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Climate change, hydro-power dams and socio-economic development in the upper Mekong River, have caused changes in surface water resources and agricultural transformation in the Vietnamese Mekong Delta (VMD). It is unpredictable and influences decision-making processes on water resource management. This study aims at applying the DAPP (Dynamic Adaptive Policy Pathways) approach to develop solution roadmaps supporting rice production in the Mekong Delta projection to 2030 under the context of uncertain changes on surface water resources. Data were collected from local officers, farmers, experts at Can Tho University, Technical University Delft, and Utrecht University using semi-structured interview techniques including workshops, group discussions, and in-depth interviews. The consolidated solutions from stakeholder’s opinions were evaluated at different socio-economic and environmental dimensions, specially focused on the time of tipping point of each solution. The study results show that two solution roadmaps were developed as sample scenarios to promote rice production in the upper area of the VMD. The determination of the solutions roadmaps based on the agreement of the stakeholders, which is a new feature of this approach in linking many stakeholders for the general development of the region. The DAPP approach could be suitable to support decision-making on surface water resources management in the context of uncertain changes. Therefore, further studies can apply this approach to other regions in the VMD which will support to reinforce and demonstrate the applicability of the DAPP approach on supporting decision-making in the face of increasingly uncertain changes in the VMD.

DNA methyltransferase inhibitors (DNMTI) like 5-Azacytidine (5-Aza) are the only disease-modifying drugs approved for the treatment of higher-risk myelodysplastic syndromes (MDS), however less than 50% of patients respond, and there are no predictors of response with clinical utility. Somatic mutations in the DNA methylation regulating gene tet-methylcytosine dioxygenase 2 (TET2) are associated with response to DNMTIs, however the mechanisms responsible for this association remain unknown. Using bisulfite padlock probes, mRNA sequencing, and hydroxymethylcytosine pull-down sequencing at several time points throughout 5-Aza treatment, we show that TET2 loss particularly influences DNA methylation (5mC) and hydroxymethylation (5hmC) patterns at erythroid gene enhancers and is associated with downregulation of erythroid gene expression in the human erythroleukemia cell line TF-1. 5-Aza disproportionately induces expression of these down-regulated genes in TET2KO cells and this effect is related to dynamic 5mC changes at erythroid gene enhancers after 5-Aza exposure. We identified differences in remethylation kinetics after 5-Aza exposure for several types of genomic regulatory elements, with distal enhancers exhibiting longer-lasting 5mC changes than other regions. This work highlights the role of 5mC and 5hmC dynamics at distal enhancers in regulating the expression of differentiation-associated gene signatures, and sheds light on how 5-Aza may be more effective in patients harboring TET2 mutations.Implications:TET2 loss in erythroleukemia cells induces hypermethylation and impaired expression of erythroid differentiation genes which can be specifically counteracted by 5-Azacytidine, providing a potential mechanism for the increased efficacy of 5-Aza in TET2-mutant patients with MDS.Visual Overview:http://mcr.aacrjournals.org/content/molcanres/19/3/451/F1.large.jpg.

A) Expression of hemoglobin genes which increase in terminally differentiated erythroid cells. B) Expression of key erythrocyte marker genes. TPM=Transcripts per kilobase million; a gene expression measure for RNAseq that normalizes for gene-length and the sample sequencing depth.

A) Dose-response curve of wild-type TF1 cell viability for three different lengths of 5-Aza treatments (1 dose per day for a total length of treatment between 1 and 3 days). Points represent replicates per condition, shaded region corresponds to 95% confidence band estimated by 4 parameter logistic regression model (EC50 and slope as parameter and lower bounded at zero; modeled with R package "drc"). B) Dose-response of DNMT1 downregulation 24 hours after the indicated 5-Aza dose. C) Time course of DNMT1 downregulation following the indicated dosage schedules.

Cells were treated with 100uM L-AA or vehicle 24hrs before DNA isolation. A) 5hmC dot blot for L-AA treated or untreated Cells. B) Standard curve of 5hmC densitometric signal vs. pmol (calculated from 5hmC standard with known 5hmC amount). C) Quantification of dot blot signal for TET2-WT and KO cells +/- L-AA.

Line plots in grid: each colored line represents a single CpG locus over time after 5-Aza treatment. Black line indicates mean value for that cluster group. Dotted black line indicates mean +/- 1 standard deviation. From left to right: clustering solutions with K=2 to K=9 clusters (derived from cutting the hierarchical cluster tree at different levels); top to bottom: the patterns of methylation in each cluster for that cluster solution. Mean silhouette width plot shows the mean silhouette width of all clusters for each individual clustering solution. K=4 clusters produced the most well-defined clusters as indicated by the peak in silhouette width.

Comprehensive characterization of cellular heterogeneity and the underlying regulatory landscapes of tissues and organs requires a highly robust and scalable method to acquire matched RNA and chromatin accessibility profiles on the same cells. Here, we describe a single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-seq2) assay, implemented with cellular combinatorial indexing. This method involves tagmentation within permeabilized and fixed single-nucleus isolates to capture accessible chromatin (AC) regions, followed by the capture and reverse transcription of RNA transcripts. Through combinatorial split pool ligations, cDNA and AC within each single nucleus become appended with a common cell barcode combination. The captured cDNA and AC are then co-amplified before splitting and enrichment into single-nucleus RNA and single-nucleus AC sequencing libraries. This protocol is compatible with both nuclei and whole cells and can be completed in 3.5 d. SNARE-seq2 permits robust generation of high-quality, joint single-cell RNA and AC sequencing libraries from hundreds of thousands of single cells per experiment.

Here we describe a computational protocol for performing quality control analysis on shallow sequencing data obtained from 10X snRNA-seq experiments. The workflow starts with raw MiSeq run folders and uses cellranger to generate count matrices. The raw count matrices are analyzed and sequencing saturation plots are generated. The saturation plots are then compared against plots from a reference set of libraries with varying qualities (bad, fair, good, great), thus allowing for the determination of sequencing requirements as well as an assessment of the overall quality of each 10X snRNA experiment.

Eyewitness misidentification accounts for 70% of verified erroneous convictions. To address this alarming phenomenon, research has focused on factors that influence likelihood of correct identification, such as the manner in which a lineup is conducted. Traditional lineups rely on overt eyewitness responses that confound two covert factors: strength of recognition memory and the criterion for deciding what memory strength is sufficient for identification. Here we describe a lineup that permits estimation of memory strength independent of decision criterion. Our procedure employs powerful techniques developed in studies of perception and memory: perceptual scaling and signal detection analysis. Using these tools, we scale memory strengths elicited by lineup faces, and quantify performance of a binary classifier tasked with distinguishing perpetrator from innocent suspect. This approach reveals structure of memory inaccessible using traditional lineups and renders accurate identifications uninfluenced by decision bias. The approach furthermore yields a quantitative index of individual eyewitness performance., Eyewitness errors contribute to wrongful convictions. Here, the authors present a lineup procedure that reveals the structure of eyewitness memory, reduces decision bias, and measures performance of individual witnesses.

Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.

10X Genomics Single Cell 3' (v3) RNA sequencing is a microdroplet-based method that permits the effective capture and sequencing of the mRNA and pre-mRNA molecules from single nuclei [1]. RNA molecules are transcribed and processed within the nucleus before exporting to ER for translation into proteins. As such, nuclear RNA is a mixture of nascent transcripts, partially or fully processed mRNA, and various non-coding RNA molecules. The total RNA content within the nucleus is roughly 10% of the RNA content in a whole cell, but has been found to accurately represent whole cell expression values in adult human tissues [2,3] including the kidney [4]. 10x Genomics Single Cell ATAC sequencing is a microdroplet-based method that allows for the effective capture, sequencing and profiling of accessible chromatin in single nuclei. Chromatin accessibility is a major determinant of gene regulation, defining the transcriptional regulatory networks that determine cellular identity and function as well as additional biological processes (e.g., differentiation, proliferation, development and responses to the extracellular environment). The 10X Multiome ATAC + Gene Expression assay permits capture of both RNA expression and epigenomic profiles from the same nuclei for a deeper understanding of cell type or state gene regulation. Here we present a modified version of the published 10X protocol [5] that we have adapted for the processing of nuclei isolated from adult human organs or tissues (e.g. kidney). Nuclei can be readily isolated from frozen tissues with a combination of chemical and physical treatments that can effectively circumvent the non-uniform or incomplete dissociation of solid tissues into single cells, as well as RNA degradation or artefacts (such as stress response) during dissociation. This 10X Multiome ATAC + Gene Expression protocol permits the generation of a molecular atlas of a human organ or tissue with comprehensive cell types and minimal processing artifacts. References 1. Chromium Single Cell 3' Reagent Kits v3 User Guide (Rev A) CG000183, support.10xgenomics.com. 2. Lake et al. (2016). Science, doi:10.1126/science.aaf1204. 3. Lake et al. (2018). Nature Biotechnology, doi:10.1038/nbt.4038. 4. Lake et al. (2019). Nature Communications, doi:10.1038/s41467-019-10861-2. 5. Chromium Next GEM Single Cell Multiome ATAC + Gene Expression (CG000338 Rev D).

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch–seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations., An examination of motor cortex in humans, marmosets and mice reveals a generally conserved cellular makeup that is likely to extend to many mammalian species, but also differences in gene expression, DNA methylation and chromatin state that lead to species-dependent specializations.

Understanding kidney disease relies upon defining the complexity of cell types and states, their associated molecular profiles, and interactions within tissue neighborhoods. We have applied multiple single-cell or -nucleus assays (>400,000 nuclei/cells) and spatial imaging technologies to a broad spectrum of healthy reference (n = 42) and disease (n = 42) kidneys. This has provided a high resolution cellular atlas of 100 cell types that include rare and novel cell populations. The multi-omic approach provides detailed transcriptomic profiles, epigenomic regulatory factors, and spatial localizations for major cell types spanning the entire kidney. We further identify and define cellular states altered in kidney injury, encompassing cycling, adaptive or maladaptive repair, transitioning and degenerative states affecting several segments. Molecular signatures of these states permitted their localization within injury neighborhoods using spatial transcriptomics, and large-scale 3D imaging analysis of ∼1.2 million neighborhoods provided linkages to active immune responses. These analyses further defined biological pathways relevant to injury niches, including signatures underlying the transition from reference to predicted maladaptive states that were associated with a decline in kidney function during chronic kidney disease. This human kidney cell atlas, including injury cell states and neighborhoods, will be a valuable resource for future studies.

10x Genomics Single Cell ATAC (v1) sequencing is a microdroplet-based method that allows for the effective capture, sequencing and profiling of accessible chromatin in single nuclei. Chromatin accessibility is a major determinant of gene regulation, defining the transcriptional regulatory networks that determine cellular identity and function as well as additional biological processes (e.g., differentiation, proliferation, development and responses to the extracellular environment). Chromatin accessibility sequencing (ATAC-seq) provides insight into the upstream regulatory landscape associated with open and accessible chromatin regions. References 1. Lake et al. (2016). Science, doi:10.1126/science.aaf1204. 2. Lake et al. (2018). Nature Biotechnology, doi:10.1038/nbt.4038. 3. Lake et al. (2019). Nature Communications, doi:10.1038/s41467-019-10861-2. 4. Chromium Single Cell ATAC Reagent Kits User Guide v1 (Rev B) CG000168, support.10xgenomics.com. 5. Lake and Zhang. Nuclei Isolation Protocol Applicable to Adult Human Kidney Tissues, kpmp.org/researcher-resources.

Neuroblastoma is a childhood cancer originating from embryonic neuronal progenitor cells and is the most common cancer diagnosed in infants. Although the majority of patients respond to initial chemotherapy, most high-risk patients suffer relapse due to therapy resistance. Here, we generated single-cell transcriptome and bulk whole-genome sequencing data of diagnosis and post-therapy samples from 23 patients diagnosed with high-risk neuroblastoma. We found two distinct adrenergic populations in the patient samples. Most tumor cells showed a mature sympathoblast phenotype whereas a small fraction was in an undifferentiated state with activation of protein translation, unfolded protein, and oxidative phosphorylation pathways. We found that therapy-resistant tumor cells in these two subpopulations had distinct characteristics. The more mature resistant subpopulation upregulated genes associated with epithelial-to-mesenchymal transition, including TWIST1, PLCB1 and CD276. The undifferentiated resistant subpopulation upregulated Ras signal transduction and TP53 pathway genes. Analysis of the immune microenvironment revealed that most tumor-associated macrophages became more immunosuppressive post-therapy via multiple newly gained signaling interactions including the complement signaling pathway. We discovered a limited infiltration of T lymphocytes in the tumor microenvironment, and chemotherapy induced an effector state with upregulated mTOR signaling and metabolism. Overall, our study revealed subpopulations of tumor cells in neuroblastoma that responded differently to induction chemotherapy. Our findings uncovered distinct molecular signatures of the resistant cells and their interactions with the immune microenvironment, paving the way for developing novel therapies for high-risk neuroblastoma. Citation Format: Yasin Uzun, Liron D. Grossmann, Chia-Hui Chen, Anusha Thadi, Chi-Yun Wu, Peng Gao, Dinh Diep, Lea Surrey, Daniel Martinez, Tasleema Patel, Qi Qiu, Sarah Johnson, Wenbao Yu, Shane Drabings, Changya Chen, Yuxuan Hu, Gregory Chen, Derek A. Oldridge, Kun Zhang, Hao Wu, Kathrin Bernt, Nancy Zhang, John M. Maris, Kai Tan. Longitudinal single-cell sequencing of high-risk neuroblastoma tumors reveals intrinsic and extrinsic mechanisms of therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6051.

BACKGROUND High-risk neuroblastoma is a pediatric cancer arising from the developing sympathetic nervous system with a 50% relapse rate that is typically fatal. At least two subpopulations of neuroblastoma cells exist that can transdifferentiate, adrenergic and mesenchymal, the latter being more resistant to chemotherapy. Mechanisms of therapy resistance are largely unknown and the cells responsible for relapse have not been identified. METHODS We used single nucleus RNA and ATAC sequencing to identify and characterize the cells that survive chemotherapy, termed here “persister cells”, from a cohort of 20 matched diagnostic and post induction chemotherapyhigh-risk neuroblastoma patients and two patient derived xenograft (PDX) models from diagnostic tumors. Eight representative cell lines derived from neuroblastomas at diagnosis were treated with standard-of-care chemotherapy, and flow cytometry was used to sort for live cells. ML120B and CRISPR-CAS9 were used to modulate NF-kB signaling. An RNA-seq dataset of 153 high-risk neuroblastoma patients was used to determine differentially activated pathways between adrenergic and mesenchymal tumors. RESULTS Residual malignant cells in the post-chemotherapy tumor samples clustered into three main groups separated by the response to therapy. The most prevalent group of persister cells in responders (N=16/20) displayed low MYC(N) activity even in the presence of MYCN amplification. This group also demonstrated decreased expression of the adrenergic core regulatory circuit genes including PHOX2B, ISL1, HAND2, along with marked activation of TNF-alpha via NF-kB signaling. High NF-kB activity was found in a subpopulation of diagnostic cells in two chemo-refractory patients. We validated decreased expression of MYCN (2-fold decrease, p CONCLUSIONS NF-kB activation is a major mediator of de novo and acquired chemotherapy resistance in high-risk neuroblastoma. We postulate that concomitant silencing of this pathway could eliminate persister cells and prevent disease relapse. Citation Format: Liron D. Grossmann, Yasin Uzun, Jarrett Lindsay, Chia-Hui Chen, Catherine Wingrove, Peng Gao, Anusha Thadi, Quinlen Marshall, Nathan M. Kendsersky, Lea Surrey, Daniel Martinez, Emily Mycek, Colleen Casey, Kateryna Krytska, Matthew Tsang, Adam Wolpaw, David N. Groff, Erin Runbeck, Jayne McDevitt, Dinh Diep, Tasleema Patel, Kathrin M. Bernt, Chi Dang, Kun Zhang, Yael P. Mosse, Kai Tan, John M. Maris. NF-kB is a master regulator of resistance to therapy in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 699.

DNA methyltransferase inhibitors (DNMTI) like 5-Azacytidine (5-Aza) are the only disease-modifying drugs approved for the treatment of higher-risk myelodysplastic syndromes (MDS), however less than 50% of patients respond, and there are no predictors of response with clinical utility. Somatic mutations in the DNA methylation regulating gene tet-methylcytosine dioxygenase 2 (TET2) are associated with response to DNMTIs, however the mechanisms responsible for this association remain unknown. Using bisulfite padlock probes, mRNA sequencing, and hydroxymethylcytosine pull-down sequencing at several time points throughout 5-Aza treatment, we show that TET2 loss particularly influences DNA methylation (5mC) and hydroxymethylation (5hmC) patterns at erythroid gene enhancers and is associated with downregulation of erythroid gene expression in the human erythroleukemia cell line TF-1. 5-Aza disproportionately induces expression of these down-regulated genes in TET2KO cells and this effect is related to dynamic 5mC changes at erythroid gene enhancers after 5-Aza exposure. We identified differences in remethylation kinetics after 5-Aza exposure for several types of genomic regulatory elements, with distal enhancers exhibiting longer-lasting 5mC changes than other regions. This work highlights the role of 5mC and 5hmC dynamics at distal enhancers in regulating the expression of differentiation-associated gene signatures, and sheds light on how 5-Aza may be more effective in patients harboring TET2 mutations. Implications: TET2 loss in erythroleukemia cells induces hypermethylation and impaired expression of erythroid differentiation genes which can be specifically counteracted by 5-Azacytidine, providing a potential mechanism for the increased efficacy of 5-Aza in TET2-mutant patients with MDS. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/19/3/451/F1.large.jpg.

The primary motor cortex (M1) is essential for voluntary fine motor control and is functionally conserved across mammals. Using high-throughput transcriptomic and epigenomic profiling of over 450,000 single nuclei in human, marmoset monkey, and mouse, we demonstrate a broadly conserved cellular makeup of this region, whose similarity mirrors evolutionary distance and is consistent between the transcriptome and epigenome. The core conserved molecular identity of neuronal and non-neuronal types allowed the generation of a cross-species consensus cell type classification and inference of conserved cell type properties across species. Despite overall conservation, many species specializations were apparent, including differences in cell type proportions, gene expression, DNA methylation, and chromatin state. Few cell type marker genes were conserved across species, providing a short list of candidate genes and regulatory mechanisms responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allowed the Patch-seq identification of layer 5 (L5) corticospinal Betz cells in non-human primate and human and characterization of their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell type diversity in M1 across mammals and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.

Ninh Kieu District is located in the center of Can Tho City, established under the Government’s Decree No. 05/2004/ND-CP. This is a modern and rich district, with a history of formation and development associated with the Mekong Delta. According to statistics in 2019, Ninh Kieu District has an average population of 280,792 people, with a population density of 9,605 people/km2 and an area of 29.23 km2. In the current period, the urbanization process in Ninh Kieu District takes place at a fast and strong pace. This has made it impossible for the urban drainage system to keep up with this change, thereby creating great difficulties for the drainage management here. In order to deal with these enormous challenges, the managers in this area need to incorporate new technologies into drainage management. This paper presents the main result of building a spatial database to support drainage management for Ninh Kieu District. This is considered as a fundamental source of data for application of GIS in urban drainage research and management in Ninh Kieu District.

10X Genomics Single Cell 3' (v3) RNA sequencing is a microdroplet-based method that permits the effective capture and sequencing of the mRNA and pre-mRNA molecules from single nuclei [1]. RNA molecules are transcribed and processed within the nucleus before exporting to ER for translation into proteins. As such, nuclear RNA is a mixture of nascent transcripts, partially or fully processed mRNA, and various non-coding RNA molecules. The total RNA content within the nucleus is roughly 10% of the RNA content in a whole cell, but has been found to accurately represent whole cell expression values in adult human tissues [2,3] including the kidney [4]. Nuclei can be readily isolated from frozen tissues with a combination of chemical and physical treatments that can effectively circumvent the non-uniform or incomplete dissociation of solid tissues into single cells, as well as RNA degradation or artefacts (such as stress response) during dissociation. Here we present a modified version of the published 10X protocol [1] that we have adapted for the processing of adult human kidney nuclei. References 1. Chromium Single Cell 3' Reagent Kits v3 User Guide (Rev A) CG000183, support.10xgenomics.com. 2. Lake et al. (2016). Science, doi:10.1126/science.aaf1204. 3. Lake et al. (2018). Nature Biotechnology, doi:10.1038/nbt.4038. 4. Lake et al. (2019). Nature Communications, doi:10.1038/s41467-019-10861-2.

Recurrent mutations implicate several epigenetic regulators in the early molecular pathobiology of myelodysplastic syndromes (MDS). We hypothesized that MDS subtypes defined by DNA methylation (DNAm) patterns could enhance our understanding of MDS disease biology and identify patients with convergent epigenetic profiles. Bisulfite padlock probe sequencing was used to measure DNAm of ∼500 000 unique cytosine guanine dinucleotides covering 140 749 nonoverlapping regulatory regions across the genome in bone marrow DNA samples from 141 patients with MDS. Application of a nonnegative matrix factorization (NMF)–based decomposition of DNAm profiles identified 5 consensus clusters described by 5 NMF components as the most stable grouping solution. Each of the 5 NMF components identified by this approach correlated with specific genetic abnormalities and categorized patients into 5 distinct methylation clusters, each largely defined by a single NMF component. Methylation clusters displayed unique differentially methylated regulatory loci enriched for active and bivalent promoters and enhancers. Two clusters were enriched for samples with complex karyotypes, although only one had an increased number of TP53 mutations. Each of the 3 most frequently mutated splicing factors, SF3B1, U2AF1, and SRSF2, was enriched in different clusters. Mutations of ASXL1, EZH2, and RUNX1 were coenriched in the SRSF2-containing cluster. In multivariate analysis, methylation cluster membership remained independently associated with overall survival. Targeted DNAm profiles identify clinically relevant subtypes of MDS not otherwise distinguished by mutations or clinical features. Patients with diverse genetic lesions can converge on common DNAm states with shared pathogenic mechanisms and clinical outcomes.

Adjacent CpG sites in mammalian genomes can be co-methylated due to the processivity of methyltransferases or demethylases. Yet discordant methylation patterns have also been observed, and found related to stochastic or uncoordinated molecular processes. We focused on a systematic search and investigation of regions in the full human genome that exhibit highly coordinated methylation. We defined 147,888 blocks of tightly coupled CpG sites, called methylation haplotype blocks (MHBs) with 61 sets of whole genome bisulfite sequencing (WGBS) data, and further validated with 101 sets of reduced representation bisulfite sequencing (RRBS) data and 637 sets of methylation array data. Using a metric called methylation haplotype load (MHL), we performed tissue-specific methylation analysis at the block level. Subsets of informative blocks were further identified for deconvolution of heterogeneous samples. Finally, we demonstrated quantitative estimation of tumor load and tissue-of-origin mapping in the circulating cell-free DNA of 59 cancer patients using methylation haplotypes.

A loss-of-function approach in mice is used to show that the methylcytosine dioxygenase Tet1 has a role in regulating meiosis and meiotic gene activation in female germ cells; Tet1 deficiency does not greatly affect genome-wide demethylation but has a more specific effect on the expression of a subset of meiotic genes. DNA methylation on cytosine is an important epigenetic modification, and the mechanisms controlling 5-methylcytosine (5mC) dynamics constitute an active area of research. The Tet family of dioxygenases can catalyse oxidation of 5mC to produce derivatives such as 5-hydroxymethylcytosine (5hmC), but little is known about the biological function of Tet proteins. Here, a loss-of-function approach in mice is used to show that Tet1 has a role in meiosis and meiotic gene activation in female germ cells. Tet1 deficiency does not greatly impact genome-wide demethylation, but has a more specific effect on the expression of a subset of meiotic genes. Meiosis is a germ-cell-specific cell division process through which haploid gametes are produced for sexual reproduction1. Before the initiation of meiosis, mouse primordial germ cells undergo a series of epigenetic reprogramming steps2,3, including the global erasure of DNA methylation at the 5-position of cytosine (5mC) in CpG-rich DNA4,5. Although several epigenetic regulators, such as Dnmt3l and the histone methyltransferases G9a and Prdm9, have been reported to be crucial for meiosis6, little is known about how the expression of meiotic genes is regulated and how their expression contributes to normal meiosis. Using a loss-of-function approach in mice, here we show that the 5mC-specific dioxygenase Tet1 has an important role in regulating meiosis in mouse oocytes. Tet1 deficiency significantly reduces female germ-cell numbers and fertility. Univalent chromosomes and unresolved DNA double-strand breaks are also observed in Tet1-deficient oocytes. Tet1 deficiency does not greatly affect the genome-wide demethylation that takes place in primordial germ cells, but leads to defective DNA demethylation and decreased expression of a subset of meiotic genes. Our study thus establishes a function for Tet1 in meiosis and meiotic gene activation in female germ cells.

Summary Tumor heterogeneity is a major barrier to effective cancer diagnosis and treatment. We recently identified cancer-specific differentially DNA-methylated regions (cDMRs) in colon cancer, which also distinguish normal tissue types from each other, suggesting that these cDMRs might be generalized across cancer types. Here we show stochastic methylation variation of the same cDMRs, distinguishing cancer from normal, in colon, lung, breast, thyroid, and Wilms tumors, with intermediate variation in adenomas. Whole genome bisulfite sequencing shows these variable cDMRs are related to loss of sharply delimited methylation boundaries at CpG islands. Furthermore, we find hypomethylation of discrete blocks encompassing half the genome, with extreme gene expression variability. Genes associated with the cDMRs and large blocks are involved in mitosis and matrix remodeling, respectively. These data suggest a model for cancer involving loss of epigenetic stability of well-defined genomic domains that underlies increased methylation variability in cancer and could contribute to tumor heterogeneity.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal human premature ageing disease, characterized by premature arteriosclerosis and degeneration of vascular smooth muscle cells (SMCs). HGPS is caused by a single point mutation in the lamin A (LMNA) gene, resulting in the generation of progerin, a truncated splicing mutant of lamin A. Accumulation of progerin leads to various ageing-associated nuclear defects including disorganization of nuclear lamina and loss of heterochromatin. Here we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with HGPS. HGPS-iPSCs show absence of progerin, and more importantly, lack the nuclear envelope and epigenetic alterations normally associated with premature ageing. Upon differentiation of HGPS-iPSCs, progerin and its ageing-associated phenotypic consequences are restored. Specifically, directed differentiation of HGPS-iPSCs to SMCs leads to the appearance of premature senescence phenotypes associated with vascular ageing. Additionally, our studies identify DNA-dependent protein kinase catalytic subunit (DNAPKcs, also known as PRKDC) as a downstream target of progerin. The absence of nuclear DNAPK holoenzyme correlates with premature as well as physiological ageing. Because progerin also accumulates during physiological ageing, our results provide an in vitro iPSC-based model to study the pathogenesis of human premature and physiological vascular ageing.

Background: Aberrant DNA methylation is an epigenetic hallmark of oncogenesis implicated in the pathogenesis of myelodysplastic syndromes (MDS). TET2 is one of the most frequently mutated genes in MDS, and is associated with net global hypermethylation in animal models. However, patients with MDS rarely present with a single somatic mutation, and thus methylation must be interpreted in the context of several mutated genes. We postulate that TET2 mutant MDS patients are not uniform and that epigenetic subgroups associated with clinical outcomes may exist. Methylation profiles at key regulatory regions could therefore serve as biomarkers in conjunction with somatic mutations. The aim of this study was to characterize the epigenetic heterogeneity of TET2 mutant MDS, and how specific methylation patterns relate to the molecular and clinical features of patients. Methods: To study methylation patterns in 96 patients with MDS, we utilized bisulfite padlock probes (BSPP), a novel enrichment technique (Deng et al. Nat Biotechnol 2009 and Diep et al. Nat Methods 2012) that targets ~500,000 unique CpGs in selected regions known to contain differentially methylated sites that have regulatory relevance, including transcription repressor CTCF binding sites, DNase I accessibility clusters, all microRNA genes, and all RefSeq gene promoters. BSPP was performed on genomic DNA purified from bone marrow mononuclear cells from untreated patients diagnosed with MDS that were genetically characterized in previous studies (Bejar et al. N Engl J Med 2011 and J Clin Oncol 2012). BSPP products were PCR amplified with primers containing individual barcodes as well as adaptors compatible with Illumina sequencers, allowing for massively parallel high throughput sequencing and digital quantification of CpG methylation. Results: Unsupervised clustering of the top 5% most variably methylated CpGs among 96 patients did not identify a correlation between gene mutations and methylation patterns. However, unsupervised analysis on the 26 patients with TET2 mutations (mean VAF 56.1%, not determined in 6 patients) sorted 20 patients into one of two discrete methylation clusters. Supervised analysis comparing these two clusters is shown in figure 1, highlighting the 9712 differentially methylated cytosines (DMCs) with >25% methylation difference and FDR-corrected p Discussion: We used BSPP to identify an epigenetic signature associated with survival among TET2 mutated patients with MDS. Unsupervised clustering identified methylation subgroups within TET2 mutants that are distinguished by differences in mutational burden, platelet count, and median overall survival. Between these two subgroups, DMCs are distributed in distinct patterns that identify regulatory regions that could serve as clinical biomarkers. Larger methylation profiling studies of MDS patients with various mutations and known treatment outcomes are ongoing. These will increase our ability to incorporate both epigenetic and genetic biomarkers into clinical practice. Disclosures Bejar: Genoptix: Consultancy, Honoraria, Patents & Royalties: No royalties; Celgene: Consultancy, Honoraria; Foundation Medicine: Consultancy.

Background: The DNA methyltransferase inhibitors, 5-azactidine and decitabine, are presumed to reverse aberrant methylation in tumor cells from patients with myelodysplastic syndromes (MDS), often resulting in hematologic improvement and even temporary eradication of the malignant clone. This suggests a causative role for abnormal DNA methylation in the development and progression of MDS. We hypothesize that patterns of DNA methylation associated with somatic mutations may serve as clinical biomarkers. We seek to determine how such somatic mutations in MDS alter the pattern of 5 methylcytosine (5mC) at key regulatory sites in the human genome. Since 5mC constitutes only 1% of total DNA bases, enrichment methods have been developed that are more cost-effective and time efficient compared to whole genome sequencing. Here we compared a novel targeted DNA enrichment approach, bisulfite padlock probes (BSPP), to a more standard enrichment method, reduced representation bisulfite sequencing (RRBS), to determine which method offers broader and more relevant coverage of the methylome in MDS tumor cells and K562 DNA-edited cell lines. Methods: We selected eight patient samples harboring somatic mutations that we anticipated would yield aberrant methylation, including mutations in TET2, IDH1, IDH2 and DNMT3A. We performed BSPP and RRBS on genomic DNA purified from bone marrow mononuclear cells. We then utilized the CRISPR/Cas9 approach to introduce mutations into the coding region in K562 myeloid lineage cells, creating DNA-edited cell lines with knockout mutations. We performed BSPP and RRBS on wild-type and mutant TET2 and DNMT3A myeloid cell lines followed by massively parallel sequencing. The resulting sequencing files were quality and adapter trimmed before being mapped to the bisulfite genome using BisReadMapper (Diep et. al 2012). Exploratory analyses including methylation beta value distribution calculation was carried out using the ÒRnBeadsÓ package for R version 3.2.1. Results: Among the eight patient samples, BSPP covered a quarter as many CpG sites as RRBS when examining regions sequenced in all samples. However, the sites captured by BSPP were sequenced with greater depth and had a higher alignment rate compared to RRBS. Only 3% of RRBS CpG sites were covered by BSPP, representing 12% of BSPP CpG sites. Concordance for 5mC in the regions covered by both techniques was high (R2 = 0.876). Compared to RRBS, genomic coverage beyond CpG islands was much greater with BSPP capture (Figure 1). Enrichment for CpG sites captured by BSPP was particularly seen in promoters, DNAse hypersensitivity sites and transcription factor binding sites. Mutant versus non-mutant methylation beta value density graphs showed similar methylation distributions when comparing data obtained from the patient samples and the K562 cell lines (Figure 2). Methylation values from BSPP data showed greater density distribution in hypomethylated regions among the patient samples and cell lines. Methylation values from RRBS data showed greater density distributions in hypermethylated regions. Discussion: RRBS approaches have failed to find pretreatment patterns of methylation associated with response, possibly due to inadequate coverage of 5mC distribution. We were able to obtain enhanced genomic coverage of the methylome utilizing BSPP compared to RRBS on both MDS patient samples and myeloid cell lines. One of the drawbacks of RRBS is its failure to capture areas with lower CpG densities that are potentially relevant to MDS pathobiology. Unlike RRBS, the padlock probes are not limited to regions adjacent to recognition sites of the restriction enzymes, allowing for parallel capture of a much broader range of genomic targets. We assessed methylation value distributions obtained by both approaches, and indeed, BSPP showed greater density distribution in hypomethylated regions in contrast to RRBS. Future work will focus on determining which of these changes in 5mC will prove the greatest predictor of response in MDS patients. Conclusions: We have shown that BSPP captures broader coverage of different regulatory regions of the genome compared to a more standard enrichment bisulfite sequencing techniques, RRBS. We have shown that BSPP is able to capture the variance in 5mC between TET2 and DNMT3A mutant and wild-type MDS patients, as well as in K562 DNA-edited cell lines. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Bejar: Genoptix Medical Laboratory: Consultancy, Honoraria, Patents & Royalties: MDS prognostic gene signature; Celgene: Consultancy, Honoraria; Alexion: Other: ad hoc advisory board.

Metabolism is vital to every aspect of cell function, yet the metabolome of induced pluripotent stem cells (iPSCs) remains largely unexplored. Here we report, using an untargeted metabolomics approach, that human iPSCs share a pluripotent metabolomic signature with embryonic stem cells (ESCs) that is distinct from their parental cells, and that is characterized by changes in metabolites involved in cellular respiration. Examination of cellular bioenergetics corroborated with our metabolomic analysis, and demonstrated that somatic cells convert from an oxidative state to a glycolytic state in pluripotency. Interestingly, the bioenergetics of various somatic cells correlated with their reprogramming efficiencies. We further identified metabolites that differ between iPSCs and ESCs, which revealed novel metabolic pathways that play a critical role in regulating somatic cell reprogramming. Our findings are the first to globally analyze the metabolome of iPSCs, and provide mechanistic insight into a new layer of regulation involved in inducing pluripotency, and in evaluating iPSC and ESC equivalence.

Targeted quantification of DNA methylation allows for interrogation of the most informative loci across many samples quickly and cost-effectively. Here we report improved bisulfite padlock probes (BSPPs) with a design algorithm to generate efficient padlock probes, a library-free protocol that dramatically reduces sample-preparation cost and time and is compatible with automation, and an efficient bioinformatics pipeline to accurately obtain both methylation levels and genotypes from sequencing of bisulfite-converted DNA.

Generation of human induced pluripotent stem cells (hiPSCs) by the expression of specific transcription factors depends on successful epigenetic reprogramming to a pluripotent state. Although hiPSCs and human embryonic stem cells (hESCs) display a similar epigenome, recent reports demonstrated the persistence of specific epigenetic marks from the somatic cell type of origin and aberrant methylation patterns in hiPSCs. However, it remains unknown whether the use of different somatic cell sources, encompassing variable levels of selection pressure during reprogramming, influences the level of epigenetic aberrations in hiPSCs. In this work, we characterized the epigenomic integrity of 17 hiPSC lines derived from six different cell types with varied reprogramming efficiencies. We demonstrate that epigenetic aberrations are a general feature of the hiPSC state and are independent of the somatic cell source. Interestingly, we observe that the reprogramming efficiency of somatic cell lines inversely correlates with the amount of methylation change needed to acquire pluripotency. Additionally, we determine that both shared and line-specific epigenetic aberrations in hiPSCs can directly translate into changes in gene expression in both the pluripotent and differentiated states. Significantly, our analysis of different hiPSC lines from multiple cell types of origin allow us to identify a reprogramming-specific epigenetic signature comprised of nine aberrantly methylated genes that is able to segregate hESC and hiPSC lines regardless of the somatic cell source or differentiation state.

Biological systems are inherently hierarchal and multiscale in time and space. A major challenge of systems biology is to describe biological systems as a computational model, which can be used to derive novel hypothesis and drive experiments leading to new knowledge. The constraint-based reconstruction and analysis approach has been successfully applied to metabolism and to the macromolecular synthesis machinery assembly. Here, we present the first integrated stoichiometric multiscale model of metabolism and macromolecular synthesis for Escherichia coli K12 MG1655, which describes the sequence-specific synthesis and function of almost 2000 gene products at molecular detail. We added linear constraints, which couple enzyme synthesis and catalysis reactions. Comparison with experimental data showed improvement of growth phenotype prediction with the multiscale model over E. coli's metabolic model alone. Many of the genes covered by this integrated model are well conserved across enterobacters and other, less related bacteria. We addressed the question of whether the bias in synonymous codon usage could affect the growth phenotype and environmental niches that an organism can occupy. We created two classes of in silico strains, one with more biased codon usage and one with more equilibrated codon usage than the wildtype. The reduced growth phenotype in biased strains was caused by tRNA supply shortage, indicating that expansion of tRNA gene content or tRNA codon recognition allow E. coli to respond to changes in codon usage bias. Our analysis suggests that in order to maximize growth and to adapt to new environmental niches, codon usage and tRNA content must co-evolve. These results provide further evidence for the mutation-selection-drift balance theory of codon usage bias. This integrated multiscale reconstruction successfully demonstrates that the constraint-based modeling approach is well suited to whole-cell modeling endeavors. Department of Energy DE-SC0002009 National Institutes of Health R0157089 info:eu-repo/grantAgreement/EC/FP7/249261

5-Hydroxymethylcytosine (5hmC) was recently found to be abundantly present in certain cell types, including embryonic stem cells. There is growing evidence that TET proteins, which convert 5-methylcytosine (5mC) to 5hmC, play important biological roles. To further understand the function of 5hmC, an analysis of the genome-wide localization of this mark is required.Here, we have generated a genome-wide map of 5hmC in human embryonic stem cells by hmeDIP-seq, in which hydroxymethyl-DNA immunoprecipitation is followed by massively parallel sequencing. We found that 5hmC is enriched in enhancers as well as in gene bodies, suggesting a potential role for 5hmC in gene regulation. Consistent with localization of 5hmC at enhancers, 5hmC was significantly enriched in histone modifications associated with enhancers, such as H3K4me1 and H3K27ac. 5hmC was also enriched in other protein-DNA interaction sites, such as OCT4 and NANOG binding sites. Furthermore, we found that 5hmC regions tend to have an excess of G over C on one strand of DNA.Our findings suggest that 5hmC may be targeted to certain genomic regions based both on gene expression and sequence composition.

TET dioxygenases successively oxidize 5-methylcytosine (5mC) in mammalian genomes to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC/5caC can be excised and repaired to regenerate unmodified cytosines by thymine-DNA glycosylase (TDG) and base excision repair (BER) pathway, but it is unclear to what extent and at which part of the genome this active demethylation process takes place. Here, we have generated genome-wide distribution maps of 5hmC/5fC/5caC using modification-specific antibodies in wild-type and Tdg-deficient mouse embryonic stem cells (ESCs). In wild-type mouse ESCs, 5fC/5caC accumulates to detectable levels at major satellite repeats but not at non-repetitive loci. In contrast, Tdg depletion in mouse ESCs causes marked accumulation of 5fC and 5caC at a large number of proximal and distal gene regulatory elements. Thus, these results reveal the first genome-wide view of iterative 5mC oxidation dynamics and indicate that TET/TDG-dependent active DNA demethylation process occurs extensively in the mammalian genome.