No abstract

A comprehensive catalogue of the mutations that drive tumorigenesis and progression is essential to understanding tumor biology and developing therapies. Protein-coding driver mutations have been well-characterized by large exome-sequencing studies, however many tumors have no mutations in protein-coding driver genes. Non-coding mutations are thought to explain many of these cases, however few non-coding drivers besides TERT promoter are known. To fill this gap, we analyzed 150,000 cis-regulatory regions in 1,844 whole cancer genomes from the ICGC-TCGA PCAWG project. Using our new method, ActiveDriverWGS, we found 41 frequently mutated regulatory elements (FMREs) enriched in non-coding SNVs and indels (FDR<0.05) characterized by aging-associated mutation signatures and frequent structural variants. Most FMREs are distal from genes, reported here for the first time and also recovered by additional driver discovery methods. FMREs were enriched in super-enhancers, H3K27ac enhancer marks of primary tumors and long-range chromatin interactions, suggesting that the mutations drive cancer by distally controlling gene expression through threedimensional genome organization. In support of this hypothesis, the chromatin interaction network of FMREs and target genes revealed associations of mutations and differential gene expression of known and novel cancer genes (e.g., CNNB1IP1, RCC1), activation of immune response pathways and altered enhancer marks. Thus distal genomic regions may include additional, infrequently mutated drivers that act on target genes via chromatin loops. Our study is an important step towards finding such regulatory regions and deciphering the somatic mutation landscape of the non-coding genome..

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.

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 &gt;100-fold overexpression of PLAU specifically in megakaryocytes. This overexpression leads to &gt;100-fold increased platelet stores of urokinase plasminogen activator (PLAU/uPA), subsequent plasmin-mediated degradation of diverse a-granule proteins, and platelet-dependent, accelerated fibrinolysis. The causative mutation is a 78kb 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 QPD participants and unaffected controls. We show that the QPD duplication leads 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 subTAD genome architecture results in a dramatic, cell-type specific blood disorder phenotype.

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is an essential and evolutionarily conserved transcription factor complex primarily involved in innate immunity and inflammation. Transposable elements (TEs) can be co-opted to innovate immune transcriptional regulatory networks; however, the extent to which TEs have contributed to the modulation of NF-κB response in different mammalian lineages is not well established. Here we performed a multi-species analysis of TEs bound by the NF-κB subunit RELA (p65) in response to the pro-inflammatory cytokine TNFα (Tumor Necrosis Factor alpha). Using endothelial cell RELA ChIP-seq data from human, mouse and cow, we found that 55 TE subfamilies were enriched within NF-κB bound regions. These RELA-bound transposons possess multiple active epigenetic features and reside near TNFα-induced genes. A prominent example of lineage-specific contribution of transposons comes from the bovine SINE subfamilies Bov-tA1/2/3 which collectively contributed over 14,000 NF-κB bound regions in cow. By comparing NF-κB binding data across species, we found several examples of NF-κB motif-bearing TEs that appeared to colonize the genome prior to the divergence of the selected mammals, including a DNA transposon MER81, whose ancestral sequence contains two intact RELA motifs. We demonstrate that one NF-κB bound MER81 element can control the TNFα-induced expression of INFGR2 (Interferon Gamma Receptor 2) in human. Lastly, the presence of RELA motifs within MER81 elements appeared to stabilize during human evolution, indicative of purifying selection acting on a subset of these NF-κB bound ancient DNA transposons. Taken together, our results implicate multiple transposons in establishing NF-κB mediated regulatory networks during mammalian evolution.

No abstract

The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor plays a prominent role in inflammation and contributes to the development of atherosclerosis. Genome-wide DNA binding assays of the human NF-κB subunit RELA (p65) have revealed tens of thousands of NF-κB binding sites and hundreds of target genes. However, the function of individual RELA binding sites and the extent to which NF-κB occupancy and function is conserved across mammals are not well understood. To better understand the function of NF-κB we characterized the genome-wide binding of RELA in primary vascular endothelial cells (ECs) isolated from the aortas of human, mouse and cow. ECs were stimulated acutely with the pro-inflammatory cytokine tumor necrosis factor alpha (TNFA) and we profiled RELA occupancy, open chromatin, select histone modifications, and RNA expression. We found ~5000 RELA binding events conserved across all three species and these highly conserved human binding events were enriched for genes controlling vascular development, apoptosis, and pro-inflammatory responses. Approximately 2000 of these highly conserved RELA binding events were also shared across multiple human cell types, revealing a conserved core of robustly bound NF-κB sites. These NF-κB binding sites were also prominent components of ~40 inflammation-induced super-enhancers (SE) common to several tissues. To gain insight into the function of individual conserved NF-κB binding sites we focused on the inflammation-induced SE proximal to the monocyte recruiting chemokine CCL2 , which we detected as a SE in all three species and across multiple cell types. We tested the functional significance of six conserved RELA binding sites comprising this SE using CRISPR/Cas9 genome editing. We found that only deletion of the most proximal upstream RELA binding site could abolish the induction of CCL2 upon TNFA treatment. This site also contains a disease associated variant that can modulate CCL2 induction. Overall, our comparative genomics assessment of NF-κB binding gives new insight into NF-κB biology and the function of conserved transcription factor binding events within mammalian super-enhancers.