NSD2 drives t(4;14) myeloma cell dependence on adenylate kinase 2 by diverting one-carbon metabolism to the epigenome.

Chromosomal translocation (4;14), an adverse prognostic factor in multiple myeloma (MM), drives overexpression of the histone methyltransferase NSD2. A genome-wide CRISPR screen in MM cells identified adenylate kinase 2 (AK2), an enzyme critical for high energy phosphate transfer from the mitochondria, as an NSD2-driven vulnerability. AK2 suppression in t(4;14) MM cells decreased NADP(H) critical for conversion of ribonucleotides to deoxyribonucleosides, leading to replication stress, DNA damage and apoptosis. Driving a large genome-wide increase in chromatin methylation, NSD2 overexpression depletes S-adenosylmethionine (SAM), compromising synthesis of creatine from its precursor guanidinoacetate. Creatine supplementation restored NADP(H) levels, reduced DNA damage and rescued AK2-deficient t(4;14) MM cells. As the creatine phosphate shuttle constitutes an alternative means for mitochondrial high energy phosphate transport, these results indicate that NSD2-driven creatine depletion underlies the hypersensitivity of t(4;14) MM cells to AK2 loss. Furthermore, AK2 depletion in t(4;14) cells impaired protein folding in the endoplasmic reticulum consistent with impaired utilization of mitochondrial ATP. Accordingly, AK2 suppression increased sensitivity of MM cells to proteasome inhibition. These findings delineate a novel mechanism in which aberrant transfer of carbon to the epigenome creates a metabolic vulnerability, with direct therapeutic implications for t(4;14) MM.

HIRA-mediated loading of histone variant H3.3 controls androgen-induced transcription by regulation of AR/BRD4 complex assembly at enhancers.

Incorporation of histone variant H3.3 comprises active territories of chromatin. Exploring the function of H3.3 in prostate cancer (PC), we found that knockout (KO) of H3.3 chaperone HIRA suppresses PC growth in vitro and in xenograft settings, deregulates androgen-induced gene expression and alters androgen receptor (AR) binding within enhancers of target genes. H3.3 affects transcription in multiple ways, including activation of p300 by phosphorylated H3.3 at Ser-31 (H3.3S31Ph), which results in H3K27 acetylation (H3K27Ac) at enhancers. In turn, H3K27Ac recruits bromodomain protein BRD4 for enhancer-promoter interaction and transcription activation. We observed that HIRA KO reduces H3.3 incorporation, diminishes H3.3S31Ph and H3K27Ac, modifies recruitment of BRD4. These results suggest that H3.3-enriched enhancer chromatin serves as a platform for H3K27Ac-mediated BRD4 recruitment, which interacts with and retains AR at enhancers, resulting in transcription reprogramming. In addition, HIRA KO deregulates glucocorticoid- (GR) driven transcription of genes co-regulated by AR and GR, suggesting a common H3.3/HIRA-dependent mechanism of nuclear receptors function. Expression of HIRA complex proteins is increased in PC compared with normal prostate tissue, especially in high-risk PC groups, and is associated with a negative prognosis. Collectively, our results demonstrate function of HIRA-dependent H3.3 pathway in regulation of nuclear receptors activity.

Exertional heat stroke causes long-term skeletal muscle epigenetic reprogramming, altered gene expression, and impaired satellite cell function in mice.

The effect of exertional heat stroke (EHS) exposure on skeletal muscles is incompletely understood. Muscle weakness is an early symptom of EHS but is not considered a major target of multiorgan injury. Previously, in a preclinical mouse model of EHS, we observed the vulnerability of limb muscles to a second EHS exposure, suggesting hidden processes contributing to declines in muscle resilience. Here, we evaluated the possible molecular origins of EHS-induced declines in muscle resilience. Female C57BL/6 mice [total n = 56; 28/condition, i.e., EHS and exercise control (EXC)] underwent forced wheel running at 37.5°C/40% relative humidity until symptom limitation (unconsciousness). EXC mice exercised identically at room temperature (22-23°C). After 1 mo of recovery, the following were assessed: 1) specific force and caffeine-induced contracture in soleus (SOL) and extensor digitorum longus (EDL) muscles; 2) transcriptome and DNA methylome responses in gastrocnemius (GAST); and 3) primary satellite cell function (proliferation and differentiation). There were no differences in specific force in either SOL or EDL from EXC. Only EHS solei exhibited lower caffeine sensitivity. EHS GAST exhibited higher RNA expression of genes encoding structural proteins of slow fibers, heat shock proteins, and myogenesis. A total of ∼2,500 differentially methylated regions of DNA that could potentially affect many cell functions were identified. Primary satellite cells exhibited suppressed proliferation rates but normal differentiation responses. Results demonstrate long-term changes in skeletal muscles 1 mo after EHS that could contribute to declines in muscle resilience. Skeletal muscle may join other, more recognized tissues considered vulnerable to long-term effects of EHS.NEW & NOTEWORTHY Exertional heat stroke (EHS) in mice induces long-term molecular and functional changes in limb muscle that could reflect a loss of "resilience" to further stress. The phenotype was characterized by altered caffeine sensitivity and suppressed satellite cell proliferative potential. This was accompanied by changes in gene expression and DNA methylation consistent with ongoing muscle remodeling and stress adaptation. We propose that EHS may induce a prolonged vulnerability of skeletal muscle to further stress or injury.

ARCA: the interactive database for arbovirus reported cases in the Americas.

BACKGROUND: Accurate case report data are essential to understand arbovirus dynamics, including spread and evolution of arboviruses such as Zika, dengue and chikungunya viruses. Giving the multi-country nature of arbovirus epidemics in the Americas, these data are not often accessible or are reported at different time scales (weekly, monthly) from different sources. RESULTS: We developed a publicly available and user-friendly database for arboviral case data in the Americas: ARCA. ARCA is a relational database that is hosted on the ARCA website. Users can interact with the database through the website by submitting queries through the website, which generates displays results and allows users to download these results in different, convenient file formats. Users can choose to view arboviral case data through a table which containscontaining the number of cases for a particular week, a plot, or through a map. CONCLUSION: Our ARCA database is a useful tool for arboviral epidemiology research allowing for complex queries, data visualization, integration, and formatting.

Ancestral Origin and Dissemination Dynamics of Reemerging Toxigenic Vibrio cholerae, Haiti.

The 2010 cholera epidemic in Haiti was thought to have ended in 2019, and the Prime Minister of Haiti declared the country cholera-free in February 2022. On September 25, 2022, cholera cases were again identified in Port-au-Prince. We compared genomic data from 42 clinical Vibrio cholerae strains from 2022 with data from 327 other strains from Haiti and 1,824 strains collected worldwide. The 2022 isolates were homogeneous and closely related to clinical and environmental strains circulating in Haiti during 2012-2019. Bayesian hypothesis testing indicated that the 2022 clinical isolates shared their most recent common ancestor with an environmental lineage circulating in Haiti in July 2018. Our findings strongly suggest that toxigenic V. cholerae O1 can persist for years in aquatic environmental reservoirs and ignite new outbreaks. These results highlight the urgent need for improved public health infrastructure and possible periodic vaccination campaigns to maintain population immunity against V. cholerae.

Optimizing viral genome subsampling by genetic diversity and temporal distribution (TARDiS) for phylogenetics.

SUMMARY: TARDiS is a novel phylogenetic tool for optimal genetic subsampling. It optimizes both genetic diversity and temporal distribution through a genetic algorithm. AVAILABILITY AND IMPLEMENTATION: TARDiS, along with example datasets and a user manual, is available at https://github.com/smarini/tardis-phylogenetics.

Decreases in different Dnmt3b activities drive distinct development of hematologic malignancies in mice.

DNA methylation regulates gene transcription and is involved in various physiological processes in mammals, including development and hematopoiesis. It is catalyzed by DNA methyltransferases including Dnmt1, Dnmt3a, and Dnmt3b. For Dnmt3b, its effects on transcription can result from its own DNA methylase activity, the recruitment of other Dnmts to mediate methylation, or transcription repression in a methylation-independent manner. Low-frequency mutations in human DNMT3B are found in hematologic malignancies including cutaneous T-cell lymphomas, hairy cell leukemia, and diffuse large B-cell lymphomas. Moreover, Dnmt3b is a tumor suppressor in oncogene-driven lymphoid and myeloid malignancies in mice. However, it is poorly understood how the different Dnmt3b activities contribute to these outcomes. We modulated Dnmt3b activity in vivo by generating Dnmt3b+/- mice expressing one wild-type allele as well as Dnmt3b+/CI and Dnmt3bCI/CI mice where one or both alleles express catalytically inactive Dnmt3bCI. We show that 43% of Dnmt3b+/- mice developed T-cell lymphomas, chronic lymphocytic leukemia, and myeloproliferation over 18 months, thus resembling phenotypes previously observed in Dnmt3a+/- mice, possibly through regulation of shared target genes. Interestingly, Dnmt3b+/CI and Dnmt3bCI/CI mice survived postnatal development and were affected by B-cell rather than T-cell malignancies with decreased penetrance. Genome-wide hypomethylation, increased expression of oncogenes such as Jdp2, STAT1, and Trip13, and p53 downregulation were major events contributing to Dnmt3b+/- lymphoma development. We conclude that Dnmt3b catalytic activity is critical to prevent B-cell transformation in vivo, whereas accessory and methylation-independent repressive functions are important to prevent T-cell transformation.

Severe Acute Respiratory Syndrome Coronavirus 2 Delta Vaccine Breakthrough Transmissibility in Alachua County, Florida.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant has caused a dramatic resurgence in infections in the United Sates, raising questions regarding potential transmissibility among vaccinated individuals. METHODS: Between October 2020 and July 2021, we sequenced 4439 SARS-CoV-2 full genomes, 23% of all known infections in Alachua County, Florida, including 109 vaccine breakthrough cases. Univariate and multivariate regression analyses were conducted to evaluate associations between viral RNA burden and patient characteristics. Contact tracing and phylogenetic analysis were used to investigate direct transmissions involving vaccinated individuals. RESULTS: The majority of breakthrough sequences with lineage assignment were classified as Delta variants (74.6%) and occurred, on average, about 3 months (104 ±â€…57.5 days) after full vaccination, at the same time (June-July 2021) of Delta variant exponential spread within the county. Six Delta variant transmission pairs between fully vaccinated individuals were identified through contact tracing, 3 of which were confirmed by phylogenetic analysis. Delta breakthroughs exhibited broad viral RNA copy number values during acute infection (interquartile range, 1.2-8.64 Log copies/mL), on average 38% lower than matched unvaccinated patients (3.29-10.81 Log copies/mL, P < .00001). Nevertheless, 49% to 50% of all breakthroughs, and 56% to 60% of Delta-infected breakthroughs exhibited viral RNA levels above the transmissibility threshold (4 Log copies/mL) irrespective of time after vaccination. CONCLUSIONS: Delta infection transmissibility and general viral RNA quantification patterns in vaccinated individuals suggest limited levels of sterilizing immunity that need to be considered by public health policies. In particular, ongoing evaluation of vaccine boosters should specifically address whether extra vaccine doses curb breakthrough contribution to epidemic spread.

Rapid Emergence and Spread of Severe Acute Respiratory Syndrome Coronavirus 2 Gamma (P.1) Variant in Haiti.

After an initial wave of coronavirus disease 2019 (COVID-19) in Haiti in summer 2020 (primarily lineage B.1), seropositivity for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin G (IgG) was ~40%. Variant P.1 (gamma) was introduced in February 2021, with an initially limited introduction followed by exponential local dissemination within this unvaccinated population with prior exposure to earlier SARS-CoV-2 lineages.

Methylscaper: an R/Shiny app for joint visualization of DNA methylation and nucleosome occupancy in single-molecule and single-cell data.

SUMMARY: Differential DNA methylation and chromatin accessibility are associated with disease development, particularly cancer. Methods that allow profiling of these epigenetic mechanisms in the same reaction and at the single-molecule or single-cell level continue to emerge. However, a challenge lies in jointly visualizing and analyzing the heterogeneous nature of the data and extracting regulatory insight. Here, we present methylscaper, a visualization framework for simultaneous analysis of DNA methylation and chromatin accessibility landscapes. Methylscaper implements a weighted principal component analysis that orders DNA molecules, each providing a record of the chromatin state of one epiallele, and reveals patterns of nucleosome positioning, transcription factor occupancy, and DNA methylation. We demonstrate methylscaper's utility on a long-read, single-molecule methyltransferase accessibility protocol for individual templates (MAPit-BGS) dataset and a single-cell nucleosome, methylation, and transcription sequencing (scNMT-seq) dataset. In comparison to other procedures, methylscaper is able to readily identify chromatin features that are biologically relevant to transcriptional status while scaling to larger datasets. AVAILABILITY AND IMPLEMENTATION: Methylscaper, is implemented in R (version > 4.1) and available on Bioconductor: https://bioconductor.org/packages/methylscaper/, GitHub: https://github.com/rhondabacher/methylscaper/, and Web: https://methylscaper.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Low-frequency variants in mildly symptomatic vaccine breakthrough infections presents a doubled-edged sword.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) has raised questions regarding vaccine protection against SARS-CoV-2 infection, transmission, and ongoing virus evolution. Twenty-three mildly symptomatic "vaccination breakthrough" infections were identified as early as January 2021 in Alachua County, Florida, among individuals fully vaccinated with either the BNT162b2 (Pfizer) or the Ad26 (Janssen/J&J) vaccines. SARS-CoV-2 genomes were successfully generated for 11 of the vaccine breakthroughs, and 878 individuals in the surrounding area and were included for reference-based phylogenetic investigation. These 11 individuals were characterized by infection with VOCs, but also low-frequency variants present within the surrounding population. Low-frequency mutations were observed, which have been more recently identified as mutations of interest owing to their location within targeted immune epitopes (P812L) and association with increased replicative capacity (L18F). We present these results to posit the nature of the efficacy of vaccines in reducing symptoms as both a blessing and a curse-as vaccination becomes more widespread and self-motivated testing reduced owing to the absence of severe symptoms, we face the challenge of early recognition of novel mutations of potential concern. This case study highlights the critical need for continued testing and monitoring of infection and transmission among individuals regardless of vaccination status.

Exertional heat stroke leads to concurrent long-term epigenetic memory, immunosuppression and altered heat shock response in female mice.

KEY POINTS: Exposure to exertional heat stroke (EHS) has been linked to increased long-term decrements of health. Epigenetic reprogramming is involved in the response to heat acclimation; however, whether the long-term effects of EHS are mediated by epigenetic reprogramming is unknown. In female mice, we observed DNA methylation reprogramming in bone marrow-derived (BMD) monocytes as early as 4 days of recovery from EHS and as late as 30 days compared with sham exercise controls. Whole blood, collected after 30 days of recovery from EHS, exhibited an immunosuppressive phenotype when challenged in vitro by lipopolysaccharide. After 30 days of recovery from EHS, BMD monocytes exhibited an altered in vitro heat shock response. The location of differentially methylated CpGs are predictive of both the immunosuppressive phenotype and altered heat shock responses. ABSTRACT: Exposure to exertional heat stroke (EHS) has been linked to increased susceptibility to a second heat stroke, infection and cardiovascular disease. Whether these clinical outcomes are mediated by an epigenetic memory is unknown. Using a preclinical mouse model of EHS, we investigated whether EHS exposure produces a lasting epigenetic memory in monocytes and whether there are phenotypic alterations that may be consistent with these epigenetic changes. Female mice underwent forced wheel running at 37.5°C/40% relative humidity until symptom limitation, characterized by CNS dysfunction. Results were compared with matched exercise controls at 22.5°C. Monocytes were isolated from bone marrow after 4 or 30 days of recovery to extract DNA and analyse methylation. Broad-ranging alterations to the DNA methylome were observed at both time points. At 30 days, very specific alterations were observed to the promoter regions of genes involved with immune responsiveness. To test whether these changes might be related to phenotype, whole blood at 30 days was challenged with lipopolysaccharide (LPS) to measure cytokine secretion; monocytes were also challenged with heat shock to quantify mRNA expression. Whole blood collected from EHS mice showed markedly attenuated inflammatory responses to LPS challenge. Furthermore, monocyte mRNA from EHS mice showed significantly altered responses to heat shock challenge. These results demonstrate that EHS leads to a unique DNA methylation pattern in monocytes and altered immune and heat shock responsiveness after 30 days. These data support the hypothesis that EHS exposure can induce long-term physiological changes that may be linked to altered epigenetic profiles.

Brain tissue transcriptomic analysis of SIV-infected macaques identifies several altered metabolic pathways linked to neuropathogenesis and poly (ADP-ribose) polymerases (PARPs) as potential therapeutic targets.

Despite improvements in antiretroviral therapy, human immunodeficiency virus type 1 (HIV-1)-associated neurocognitive disorders (HAND) remain prevalent in subjects undergoing therapy. HAND significantly affects individuals' quality of life, as well as adherence to therapy, and, despite the increasing understanding of neuropathogenesis, no definitive diagnostic or prognostic marker has been identified. We investigated transcriptomic profiles in frontal cortex tissues of Simian immunodeficiency virus (SIV)-infected Rhesus macaques sacrificed at different stages of infection. Gene expression was compared among SIV-infected animals (n = 11), with or without CD8+ lymphocyte depletion, based on detectable (n = 6) or non-detectable (n = 5) presence of the virus in frontal cortex tissues. Significant enrichment in activation of monocyte and macrophage cellular pathways was found in animals with detectable brain infection, independently from CD8+ lymphocyte depletion. In addition, transcripts of four poly (ADP-ribose) polymerases (PARPs) were up-regulated in the frontal cortex, which was confirmed by real-time polymerase chain reaction. Our results shed light on involvement of PARPs in SIV infection of the brain and their role in SIV-associated neurodegenerative processes. Inhibition of PARPs may provide an effective novel therapeutic target for HIV-related neuropathology.

A Bayesian data fusion based approach for learning genome-wide transcriptional regulatory networks.

BACKGROUND: Reverse engineering of transcriptional regulatory networks (TRN) from genomics data has always represented a computational challenge in System Biology. The major issue is modeling the complex crosstalk among transcription factors (TFs) and their target genes, with a method able to handle both the high number of interacting variables and the noise in the available heterogeneous experimental sources of information. RESULTS: In this work, we propose a data fusion approach that exploits the integration of complementary omics-data as prior knowledge within a Bayesian framework, in order to learn and model large-scale transcriptional networks. We develop a hybrid structure-learning algorithm able to jointly combine TFs ChIP-Sequencing data and gene expression compendia to reconstruct TRNs in a genome-wide perspective. Applying our method to high-throughput data, we verified its ability to deal with the complexity of a genomic TRN, providing a snapshot of the synergistic TFs regulatory activity. Given the noisy nature of data-driven prior knowledge, which potentially contains incorrect information, we also tested the method's robustness to false priors on a benchmark dataset, comparing the proposed approach to other regulatory network reconstruction algorithms. We demonstrated the effectiveness of our framework by evaluating structural commonalities of our learned genomic network with other existing networks inferred by different DNA binding information-based methods. CONCLUSIONS: This Bayesian omics-data fusion based methodology allows to gain a genome-wide picture of the transcriptional interplay, helping to unravel key hierarchical transcriptional interactions, which could be subsequently investigated, and it represents a promising learning approach suitable for multi-layered genomic data integration, given its robustness to noisy sources and its tailored framework for handling high dimensional data.

Multiplex mapping of chromatin accessibility and DNA methylation within targeted single molecules identifies epigenetic heterogeneity in neural stem cells and glioblastoma.

Human tumors are comprised of heterogeneous cell populations that display diverse molecular and phenotypic features. To examine the extent to which epigenetic differences contribute to intratumoral cellular heterogeneity, we have developed a high-throughput method, termed MAPit-patch. The method uses multiplexed amplification of targeted sequences from submicrogram quantities of genomic DNA followed by next generation bisulfite sequencing. This provides highly scalable and simultaneous mapping of chromatin accessibility and DNA methylation on single molecules at high resolution. Long sequencing reads from targeted regions maintain the structural integrity of epigenetic information and provide substantial depth of coverage, detecting for the first time minority subpopulations of epigenetic configurations formerly obscured by existing genome-wide and population-ensemble methodologies. Analyzing a cohort of 71 promoters of genes with exons commonly mutated in cancer, MAPit-patch uncovered several differentially accessible and methylated promoters that are associated with altered gene expression between neural stem cell (NSC) and glioblastoma (GBM) cell populations. In addition, considering each promoter individually, substantial epigenetic heterogeneity was observed across the sequenced molecules, indicating the presence of epigenetically distinct cellular subpopulations. At the divergent MLH1/EPM2AIP1 promoter, a locus with three well-defined, nucleosome-depleted regions (NDRs), a fraction of promoter copies with inaccessible chromatin was detected and enriched upon selection of temozolomide-tolerant GBM cells. These results illustrate the biological relevance of epigenetically distinct subpopulations that in part underlie the phenotypic heterogeneity of tumor cell populations. Furthermore, these findings show that alterations in chromatin accessibility without accompanying changes in DNA methylation may constitute a novel class of epigenetic biomarker.

The murine Pbx1-d lupus susceptibility allele accelerates mesenchymal stem cell differentiation and impairs their immunosuppressive function.

Pre­B cell leukemia homeobox 1 (Pbx1)-d is a dominant-negative splice isoform of the gene Pbx1 that corresponds to the NZM2410 lupus susceptibility locus Sle1a1. Pbx1 is required to maintain stem cell self-renewal, including that of mesenchymal stem cells (MSCs). MSCs have immunosuppressive functions that require stem cell maintenance. We tested the hypothesis that the expression of Pbx1-d favors MSC differentiation and impairs their immunosuppressive functions. We demonstrate that Sle1a1 MSCs express high levels of Pbx1-d as compared with congenic C57BL/6J (B6) MSCs. Sle1a1 MSCs grew faster and differentiated significantly more rapidly into osteoblasts than did B6 MSCs. This corresponded to a significant decrease in the expression of genes associated with stemness and an increase in the expression of genes associated with differentiation. Additionally, Sle1a1 MSCs express a gene expression profile associated with an enhanced innate immunity and inflammation. Suppression of Ig production from TLR-activated B6 B cells and IL-2 secretion from activated B6 CD4+ T cells was significantly impaired in Sle1a1 MSCs as compared with B6 MSCs. B6.Sle1a1 MSCs showed intermediate activity in suppressing lupus immunophenotypes in three different mouse models. Taken together, these data suggest that the expression of the lupus susceptibility allele Pbx1-d isoform impairs MSC functions, which may contribute to lupus pathogenesis both through a defective immunosuppression and the promotion of a proinflammatory environment.

BigQ: a NoSQL based framework to handle genomic variants in i2b2.

BACKGROUND: Precision medicine requires the tight integration of clinical and molecular data. To this end, it is mandatory to define proper technological solutions able to manage the overwhelming amount of high throughput genomic data needed to test associations between genomic signatures and human phenotypes. The i2b2 Center (Informatics for Integrating Biology and the Bedside) has developed a widely internationally adopted framework to use existing clinical data for discovery research that can help the definition of precision medicine interventions when coupled with genetic data. i2b2 can be significantly advanced by designing efficient management solutions of Next Generation Sequencing data. RESULTS: We developed BigQ, an extension of the i2b2 framework, which integrates patient clinical phenotypes with genomic variant profiles generated by Next Generation Sequencing. A visual programming i2b2 plugin allows retrieving variants belonging to the patients in a cohort by applying filters on genomic variant annotations. We report an evaluation of the query performance of our system on more than 11 million variants, showing that the implemented solution scales linearly in terms of query time and disk space with the number of variants. CONCLUSIONS: In this paper we describe a new i2b2 web service composed of an efficient and scalable document-based database that manages annotations of genomic variants and of a visual programming plug-in designed to dynamically perform queries on clinical and genetic data. The system therefore allows managing the fast growing volume of genomic variants and can be used to integrate heterogeneous genomic annotations.

The MAPPER2 Database: a multi-genome catalog of putative transcription factor binding sites.

The mapper(2) Database (http://genome.ufl.edu/mapperdb) is a component of mapper(2), a web-based system for the analysis of transcription factor binding sites in multiple genomes. The database contains predicted binding sites identified in the promoters of all human, mouse and Drosophila genes using 1017 probabilistic models representing over 600 different transcription factors. In this article we outline the current contents of the database and we describe its web-based user interface in detail. We then discuss ongoing work to extend the database contents to experimental data and to add analysis capabilities. Finally, we provide information about recent improvements to the hardware and software platform that mapper(2) is based on.

Single-molecule long-read methylation profiling reveals regional DNA methylation regulated by Elongator Complex Subunit 2 in Arabidopsis roots experiencing spaceflight.

BACKGROUND: The Advanced Plant Experiment-04 - Epigenetic Expression (APEX-04-EpEx) experiment onboard the International Space Station examined the spaceflight-altered cytosine methylation in two genetic lines of Arabidopsis thaliana, wild-type Col-0 and the mutant elp2-5, which is deficient in an epigenetic regulator Elongator Complex Subunit 2 (ELP2). Whole-genome bisulfite sequencing (WGBS) revealed distinct spaceflight associated methylation differences, presenting the need to explore specific space-altered methylation at single-molecule resolution to associate specific changes over large regions of spaceflight related genes. To date, tools of multiplexed targeted DNA methylation sequencing remain limited for plant genomes. RESULTS: To provide methylation data at single-molecule resolution, Flap-enabled next-generation capture (FENGC), a novel targeted multiplexed DNA capture and enrichment technique allowing cleavage at any specified sites, was applied to survey spaceflight-altered DNA methylation in genic regions of interest. The FENGC capture panel contained 108 targets ranging from 509 to 704 nt within the promoter or gene body regions of gene targets derived from spaceflight whole-genome data sets. In addition to genes with significant changes in expression and average methylation levels between spaceflight and ground control, targets with space-altered distributions of the proportion of methylated cytosines per molecule were identified. Moreover, trends of co-methylation of different cytosine contexts were exhibited in the same DNA molecules. We further identified significant DNA methylation changes in three previously biological process-unknown genes, and loss-of-function mutants of two of these genes (named as EMO1 and EMO2 for ELP2-regulated Methylation in Orbit 1 and 2) showed enhanced root growth rate. CONCLUSIONS: FENGC simplifies and reduces the cost of multiplexed, targeted, single-molecule profiling of methylation in plants, providing additional resolution along each DNA molecule that is not seen in population-based short-read data such as WGBS. This case study has revealed spaceflight-altered regional modification of cytosine methylation occurring within single DNA molecules of cell subpopulations, which were not identified by WGBS. The single-molecule survey by FENGC can lead to identification of novel functional genes. The newly identified EMO1 and EMO2 are root growth regulators which may be epigenetically involved in plant adaptation to spaceflight.

Genome-wide methylation profiling of Peripheral T-cell lymphomas identifies TRIP13 as a critical driver of tumor proliferation and survival.

Cytosine methylation of genomic DNA contributes to the regulation of gene expression and is involved in normal development including hematopoiesis in mammals. It is catalyzed by the family of DNA methyltransferases (DNMTs) that include DNMT1, DNMT3A, and DNMT3B. Peripheral T-cell lymphomas (PTCLs) represent a diverse group of aggressive mature T-cell malignancies accounting for approximately 10-15% of non-Hodgkin lymphoma cases in the US. PTCLs exhibit a broad spectrum of clinical, histological, and immunophenotypic features with poor prognosis and inadequately understood molecular pathobiology. To better understand the molecular landscape and identify candidate genes involved in disease maintenance, we used high-resolution Whole Genome Bisulfite Sequencing (WGBS) and RNA-seq to profile DNA methylation and gene expression of PTCLs and normal T-cells. We found that the methylation patterns in PTCLs are deregulated and heterogeneous but share 767 hypo- and 567 hypermethylated differentially methylated regions (DMRs) along with 231 genes up- and 91 genes downregulated in all samples suggesting a potential association with tumor development. We further identified 39 hypomethylated promoters associated with increased gene expression in the majority of PTCLs. This putative oncogenic signature included the TRIP13 (thyroid hormone receptor interactor 13) gene whose both genetic and pharmacologic inactivation, inhibited cellular growth of PTCL cell lines by inducing G2-M arrest accompanied by apoptosis suggesting that such an approach might be beneficial in human lymphoma treatment. Altogether we show that human PTCLs are characterized by a large number of recurrent methylation alterations, and demonstrated that TRIP13 is critical for PTCL maintenance in vitro.