Tissue-specific Super-enhancers Research Articles

Genetic Predisposition to Colon and Rectal Adenocarcinoma Is Mediated by a Super-enhancer Polymorphism Coactivating CD9 and PLEKHG6.

Genome-wide association studies (GWAS) have identified dozens of loci associated with colon and rectal adenocarcinoma risk. As tissue-specific super-enhancers (SE) play important roles in tumorigenesis, we systematically investigate SEs and inner variants in established GWAS loci to decipher the underlying biological mechanisms. Through a comprehensive bioinformatics analysis on multi-omics data, we screen potential single-nucleotide polymorphisms (SNP) in cancer-specific SEs, and then subject them to a two-stage case-control study containing 4,929 cases and 7,083 controls from the Chinese population. A series of functional assays, including reporter gene assays, electrophoretic mobility shift assays (EMSA), CRISPR-Cas9 genome editing, chromosome conformation capture (3C) assays, and cell proliferation experiments, are performed to characterize the variant's molecular consequence and target genes. The SNP rs11064124 in 12p13.31 is found significantly associated with the risk of colon and rectal adenocarcinoma with an odds ratio (OR) of 0.87 [95% confidence interval (CI), 0.82-0.92, P = 8.67E-06]. The protective rs11064124-G weakens the binding affinity with vitamin D receptor (VDR) and increases the enhancer's activity and interactions with two target genes' promoters, thus coactivating the transcription of CD9 and PLEKHG6, which are both putative tumor suppressor genes for colon and rectal adenocarcinoma. Our integrative study highlights an SE polymorphism rs11064124 and two susceptibility genes CD9 and PLEKHG6 in 12p13.31 for colon and rectal adenocarcinoma. These findings suggest a novel insight for genetic pathogenesis of colon and rectal adenocarcinoma, involving transcriptional coactivation of diverse susceptibility genes via the SE element as a gene regulation hub.

Abstract 5211: Super enhancer-associated master transcription factor SOX4 suppresses anti-tumor immunity in NAFLD-associated hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) has become a prominent global health threat due to its occurrence and lethality. Increasing prevalence of non-alcoholic fatty liver disease (NAFLD) have been found culpable for HCC initiation, via disruption of the liver microenvironment. Super enhancers (SEs) and master transcription factors (TFs) translate microenvironmental changes into chromatin remodelling and activation, which subsequently disrupt the transcriptome profile and initiate oncogenesis. This project aims at profiling the SE status and unveiling the master regulators involved in diet-induced HCC progression. Nanoscale chromatin immunoprecipitation sequencing (nano ChIP-seq) against histone mark H3K27ac in 10 pairs of primary human NAFLD-HCC tumors and adjacent non-tumor tissues revealed potential tissue-specific SEs (averaged 541 and 512 per HCC tumor and non-tumor tissues, respectively). Global mRNA profile was detected by RNA sequencing (RNA-seq) to support the enhancer-target gene transcription axes in the enriched metabolic and immune response pathways. A non-alcoholic steatohepatitis-induced HCC (NASH-HCC) mouse model was established by subcutaneous injection of a diabetogenic agent streptozotocin (STZ) (200 μg) two days after birth followed by a 19-week high-fat high-carbohydrate (HFHC) diet from the age of 4 weeks. Another carcinogen diethylnitrosamine-induced HCC (DEN-HCC) mouse model was established by intraperitoneal injection of DEN (25 mg/kg) followed by a 22-week HFHC diet from the age of 6 weeks. Tumor-enabling SEs were profiled in primary human NAFLD-HCC tissues and a network of master TFs were identified using integrated bioinformatic analysis. Intriguingly, tumor-enabling SEs target critical genes involved in PDGFB signalling and NAFLD pathogenesis, which are significantly correlated with high master TF SOX4 expression. Notable overexpression of Sox4 and Pdgfb was also observed in liver tumors in two HCC mouse models, significantly correlating with the CD11b+ CD11c+ IL10+ IDO+ tolerogenic dendritic cell population. Integrated analysis of ChIP-seq and RNA-seq in primary human tissues revealed a super enhancer-associated master regulatory network in NAFLD-HCC development. Our findings suggest that epigenetic regulation of the SOX4 and PDGFB may contribute to an immunosuppressive liver tumor environment, providing insights for novel therapeutic strategy against this rapidly-increasing dreadful disease. This project is supported by Collaborative Research Fund C4017-14G and the Focused Innovations Scheme 1907309. Citation Format: Ka Wing Cheung, Feng Wu, Wenshu Tang, Weiqin Yang, Jingying Zhou, Patrick Tan, Yuk Lap Kevin Yip, Ka Fai To, Sze Lok Alfred Cheng. Super enhancer-associated master transcription factor SOX4 suppresses anti-tumor immunity in NAFLD-associated hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5211.

Dissecting Tissue-Specific Super-Enhancers by Integrating Genome-Wide Analyses and CRISPR/Cas9 Genome Editing.

Recent advances in genome-wide sequencing technologies have provided researchers with unprecedented opportunities to discover the genomic structures of gene regulatory units in living organisms. In particular, the integration of ChIP-seq, RNA-seq, and DNase-seq techniques has facilitated the mapping of a new class of regulatory elements. These elements, called super-enhancers, can regulate cell-type-specific gene sets and even fine-tune gene expression regulation in response to external stimuli, and have become a hot topic in genome biology. However, there is scant genetic evidence demonstrating their unique biological relevance and the mechanisms underlying these biological functions. In this review, we describe a robust genome-wide strategy for mapping cell-type-specific enhancers or super-enhancers in the mammary genome. In this strategy, genome-wide screening of active enhancer clusters that are co-occupied by mammary-enriched transcription factors, co-factors, and active enhancer marks is used to identify bona fide mammary tissue-specific super-enhancers. The in vivo function of these super-enhancers and their associated regulatory elements may then be investigated in various ways using the advanced CRISPR/Cas9 genome-editing technology. Based on our experience targeting various mammary genomic sites using CRISPR/Cas9 in mice, we comprehensively discuss the molecular consequences of the different targeting methods, such as the number of gRNAs and the dependence on their simultaneous or sequential injections. We also mention the considerations that are essential for obtaining accurate results and shed light on recent progress that has been made in developing modified CRISPR/Cas9 genome-editing techniques. In the future, the coupling of advanced genome-wide sequencing and genome-editing technologies could provide new insights into the complex genetic regulatory networks involved in mammary-gland development.

Abstract 5381: Understanding the genesis and oncogenic consequences of tandem duplicator phenotypes in human cancers

Abstract Genome-scale instability configurations in cancer genomes are commonly found across different types of cancers. We and others have described an enrichment of head-to-tail somatic segmental tandem duplications (TDs) in primarily breast and ovarian cancers, a configuration known as the Tandem Duplicator Phenotype (TDP). Here, we perform a meta-analysis of 2,717 human cancer genomes, representing 30 different tumor types and refine the TDP classification based on the identification of highly specific and recurrent TD span profiles. Overall, we identify the TDP as a highly recurrent chromosomal instability configuration, occurring in 14% of the cancer genomes examined. The TDP is remarkably prevalent in triple negative breast cancer (50%), ovarian cancer (55%), and uterine cancers (45%), but is infrequent in most other cancer types. In TDP tumors, TD span sizes can be classified into three major discrete intervals with modal values of 10 Kb, 230 Kb, and 1.7 Mb. TDPs with a dominant TD modal span size of 10 Kb are strongly associated with conjoint TP53 mutations and BRCA1 deficiency across different tumor types. TDPs with larger span sizes (230 Kb and 1.7 Mb) are associated with CCNE1 pathway activation and CRKRS/CDK12 gene disruptions. We proved the driver role of the conjoint abrogation of the BRCA1 and TP53 genes for the induction of TDP cancers with the 10 Kb predominant span peak by generating mammary cancers with a Tandem Duplicator Phenotype of the ~10 Kb TD span configuration type in genetically modified mouse models harboring deleterious mutations in only these same two genes. Lastly, we describe how heterogeneous combinations of tumor suppressors and chromatin topologically associating domains are disrupted, and oncogene and tissue-specific super enhancers are duplicated as a consequence of TD formation in TDP tumors. In summary, our work unifies a number of observations around a specific cancer genomic signature, shows how a number of genetic drivers converge on creating the tandem duplicator phenotype, and how the downstream consequences of the TDs generate oncogenic diversity that allows for further formation of genetic subgroups. The implications of TDP-based stratification for therapy response are currently under investigation. Citation Format: Francesca Menghi, Floris Barthel, Vinod Yadav, Ming Tang, Bo Ji, Gregory Carter, Jos Jonkers, Roel Verhaak, Ralph Scully, Ed Liu. Understanding the genesis and oncogenic consequences of tandem duplicator phenotypes in human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5381.

Abstract CN05-01: DNA damage signatures and practical use

Abstract A cancer genome is a historical account of the mutagenic activity that has occurred throughout the development of the tumor. Indeed, every mutation matters. While driver mutations were the main focus of cancer research for a long time, passenger mutational signatures, the imprints of DNA damage and DNA repair processes that have been operative during tumorigenesis, are also biologically informative (1-5). We previously outlined the methods for identifying and quantifying base substitution mutational signatures present in primary human cancers (1-5). Recently, the intellectual framework of mutational signatures was extended to include six novel rearrangement signatures (6). Exploring this in >2,500 whole genome-sequenced tumors of multiple tumor types reveals how our early (and rather simplistic) thinking of mutational signatures requires critical reevaluation. There are more nuances than previously appreciated. Diving into the detail of individual mutational signatures (7), we reveal intriguing mechanistic insights into the DNA damage and repair processes that mark the landscape of cancer genomes. In some instances, our findings invite more thoughtful consideration of that relatively binary distinction between drivers and passengers. Furthermore, we demonstrate why our whole-genome profiling methods could assist in taking things to a clinical level (6-8), with mutational signatures forming an additional weapon in the arsenal of cancer diagnostics and therapeutic stratification, in the modern war against cancer.

Corrigendum: A somatic-mutational process recurrently duplicates germline susceptibility loci and tissue-specific super-enhancers in breast cancers.

This corrects the article DOI: 10.1038/ng.3771.

Abstract LB-262: A somatic-mutational process in breast cancer genomes recurrently duplicates germline susceptibility loci and tissue-specific super-enhancers

Abstract Somatic rearrangements are a part of mutational landscape of human cancer genomes. We systematically investigated the distribution of the somatic rearrangements in 560 breast cancer genomes and highlight 33 rearrangement hotspots associated with a signature of Homologous Recombinational repair deficiency, characterized mainly by large (>100kb) tandem duplications. Remarkably, these tandem duplication hotspots are enriched for breast cancer germline susceptibility loci (OR 4.28) and breast-specific “super-enhancer” regulatory elements (OR 3.54). They likely represent sites of selective susceptibility to double-strand break damage due to high transcriptional activity. However, through incrementally increasing the number of copies of these functionally active loci, they could inadvertently come under some level of selective pressure. Indeed, transcriptomic consequences range from strong individual oncogene effects through to weak but quantifiable multigene expression effects. We thus present a passenger somatic rearrangement mutational process that has the ability to create secondary driver events. It exerts its influence through coding sequences and non-coding regulatory elements, contributing a continuum of driver consequences, from modest through to strong effects, making this mutational process a particularly deleterious one. Citation Format: Dominik Glodzik, Sandro Morganella, Helen Davies, Serena Nik-Zainal, ICGC Breast Cancer Consortium. A somatic-mutational process in breast cancer genomes recurrently duplicates germline susceptibility loci and tissue-specific super-enhancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-262. doi:10.1158/1538-7445.AM2017-LB-262

Bromodomain protein 4 discriminates tissue-specific super-enhancers containing disease-specific susceptibility loci in prostate and breast cancer

BackgroundEpigenetic information can be used to identify clinically relevant genomic variants single nucleotide polymorphisms (SNPs) of functional importance in cancer development. Super-enhancers are cell-specific DNA elements, acting to determine tissue or cell identity and driving tumor progression. Although previous approaches have been tried to explain risk associated with SNPs in regulatory DNA elements, so far epigenetic readers such as bromodomain containing protein 4 (BRD4) and super-enhancers have not been used to annotate SNPs. In prostate cancer (PC), androgen receptor (AR) binding sites to chromatin have been used to inform functional annotations of SNPs.ResultsHere we establish criteria for enhancer mapping which are applicable to other diseases and traits to achieve the optimal tissue-specific enrichment of PC risk SNPs. We used stratified Q-Q plots and Fisher test to assess the differential enrichment of SNPs mapping to specific categories of enhancers. We find that BRD4 is the key discriminant of tissue-specific enhancers, showing that it is more powerful than AR binding information to capture PC specific risk loci, and can be used with similar effect in breast cancer (BC) and applied to other diseases such as schizophrenia.ConclusionsThis is the first study to evaluate the enrichment of epigenetic readers in genome-wide associations studies for SNPs within enhancers, and provides a powerful tool for enriching and prioritizing PC and BC genetic risk loci. Our study represents a proof of principle applicable to other diseases and traits that can be used to redefine molecular mechanisms of human phenotypic variation.

A somatic-mutational process recurrently duplicates germline susceptibility loci and tissue-specific super-enhancers in breast cancers.

Somatic rearrangements contribute to the mutagenized landscape of cancer genomes. Here, we systematically interrogated rearrangements in 560 breast cancers by using a piecewise constant fitting approach. We identified 33 hotspots of large (>100 kb) tandem duplications, a mutational signature associated with homologous-recombination-repair deficiency. Notably, these tandem-duplication hotspots were enriched in breast cancer germline susceptibility loci (odds ratio (OR) = 4.28) and breast-specific 'super-enhancer' regulatory elements (OR = 3.54). These hotspots may be sites of selective susceptibility to double-strand-break damage due to high transcriptional activity or, through incrementally increasing copy number, may be sites of secondary selective pressure. The transcriptomic consequences ranged from strong individual oncogene effects to weak but quantifiable multigene expression effects. We thus present a somatic-rearrangement mutational process affecting coding sequences and noncoding regulatory elements and contributing a continuum of driver consequences, from modest to strong effects, thereby supporting a polygenic model of cancer development.

Abstract 4634: Variants within super-enhancer regulatory elements associate with epithelial ovarian cancer risk

Abstract Background: Cell-type-specific super enhancers, or large clusters of mediator-rich enhancers, are highly transcribed regions of the genome with key functions in the maintenance of cell identity and control. These regulatory elements are easily perturbed and enriched for disease-associated sequence variation. Identification of disease-associated super-enhancers and investigation of their sequence variants may help identify important, biologically relevant associations previously overlooked by genome-wide studies. Thus, we sought to identify variants within ovarian tissue specific super-enhancers and investigate their association with epithelial ovarian cancer (EOC) susceptibility. Methods: We utilized genotype data from a European population of ∼11,000 invasive EOC cases and 22,000 healthy controls from the Ovarian Cancer Association Consortium. A public catalog of 478 super-enhancers, identified by peak histone H3K27ac ChIP-seq signal in ovarian tissue, was used to identify 72,116 variants located within 344 super-enhancer regions. Unconditional logistic regression (log-additive model) was used to assess individual SNP-EOC risk associations. Additionally, we reviewed associations within 500 kb of super-enhancer regions to identify proximal associated variants. When LD structure indicated, we employed conditional analysis to determine independence of the super-enhancer signal. Gene-EOC risk associations were assessed with the Admixture Maximum Likelihood (AML) test and Sequence-Kernel Association Test (SKAT) for the combined effect of common and rare (MAF<0.01) variants. Models were adjusted for population stratification using the first five principal components. To adjust for multiple comparisons, a false discovery rate of 10% was used. Results: The most significant SNP observed was rs147487657 (p = 7.7×10−6) on locus 2q37.3 in the HDAC4 gene and there were an additional 12 SNPs with p<1.0×10−4, although not significant at 10% FDR. Gene-level analyses identified two super-enhancers significantly associated with EOC risk. An enhancer at the previously reported 2q31 locus, containing HOXD8 and HOXD-AS2, associated (SKAT p = 4.3×10−5; AML p<0.001) with the top SNP located within the 5′UTR of HOXD8 transcription factor (rs847163 (T>C); p<2.08×10−5). Conditional analysis did not identify an independent association within the super-enhancer region. The second EOC-associated super-enhancer identified was at locus 1q32 (SKAT p = 0.00052, AML p = 0.001) containing the CSRP1 gene. There were 6 SNPs with p<0.001 and 16 SNPs with p<0.01, all were inversely associated with risk and consistent with the top hit at rs927901 T>C (OR = 0.94; 95% CI: 0.90-0.97; p = 2.1×10−4). Associations varied by histologic subtype, possibly due to variance in disease relevant super-enhancer cell-type.Conclusion: These findings suggest germline variation within super-enhancer regions may contribute to EOC susceptibility Citation Format: Brett M. Reid, Jennifer Permuth-Wey, Y. Ann Chen, Hui-Yi Lin, Alvaro Monteiro, Zhihua Chen, Andrew Berchuck, Georgia Chenevix-Trench, Jennifer Doherty, Simon Gayther, Ellen L. Goode, Edwin Iversen, Leigh Pearce, Paul Pharoah, Catherine Phelan, Susan Ramus, Mary Anne Rossing, Joellen Schildkraut, Thomas Sellers, on behalf of the Ovarian Cancer Association Consortium. Variants within super-enhancer regulatory elements associate with epithelial ovarian cancer risk. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4634. doi:10.1158/1538-7445.AM2015-4634