DNA methylation loss occurs frequently in cancer genomes, primarily within lamina-associated, late-replicating regions termed partially methylated domains (PMDs). We profiled 39 diverse primary ...tumors and 8 matched adjacent tissues using whole-genome bisulfite sequencing (WGBS) and analyzed them alongside 343 additional human and 206 mouse WGBS datasets. We identified a local CpG sequence context associated with preferential hypomethylation in PMDs. Analysis of CpGs in this context ('solo-WCGWs') identified previously undetected PMD hypomethylation in almost all healthy tissue types. PMD hypomethylation increased with age, beginning during fetal development, and appeared to track the accumulation of cell divisions. In cancer, PMD hypomethylation depth correlated with somatic mutation density and cell cycle gene expression, consistent with its reflection of mitotic history and suggesting its application as a mitotic clock. We propose that late replication leads to lifelong progressive methylation loss, which acts as a biomarker for cellular aging and which may contribute to oncogenesis.
Esophageal squamous cell carcinoma (ESCC) is among the most common malignancies, but little is known about its spatial intratumoral heterogeneity (ITH) and temporal clonal evolutionary processes. To ...address this, we performed multiregion whole-exome sequencing on 51 tumor regions from 13 ESCC cases and multiregion global methylation profiling for 3 of these 13 cases. We found an average of 35.8% heterogeneous somatic mutations with strong evidence of ITH. Half of the driver mutations located on the branches of tumor phylogenetic trees targeted oncogenes, including PIK3CA, NFE2L2 and MTOR, among others. By contrast, the majority of truncal and clonal driver mutations occurred in tumor-suppressor genes, including TP53, KMT2D and ZNF750, among others. Interestingly, phyloepigenetic trees robustly recapitulated the topological structures of the phylogenetic trees, indicating a possible relationship between genetic and epigenetic alterations. Our integrated investigations of spatial ITH and clonal evolution provide an important molecular foundation for enhanced understanding of tumorigenesis and progression in ESCC.
Cardiosphere-derived cells (CDCs) confer cardioprotection in acute myocardial infarction by distinctive macrophage (Mϕ) polarization. Here we demonstrate that CDC-secreted exosomes (CDC
) ...recapitulate the cardioprotective effects of CDC therapy known as cellular postconditioning.
Rats and pigs underwent myocardial infarction induced by ischemia/reperfusion before intracoronary infusion of CDC
, inert fibroblast exosomes (Fb
; control), or vehicle. Two days later, infarct size was quantified. Macrophages were isolated from cardiac tissue or bone marrow for downstream analyses. RNA sequencing was used to determine exosome content and alterations in gene expression profiles in Mϕ.
Administration of CDC
but not Fb
after reperfusion reduces infarct size in rat and pig models of myocardial infarction. Furthermore, CDC
reduce the number of CD68+ Mϕ within infarcted tissue and modify the polarization state of Mϕ so as to mimic that induced by CDCs. CDC
are enriched in several miRNAs (including miR-146a, miR-181b, and miR-126) relative to Fb
. Reverse pathway analysis of whole-transcriptome data from CDC
-primed Mϕ implicated miR-181b as a significant (
=1.3x10
) candidate mediator of CDC-induced Mϕ polarization, and PKCδ (protein kinase C δ) as a downstream target. Otherwise inert Fb
loaded selectively with miR-181b alter Mϕ phenotype and confer cardioprotective efficacy in a rat model of myocardial infarction. Adoptive transfer of PKCδ-suppressed Mϕ recapitulates cardioprotection.
Our data support the hypothesis that exosomal transfer of miR-181b from CDCs into Mϕ reduces PKCδ transcript levels and underlies the cardioprotective effects of CDCs administered after reperfusion.
DNA methylation and nucleosome positioning work together to generate chromatin structures that regulate gene expression. Nucleosomes are typically mapped using nuclease digestion requiring ...significant amounts of material and varying enzyme concentrations. We have developed a method (NOMe-seq) that uses a GpC methyltransferase (M.CviPI) and next generation sequencing to generate a high resolution footprint of nucleosome positioning genome-wide using less than 1 million cells while retaining endogenous DNA methylation information from the same DNA strand. Using a novel bioinformatics pipeline, we show a striking anti-correlation between nucleosome occupancy and DNA methylation at CTCF regions that is not present at promoters. We further show that the extent of nucleosome depletion at promoters is directly correlated to expression level and can accommodate multiple nucleosomes and provide genome-wide evidence that expressed non-CpG island promoters are nucleosome-depleted. Importantly, NOMe-seq obtains DNA methylation and nucleosome positioning information from the same DNA molecule, giving the first genome-wide DNA methylation and nucleosome positioning correlation at the single molecule, and thus, single cell level, that can be used to monitor disease progression and response to therapy.
The mesoderm- and epithelial-mesenchymal transition-associated transcription factor FOXC1 is specifically overexpressed in basal-like breast cancer (BLBC), but its biochemical function is not ...understood. Here, we demonstrate that FOXC1 controls cancer stem cell (CSC) properties enriched in BLBC cells via activation of Smoothened (SMO)-independent Hedgehog (Hh) signaling. This non-canonical activation of Hh is specifically mediated by Gli2. Furthermore, we show that the N-terminal domain of FOXC1 (aa 1–68) binds directly to an internal region (aa 898–1168) of Gli2, enhancing the DNA-binding and transcription-activating capacity of Gli2. FOXC1 expression correlates with that of Gli2 and its targets in human breast cancers. Moreover, FOXC1 overexpression reduces sensitivity to anti-Hedgehog (Hh) inhibitors in BLBC cells and xenograft tumors. Together, these findings reveal FOXC1-mediated non-canonical Hh signaling that determines the BLBC stem-like phenotype and anti-Hh sensitivity, supporting inhibition of FOXC1 pathways as potential approaches for improving BLBC treatment.
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•FOXC1 controls CSC properties in BLBC•SMO-independent Gli2 activity mediates the regulation of CSC properties by FOXC1•FOXC1 directly binds Gli2 and potentiates Gli2 DNA-binding activity•FOXC1 renders BLBC cells refractory to anti-Hh inhibitors
Han et al. find that FOXC1 binds directly to Gli2, activating SMO-independent hedgehog signaling and leading to the induction of cancer stem cell properties and anti-SMO drug resistance in basal-like breast cancer cells.
Abstract
The Oxford Nanopore (ONT) platform provides portable and rapid genome sequencing, and its ability to natively profile DNA methylation without complex sample processing is attractive for ...point-of-care real-time sequencing. We recently demonstrated ONT shallow whole-genome sequencing to detect copy number alterations (CNAs) from the circulating tumor DNA (ctDNA) of cancer patients. Here, we show that cell type and cancer-specific methylation changes can also be detected, as well as cancer-associated fragmentation signatures. This feasibility study suggests that ONT shallow WGS could be a powerful tool for liquid biopsy.
Graphical Abstract
DNA methylation has been used to identify functional changes at transcriptional enhancers and other cis-regulatory modules (CRMs) in tumors and other disease tissues. Our R/Bioconductor package ELMER ...(Enhancer Linking by Methylation/Expression Relationships) provides a systematic approach that reconstructs altered gene regulatory networks (GRNs) by combining enhancer methylation and gene expression data derived from the same sample set.
We present a completely revised version 2 of ELMER that provides numerous new features including an optional web-based interface and a new Supervised Analysis mode to use pre-defined sample groupings. We show that Supervised mode significantly increases statistical power and identifies additional GRNs and associated Master Regulators, such as SOX11 and KLF5 in Basal-like breast cancer.
ELMER v.2 is available as an R/Bioconductor package at http://bioconductor.org/packages/ELMER/.
Supplementary data are available at Bioinformatics online.
Colorectal cancer (CRC) is a leading cause of cancer-related deaths in the United States. Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with ...increased risk for CRC. A molecular understanding of the functional consequences of this genetic variation has been complicated because each GWAS SNP is a surrogate for hundreds of other SNPs, most of which are located in non-coding regions. Here we use genomic and epigenomic information to test the hypothesis that the GWAS SNPs and/or correlated SNPs are in elements that regulate gene expression, and identify 23 promoters and 28 enhancers. Using gene expression data from normal and tumour cells, we identify 66 putative target genes of the risk-associated enhancers (10 of which were also identified by promoter SNPs). Employing CRISPR nucleases, we delete one risk-associated enhancer and identify genes showing altered expression. We suggest that similar studies be performed to characterize all CRC risk-associated enhancers.
Understanding the intratumoral heterogeneity of hepatocellular carcinoma is instructive for developing personalized therapy and identifying molecular biomarkers. Here we applied whole-exome ...sequencing to 69 samples from 11 patients to resolve the genetic architecture of subclonal diversification. Spatial genomic diversity was found in all 11 hepatocellular carcinoma cases, with 29% of driver mutations being heterogeneous, including TERT, ARID1A, NOTCH2, and STAG2. Similar with other cancer types, TP53 mutations were always shared between all tumor regions, that is, located on the "trunk" of the evolutionary tree. In addition, we found that variants within several drug targets such as KIT, SYK, and PIK3CA were mutated in a fully clonal manner, indicating their therapeutic potentials for hepatocellular carcinoma. Temporal dissection of mutational signatures suggested that mutagenic processes associated with exposure to aristolochic acid and aflatoxin might play a more important role in early, as opposed to late, stages of hepatocellular carcinoma development. Moreover, we observed extensive intratumoral epigenetic heterogeneity in hepatocellular carcinoma based on multiple independent analytical methods and showed that intratumoral methylation heterogeneity might play important roles in the biology of hepatocellular carcinoma cells. Our results also demonstrated prominent heterogeneity of intratumoral methylation even in a stable hepatocellular carcinoma genome. Together, these findings highlight widespread intratumoral heterogeneity at both the genomic and epigenomic levels in hepatocellular carcinoma and provide an important molecular foundation for better understanding the pathogenesis of this malignancy.
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