Modulation of enhancer looping and differential gene targeting by Epstein-Barr virus transcription factors directs cellular reprogramming

Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of enhancer-promoter interactions by viral transcription factors

Systematic search for enhancer elements and somatic allelic imbalance at seven low-penetrance colorectal cancer predisposition loci

Peer reviewe

Evaluation of Allele-Specific Somatic Changes of Genome-Wide Association Study Susceptibility Alleles in Human Colorectal Cancers

Tumors frequently exhibit loss of tumor suppressor genes or allelic gains of activated oncogenes. A significant proportion of cancer susceptibility loci in the mouse show somatic losses or gains consistent with the presence of a tumor susceptibility or resistance allele. Thus, allele-specific somatic gains or losses at loci may demarcate the presence of resistance or susceptibility alleles. The goal of this study was to determine if previously mapped susceptibility loci for colorectal cancer show evidence of allele-specific somatic events in colon tumors.We performed quantitative genotyping of 16 single nucleotide polymorphisms (SNPs) showing statistically significant association with colorectal cancer in published genome-wide association studies (GWAS). We genotyped 194 paired normal and colorectal tumor DNA samples and 296 paired validation samples to investigate these SNPs for allele-specific somatic gains and losses. We combined analysis of our data with published data for seven of these SNPs.No statistically significant evidence for allele-specific somatic selection was observed for the tested polymorphisms in the discovery set. The rs6983267 variant, which has shown preferential loss of the non-risk T allele and relative gain of the risk G allele in previous studies, favored relative gain of the G allele in the combined discovery and validation samples (corrected p-value = 0.03). When we combined our data with published allele-specific imbalance data for this SNP, the G allele of rs6983267 showed statistically significant evidence of relative retention (p-value = 2.06×10(-4)).Our results suggest that the majority of variants identified as colon cancer susceptibility alleles through GWAS do not exhibit somatic allele-specific imbalance in colon tumors. Our data confirm previously published results showing allele-specific imbalance for rs6983267. These results indicate that allele-specific imbalance of cancer susceptibility alleles may not be a common phenomenon in colon cancer

Association studies on 11 published colorectal cancer risk loci

Colorectal cancer (CRC) is the third most common cancer type in the Western world. Over one million patients are diagnosed worldwide yearly. A family history of CRC is a major risk factor for CRC. The total genetic contribution to disease development is estimated to be 35%. High-risk syndromes caused by known genes such as familial adenomatous polyposis (FAP) and Lynch Syndrome (LS) explain less than 5% of that number. Recently, several genome-wide association studies (GWAS) have independently found numerous loci at which common single-nucleotide polymorphisms (SNPs) modestly influence the risk of developing colorectal cancer. In total, germline mutations in known genes and moderate- and low risk variants are today suggested to explain 10-15% of the total genetic burden. Hence, predisposed genetic factor are still left to be found. The aim of paper I was to investigate if 11 published loci reported to be associated with an increased or decreased risk of colorectal cancer could be confirmed in a Swedish-based cohort. The cohort was composed of 1786 cases and 1749 controls that were genotyped and analyzed statistically. Genotype– phenotype analysis, for all 11 SNPs and sex, age of onset, family history of CRC and tumor location, was performed. Of 11 loci, 5 showed statistically significant odds ratios similar to previously published findings. Most of the remaining loci showed similar OR to previous publications. Four statistically significant genotype–phenotype associations were reported. The aim of paper II was to further study these 11 SNPs and their possible correlation with morphological features in tumors. We analyzed 15 histological features in 1572 CRC cases. Five SNPs showed statistically significant associations with morphological parameters. The parameters were poor differentiation, mucin production, decreased frequency of Crohn-like peritumoral reaction and desmoplastic response. The aim of paper III was to identify new CRC loci using a genome wide linkage analysis. We used 121 non-FAP/LS colorectal cancer families and genotyped 600 subjects using SNP array chips. No statistically significant result was found. However, suggestive linkage was found in the parametric analysis. This was observed in a recessive model for high-risk families, at locus 9q31.1 (HLOD=2.2) and for moderate-risk families, at locus Xp22.33 (LOD=2.2 and HLOD=2.5). Using families with early-onset, recessive analysis suggested one locus on 4p16.3 (LOD=2.2) and one on 17p13.2 (LOD/HLOD=2.0). Our linkage study adds support for the previously suggested region on chromosome 9 and suggests three additional loci to be involved in colorectal cancer risk. It is debated whether CRC is a single entity or two different entities, colon- and rectal cancer. Studies have recognized their molecular differences. The aim of paper IV was to identify novel colon- and rectal loci. We performed a genome wide linkage analysis using 32 colon- and 56 rectal cancer families. No LOD or HLOD score above three was observed. However, results close to three could be demonstrated. A maximum HLOD= 2.49 at locus 6p21.1-p12.1 and HLOD= 2.55 at locus 18p11.2 was observed for the colon- and rectal cancer families respectively. Exome sequencing was done, on colon and rectal patients, in these regions of interest. We report 25 variants mutated in family members on chromosome 6 and 27 variants on chromosome 18. Further studies are ongoing to elucidate the importance of these variants

Comprehensive evaluation of coding region point mutations in microsatellite-unstable colorectal cancer

Microsatellite instability (MSI) leads to accumulation of an excessive number of mutations in the genome, mostly small insertions and deletions. MSI colorectal cancers (CRCs), however, also contain more point mutations than microsatellite-stable (MSS) tumors, yet they have not been as comprehensively studied. To identify candidate driver genes affected by point mutations in MSI CRC, we ranked genes based on mutation significance while correcting for replication timing and gene expression utilizing an algorithm, MutSigCV. Somatic point mutation data from the exome kit-targeted area from 24 exome-sequenced sporadic MSI CRCs and respective normals, and 12 whole-genome-sequenced sporadic MSI CRCs and respective normals were utilized. The top 73 genes were validated in 93 additional MSI CRCs. The MutSigCV ranking identified several well-established MSI CRC driver genes and provided additional evidence for previously proposed CRC candidate genes as well as shortlisted genes that have to our knowledge not been linked to CRC before. Two genes, SMARCB1 and STK38L, were also functionally scrutinized, providing evidence of a tumorigenic role, for SMARCB1 mutations in particular. © 2018 The Authors. Published under the terms of the CC BY 4.0 licensePeer reviewe

A Novel Test for Gene-Ancestry Interactions in Genome-Wide Association Data

Genome-wide association study (GWAS) data on a disease are increasingly available from multiple related populations. In this scenario, meta-analyses can improve power to detect homogeneous genetic associations, but if there exist ancestry-specific effects, via interactions on genetic background or with a causal effect that co-varies with genetic background, then these will typically be obscured. To address this issue, we have developed a robust statistical method for detecting susceptibility gene-ancestry interactions in multi-cohort GWAS based on closely-related populations. We use the leading principal components of the empirical genotype matrix to cluster individuals into “ancestry groups” and then look for evidence of heterogeneous genetic associations with disease or other trait across these clusters. Robustness is improved when there are multiple cohorts, as the signal from true gene-ancestry interactions can then be distinguished from gene-collection artefacts by comparing the observed interaction effect sizes in collection groups relative to ancestry groups. When applied to colorectal cancer, we identified a missense polymorphism in iron-absorption gene CYBRD1 that associated with disease in individuals of English, but not Scottish, ancestry. The association replicated in two additional, independently-collected data sets. Our method can be used to detect associations between genetic variants and disease that have been obscured by population genetic heterogeneity. It can be readily extended to the identification of genetic interactions on other covariates such as measured environmental exposures. We envisage our methodology being of particular interest to researchers with existing GWAS data, as ancestry groups can be easily defined and thus tested for interactions

Germline Variation Controls the Architecture of Somatic Alterations in Tumors

Studies have suggested that somatic events in tumors can depend on an individual's constitutional genotype. We used squamous cell carcinomas (SCC) of the skin, which arise in high multiplicity in organ transplant recipients, as a model to compare the pattern of somatic alterations within and across individuals. Specifically, we performed array comparative genomic hybridization on 104 tumors from 25 unrelated individuals who each had three or more independently arisen SCCs and compared the profiles occurring within patients to profiles of tumors across a larger set of 135 patients. In general, chromosomal aberrations in SCCs were more similar within than across individuals (two-sided exact-test p-value ), consistent with the notion that the genetic background was affecting the pattern of somatic changes. To further test this possibility, we performed allele-specific imbalance studies using microsatellite markers mapping to 14 frequently aberrant regions of multiple independent tumors from 65 patients. We identified nine loci which show evidence of preferential allelic imbalance. One of these loci, 8q24, corresponded to a region in which multiple single nucleotide polymorphisms have been associated with increased cancer risk in genome-wide association studies (GWAS). We tested three implicated variants and identified one, rs13281615, with evidence of allele-specific imbalance (p-value = 0.012). The finding of an independently identified cancer susceptibility allele with allele-specific imbalance in a genomic region affected by recurrent DNA copy number changes suggest that it may also harbor risk alleles for SCC. Together these data provide strong evidence that the genetic background is a key driver of somatic events in cancer, opening an opportunity to expand this approach to identify cancer risk alleles

Multiple Wnt/ß-Catenin Responsive Enhancers Align with the MYC Promoter through Long-Range Chromatin Loops

Inappropriate activation of c-Myc (MYC) gene expression by the Wnt/ß-catenin signaling pathway is required for colorectal carcinogenesis. The elevated MYC levels in colon cancer cells are attributed in part to ß-catenin/TCF4 transcription complexes that are assembled at proximal Wnt/ß-catenin responsive enhancers (WREs). Recent studies suggest that additional WREs that control MYC expression reside far upstream of the MYC transcription start site. Here, I report the characterization of five novel WREs that localize to a region over 400 kb upstream from MYC. These WREs harbor nucleosomes with post-translational histone modifications that demarcate enhancer and gene promoter regions. Using quantitative chromatin conformation capture, I show that the distal WREs are aligned with the MYC promoter through large chromatin loops. The chromatin loops are not restricted to colon cancer cells, but are also found in kidney epithelial and lung fibroblast cell lines that lack de-regulated Wnt signaling and nuclear ß-catenin/TCF4 complexes. While each chromatin loop is detected in quiescent cells, the positioning of three of the five distal enhancers with the MYC promoter is induced by serum mitogens. These findings suggest that the architecture of the MYC promoter is comprised of distal elements that are juxtaposed through large chromatin loops and that ß-catenin/TCF4 complexes utilize this conformation to activate MYC expression in colon cancer cells

Identification of β-catenin binding regions in colon cancer cells using ChIP-Seq

Deregulation of the Wnt/β-catenin signaling pathway is a hallmark of colon cancer. Mutations in the adenomatous polyposis coli (APC) gene occur in the vast majority of colorectal cancers and are an initiating event in cellular transformation. Cells harboring mutant APC contain elevated levels of the β-catenin transcription coactivator in the nucleus which leads to abnormal expression of genes controlled by β-catenin/T-cell factor 4 (TCF4) complexes. Here, we use chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-Seq) to identify β-catenin binding regions in HCT116 human colon cancer cells. We localized 2168 β-catenin enriched regions using a concordance approach for integrating the output from multiple peak alignment algorithms. Motif discovery algorithms found a core TCF4 motif (T/A–T/A–C–A–A–A–G), an extended TCF4 motif (A/T/G–C/G–T/A–T/A–C–A–A–A–G) and an AP-1 motif (T–G–A–C/T–T–C–A) to be significantly represented in β-catenin enriched regions. Furthermore, 417 regions contained both TCF4 and AP-1 motifs. Genes associated with TCF4 and AP-1 motifs bound β-catenin, TCF4 and c-Jun in vivo and were activated by Wnt signaling and serum growth factors. Our work provides evidence that Wnt/β-catenin and mitogen signaling pathways intersect directly to regulate a defined set of target genes

Characterization of the association between 8q24 and colon cancer: gene-environment exploration and meta-analysis

<p>Abstract</p> <p>Background</p> <p>Genome-wide association studies and subsequent replication studies have shown that single nucleotide polymorphisms (SNPs) in the chromosomal region 8q24 are associated with colorectal cancer susceptibility.</p> <p>Methods</p> <p>We examined 11 SNP markers in the 8q24 region between 128.47 and 128.54 Mb, using a total of 1,987 colon cases and 2,339 controls who self-reported as white from two independent, well-characterized study populations. Analysis was performed separately within each study, and combined using random effects meta-analysis. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs) and to test for effect modification by known colon cancer risk factors. We also performed a meta-analysis combining our results with previous studies.</p> <p>Results</p> <p>We observed evidence of association for four SNPs in low to high linkage disequilibrium (r²ranging from 0.18 to 0.93) localized in a 16.2 kb region defined by rs10505477 and rs1056368. The combined results for our two studies of colon cancer showed an OR of 1.10 (95% CI: 1.01-1.20, P_trend= 0.023), and a meta-analysis of our results with previously reported studies of colon and colorectal cancer strongly support the association for this SNP (combined OR for rs6983267 = 1.21, 95% CI: 1.18-1.24, p = 5.5 × 10^-44). We did not observe any notable evidence of effect modification by known colon cancer risk factors, and risk did not differ significantly by tumor site or stage.</p> <p>Conclusions</p> <p>Our study confirms the association between polymorphisms on chromosome 8q24 and colon cancer risk and suggests that the susceptibility locus in region 8q24 is not strongly modified by various lifestyle, environmental, and demographic risk factors for colon cancer.</p