive X chromosome with or without XIST RNA coating (XiXIST+Xa;XiXa), or an Xa and an eroded Xi (XeXa) where the Xi no longer expresses XIST RNA and has partially reactivated. Here, we established XiXa, XeXa, and XaXa ESC lines and followed their X chromosome state during differentiation. Surprisingly, we found that the X state pre-existing in primed ESCs is maintained in differentiated cells. Consequently, differentiated XeXa and XaXa cells lacked XIST, did not induce X inactivation, and displayed higher X-linked gene expression than XiXa cells. These results demonstrate that X chromosome dosage compensation is not required for ESC differentiation. Our data imply that XiXIST+Xa ESCs are most suited for downstream applications and show that all other X states are abnormal byproducts of our ESC derivation and propagation method.

Whole-genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS) are widely used for measuring DNA methylation levels on a genome-wide scale. Both methods have limitations: WGBS is expensive and prohibitive for most large-scale projects; RRBS only interrogates 6–12% of the CpGs in the human genome. Here, we introduce methylation-sensitive restriction enzyme bisulfite sequencing (MREBS) which has the reduced sequencing requirements of RRBS, but significantly expands the coverage of CpG sites in the genome. We built a multiple regression model that combines the two features of MREBS: the bisulfite conversion ratios of single cytosines (as in WGBS and RRBS) as well as the number of reads that cover each locus (as in MRE-seq). This combined approach allowed us to estimate differential methylation across 60% of the genome using read count data alone, and where counts were sufficiently high in both samples (about 1.5% of the genome), our estimates were significantly improved by the single CpG conversion information. We show that differential DNA methylation values based on MREBS data correlate well with those based on WGBS and RRBS. This newly developed technique combines the sequencing cost of RRBS and DNA methylation estimates on a portion of the genome similar to WGBS, making it ideal for large-scale projects of mammalian genomes.

Background: ACT of TCR transgenic T cells recognizing shared tumor antigens is associated with robust initial response rates, but frequent disease relapse. This usually occurs in the setting of poor long-term persistence of cells expressing the transgenic TCR, generated most frequently using a murine stem cell virus (MSCV) gamma retroviral vector. Patients and Methods: Between 2009 and 2016, 24 patients were treated at UCLA using transgenic TCR ACT directed against the tumor antigens MART-1 or NY-ESO-1 for the treatment of melanoma or sarcoma, respectively. We analyzed 16 of these patients' cell therapy products for in vivo persistence and expression of the transgenic TCR at baseline and over time. Transgenic TCR DNA, RNA, and surface protein expression were assessed over time via TCR sequencing, qRT-PCR, and MHC dextramer analysis via FACS, respectively. DNA methylation status of the MSCV 5'LTR promoter region was determined via targeted bisulfite sequencing. Custom biotinylated RNA probes that target the entire MSCV vector were utilized to assess whole vector methylation over time, as well as integration sites within the host genome. Results: Despite overall strong persistence of the transgenic TCR at the DNA level in all patients over time, its expression was profoundly decreased over time at the RNA and protein levels. In a subset of these patients (N=6), the degree of TCR surface expression at day +70 was Conclusions: Progressive acquisition of DNA methylation within the MSCV retroviral vector is associated with suppression of expression of the encoded transgenic TCR. These results have significant implications for the design of future transgenic cell therapies and their associated viral vectors. Citation Format: Theodore S. Nowicki, Colin Farrell, Marco Morselli, Liudmilla Rubbi, Katie Campbell, Beata Berent-Maoz, Begonya Comin-Anduix, Matteo Pellegrini, Antoni Ribas. Epigenetic suppression of transgenic T-cell receptor (TCR) expression in adoptive cell transfer (ACT) therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6590.

Inherent genetic programming and environmental factors affect fetal growth in utero. Epidemiologic data in growth-altered fetuses, either intrauterine growth restricted (IUGR) or large for gestational age (LGA), demonstrate that these newborns are at increased risk of cardiometabolic disease in adulthood. There is growing evidence that the in utero environment leads to epigenetic modification, contributing to eventual risk of developing heart disease or diabetes. In this study, we used reduced representation bisulfite sequencing to examine genome-wide DNA methylation variation in placental samples from offspring born IUGR, LGA, and appropriate for gestational age (AGA) and to identify differential methylation of genes important for conferring risk of cardiometabolic disease. We found that there were distinct methylation signatures for IUGR, LGA, and AGA groups and identified over 500 differentially methylated genes (DMGs) among these group comparisons. Functional and gene network analyses revealed expected relationships of DMGs to placental physiology and transport, but also identified novel pathways with biologic plausibility and potential clinical importance to cardiometabolic disease. Specific loci for DMGs of interest had methylation patterns that were strongly associated with anthropometric presentations. We further validated altered gene expression of these specific DMGs contributing to vascular and metabolic diseases (SLC36A1, PTPRN2, CASZ1, IL10), thereby establishing transcriptional effects toward assigning functional significance. Our results suggest that the gene expression and methylation state of the human placenta are related and sensitive to the intrauterine environment, as it affects fetal growth patterns. We speculate that these observed changes may affect risk for offspring in developing adult cardiometabolic disease.

Transposable elements constitute an important fraction of eukaryotic genomes. Given their mutagenic potential, host-genomes have evolved epigenetic defense mechanisms to limit their expansion. In fungi, epigenetic modifications have been widely studied in ascomycetes, although we lack a global picture of the epigenetic landscape in basidiomycetes. In this study, we analysed the genome-wide epigenetic and transcriptional patterns of the white-rot basidiomycete Pleurotus ostreatus throughout its life cycle. Our results performed by using high-throughput sequencing analyses revealed that strain-specific DNA methylation profiles are primarily involved in the repression of transposon activity and suggest that 21 nt small RNAs play a key role in transposon silencing. Furthermore, we provide evidence that transposon-associated DNA methylation, but not sRNA production, is directly involved in the silencing of genes surrounded by transposons. Remarkably, we found that nucleus-specific methylation levels varied in dikaryotic strains sharing identical genetic complement but different subculture conditions. Finally, we identified key genes activated in the fruiting process through the comparative analysis of transcriptomes. This study provides an integrated picture of epigenetic defense mechanisms leading to the transcriptional silencing of transposons and surrounding genes in basidiomycetes. Moreover, our findings suggest that transcriptional but not methylation reprogramming triggers fruitbody development in P. ostreatus. This work was supported by Spanish National Research Plan (Project AGL2014-55971 R) and FEDER funds; Public University of Navarre. AB and LLV hold a PhD scholarship from the Public University of Navarre, RC holds a FPI-PhD scholarship from the Ministry of Economy and Competitiveness and M.M. holds a Philip Whitcome and Dissertation Year Fellowship. M.P. and M.M. were supported by a cooperative agreement with the US Department of Energy Office of Science, Office of Biological and Environmental Research programme under Award DE-FC02-02ER63421.

Most epigenome-wide association studies to date have been conducted in blood. However, metabolic syndrome is mediated by a dysregulation of adiposity and therefore it is critical to study adipose tissue in order to understand the effects of this syndrome on epigenomes. To determine if natural variation in DNA methylation was associated with metabolic syndrome traits, we profiled global methylation levels in subcutaneous abdominal adipose tissue. We measured association between 32 clinical traits related to diabetes and obesity in 201 people from the Metabolic Syndrome In Men cohort. We performed epigenome-wide association studies between DNA methylation levels and traits, and identified associations for 13 clinical traits in 21 loci. We prioritized candidate genes in these loci using eQTL, and identified 18 high confidence candidate genes, including known and novel genes associated with diabetes and obesity traits. Using methylation deconvolution, we examined which cell types may be mediating the associations, and concluded that most of the loci we identified were specific to adipocytes. We determined whether the abundance of cell types varies with metabolic traits, and found that macrophages increased in abundance with the severity of metabolic syndrome traits. Finally, we developed a DNA methylation based biomarker to assess type II diabetes risk in adipose tissue. In conclusion, our results demonstrate that profiling DNA methylation in adipose tissue is a powerful tool for understanding the molecular effects of metabolic syndrome on adipose tissue, and can be used in conjunction with traditional genetic analyses to further characterize this disorder.

Whole-genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS) are widely used for measuring DNA methylation levels on a genome-wide scale(1). Both methods have limitations: WGBS is expensive and prohibitive for most large-scale projects; RRBS only interrogates 6-12% of the CpGs in the human genome(16,19). Here, we introduce methylation-sensitive restriction enzyme bisulfite sequencing (MREBS) which has the reduced sequencing requirements of RRBS, but significantly expands the coverage of CpG sites in the genome. We built a multiple regression model that combines the two features of MREBS: the bisulfite conversion ratios of single cytosines (as in WGBS and RRBS) as well as the number of reads that cover each locus (as in MRE-seq(12)). This combined approach allowed us to estimate differential methylation across 60% of the genome using read count data alone, and where counts were sufficiently high in both samples (about 1.5% of the genome), our estimates were significantly improved by the single CpG conversion information. We show that differential DNA methylation values based on MREBS data correlate well with those based on WGBS and RRBS. This newly developed technique combines the sequencing cost of RRBS and DNA methylation estimates on a portion of the genome similar to WGBS, making it ideal for large-scale projects of mammalian genomes.

Ribosome biogenesis in Saccharomyces cerevisiae involves a regulon of >200 genes (Ribi genes) coordinately regulated in response to nutrient availability and cellular growth rate. Two cis-acting elements called PAC and RRPE are known to mediate Ribi gene repression in response to nutritional downshift. Here, we show that most Ribi gene promoters also contain binding sites for one or more General Regulatory Factors (GRFs), most frequently Abf1 and Reb1, and that these factors are enriched in vivo at Ribi promoters. Abf1/Reb1/Tbf1 promoter association was required for full Ribi gene expression in rich medium and for its modulation in response to glucose starvation, characterized by a rapid drop followed by slow recovery. Such a response did not entail changes in Abf1 occupancy, but it was paralleled by a quick increase, followed by slow decrease, in Rpd3L histone deacetylase occupancy. Remarkably, Abf1 site disruption also abolished Rpd3L complex recruitment in response to starvation. Extensive mutational analysis of the DBP7 promoter revealed a complex interplay of Tbf1 sites, PAC and RRPE in the transcriptional regulation of this Ribi gene. Our observations point to GRFs as new multifaceted players in Ribi gene regulation both during exponential growth and under repressive conditions.

Maternal nutrient restriction causes the development of adult onset chronic diseases in the intrauterine growth restricted (IUGR) fetus. Investigations in mice have shown that either protein or calorie restriction during pregnancy leads to glucose intolerance, increased fat mass, and hypercholesterolemia in adult male offspring. Some of these phenotypes are shown to persist in successive generations. The molecular mechanisms underlying IUGR remain unclear. The placenta is a critical organ for mediating changes in the environment and the development of embryos. To shed light on molecular mechanisms that might affect placental responses to differing environments we examined placentas from mice that had been exposed to different diets. We measured gene expression and whole genome DNA methylation in both male and female placentas of mice exposed to either caloric restriction or ad libitum diets. We observed several differentially expressed pathways associated with IUGR phenotypes and, most importantly, a significant decrease in the overall methylation between these groups as well as sex-specific effects that are more pronounced in males. In addition, a set of significantly differentially methylated genes that are enriched for known imprinted genes were identified, suggesting that imprinted loci may be particularly susceptible to diet effects. Lastly, we identified several differentially methylated microRNAs that target genes associated with immunological, metabolic, gastrointestinal, cardiovascular, and neurological chronic diseases, as well as genes responsible for transplacental nutrient transfer and fetal development.

Cytosine methylation regulates the length and stability of telomeres, which can affect a wide variety of biological features, including cell differentiation, development, or illness. Although it is well established that subtelomeric regions are methylated, the presence of methylated cytosines at telomeres has remained controversial. Here, we have analyzed multiple bisulfite sequencing studies to address the methylation status of Arabidopsis thaliana telomeres. We found that the levels of estimated telomeric DNA methylation varied among studies. Interestingly, we estimated higher levels of telomeric DNA methylation in studies that produced C-rich telomeric strands with lower efficiency. However, these high methylation estimates arose due to experimental limitations of the bisulfite technique. We found a similar phenomenon for mitochondrial DNA: The levels of mitochondrial DNA methylation detected were higher in experiments with lower mitochondrial read production efficiencies. Based on experiments with high telomeric C-rich strand production efficiencies, we concluded that Arabidopsis telomeres are not methylated, which was confirmed by methylation-dependent restriction enzyme analyses. Thus, our studies indicate that telomeres are refractory to de novo DNA methylation by the RNA-directed DNA methylation machinery. This result, together with previously reported data, reveals that subtelomeric DNA methylation controls the homeostasis of telomere length.

We have investigated transcriptional and epigenetic differences in peripheral blood mononuclear cells (PBMCs) of monozygotic female twins discordant in the diagnosis of amyotrophic lateral sclerosis (ALS). Exploring DNA methylation differences by reduced representation bisulfite sequencing (RRBS), we determined that, over time, the ALS twin developed higher abundances of the CD14 macrophages and lower abundances of T cells compared to the non-ALS twin. Higher macrophage signature in the ALS twin was also shown by RNA sequencing (RNA-seq). Moreover, the twins differed in the methylome at loci near several genes, including EGFR and TNFRSF11A, and in the pathways related to the tretinoin and H3K27me3 markers. We also tested cytokine production by PBMCs. The ALS twin's PBMCs spontaneously produced IL-6 and TNF-α, whereas PBMCs of the healthy twin produced these cytokines only when stimulated by superoxide dismutase (SOD)-1. These results and flow cytometric detection of CD45 and CD127 suggest the presence of memory T cells in both twins, but effector T cells only in the ALS twin. The ALS twin's PBMC supernatants, but not the healthy twin's, were toxic to rat cortical neurons, and this toxicity was strongly inhibited by an IL-6 receptor antibody (tocilizumab) and less well by TNF-α and IL-1β antibodies. The putative neurotoxicity of IL-6 and TNF-α is in agreement with a high expression of these cytokines on infiltrating macrophages in the ALS spinal cord. We hypothesize that higher macrophage abundance and increased neurotoxic cytokines have a fundamental role in the phenotype and treatment of certain individuals with ALS.-Lam, L., Chin, L., Halder, R. C., Sagong, B., Famenini, S., Sayre, J., Montoya, D., Rubbi L., Pellegrini, M., Fiala, M. Epigenetic changes in T-cell and monocyte signatures and production of neurotoxic cytokines in ALS patients.

SummaryDNA methylation is one of a number of modes of epigenetic gene regulation. Here, we profile the DNA methylome, transcriptome, and global occupancy of histone modifications (H3K4me1, H3K4me3, H3K27me3, and H3K27ac) in a series of mouse embryonic stem cells (mESCs) with varying DNA methylation levels to study the effects of DNA methylation on deposition of histone modifications. We find that genome-wide DNA demethylation alters occupancy of histone modifications at both promoters and enhancers. This is reversed upon remethylation by Dnmt expression. DNA methylation promotes H3K27me3 deposition at bivalent promoters, while opposing H3K27me3 at silent promoters. DNA methylation also reversibly regulates H3K27ac and H3K27me3 at previously identified tissue-specific enhancers. These effects require DNMT catalytic activity. Collectively, our data show that DNA methylation is essential and instructive for deposition of specific histone modifications across regulatory regions, which together influences gene expression patterns in mESCs.

Mammalian tissues calcify with age and injury. Analogous to bone formation, osteogenic cells are thought to be recruited to the affected tissue and induce mineralization. In the heart, calcification of cardiac muscle leads to conduction system disturbances and is one of the most common pathologies underlying heart blocks. However the cell identity and mechanisms contributing to pathological heart muscle calcification remain unknown. Using lineage tracing, murine models of heart calcification and invivo transplantation assays, we show that cardiac fibroblasts (CFs) adopt an osteoblast cell-like fate and contribute directly to heart muscle calcification. Small-molecule inhibition of ENPP1, an enzyme that is induced upon injury and regulates bone mineralization, significantly attenuated cardiac calcification. Inhibitors of bone mineralization completely prevented ectopic cardiac calcification and improved post injury heart function. Taken together, these findings highlight the plasticity of fibroblasts in contributing to ectopic calcification and identify pharmacological targets for therapeutic development.

The process of domestication can exert intense trait-targeted selection on genes and regulatory regions. Specifically, rapid shifts in the structure and sequence of genomic regulatory elements could provide an explanation for the extensive, and sometimes extreme, variation in phenotypic traits observed in domesticated species. Here, we explored methylation differences from >24000 cytosines distributed across the genomes of the domesticated dog (Canis familiaris) and the grey wolf (Canis lupus). PCA and model-based cluster analyses identified two primary groups, domestic vs. wild canids. A scan for significantly differentially methylated sites (DMSs) revealed species-specific patterns at 68 sites after correcting for cell heterogeneity, with weak yet significant hypermethylation typical of purebred dogs when compared to wolves (59% and 58%, P66%) of differentially methylated regions contained or were associated with repetitive elements, indicative of a genotype-mediated trend. However, DMSs were also often linked to functionally relevant genes (e.g. neurotransmitters). Finally, we utilized known genealogical relationships among Yellowstone wolves to survey transmission stability of methylation marks, from which we found a substantial fraction that demonstrated high heritability (both H(2) and h(2) >0.99). These analyses provide a unique epigenetic insight into the molecular consequences of recent selection and radiation of our most ancient domesticated companion, the dog. These findings suggest selection has acted on methylation patterns, providing a new genomic perspective on phenotypic diversification in domesticated species.

Rationale: Only a small portion of the known heritability of cardiovascular diseases, such as heart failure, can be explained based on single-gene mutations. Chromatin structure and regulation provide a substrate through which genetic differences in noncoding regions may affect cellular function and response to disease, but the mechanisms are unknown. Objective: We conducted genome-wide measurements of DNA methylation in different strains of mice that are susceptible and resistant to isoproterenol-induced dysfunction to test the hypothesis that this epigenetic mark may play a causal role in the development of heart failure. Methods and Results: BALB/cJ and BUB/BnJ mice, determined to be susceptible and resistant to isoproterenol-induced heart failure, respectively, were administered the drug for 3 weeks via osmotic minipump. Reduced representational bisulfite sequencing was then used to compare the differences between the cardiac DNA methylomes in the basal state between strains and then after isoproterenol treatment. Single-base resolution DNA methylation measurements were obtained and revealed a bimodal distribution of methylation in the heart, enriched in lone intergenic CpGs and depleted from CpG islands around genes. Isoproterenol induced global decreases in methylation in both strains; however, the basal methylation pattern between strains shows striking differences that may be predictive of disease progression before environmental stress. The global correlation between promoter methylation and gene expression (as measured by microarray) was modest and revealed itself only with focused analyses of transcription start site and gene body regions (in contrast to when gene methylation was examined in toto). Modules of comethylated genes displayed correlation with other protein-based epigenetic marks, supporting the hypothesis that chromatin modifications act in a combinatorial manner to specify transcriptional phenotypes in the heart. Conclusions: This study provides the first single-base resolution map of the mammalian cardiac DNA methylome and the first case–control analysis of the changes in DNA methylation with heart failure. The findings demonstrate marked genetic differences in DNA methylation that are associated with disease progression.

Transcranial direct current stimulation (tDCS) has been shown to modulate neuroplasticity. Beneficial effects are observed in patients with psychiatric disorders and enhancement of brain performance in healthy individuals has been observed following tDCS. However, few studies have attempted to elucidate the underlying molecular mechanisms of tDCS in the brain. This study was conducted to assess the impact of tDCS on gene expression within the rat cerebral cortex. Anodal tDCS was applied at 3 different intensities followed by RNA-sequencing and analysis. In each current intensity, approximately 1,000 genes demonstrated statistically significant differences compared to the sham group. A variety of functional pathways, biological processes, and molecular categories were found to be modified by tDCS. The impact of tDCS on gene expression was dependent on current intensity. Results show that inflammatory pathways, antidepressant-related pathways (GTP signaling, calcium ion binding, and transmembrane/signal peptide pathways), and receptor signaling pathways (serotonergic, adrenergic, GABAergic, dopaminergic, and glutamate) were most affected. Of the gene expression profiles induced by tDCS, some changes were observed across multiple current intensities while other changes were unique to a single stimulation intensity. This study demonstrates that tDCS can modify the expression profile of various genes in the cerebral cortex and that these tDCS-induced alterations are dependent on the current intensity applied.

BCR-ABL1 is a fusion tyrosine kinase, which causes multiple types of leukemia. We used an integrated proteomic approach that includes label-free quantitative protein complex and phosphorylation profiling by mass spectrometry to systematically characterize the proximal signaling network of this oncogenic kinase. The proximal BCR-ABL1 signaling network shows a modular and layered organization with an inner core of three leukemia transformation-relevant adaptor protein complexes (Grb2/Gab2/Shc1 complex, CrkI complex and Dok1/Dok2 complex). We introduced an 'interaction directionality' analysis, which annotates static protein networks with information on the directionality of phosphorylation-dependent interactions. In this analysis, the observed network structure was consistent with a step-wise phosphorylation-dependent assembly of the Grb2/Gab2/Shc1 and the Dok1/Dok2 complexes on the BCR-ABL1 core. The CrkI complex demonstrated a different directionality, which supports a candidate assembly on the Nedd9 (Hef1, CasL) scaffold. As adaptor protein family members can compensate for each other in leukemic transformation, we compared members of the Dok and Crk protein families and found both overlapping and differential binding patterns. We identified an additional level of regulation for the CrkII protein via binding to 14-3-3 proteins, which was independent from its inhibitory phosphorylation. We also identified novel components of the inner core complexes, including the kinases Pragmin (Sgk223) and Lrrk1 (Lrrk2 paralog). Pragmin was found as a component of the CrkI complex and is a potential link between BCR-ABL1/CrkI and RhoA signaling. Lrrk1 is an unusual kinase with a GTPase domain. We detected Lrrk1 as a component of the Grb2/Gab2/Shc1 complex and found that it functionally interacts with the regulator of small GTPases Arap1 (Centd2) and possibly participates in the mitogen-activated protein kinase response to cellular stresses. This modular and phosphorylation-driven interaction network provides a framework for the integration of pleiotropic signaling effects of BCR-ABL1 toward leukemic transformation.

The extensively studied yeast GAL1-10 gene cluster is tightly regulated by environmental sugar availability. Unexpectedly, under repressive conditions the 3' region of the GAL10 coding sequence is trimethylated by Set1 on histone H3 K4, normally characteristic of 5' regions of actively transcribed genes. This reflects transcription of a long noncoding RNA (GAL10-ncRNA) that is reciprocal to GAL1 and GAL10 mRNAs and driven by the DNA-binding protein Reb1. Point mutations in predicted Reb1-binding sites abolished Reb1 binding and ncRNA synthesis. The GAL10-ncRNA is transcribed approximately once every 50 min and targeted for degradation by the TRAMP and exosome complexes, resulting in low steady-state levels (approximately one molecule per 14 cells). GAL10-ncRNA transcription recruits the methyltransferase Set2 and histone deacetylation activities in cis, leading to stable changes in chromatin structure. These chromatin modifications act principally through the Rpd3S complex to aid glucose repression of GAL1-10 at physiologically relevant sugar concentrations.