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Non-uniform dynamics of single CpGs among CLL progression

Table of correlation between time points within one sample and number of hyper- or hypomethylated CpGs for all CLL (top) and naïve and memory B cell samples (bottom)

Supplementary Figures 1-7 and matching legends

Most human cancers converge to a deregulated methylome with reduced global levels and elevated methylation at select CpG islands. To investigate the emergence and dynamics of the cancer methylome, we characterized genome-wide DNA methylation in preneoplastic monoclonal B-cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL), including serial samples collected across disease course. We detected the aberrant tumor-associated methylation landscape at CLL diagnosis and found no significant differentially methylated regions in the high-count MBL-to-CLL transition. Patient methylomes showed remarkable stability with natural disease and posttherapy progression. Single CLL cells were consistently aberrantly methylated, indicating a homogeneous transition to the altered epigenetic state and a distinct expression profile together with MBL cells compared with normal B cells. Our longitudinal analysis reveals the cancer methylome to emerge early, which may provide a platform for subsequent genetically driven growth dynamics, and, together with its persistent presence, suggests a central role in disease onset.Significance:DNA methylation data from a large cohort of patients with MBL and CLL show that epigenetic transformation emerges early and persists throughout disease stages with limited subsequent changes. Our results indicate an early role for this aberrant landscape in the normal-to-preneoplastic transition that may reflect a pan-cancer mechanism.See related commentary by Rossi, p. 6.This article is highlighted in the In This Issue feature, p. 1

Epigenetic alterations, such as promoter hypermethylation, may drive cancer through tumor suppressor gene inactivation. However, we have limited ability to differentiate driver DNA methylation (DNAme) changes from passenger events. We developed DNAme driver inference–MethSig–accounting for the varying stochastic hypermethylation rate across the genome and between samples. We applied MethSig to bisulfite sequencing data of chronic lymphocytic leukemia (CLL), multiple myeloma, ductal carcinoma in situ, glioblastoma, and to methylation array data across 18 tumor types in TCGA. MethSig resulted in well-calibrated quantile–quantile plots and reproducible inference of likely DNAme drivers with increased sensitivity/specificity compared with benchmarked methods. CRISPR/Cas9 knockout of selected candidate CLL DNAme drivers provided a fitness advantage with and without therapeutic intervention. Notably, DNAme driver risk score was closely associated with adverse outcome in independent CLL cohorts. Collectively, MethSig represents a novel inference framework for DNAme driver discovery to chart the role of aberrant DNAme in cancer.Significance:MethSig provides a novel statistical framework for the analysis of DNA methylation changes in cancer, to specifically identify candidate DNA methylation driver genes of cancer progression and relapse, empowering the discovery of epigenetic mechanisms that enhance cancer cell fitness.This article is highlighted in the In This Issue feature, p. 2113

Copy number changes in MethSig CLL DNAme drivers.

Supplementary Figure S1 to S7 and Supplementary Notes.

Chronic lymphocytic leukemia (CLL) is characterized by disordered DNA methylation, suggesting these epigenetic changes might play a critical role in disease onset and progression. The methyltransferase DNMT3A is a key regulator of DNA methylation. Although DNMT3A somatic mutations in CLL are rare, we found that low DNMT3A expression is associated with more aggressive disease. A conditional knockout mouse model showed that homozygous depletion of Dnmt3a from B cells results in the development of CLL with 100% penetrance at a median age of onset of 5.3 months, and heterozygous Dnmt3a depletion yields a disease penetrance of 89% with a median onset at 18.5 months, confirming its role as a haploinsufficient tumor suppressor. B1a cells were confirmed as the cell of origin of disease in this model, and Dnmt3a depletion resulted in focal hypomethylation and activation of Notch and Myc signaling. Amplification of chromosome 15 containing the Myc gene was detected in all CLL mice tested, and infiltration of high-Myc–expressing CLL cells in the spleen was observed. Notably, hyperactivation of Notch and Myc signaling was exclusively observed in the Dnmt3a CLL mice, but not in three other CLL mouse models tested (Sf3b1-Atm, Ikzf3, and MDR), and Dnmt3a-depleted CLL were sensitive to pharmacologic inhibition of Notch signaling in vitro and in vivo. Consistent with these findings, human CLL samples with lower DNMT3A expression were more sensitive to Notch inhibition than those with higher DNMT3A expression. Altogether, these results suggest that Dnmt3a depletion induces CLL that is highly dependent on activation of Notch and Myc signaling.Significance:Loss of DNMT3A expression is a driving event in CLL and is associated with aggressive disease, activation of Notch and Myc signaling, and enhanced sensitivity to Notch inhibition.

Epigenetic alterations, such as promoter hypermethylation, may drive cancer through tumor suppressor gene inactivation. However, we have limited ability to differentiate driver DNA methylation (DNAme) changes from passenger events. We developed DNAme driver inference–MethSig–accounting for the varying stochastic hypermethylation rate across the genome and between samples. We applied MethSig to bisulfite sequencing data of chronic lymphocytic leukemia (CLL), multiple myeloma, ductal carcinoma in situ, glioblastoma, and to methylation array data across 18 tumor types in TCGA. MethSig resulted in well-calibrated quantile–quantile plots and reproducible inference of likely DNAme drivers with increased sensitivity/specificity compared with benchmarked methods. CRISPR/Cas9 knockout of selected candidate CLL DNAme drivers provided a fitness advantage with and without therapeutic intervention. Notably, DNAme driver risk score was closely associated with adverse outcome in independent CLL cohorts. Collectively, MethSig represents a novel inference framework for DNAme driver discovery to chart the role of aberrant DNAme in cancer. Significance: MethSig provides a novel statistical framework for the analysis of DNA methylation changes in cancer, to specifically identify candidate DNA methylation driver genes of cancer progression and relapse, empowering the discovery of epigenetic mechanisms that enhance cancer cell fitness. This article is highlighted in the In This Issue feature, p. 2113

Chronic lymphocytic leukemia (CLL) is characterized by disordered DNA methylation, suggesting these epigenetic changes might play a critical role in disease onset and progression. The methyltransferase DNMT3A is a key regulator of DNA methylation. Although DNMT3A somatic mutations in CLL are rare, we found that low DNMT3A expression is associated with more aggressive disease. A conditional knockout mouse model showed that homozygous depletion of Dnmt3a from B cells results in the development of CLL with 100% penetrance at a median age of onset of 5.3 months, and heterozygous Dnmt3a depletion yields a disease penetrance of 89% with a median onset at 18.5 months, confirming its role as a haploinsufficient tumor suppressor. B1a cells were confirmed as the cell of origin of disease in this model, and Dnmt3a depletion resulted in focal hypomethylation and activation of Notch and Myc signaling. Amplification of chromosome 15 containing the Myc gene was detected in all CLL mice tested, and infiltration of high-Myc–expressing CLL cells in the spleen was observed. Notably, hyperactivation of Notch and Myc signaling was exclusively observed in the Dnmt3a CLL mice, but not in three other CLL mouse models tested (Sf3b1-Atm, Ikzf3, and MDR), and Dnmt3a-depleted CLL were sensitive to pharmacologic inhibition of Notch signaling in vitro and in vivo. Consistent with these findings, human CLL samples with lower DNMT3A expression were more sensitive to Notch inhibition than those with higher DNMT3A expression. Altogether, these results suggest that Dnmt3a depletion induces CLL that is highly dependent on activation of Notch and Myc signaling. Significance: Loss of DNMT3A expression is a driving event in CLL and is associated with aggressive disease, activation of Notch and Myc signaling, and enhanced sensitivity to Notch inhibition.

Genetic and epigenetic intra-tumoral heterogeneity cooperate to shape the evolutionary course of cancer1. Chronic lymphocytic leukaemia (CLL) is a highly informative model for cancer evolution as it undergoes substantial genetic diversification and evolution after therapy2,3. The CLL epigenome is also an important disease-defining feature4,5, and growing populations of cells in CLL diversify by stochastic changes in DNA methylation known as epimutations6. However, previous studies using bulk sequencing methods to analyse the patterns of DNA methylation were unable to determine whether epimutations affect CLL populations homogeneously. Here, to measure the epimutation rate at single-cell resolution, we applied multiplexed single-cell reduced-representation bisulfite sequencing to B cells from healthy donors and patients with CLL. We observed that the common clonal origin of CLL results in a consistently increased epimutation rate, with low variability in the cell-to-cell epimutation rate. By contrast, variable epimutation rates across healthy B cells reflect diverse evolutionary ages across the trajectory of B cell differentiation, consistent with epimutations serving as a molecular clock. Heritable epimutation information allowed us to reconstruct lineages at high-resolution with single-cell data, and to apply this directly to patient samples. The CLL lineage tree shape revealed earlier branching and longer branch lengths than in normal B cells, reflecting rapid drift after the initial malignant transformation and a greater proliferative history. Integration of single-cell bisulfite sequencing analysis with single-cell transcriptomes and genotyping confirmed that genetic subclones mapped to distinct clades, as inferred solely on the basis of epimutation information. Finally, to examine potential lineage biases during therapy, we profiled serial samples during ibrutinib-associated lymphocytosis, and identified clades of cells that were preferentially expelled from the lymph node after treatment, marked by distinct transcriptional profiles. The single-cell integration of genetic, epigenetic and transcriptional information thus charts the lineage history of CLL and its evolution with therapy. A single-cell approach is used to follow the heritable stochastic changes to DNA methylation that occur in primary chronic lymphocytic leukaemia and healthy B cells, allowing the tracing of cell lineage histories and evolution during treatment with ibrutinib.

Metagenomic sequencing has the potential to transform microbial detection and characterization, but new tools are needed to improve its sensitivity. Here we present CATCH, a computational method to enhance nucleic acid capture for enrichment of diverse microbial taxa. CATCH designs optimal probe sets, with a specified number of oligonucleotides, that achieve full coverage of, and scale well with, known sequence diversity. We focus on applying CATCH to capture viral genomes in complex metagenomic samples. We design, synthesize, and validate multiple probe sets, including one that targets the whole genomes of the 356 viral species known to infect humans. Capture with these probe sets enriches unique viral content on average 18-fold, allowing us to assemble genomes that could not be recovered without enrichment, and accurately preserves within-sample diversity. We also use these probe sets to recover genomes from the 2018 Lassa fever outbreak in Nigeria and to improve detection of uncharacterized viral infections in human and mosquito samples. The results demonstrate that CATCH enables more sensitive and cost-effective metagenomic sequencing.

Most human cancers converge to a deregulated methylome with reduced global levels and elevated methylation at select CpG islands. To investigate the emergence and dynamics of the cancer methylome, we characterized genome-wide DNA methylation in preneoplastic monoclonal B-cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL), including serial samples collected across disease course. We detected the aberrant tumor-associated methylation landscape at CLL diagnosis and found no significant differentially methylated regions in the high-count MBL-to-CLL transition. Patient methylomes showed remarkable stability with natural disease and posttherapy progression. Single CLL cells were consistently aberrantly methylated, indicating a homogeneous transition to the altered epigenetic state and a distinct expression profile together with MBL cells compared with normal B cells. Our longitudinal analysis reveals the cancer methylome to emerge early, which may provide a platform for subsequent genetically driven growth dynamics, and, together with its persistent presence, suggests a central role in disease onset. Significance: DNA methylation data from a large cohort of patients with MBL and CLL show that epigenetic transformation emerges early and persists throughout disease stages with limited subsequent changes. Our results indicate an early role for this aberrant landscape in the normal-to-preneoplastic transition that may reflect a pan-cancer mechanism. See related commentary by Rossi, p. 6. This article is highlighted in the In This Issue feature, p. 1

Phylogenetics of superspreading One important characteristic of coronavirus epidemiology is the occurrence of superspreading events. These are marked by a disproportionate number of cases originating from often-times asymptomatic individuals. Using a rich sequence dataset from the early stages of the Boston outbreak, Lemieux et al. identified superspreading events in specific settings and analyzed them phylogenetically (see the Perspective by Alizon). Using ancestral trait inference, the authors identified several importation events, further investigated the context and contribution of particular superspreading events to the establishment of local and wider SARS-CoV-2 transmission, and used viral phylogenies to describe sustained transmission. Science , this issue p. eabe3261 ; see also p. 574

Leukemic relapse remains a major barrier to successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) for aggressive hematologic malignancies. The basis for relapse of advanced lymphoid malignancies remains incompletely understood and may involve escape from the graft-versus-leukemia (GvL) effect. We hypothesized that for patients with chronic lymphocytic leukemia (CLL) treated with allo-HSCT, leukemic cell-intrinsic features influence transplant outcomes by directing the evolutionary trajectories of CLL cells. Integrated genetic, transcriptomic, and epigenetic analyses of CLL cells from 10 patients revealed that the clinical kinetics of post-HSCT relapse are shaped by distinct molecular dynamics. Early relapses after allo-HSCT exhibited notable genetic stability; single CLL cell transcriptional analysis demonstrated a cellular heterogeneity that was static over time. In contrast, CLL cells relapsing late after allo-HSCT displayed notable genetic evolution and evidence of neoantigen depletion, consistent with marked single-cell transcriptional shifts that were unique to each patient. We observed a greater rate of epigenetic change for late relapses not seen in early relapses or relapses after chemotherapy alone, suggesting that the selection pressures of the GvL bottleneck are unlike those imposed by chemotherapy. No selective advantage for human leukocyte antigen (HLA) loss was observed, even when present in pretransplant subpopulations. Gain of stem cell modules was a common signature associated with leukemia relapse regardless of posttransplant relapse kinetics. These data elucidate the biological pathways that underlie GvL resistance and posttransplant relapse.

SARS-CoV-2 has caused a severe, ongoing outbreak of COVID-19 in Massachusetts with 111,070 confirmed cases and 8,433 deaths as of August 1, 2020. To investigate the introduction, spread, and epidemiology of COVID-19 in the Boston area, we sequenced and analyzed 772 complete SARS-CoV-2 genomes from the region, including nearly all confirmed cases within the first week of the epidemic and hundreds of cases from major outbreaks at a conference, a nursing facility, and among homeless shelter guests and staff. The data reveal over 80 introductions into the Boston area, predominantly from elsewhere in the United States and Europe. We studied two superspreading events covered by the data, events that led to very different outcomes because of the timing and populations involved. One produced rapid spread in a vulnerable population but little onward transmission, while the other was a major contributor to sustained community transmission, including outbreaks in homeless populations, and was exported to several other domestic and international sites. The same two events differed significantly in the number of new mutations seen, raising the possibility that SARS-CoV-2 superspreading might encompass disparate transmission dynamics. Our results highlight the failure of measures to prevent importation into MA early in the outbreak, underscore the role of superspreading in amplifying an outbreak in a major urban area, and lay a foundation for contact tracing informed by genetic data.

Early detection of infection with SARS-CoV-2 is key to managing the current global pandemic, as evidence shows the virus is most contagious on or before symptom onset. Here, we introduce a low-cost, high-throughput method for diagnosing and studying SARS-CoV-2 infection. Dubbed Pathogen-Oriented Low-Cost Assembly & Re-Sequencing (POLAR), this method amplifies the entirety of the SARS-CoV-2 genome. This contrasts with typical RT-PCR-based diagnostic tests, which amplify only a few loci. To achieve this goal, we combine a SARS-CoV-2 enrichment method developed by the ARTIC Network (https://artic.network/) with short-read DNA sequencing andde novogenome assembly. Using this method, we can reliably (>95% accuracy) detect SARS-CoV-2 at a concentration of 84 genome equivalents per milliliter (GE/mL). Almost all diagnostic methods currently authorized for use by the United States Food and Drug Administration with the Coronavirus Disease 2019 (COVID-19) Emergency Use Authorization require larger concentrations of the virus to achieve this degree of accuracy. In addition, we can reliably assemble the SARS-CoV-2 genome in the sample, often with no gaps and perfect accuracy. The genotypic data contained in these genome assemblies enable the more effective analysis of disease spread than is possible with an ordinary binary diagnostic. These data can also help identify vaccine and drug targets. Finally, we show that the diagnoses obtained using POLAR of both positive and negative clinical nasopharyngeal swab samples 100% match the diagnoses obtained in a clinical diagnostic lab using the Center for Disease Control’s 2019-Novel Coronavirus test. Using POLAR, a single person can manually process 192 samples over an 8- hour experiment at the cost of ∼$36 per patient (as of December 7th, 2022), enabling a 24-hour turnaround with sequencing and data analysis time. We anticipate that further testing and refinement will allow greater sensitivity in this approach.

Resistance to the graft-versus-leukemia (GvL) effect remains the major barrier to successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) for aggressive hematologic malignancies. The basis of GvL resistance for advanced lymphoid malignancies remains incompletely understood. We hypothesized that for patients with chronic lymphocytic leukemia (CLL) treated with allo-HSCT, leukemic cell-intrinsic features shape GvL outcomes by directing the evolutionary trajectories of CLL cells. Integrated genetic, transcriptomic and epigenetic analyses of CLL cells from 10 patients revealed that the clinical kinetics of post- HSCT relapse are shaped by distinct molecular dynamics and suggest that the selection pressures of the GvL bottleneck are unlike those imposed by chemotherapy. No selective advantage for HLA loss was observed, even when present in pre-transplant subpopulations. Regardless of post-transplant relapse kinetics, gain of stem cell modules was a common signature associated with leukemia relapse. These data elucidate the biological pathways that underlie GvL resistance and post-transplant relapse.One Sentence SummaryWe find that the clinical kinetics of chronic lymphocytic leukemia relapse after stem cell transplant are underwritten by distinct genetic and epigenetic evolutionary trajectories and suggest that the selection pressures of the post-transplant, immunologic bottleneck are unlike those imposed by chemotherapy.

In diploid cells, maternal and paternal copies of genes usually have similar transcriptional activity. Mammalian allele-specific epigenetic mechanisms such as X-chromosome inactivation (XCI) and imprinting were historically viewed as rare exceptions to this rule. The discovery of mitotically stable monoallelic autosomal expression (MAE) a decade ago revealed an additional allele-specific mode regulating thousands of mammalian genes. However, despite its prevalence, the mechanistic basis of MAE remains unknown. To uncover the mechanism of MAE maintenance, we devised a small-molecule screen for reactivation of silenced alleles across multiple loci using targeted RNA sequencing. Contrary to previous reports, we identified DNA methylation as a key mechanism of MAE mitotic maintenance. In contrast with the binary choice of the active allele in XCI, stringent transcriptome-wide analysis revealed MAE as a regulatory mode with tunable control of allele-specific expression, dependent on the extent of DNA methylation. In a subset of MAE genes, allelic imbalance was insensitive to changes in DNA methylation, implicating additional mechanisms in MAE maintenance in these loci. Our findings identify a key mechanism of MAE maintenance, reveal tunability of this mode of gene regulation, and provide the essential platform for probing the biological role of MAE in development and disease.

In normal mammalian development cytosine methylation is essential and is directed to specific regions of the genome. Despite notable advances through mapping its genome-wide distribution, studying the direct contribution of DNA methylation to gene and genome regulation has been limited by the lack of tools for its precise manipulation. Thus, combining the targeting capability of the CRISPR–Cas9 system with an epigenetic modifier has attracted interest in the scientific community. In contrast to profiling the genome-wide cleavage of a nuclease competent Cas9, tracing the global activity of a dead Cas9 (dCas9) methyltransferase fusion protein is challenging within a highly methylated genome. Here, we report the generation and use of an engineered, methylation depleted but maintenance competent mouse ES cell line and find surprisingly ubiquitous nuclear activity of dCas9-methyltransferases. Subsequent experiments in human somatic cells refine these observations and point to an important difference between genetic and epigenetic editing tools that require unique experimental considerations., Catalytically inactive Cas9 fused to a methyltransferase has emerged as a promising epigenome modifying tool. Here the authors generate a methylation depleted but maintenance competent mouse ES cell line and find ubiquitous off-target activity.

Transcription factors (TFs) direct developmental transitions by binding to target DNA sequences, influencing gene expression and establishing complex gene-regultory networks. To systematically determine the molecular components that enable or constrain TF activity, we investigated the genomic occupancy of FOXA2, GATA4 and OCT4 in several cell types. Despite their classification as pioneer factors, all three TFs exhibit cell-type-specific binding, even when supraphysiologically and ectopically expressed. However, FOXA2 and GATA4 can be distinguished by low enrichment at loci that are highly occupied by these factors in alternative cell types. We find that expression of additional cofactors increases enrichment at a subset of these sites. Finally, FOXA2 occupancy and changes to DNA accessibility can occur in G1-arrested cells, but subsequent loss of DNA methylation requires DNA replication.

Concomitant activation of the Wnt pathway and suppression of Mapk signalling by two small molecule inhibitors (2i) in the presence of leukaemia inhibitory factor (LIF) (hereafter termed 2i/L) induces a naive state in mouse embryonic stem (ES) cells that resembles the inner cell mass (ICM) of the pre-implantation embryo. Since the ICM exists only transiently in vivo, it remains unclear how sustained propagation of naive ES cells in vitro affects their stability and functionality. Here we show that prolonged culture of male mouse ES cells in 2i/L results in irreversible epigenetic and genomic changes that impair their developmental potential. Furthermore, we find that female ES cells cultured in conventional serum plus LIF medium phenocopy male ES cells cultured in 2i/L. Mechanistically, we demonstrate that the inhibition of Mek1/2 is predominantly responsible for these effects, in part through the downregulation of DNA methyltransferases and their cofactors. Finally, we show that replacement of the Mek1/2 inhibitor with a Src inhibitor preserves the epigenetic and genomic integrity as well as the developmental potential of ES cells. Taken together, our data suggest that, although short-term suppression of Mek1/2 in ES cells helps to maintain an ICM-like epigenetic state, prolonged suppression results in irreversible changes that compromise their developmental potential.

The field of cancer genomics has been empowered by increasingly sophisticated inference tools to distinguish driver mutations from the vastly greater number of passenger mutations. Epigenetic alterations such as promoter DNA hypermethylation have been shown to drive cancer through inactivation of tumor suppressor genes (TSGs), but growing malignant populations also accrue pervasive stochastic epigenetic changes in DNA methylation (DNAme), most of which likely carry little functional impact. Unlike with somatic mutations, we have limited ability to robustly differentiate driver DNAme changes (DNAme drivers) from stochastic, passenger DNAme changes. To address this challenge, we developed MethSig, a statistical inference framework that accounts for the varying stochastic hypermethylation rates across the genome and between samples. MethSig estimates expected background DNAme changes, thereby allowing the identification of epigenetically disrupted loci, where observed hypermethylation significantly exceeds expectation, potentially reflecting positive selection (Fig. 1a). We applied MethSig to reduced representation bisulfite sequencing (RRBS) data of chronic lymphocytic leukemia (CLL) cohorts, which include 304 CLLs collected in a prospective clinical trial (CLL8) and 103 CLLs in a previously published study (CLL-DFCI, Landau et al., 2014), as well as other malignancies where RRBS data is available, including ductal carcinoma in situ (Abba et al., 2015) and multiple myeloma. Area under the receiver operating characteristic curve (AUROC) was used to evaluate sensitivity and specificity of methods in the inference of likely DNAme drivers. We identified two key features that are likely to be strongly associated with true candidate DNAme drivers: gene silencing in relation to promoter hypermethylation and association with clinical outcome. MethSig qualitatively improved ROC across those clinical and biological read outs (0.955 of MethSig, 95% confidence interval [CI] 0.945 - 0.965, versus 0.703 of benchmarked methods, 95% CI 0.669 - 0.737, Fig. 1b used CLL8 as an example). We identified 189 candidate DNAme drivers in CLL, which include known TSGs, and are enriched in genes hypermethylated or inactivated across cancer types. To further validate MethSig's inferences, selected CLL candidate DNAme drivers (DUSP22, RPRM) underwent CRISPR/Cas9 knockout (KO) in CLL cells and stable KO clones were generated through single-cell cloning to eliminate genetic heterogeneity effect. The RPRM and DUSP22 KO clones showed faster growth without treatment (Fig. 1c) and superior fitness in ibrutinib/fludarabine treatment compared with controls (Fig. 1d). Notably, we observed a gene dose effect in the RPRM KO clones (Fig. 1c-d, greater growth of the bi-allelic compared to mono-allelic KO). Elastic net regression with a Cox proportional hazards model was used to evaluate DNAme drivers' contribution to the prediction of failure-free survival after treatment (FFS; failure defined as retreatment or death) and a rigorous training (CLL8) and validation (an independent cohort CLL-DFCI) cohort study design was implemented to safeguard from overfitting and poor generalizability. DNAme drivers were found to be associated with shorter FFS in independent CLL cohorts (Fig. 1e-f). A regression model including established CLL risk indicators (IGHV unmutated status, del[17p] or TP53 mutation) showed an adjusted hazard ratio of 2.3 (95% CI 1.6 - 3.3, P = 2 × 10-6) in CLL8 cohort and 3.2 (95% CI 1.2 - 8.8, P = 0.02) in CLL-DFCI cohort for patients with high risk. Application of MethSig to CLL relapsed after chemoimmunotherapy further identified relapse-specific DNAme drivers, enriched in TP53 targets as well as DNA damage pathway, which indicates that CLL relapse after chemotherapy may follow an alternative path compared to CLL progression in the absence of therapy, offering novel insights for therapeutic strategies to address drug-resistant or relapsed cancer. Collectively, our data support a novel framework for the analysis of DNAme changes in cancer to specifically identify DNAme drivers of disease progression and relapse, empowering the discovery of epigenetic mechanisms that enhance cancer cell fitness. This work addressed a central gap between cancer epigenetics and cancer genetics, where such tools have had a transformative impact in precision oncology and cancer gene discovery. Disclosures Tausch: Janssen-Cilag: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding. Fink:AbbVie: Other: travel grants; Janssen: Honoraria; Celgene: Research Funding. Fischer:AbbVie: Honoraria; F. Hoffmann-La Roche: Honoraria, Other: travel grants. Gnirke:FL67 Inc.: Consultancy. Moreaux:Diag2Tec: Consultancy. Hallek:Celgene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Mundipharma: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Roche: Consultancy, Research Funding. Stilgenbauer:Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding; Genentech: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding; Genzyme: Consultancy, Honoraria, Other: travel support, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other, Research Funding; F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Other: travel support, Research Funding; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Novartis: Consultancy, Honoraria, Other, Research Funding; Mundipharma: Consultancy, Honoraria, Other, Research Funding. Wu:BionTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding. Elemento:Acuamark: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; OneThree Biotech: Current equity holder in private company, Other: Cofounder; Owkin: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Sanofi: Research Funding; Eli Lilly: Research Funding; Volastra Therapeutics: Current equity holder in private company, Other: Cofounder; Freenome: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Genetic Intelligence: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding. Landau:Bristol Myers Squibb: Research Funding; Illumina: Research Funding.

Cancer evolution is fueled by epigenetic as well as genetic diversity. In chronic lymphocytic leukemia (CLL), intra-tumoral DNA methylation (DNAme) heterogeneity empowers evolution. Here, to comprehensively study the epigenetic dimension of cancer evolution, we integrate DNAme analysis with histone modification mapping and single cell analyses of RNA expression and DNAme in 22 primary CLL and 13 healthy donor B lymphocyte samples. Our data reveal corrupted coherence across different layers of the CLL epigenome. This manifests in decreased mutual information across epigenetic modifications and gene expression attributed to cell-to-cell heterogeneity. Disrupted epigenetic-transcriptional coordination in CLL is also reflected in the dysregulation of the transcriptional output as a function of the combinatorial chromatin states, including incomplete Polycomb-mediated gene silencing. Notably, we observe unexpected co-mapping of typically mutually exclusive activating and repressing histone modifications, suggestive of intra-tumoral epigenetic diversity. Thus, CLL epigenetic diversification leads to decreased coordination across layers of epigenetic information, likely reflecting an admixture of cells with diverging cellular identities., In chronic lymphocytic leukemia (CLL), evolution is driven by transcriptional and epigenetic heterogeneity. Here, the authors integrate epigenomic analyses to show how intra-tumoral epigenetic diversity results in divergent chromatin states in CLL cells, increasing cell-to-cell transcriptional heterogeneity.

Mammalian cells stably maintain high levels of DNA methylation despite expressing both positive (DNMT3A/B) and negative (TET1–3) regulators. Here, we used wildtype and TET triple knockout human embryonic stem cells (ESCs), generated DNMT3-null as well as TET and DNMT3-null pentuple knockouts, and compared methylation patterns using whole genome bisulfite sequencing (WGBS). The greatest impact on global methylation levels was observed in DNMT3-deficient cells, including reproducible focal demethylation at thousands of normally methylated loci. This demethylation depends upon TET expression and only occurs when both DNMT3s are absent. Dynamic loci are enriched for hydroxymethylcytosine and overlap with subsets of putative somatic enhancers that are methylated in ESCs and can be activated upon differentiation. We observe similar dynamics in mouse ESCs that were less frequenct in epiblast stem cells (EpiSCs) and scarce in somatic tissues, suggesting a conserved pluripotency-linked mechanism. Taken together, our data reveal tightly regulated competition between DNMT3s and TETs at thousands of somatic regulatory sequences within pluripotent cells.

Mucin-1 kidney disease, previously described as medullary cystic kidney disease type 1 (MCKD1, OMIM 174000), is an autosomal dominant tubulointerstitial kidney disease recently shown to be caused by a single-base insertion within the variable number tandem repeat region of the MUC1 gene. Because of variable age of disease onset and often subtle signs and symptoms, clinical diagnosis of mucin-1 kidney disease and differentiation from other forms of hereditary kidney disease have been difficult. The causal insertion resides in a variable number tandem repeat region with high GC content, which has made detection by standard next-generation sequencing impossible to date. The inherently difficult nature of this mutation required an alternative method for routine detection and clinical diagnosis of the disease. We therefore developed and validated a mass spectrometry–based probe extension assay with a series of internal controls to detect the insertion event using 24 previously characterized positive samples from patients with mucin-1 kidney disease and 24 control samples known to be wild type for the variant. Validation results indicate an accurate and reliable test for clinically establishing the molecular diagnosis of mucin-1 kidney disease with 100% sensitivity and specificity across 275 tests called., Carlos Slim Foundation. Slim Initiative for Genomic Medicine

Hi-Culfite, a protocol combining Hi-C and whole-genome bisulfite sequencing (WGBS), determines chromatin contacts and DNA methylation simultaneously. Hi-Culfite also reveals relationships that cannot be seen when the two assays are performed separately. For instance, we show that loci associated with open chromatin exhibit context-sensitive methylation: when their spatial neighbors lie in closed chromatin, they are much more likely to be methylated.

DNA methylation plays an essential role in mammalian genomes and expression of the responsible enzymes is tightly controlled. Deregulation of the de novo DNA methyltransferase DNMT3B is frequently observed across cancer types, yet little is known about its ectopic genomic targets. Here, we used an inducible transgenic mouse model to delineate rules for abnormal DNMT3B targeting, as well as the constraints of its activity across different cell types. Our results explain the preferential susceptibility of certain CpG islands to aberrant methylation and point to transcriptional state and the associated chromatin landscape as the strongest predictors. Although DNA methylation and H3K27me3 are usually non-overlapping at CpG islands, H3K27me3 can transiently co-occur with DNMT3B-induced DNA methylation. Our genome-wide data combined with ultra-deep locus-specific bisulfite sequencing suggest a distributive activity of ectopically expressed Dnmt3b that leads to discordant CpG island hypermethylation and provides new insights for interpreting the cancer methylome.

Advances in stem cell science allow the production of different cell types in vitro either through the recapitulation of developmental processes, often termed “directed differentiation,” or the forced expression of lineage-specific transcription factors. Although cells produced by both approaches are increasingly used in translational applications, their quantitative similarity to their primary counterparts remains largely unresolved. To investigate the similarity between in vitro-derived and primary cell types, we harvested and purified mouse spinal motor neurons and compared them to motor neurons produced by transcription factor-mediated lineage conversion of fibroblasts or directed differentiation of pluripotent stem cells. To enable unbiased analysis of these motor neuron types and their cells of origin, we then subjected them to whole transcriptome and DNA methylome analysis by RNA sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS). Despite major differences in methodology, lineage conversion and directed differentiation both produce cells that closely approximate the primary motor neuron state. However, we identify differences in Fas signaling, the Hox code, and synaptic gene expression between lineage-converted and directed differentiation motor neurons that affect their utility in translational studies.

Cytosine methylation is widespread among organisms and essential for mammalian development. In line with early postulations of an epigenetic role in gene regulation, symmetric CpG methylation can be mitotically propagated over many generations with extraordinarily high fidelity. Here, we combine BrdU labeling and immunoprecipitation with genome-wide bisulfite sequencing to explore the inheritance of cytosine methylation onto newly replicated DNA in human cells. Globally, we observe a pronounced lag between the copying of genetic and epigenetic information in embryonic stem cells that is reconsolidated within hours to accomplish faithful mitotic transmission. Populations of arrested cells show a global reduction of lag-induced intermediate CpG methylation when compared to proliferating cells, whereas sites of transcription factor engagement appear cell-cycle invariant. Alternatively, the cancer cell line HCT116 preserves global epigenetic heterogeneity independently of cell-cycle arrest. Taken together, our data suggest that heterogeneous methylation largely reflects asynchronous proliferation, but is intrinsic to actively engaged cis-regulatory elements and cancer.

Metagenomic sequencing has the potential to transform microbial detection and characterization, but new tools are needed to improve its sensitivity. We developed CATCH (Compact Aggregation of Targets for Comprehensive Hybridization), a computational method to enhance nucleic acid capture for enrichment of diverse microbial taxa. CATCH designs compact probe sets that achieve full coverage of known sequence diversity and that scale well with this diversity. To illustrate applications of CATCH, we focused on capturing viral genomes. We designed, synthesized, and validated multiple probe sets, including one that targets whole genomes of the 356 viral species known to infect humans. Capture with these probe sets enriched unique viral content on average 18× and allowed us to assemble genomes that we could not otherwise recover, while accurately preserving within-sample diversity. We used this approach to recover genomes from the 2018 Lassa fever outbreak in Nigeria and to improve detection of viral infections in samples with unknown content. Together, this work demonstrates a path toward more sensitive, cost-effective metagenomic sequencing.

CTLA-4 immune checkpoint blockade is clinically effective in a subset of patients with metastatic melanoma. We identify a subcluster of MAGE-A cancer-germline antigens, located within a narrow 75 kb region of chromosome Xq28, that predicts resistance uniquely to blockade of CTLA-4, but not PD-1. We validate this gene expression signature in an independent anti-CTLA-4-treated cohort and show its specificity to the CTLA-4 pathway with two independent anti-PD-1-treated cohorts. Autophagy, a process critical for optimal anti-cancer immunity, has previously been shown to be suppressed by the MAGE-TRIM28 ubiquitin ligase in vitro. We now show that the expression of the key autophagosome component LC3B and other activators of autophagy are negatively associated with MAGE-A protein levels in human melanomas, including samples from patients with resistance to CTLA-4 blockade. Our findings implicate autophagy suppression in resistance to CTLA-4 blockade in melanoma, suggesting exploitation of autophagy induction for potential therapeutic synergy with CTLA-4 inhibitors.

Acquisition and storage of Latimeria chalumnae samples was supported by grants from the African Coelacanth Ecosystem Programme of the South African National Department of Science and Technology. Generation of the Latimeria chalumnae and Protopterus annectens sequences by the Broad Institute of the Massachusetts Institute of Technology (MIT) and Harvard University was supported by grants from the National Human Genome Research Institute (NHGRI). K.L.T. is the recipient of a EURYI award from the European Science Foundation. We would also like to thank the Genomics Sequencing Platform of the Broad Institute for sequencing the L. chalumnae genome and L. chalumnae and P. annectens transcriptomes, S. Ahamada, R. Stobbs and the Association pour le Protection de Gombesa (APG) for their help in obtaining coelacanth samples, Y. Zhao for the use of data from Rana chensinensis, and L. Gaffney, C. Hamilton and J. Westlund for assistance with figure preparation. 10; International audience; The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.

DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Here we inactivated all three catalytically active DNA methyltransferases (DNMTs) in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing to further investigate the roles and genomic targets of these enzymes. Disruption of DNMT3A or DNMT3B individually as well as of both enzymes in tandem results in viable, pluripotent cell lines with distinct effects on the DNA methylation landscape, as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to findings in mouse, deletion of DNMT1 resulted in rapid cell death in human ESCs. To overcome this immediate lethality, we generated a doxycycline-responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1-mutant lines. However, doxycycline-mediated repression of exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive cell death. Our data provide a comprehensive characterization of DNMT-mutant ESCs, including single-base genome-wide maps of the targets of these enzymes.

Notch-mediated expansion of cord blood progenitors: maintenance of transcriptional and epigenetic fidelity

Summary Pluripotent stem cells provide a powerful system to dissect the underlying molecular dynamics that regulate cell fate changes during mammalian development. Here we report the integrative analysis of genome wide binding data for 38 transcription factors with extensive epigenome and transcriptional data across the differentiation of human embryonic stem cells to the three germ layers. We describe core regulatory dynamics and show the lineage specific behavior of selected factors. In addition to the orchestrated remodeling of the chromatin landscape, we find that the binding of several transcription factors is strongly associated with specific loss of DNA methylation in one germ layer and in many cases a reciprocal gain in the other layers. Taken together, our work shows context-dependent rewiring of transcription factor binding, downstream signaling effectors, and the epigenome during human embryonic stem cell differentiation.

The 2013-2016 West African epidemic caused by the Ebola virus was of unprecedented magnitude, duration and impact. Here we reconstruct the dispersal, proliferation and decline of Ebola virus throughout the region by analysing 1,610 Ebola virus genomes, which represent over 5% of the known cases. We test the association of geography, climate and demography with viral movement among administrative regions, inferring a classic 'gravity' model, with intense dispersal between larger and closer populations. Despite attenuation of international dispersal after border closures, cross-border transmission had already sown the seeds for an international epidemic, rendering these measures ineffective at curbing the epidemic. We address why the epidemic did not spread into neighbouring countries, showing that these countries were susceptible to substantial outbreaks but at lower risk of introductions. Finally, we reveal that this large epidemic was a heterogeneous and spatially dissociated collection of transmission clusters of varying size, duration and connectivity. These insights will help to inform interventions in future epidemics. ispartof: Nature vol:544 issue:7650 pages:309- ispartof: location:England status: published

Zika virus (ZIKV) is currently causing an unprecedented pandemic linked to severe congenital syndromes1,2. In July 2016, mosquito-borne ZIKV transmission was first reported in the continental United States and since then, hundreds of locally acquired infections have been described3. To gain insights into the timing, source, and likely route(s) of introduction into the United States, we tracked the virus from its first detection in Miami, Florida by direct sequencing of ZIKV genomes from infected patients and Aedes aegypti mosquitoes. We detected at least four distinct ZIKV introductions and estimate that 11-52 introductions contributed to the outbreak in Florida. Furthermore, our data suggests that ZIKV transmission likely started in the spring of 2016 - several months before initial detection. By analyzing epidemiological, surveillance, and genetic data, we discovered that several spatially distinct ZIKV transmission zones were likely portions of the same outbreak, rather than isolated events. Our analyses show that most introductions are linked to the Caribbean, which is supported by the high incidence rates and traffic, especially via cruises, from the region into Miami. By comparing mosquito abundance and travel capacity across the United States, we find that southern Florida is especially vulnerable to ZIKV introductions and at risk of repeat occurrences. By tracking the virus from its initial introduction into the United States, we provide a deeper understanding of how ZIKV initiates and sustains transmission in new regions.

In mammals, the canonical somatic DNA methylation landscape is established upon specification of the embryo proper and subsequently disrupted within many cancer types1–4. However, the underlying mechanisms that direct this genome-scale transformation remain elusive, with no clear model for its systematic acquisition or potential developmental utility5,6. Here, we analysed global remethylation from the mouse preimplantation embryo into the early epiblast and extraembryonic ectoderm. We show that these two states acquire highly divergent genomic distributions with substantial disruption of bimodal, CpG density-dependent methylation in the placental progenitor7,8. The extraembryonic epigenome includes specific de novo methylation at hundreds of embryonically protected CpG island promoters, particularly those that are associated with key developmental regulators and are orthologously methylated across most human cancer types9. Our data suggest that the evolutionary innovation of extraembryonic tissues may have required co-option of DNA methylation-based suppression as an alternative to regulation by Polycomb-group proteins, which coordinate embryonic germ-layer formation in response to extraembryonic cues10. Moreover, we establish that this decision is made deterministically, downstream of promiscuously used—and frequently oncogenic—signalling pathways, via a novel combination of epigenetic cofactors. Methylation of developmental gene promoters during tumorigenesis may therefore reflect the misappropriation of an innate trajectory and the spontaneous reacquisition of a latent, developmentally encoded epigenetic landscape.