Candidate Cancer Driver Mutations in Distal Regulatory Elements and Long-Range Chromatin Interaction Networks.

A comprehensive catalog of cancer driver mutations is essential for understanding tumorigenesis and developing therapies. Exome-sequencing studies have mapped many protein-coding drivers, yet few non-coding drivers are known because genome-wide discovery is challenging. We developed a driver discovery method, ActiveDriverWGS, and analyzed 120,788 cis-regulatory modules (CRMs) across 1,844 whole tumor genomes from the ICGC-TCGA PCAWG project. We found 30 CRMs with enriched SNVs and indels (FDR < 0.05). These frequently mutated regulatory elements (FMREs) were ubiquitously active in human tissues, showed long-range chromatin interactions and mRNA abundance associations with target genes, and were enriched in motif-rewiring mutations and structural variants. Genomic deletion of one FMRE in human cells caused proliferative deficiencies and transcriptional deregulation of cancer genes CCNB1IP1, CDH1, and CDKN2B, validating observations in FMRE-mutated tumors. Pathway analysis revealed further sub-significant FMREs at cancer genes and processes, indicating an unexplored landscape of infrequent driver mutations in the non-coding genome.

Enhancer-gene rewiring in the pathogenesis of Quebec platelet disorder.

Quebec platelet disorder (QPD) is an autosomal dominant bleeding disorder with a unique, platelet-dependent, gain-of-function defect in fibrinolysis, without systemic fibrinolysis. The hallmark feature of QPD is a >100-fold overexpression of PLAU, specifically in megakaryocytes. This overexpression leads to a >100-fold increase in platelet stores of urokinase plasminogen activator (PLAU/uPA); subsequent plasmin-mediated degradation of diverse α-granule proteins; and platelet-dependent, accelerated fibrinolysis. The causative mutation is a 78-kb tandem duplication of PLAU. How this duplication causes megakaryocyte-specific PLAU overexpression is unknown. To investigate the mechanism that causes QPD, we used epigenomic profiling, comparative genomics, and chromatin conformation capture approaches to study PLAU regulation in cultured megakaryocytes from participants with QPD and unaffected controls. QPD duplication led to ectopic interactions between PLAU and a conserved megakaryocyte enhancer found within the same topologically associating domain (TAD). Our results support a unique disease mechanism whereby the reorganization of sub-TAD genome architecture results in a dramatic, cell-type-specific blood disorder phenotype.

CpG Methylation, a Parent-of-Origin Effect for Maternal-Biased Transmission of Congenital Myotonic Dystrophy.

CTG repeat expansions in DMPK cause myotonic dystrophy (DM1) with a continuum of severity and ages of onset. Congenital DM1 (CDM1), the most severe form, presents distinct clinical features, large expansions, and almost exclusive maternal transmission. The correlation between CDM1 and expansion size is not absolute, suggesting contributions of other factors. We determined CpG methylation flanking the CTG repeat in 79 blood samples from 20 CDM1-affected individuals; 21, 27, and 11 individuals with DM1 but not CDM1 (henceforth non-CDM1) with maternal, paternal, and unknown inheritance; and collections of maternally and paternally derived chorionic villus samples (7 CVSs) and human embryonic stem cells (4 hESCs). All but two CDM1-affected individuals showed high levels of methylation upstream and downstream of the repeat, greater than non-CDM1 individuals (p = 7.04958 × 10-12). Most non-CDM1 individuals were devoid of methylation, where one in six showed downstream methylation. Only two non-CDM1 individuals showed upstream methylation, and these were maternally derived childhood onset, suggesting a continuum of methylation with age of onset. Only maternally derived hESCs and CVSs showed upstream methylation. In contrast, paternally derived samples (27 blood samples, 3 CVSs, and 2 hESCs) never showed upstream methylation. CTG tract length did not strictly correlate with CDM1 or methylation. Thus, methylation patterns flanking the CTG repeat are stronger indicators of CDM1 than repeat size. Spermatogonia with upstream methylation may not survive due to methylation-induced reduced expression of the adjacent SIX5, thereby protecting DM1-affected fathers from having CDM1-affected children. Thus, DMPK methylation may account for the maternal bias for CDM1 transmission, larger maternal CTG expansions, age of onset, and clinical continuum, and may serve as a diagnostic indicator.

Regression analysis of combined gene expression regulation in acute myeloid leukemia.

Gene expression is a combinatorial function of genetic/epigenetic factors such as copy number variation (CNV), DNA methylation (DM), transcription factors (TF) occupancy, and microRNA (miRNA) post-transcriptional regulation. At the maturity of microarray/sequencing technologies, large amounts of data measuring the genome-wide signals of those factors became available from Encyclopedia of DNA Elements (ENCODE) and The Cancer Genome Atlas (TCGA). However, there is a lack of an integrative model to take full advantage of these rich yet heterogeneous data. To this end, we developed RACER (Regression Analysis of Combined Expression Regulation), which fits the mRNA expression as response using as explanatory variables, the TF data from ENCODE, and CNV, DM, miRNA expression signals from TCGA. Briefly, RACER first infers the sample-specific regulatory activities by TFs and miRNAs, which are then used as inputs to infer specific TF/miRNA-gene interactions. Such a two-stage regression framework circumvents a common difficulty in integrating ENCODE data measured in generic cell-line with the sample-specific TCGA measurements. As a case study, we integrated Acute Myeloid Leukemia (AML) data from TCGA and the related TF binding data measured in K562 from ENCODE. As a proof-of-concept, we first verified our model formalism by 10-fold cross-validation on predicting gene expression. We next evaluated RACER on recovering known regulatory interactions, and demonstrated its superior statistical power over existing methods in detecting known miRNA/TF targets. Additionally, we developed a feature selection procedure, which identified 18 regulators, whose activities clustered consistently with cytogenetic risk groups. One of the selected regulators is miR-548p, whose inferred targets were significantly enriched for leukemia-related pathway, implicating its novel role in AML pathogenesis. Moreover, survival analysis using the inferred activities identified C-Fos as a potential AML prognostic marker. Together, we provided a novel framework that successfully integrated the TCGA and ENCODE data in revealing AML-specific regulatory program at global level.

Epigenetic control of cellular crosstalk defines gastrointestinal organ fate and function.

Epithelial-mesenchymal signaling in the gastrointestinal system is vital in establishing regional identity during organogenesis and maintaining adult stem cell homeostasis. Although recent work has demonstrated that Wnt ligands expressed by mesenchymal cells are required during gastrointestinal development and stem cell homeostasis, epigenetic mechanisms driving spatiotemporal control of crosstalk remain unknown. Here, we demonstrate that gastrointestinal mesenchymal cells control epithelial fate and function through Polycomb Repressive Complex 2-mediated chromatin bivalency. We find that while key lineage-determining genes possess tissue-specific chromatin accessibility, Polycomb Repressive Complex 2 controls Wnt expression in mesenchymal cells without altering accessibility. We show that reduction of mesenchymal Wnt secretion rescues gastrointestinal fate and proliferation defects caused by Polycomb Repressive Complex 2 loss. We demonstrate that mesenchymal Polycomb Repressive Complex 2 also regulates niche signals to maintain stem cell function in the adult intestine. Our results highlight a broadly permissive chromatin architecture underlying regionalization in mesenchymal cells, then demonstrate further how chromatin architecture in niches can influence the fate and function of neighboring cells.

Loss of Epigenetic Regulation Disrupts Lineage Integrity, Induces Aberrant Alveogenesis, and Promotes Breast Cancer.

Systematically investigating the scores of genes mutated in cancer and discerning disease drivers from inconsequential bystanders is a prerequisite for precision medicine but remains challenging. Here, we developed a somatic CRISPR/Cas9 mutagenesis screen to study 215 recurrent "long-tail" breast cancer genes, which revealed epigenetic regulation as a major tumor-suppressive mechanism. We report that components of the BAP1 and COMPASS-like complexes, including KMT2C/D, KDM6A, BAP1, and ASXL1/2 ("EpiDrivers"), cooperate with PIK3CAH1047R to transform mouse and human breast epithelial cells. Mechanistically, we find that activation of PIK3CAH1047R and concomitant EpiDriver loss triggered an alveolar-like lineage conversion of basal mammary epithelial cells and accelerated formation of luminal-like tumors, suggesting a basal origin for luminal tumors. EpiDriver mutations are found in ∼39% of human breast cancers, and ∼50% of ductal carcinoma in situ express casein, suggesting that lineage infidelity and alveogenic mimicry may significantly contribute to early steps of breast cancer etiology. SIGNIFICANCE: Infrequently mutated genes comprise most of the mutational burden in breast tumors but are poorly understood. In vivo CRISPR screening identified functional tumor suppressors that converged on epigenetic regulation. Loss of epigenetic regulators accelerated tumorigenesis and revealed lineage infidelity and aberrant expression of alveogenesis genes as potential early events in tumorigenesis. This article is highlighted in the In This Issue feature, p. 2711.

A noncoding single-nucleotide polymorphism at 8q24 drives IDH1-mutant glioma formation.

Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, we focus on the single-nucleotide polymorphism rs55705857, which confers a sixfold greater risk of isocitrate dehydrogenase (IDH)-mutant low-grade glioma (LGG). We reveal that rs55705857 itself is the causal variant and is associated with molecular pathways that drive LGG. Mechanistically, we show that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. Mutating the orthologous mouse rs55705857 locus accelerated tumor development in an Idh1R132H-driven LGG mouse model from 472 to 172 days and increased penetrance from 30% to 75%. Our work reveals mechanisms of the heritable predisposition to lethal glioma in ~40% of LGG patients.

Maternal obesity persistently alters cardiac progenitor gene expression and programs adult-onset heart disease susceptibility.

OBJECTIVE: Heart disease risk can be programmed by intrauterine exposure to obesity. Dysregulating key transcription factors in cardiac progenitors can cause subsequent adult-onset heart disease. In this study, we investigated the transcriptional pathways that are altered in the embryonic heart and linked to heart disease risk in offspring exposed to obesity during pregnancy. METHODS: Female mice were fed an obesogenic diet and mated with males fed a control diet. Heart function and genome-wide gene expression were analyzed in adult offspring born to obese and lean mice at baseline and in response to stress. Cross-referencing with genes dysregulated genome-wide in cardiac progenitors from embryos of obese mice and human fetal hearts revealed the transcriptional events associated with adult-onset heart disease susceptibility. RESULTS: We found that adult mice born to obese mothers develop mild heart dysfunction consistent with early stages of disease. Accordingly, hearts of these mice dysregulated genes controlling extracellular matrix remodeling, metabolism, and TGF-ß signaling, known to control heart disease progression. These pathways were already dysregulated in cardiac progenitors in embryos of obese mice. Moreover, in response to cardiovascular stress, the heart of adults born to obese dams developed exacerbated myocardial remodeling and excessively activated regulators of cell-extracellular matrix interactions but failed to activate metabolic regulators. Expression of developmentally regulated genes was altered in cardiac progenitors of embryos of obese mice and human hearts of fetuses of obese donors. Accordingly, the levels of Nkx2-5, a key regulator of heart development, inversely correlated with maternal body weight in mice. Furthermore, Nkx2-5 target genes were dysregulated in cardiac progenitors and persistently in adult hearts born to obese mice and human hearts from pregnancies affected by obesity. CONCLUSIONS: Obesity during pregnancy alters Nkx2-5-controlled transcription in differentiating cardiac progenitors and persistently in the adult heart, making the adult heart vulnerable to dysregulated stress responses.

Conserved regulatory logic at accessible and inaccessible chromatin during the acute inflammatory response in mammals.

The regulatory elements controlling gene expression during acute inflammation are not fully elucidated. Here we report the identification of a set of NF-κB-bound elements and common chromatin landscapes underlying the acute inflammatory response across cell-types and mammalian species. Using primary vascular endothelial cells (human/mouse/bovine) treated with the pro-inflammatory cytokine, Tumor Necrosis Factor-α, we identify extensive (~30%) conserved orthologous binding of NF-κB to accessible, as well as nucleosome-occluded chromatin. Regions with the highest NF-κB occupancy pre-stimulation show dramatic increases in NF-κB binding and chromatin accessibility post-stimulation. These 'pre-bound' regions are typically conserved (~56%), contain multiple NF-κB motifs, are utilized by diverse cell types, and overlap rare non-coding mutations and common genetic variation associated with both inflammatory and cardiovascular phenotypes. Genetic ablation of conserved, 'pre-bound' NF-κB regions within the super-enhancer associated with the chemokine-encoding CCL2 gene and elsewhere supports the functional relevance of these elements.

Thrombin generation abnormalities in Quebec platelet disorder.

INTRODUCTION: Calibrated automated thrombograms (CAT) with platelet-poor (PPP) and platelet-rich plasma (PRP) have provided useful insights on bleeding disorders. We used CAT to assess thrombin generation (TG) in Quebec platelet disorder (QPD)-a bleeding disorder caused by a PLAU duplication mutation that increases platelet (but not plasma) urokinase plasminogen activator (uPA), leading to intraplatelet (but not systemic) plasmin generation that degrades α-granule proteins and causes platelet (but not plasma) factor V (FV) deficiency. METHODS: Calibrated automated thrombograms was used to test QPD (n = 7) and control (n = 22) PPP and PRP, with or without added tranexamic acid (TXA). TG endpoints were evaluated for relationships to platelet FV and uPA, plasma FV and tissue factor pathway inhibitor (TFPI) levels, and bleeding scores. RESULTS: Quebec platelet disorder PPP TG was normal whereas QPD PRP had reduced endogenous thrombin potential and peak thrombin concentrations (P values < .01), proportionate to the platelet FV deficiency (R2  ≥ 0.81), but unrelated to platelet uPA, plasma FV, or bleeding scores. QPD TG abnormalities were not associated with TFPI abnormalities and were not reproduced by adding uPA to control PRP. TXA increased QPD and control PRP TG more than PPP TG, but it did not fully correct QPD PRP TG abnormalities or improve TG by plasminogen-deficient plasma. CONCLUSION: Quebec platelet disorder results in a platelet-specific TG defect, proportionate to the loss of platelet FV, that is improved but not fully corrected by TXA. Our study provides an interesting example of why it is important to assess both PRP and PPP TG in bleeding disorders.

Sufu- and Spop-mediated downregulation of Hedgehog signaling promotes beta cell differentiation through organ-specific niche signals.

Human embryonic stem cell-derived beta cells offer a promising cell-based therapy for diabetes. However, efficient stem cell to beta cell differentiation has proven difficult, possibly due to the lack of cross-talk with the appropriate mesenchymal niche. To define organ-specific niche signals, we isolated pancreatic and gastrointestinal stromal cells, and analyzed their gene expression during development. Our genetic studies reveal the importance of tightly regulated Hedgehog signaling in the pancreatic mesenchyme: inactivation of mesenchymal signaling leads to annular pancreas, whereas stroma-specific activation of signaling via loss of Hedgehog regulators, Sufu and Spop, impairs pancreatic growth and beta cell genesis. Genetic rescue and transcriptome analyses show that these Sufu and Spop knockout defects occur through Gli2-mediated activation of gastrointestinal stromal signals such as Wnt ligands. Importantly, inhibition of Wnt signaling in organoid and human stem cell cultures significantly promotes insulin-producing cell generation, altogether revealing the requirement for organ-specific regulation of stromal niche signals.

The duplication mutation of Quebec platelet disorder dysregulates PLAU, but not C10orf55, selectively increasing production of normal PLAU transcripts by megakaryocytes but not granulocytes.

Quebec Platelet disorder (QPD) is a unique bleeding disorder that markedly increases urokinase plasminogen activator (uPA) in megakaryocytes and platelets but not in plasma or urine. The cause is tandem duplication of a 78 kb region of chromosome 10 containing PLAU (the uPA gene) and C10orf55, a gene of unknown function. QPD increases uPA in platelets and megakaryocytes >100 fold, far more than expected for a gene duplication. To investigate the tissue-specific effect that PLAU duplication has on gene expression and transcript structure in QPD, we tested if QPD leads to: 1) overexpression of normal or unique PLAU transcripts; 2) increased uPA in leukocytes; 3) altered levels of C10orf55 mRNA and/or protein in megakaryocytes and leukocytes; and 4) global changes in megakaryocyte gene expression. Primary cells and cultured megakaryocytes from donors were prepared for quantitative reverse polymerase chain reaction analyses, RNA-seq and protein expression analyses. Rapidly isolated blood leukocytes from QPD subjects showed only a 3.9 fold increase in PLAU transcript levels, in keeping with the normal to minimally increased uPA in affinity purified, QPD leukocytes. All subjects had more uPA in granulocytes than monocytes and minimal uPA in lymphocytes. QPD leukocytes expressed PLAU alleles in proportions consistent with an extra copy of PLAU on the disease chromosome, unlike QPD megakaryocytes. QPD PLAU transcripts were consistent with reference gene models, with a much higher proportion of reads originating from the disease chromosome in megakaryocytes than granulocytes. QPD and control megakaryocytes contained minimal reads for C10orf55, and C10orf55 protein was not increased in QPD megakaryocytes or platelets. Finally, our QPD megakaryocyte transcriptome analysis revealed a global down regulation of the interferon type 1 pathway. We suggest that the low endogenous levels of uPA in blood are actively regulated, and that the regulatory mechanisms are disrupted in QPD in a megakaryocyte-specific manner.

RNAseq analysis for the diagnosis of muscular dystrophy.

The precise genetic cause remains elusive in nearly 50% of patients with presumed neurogenetic disease, representing a significant barrier for clinical care. This is despite significant advances in clinical genetic diagnostics, including the application of whole-exome sequencing and next-generation sequencing-based gene panels. In this study, we identify a deep intronic mutation in the DMD gene in a patient with muscular dystrophy using both conventional and RNAseq-based transcriptome analyses. The implications of our data are that noncoding mutations likely comprise an important source of unresolved genetic disease and that RNAseq is a powerful platform for detecting such mutations.

Topoisomerase II beta interacts with cohesin and CTCF at topological domain borders.

BACKGROUND: Type II DNA topoisomerases (TOP2) regulate DNA topology by generating transient double stranded breaks during replication and transcription. Topoisomerase II beta (TOP2B) facilitates rapid gene expression and functions at the later stages of development and differentiation. To gain new insight into the genome biology of TOP2B, we used proteomics (BioID), chromatin immunoprecipitation, and high-throughput chromosome conformation capture (Hi-C) to identify novel proximal TOP2B protein interactions and characterize the genomic landscape of TOP2B binding at base pair resolution. RESULTS: Our human TOP2B proximal protein interaction network included members of the cohesin complex and nucleolar proteins associated with rDNA biology. TOP2B associates with DNase I hypersensitivity sites, allele-specific transcription factor (TF) binding, and evolutionarily conserved TF binding sites on the mouse genome. Approximately half of all CTCF/cohesion-bound regions coincided with TOP2B binding. Base pair resolution ChIP-exo mapping of TOP2B, CTCF, and cohesin sites revealed a striking structural ordering of these proteins along the genome relative to the CTCF motif. These ordered TOP2B-CTCF-cohesin sites flank the boundaries of topologically associating domains (TADs) with TOP2B positioned externally and cohesin internally to the domain loop. CONCLUSIONS: TOP2B is positioned to solve topological problems at diverse cis-regulatory elements and its occupancy is a highly ordered and prevalent feature of CTCF/cohesin binding sites that flank TADs.