Multiple regulatory elements distant from their targets on the linear genome can influence the expression of a single gene through chromatin looping. Chromosome conformation capture implemented in ...Hi-C allows for genome-wide agnostic characterization of chromatin contacts. However, detection of functional enhancer-promoter interactions is precluded by its effective resolution that is determined by both restriction fragmentation and sensitivity of the experiment. Here we develop a capture Hi-C (cHi-C) approach to allow an agnostic characterization of these physical interactions on a genome-wide scale. Single-nucleotide polymorphisms associated with complex diseases often reside within regulatory elements and exert effects through long-range regulation of gene expression. Applying this cHi-C approach to 14 colorectal cancer risk loci allows us to identify key long-range chromatin interactions in cis and trans involving these loci.
Approximately 15% of colorectal cancers exhibit microsatellite instability (MSI), which leads to accumulation of large numbers of small insertions and deletions (indels). Genes that provide growth ...advantage to cells via loss-of-function mutations in microsatellites are called MSI target genes. Several criteria to define these genes have been suggested, one of them being simple mutation frequency. Microsatellite mutation rate, however, depends on the length and nucleotide context of the microsatellite. Therefore, assessing the general impact of mismatch repair deficiency on the likelihood of mutation events is paramount when following this approach. To identify MSI target genes, we developed a statistical model for the somatic background indel mutation rate of microsatellites to assess mutation significance. Exome sequencing data of 24 MSI colorectal cancers revealed indels at 54 million mononucleotide microsatellites of three or more nucleotides in length. The top 105 microsatellites from 71 genes were further analyzed in 93 additional MSI colorectal cancers. Mutation significance and estimated clonality of mutations determined the most likely MSI target genes to be the aminoadipate-semialdehyde dehydrogenase
and the solute transporter
Our findings offer a systematic profiling of the somatic background mutation rate in protein-coding mononucleotide microsatellites, allowing a full cataloging of the true targets of MSI in colorectal cancer.
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Background The nuclear factor κ light-chain enhancer of activated B cells (NF-κB) signaling pathway is a key regulator of immune responses. Accordingly, mutations in several NF-κB pathway genes cause ...immunodeficiency. Objective We sought to identify the cause of disease in 3 unrelated Finnish kindreds with variable symptoms of immunodeficiency and autoinflammation. Methods We applied genetic linkage analysis and next-generation sequencing and functional analyses of NFKB1 and its mutated alleles. Results In all affected subjects we detected novel heterozygous variants in NFKB1 , encoding for p50/p105. Symptoms in variant carriers differed depending on the mutation. Patients harboring a p.I553M variant presented with antibody deficiency, infection susceptibility, and multiorgan autoimmunity. Patients with a p.H67R substitution had antibody deficiency and experienced autoinflammatory episodes, including aphthae, gastrointestinal disease, febrile attacks, and small-vessel vasculitis characteristic of Behçet disease. Patients with a p.R157X stop-gain experienced hyperinflammatory responses to surgery and showed enhanced inflammasome activation. In functional analyses the p.R157X variant caused proteasome-dependent degradation of both the truncated and wild-type proteins, leading to a dramatic loss of p50/p105. The p.H67R variant reduced nuclear entry of p50 and showed decreased transcriptional activity in luciferase reporter assays. The p.I553M mutation in turn showed no change in p50 function but exhibited reduced p105 phosphorylation and stability. Affinity purification mass spectrometry also demonstrated that both missense variants led to altered protein-protein interactions. Conclusion Our findings broaden the scope of phenotypes caused by mutations in NFKB1 and suggest that a subset of autoinflammatory diseases, such as Behçet disease, can be caused by rare monogenic variants in genes of the NF-κB pathway.
Efforts are being directed to systematically analyze the non-coding regions of the genome for cancer-driving mutations
. cis-regulatory elements (CREs) represent a highly enriched subset of the ...non-coding regions of the genome in which to search for such mutations. Here we use high-throughput chromosome conformation capture techniques (Hi-C) for 19,023 promoter fragments to catalog the regulatory landscape of colorectal cancer in cell lines, mapping CREs and integrating these with whole-genome sequence and expression data from The Cancer Genome Atlas
. We identify a recurrently mutated CRE interacting with the ETV1 promoter affecting gene expression. ETV1 expression influences cell viability and is associated with patient survival. We further refine our understanding of the regulatory effects of copy-number variations, showing that RASL11A is targeted by a previously identified enhancer amplification
. This study reveals new insights into the complex genetic alterations driving tumor development, providing a paradigm for employing chromosome conformation capture to decipher non-coding CREs relevant to cancer biology.
The gene desert upstream of the
oncogene on chromosome 8q24 contains susceptibility loci for several major forms of human cancer. The region shows high conservation between human and mouse and ...contains multiple
enhancers that are activated in tumor cells. However, the role of this region in normal development has not been addressed. Here we show that a 538 kb deletion of the entire
upstream super-enhancer region in mice results in 50% to 80% decrease in
expression in multiple tissues. The mice are viable and show no overt phenotype. However, they are resistant to tumorigenesis, and most normal cells isolated from them grow slowly in culture. These results reveal that only cells whose MYC activity is increased by serum or oncogenic driver mutations depend on the 8q24 super-enhancer region, and indicate that targeting the activity of this element is a promising strategy of cancer chemoprevention and therapy.
Mechanical forces in a constrained cellular environment were recently established as a facilitator of chromosomal damage. Whether this could contribute to tumorigenesis is not known. Uterine ...leiomyomas are common neoplasms that display relatively few chromosomal aberrations. We hypothesized that if mechanical forces contribute to chromosomal damage, signs of this could be seen in uterine leiomyomas from parous women. We examined the karyotypes of 1946 tumors, and found a striking overrepresentation of chromosomal damage associated with parity. We then subjected myometrial cells to physiological forces similar to those encountered during pregnancy, and found this to cause DNA breaks and a DNA repair response. While mechanical forces acting in constrained cellular environments may thus contribute to neoplastic degeneration, and genesis of uterine leiomyoma, further studies are needed to prove possible causality of the observed association. No evidence for progression to malignancy was found.
The growth of human cancer cells is driven by aberrant enhancer and gene transcription activity. Here, we use transient transcriptome sequencing (TT‐seq) to map thousands of transcriptionally active ...putative enhancers in fourteen human cancer cell lines covering seven types of cancer. These enhancers were associated with cell type‐specific gene expression, enriched for genetic variants that predispose to cancer, and included functionally verified enhancers. Enhancer–promoter (E–P) pairing by correlation of transcription activity revealed ~ 40,000 putative E–P pairs, which were depleted for housekeeping genes and enriched for transcription factors, cancer‐associated genes, and 3D conformational proximity. The cell type specificity and transcription activity of target genes increased with the number of paired putative enhancers. Our results represent a rich resource for future studies of gene regulation by enhancers and their role in driving cancerous cell growth.
SYNOPSIS
TT‐seq measures genome‐wide enhancer transcription with high sensitivity in human cancer cells. The resulting comprehensive annotation of putative enhancers and enhancer‐promoter pairs serves as a valuable resource for studying enhancer function.
TT‐seq maps thousands of transcriptionally active, putative enhancers in fourteen human cancer cell lines covering seven types of cancer.
Enhancer‐promoter (E–P) pairing by correlation of transcription activity reveals ~ 40,000 putative E–P pairs.
A catalog comprising candidate enhancers with cancer‐associated somatic mutations and putative E–P pairs involving cancer‐associated genes is generated.
This resource serves as a valuable tool to study gene regulation by enhancers.
TT‐seq measures genome‐wide enhancer transcription with high sensitivity in human cancer cells. The resulting comprehensive annotation of putative enhancers and enhancer‐promoter pairs serves as a valuable resource for studying enhancer function.
Genome-wide association studies have identified thousands of SNPs associated with predisposition to various diseases, including prostate cancer. However, the mechanistic roles of these SNPs remain ...poorly defined, particularly for noncoding polymorphisms. Here we find that the prostate cancer risk-associated SNP rs339331 at 6q22 lies within a functional HOXB13-binding site. The risk-associated T allele at rs339331 increases binding of HOXB13 to a transcriptional enhancer, conferring allele-specific upregulation of the rs339331-associated gene RFX6. Suppression of RFX6 diminishes prostate cancer cell proliferation, migration and invasion. Clinical data indicate that RFX6 upregulation in human prostate cancers correlates with tumor progression, metastasis and risk of biochemical relapse. Finally, we observe a significant association between the risk-associated T allele at rs339331 and increased RFX6 mRNA levels in human prostate tumors. Together, our results suggest that rs339331 affects prostate cancer risk by altering RFX6 expression through a functional interaction with the prostate cancer susceptibility gene HOXB13.
Most transcription factors (TFs) can bind to a population of sequences closely related to a single optimal site. However, some TFs can bind to two distinct sequences that represent two local optima ...in the Gibbs free energy of binding (ΔG). To determine the molecular mechanism behind this effect, we solved the structures of human HOXB13 and CDX2 bound to their two optimal DNA sequences, CAATAAA and TCGTAAA. Thermodynamic analyses by isothermal titration calorimetry revealed that both sites were bound with similar ΔG. However, the interaction with the CAA sequence was driven by change in enthalpy (ΔH), whereas the TCG site was bound with similar affinity due to smaller loss of entropy (ΔS). This thermodynamic mechanism that leads to at least two local optima likely affects many macromolecular interactions, as ΔG depends on two partially independent variables ΔH and ΔS according to the central equation of thermodynamics, ΔG = ΔH - TΔS.
Cancer is the most complex genetic disease known, with mutations implicated in more than 250 genes. However, it is still elusive which specific mutations found in human patients lead to ...tumorigenesis. Here we show that a combination of oncogenes that is characteristic of liver cancer (CTNNB1, TERT, MYC) induces senescence in human fibroblasts and primary hepatocytes. However, reprogramming fibroblasts to a liver progenitor fate, induced hepatocytes (iHeps), makes them sensitive to transformation by the same oncogenes. The transformed iHeps are highly proliferative, tumorigenic in nude mice, and bear gene expression signatures of liver cancer. These results show that tumorigenesis is triggered by a combination of three elements: the set of driver mutations, the cellular lineage, and the state of differentiation of the cells along the lineage. Our results provide direct support for the role of cell identity as a key determinant in transformation and establish a paradigm for studying the dynamic role of oncogenic drivers in human tumorigenesis.
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