Encyclopedia Of DNA Elements Research Articles

Computational Probing the Methylation Sites Related to EGFR Inhibitor-Responsive Genes.

The emergence of drug resistance is one of the main obstacles to the treatment of lung cancer patients with EGFR inhibitors. Here, to further understand the mechanism of EGFR inhibitors in lung cancer and offer novel therapeutic targets for anti-EGFR-inhibitor resistance via the deep mining of pharmacogenomics data, we associated DNA methylation with drug sensitivities for uncovering the methylation sites related to EGFR inhibitor sensitivity genes. Specifically, we first introduced a grouped regularized regression model (Group Least Absolute Shrinkage and Selection Operator, group lasso) to detect the genes that were closely related to EGFR inhibitor effectiveness. Then, we applied the classical regression model (lasso) to identify the methylation sites associated with the above drug sensitivity genes. The new model was validated on the well-known cancer genomics resource: CTRP. GeneHancer and Encyclopedia of DNA Elements (ENCODE) database searches indicated that the predicted methylation sites related to EGFR inhibitor sensitivity genes were related to regulatory elements. Moreover, the correlation analysis on sensitivity genes and predicted methylation sites suggested that the methylation sites located in the promoter region were more correlated with the expression of EGFR inhibitor sensitivity genes than those located in the enhancer region and the TFBS. Meanwhile, we performed differential expression analysis of genes and predicted methylation sites and found that changes in the methylation level of some sites may affect the expression of the corresponding EGFR inhibitor-responsive genes. Therefore, we supposed that the effectiveness of EGFR inhibitors in lung cancer may be improved by methylation modification in their sensitivity genes.

Chromatin Landscapes of Human Lung Cells Predict Potentially Functional Chronic Obstructive Pulmonary Disease Genome-Wide Association Study Variants

Genome-wide association studies (GWASs) have identified dozens of loci associated with risk of chronic obstructive pulmonary disease (COPD). However, identifying the causal variants and their functional role in the appropriate cell type remains a major challenge. We aimed to identify putative causal variants in 82 GWAS loci associated with COPD susceptibility and predict the regulatory impact of these variants in lung-cell types. We used an integrated approach featuring statistical fine mapping, open chromatin profiling, and machine learning to identify functional variants. We generated chromatin accessibility data using the Assay for Transposase-Accessible Chromatin with High-Throughput Sequencing (ATAC-seq) for human primary lung-cell types implicated in COPD pathobiology. We then evaluated the enrichment of COPD risk variants in lung-specific open chromatin regions and generated cell type-specific regulatory predictions for >6,500 variants corresponding to 82 COPD GWAS loci. Integration of the fine-mapped variants with lung open chromatin regions helped prioritize 22 variants in putative regulatory elements with potential functional effects. Comparison with functional predictions from 222 Encyclopedia of DNA Elements (ENCODE) cell samples revealed cell type-specific regulatory effects of COPD variants in the lung epithelium, endothelium, and immune cells. We identified potential causal variants for COPD risk by integrating fine mapping in GWAS loci with cell-specific regulatory profiling, highlighting the importance of leveraging the chromatin status in relevant cell types to predict the molecular effects of risk variants in lung disease.

Simultaneous Expression of Long Non-Coding RNA FAL1 and Extracellular Matrix Protein 1 Defines Tumour Behaviour in Young Patients with Papillary Thyroid Cancer.

Simple SummaryFAL1 upregulation has been reported in many types of human cancers. The up-regulatory mechanism was identified in ovarian cancer but was not investigated in other type of cancers. Using The Cancer Genome Atlas (TCGA) database, we identified simultaneous upregulation of FAL1 adjacent to chromosome 1q21.3. Among 53 putative transcription factors for FAL1 and neighbouring genes, we selected c-JUN and JUND as the best candidates. This simultaneous upregulation defines molecular biological features representing RAS-driven PTC-enriched immune-related gene sets. These findings suggest that the simultaneous upregulation might be a potential diagnostic and therapeutic target for RAS-driven PTC.We investigated the regulatory mechanism of FAL1 and unravelled the molecular biological features of FAL1 upregulation in papillary thyroid cancer (PTC). Correlation analyses of FAL1 and neighbouring genes adjacent to chromosome 1q21.3 were performed. Focal amplification was performed using data from copy number alterations in The Cancer Genome Atlas (TCGA) database. To identify putative transcriptional factors, PROMO and the Encyclopaedia of DNA Elements (ENCODE) were used. To validate c-JUN and JUND as master transcription factors for FAL1 and ECM1, gene set enrichment analysis was performed according to FAL1 and ECM1 expression. Statistical analyses of the molecular biological features of FAL1- and ECM1-upregulated PTCs were conducted. FAL1 expression significantly correlated with that of neighbouring genes. Focal amplification of chromosome 1q21.3 was observed in ovarian cancer but not in thyroid carcinoma. However, PROMO suggested 53 transcription factors as putative common transcriptional factors for FAL1 and ECM1 simultaneously. Among them, we selected c-JUN and JUND as the best candidates based on ENCODE results. The expression of target genes of JUND simultaneously increased in FAL1- and ECM1-upregulated PTCs, especially in young patients. The molecular biological features represented RAS-driven PTC and simultaneously enriched immune-related gene sets. FAL1 and ECM1 expression frequently increased simultaneously and could be operated by JUND. The simultaneous upregulation might be a potential diagnostic and therapeutic target for RAS-driven PTC.

5-Hydroxymethylcytosine of Circulating Cell-Free DNA in Plasma: A Novel Non-Invasive Marker for Progression and Prognosis in Multiple Myeloma

Background: Multiple myeloma (MM) universally features profound disruption of their epigenomes with gene-specific DNA hypermethylation linked with clinically aggressive subtypes and prognosis. To date, DNA methylation (i.e., 5-methycytosine) is the most extensively studied cytosine modification. No study has characterized progression and prognostic significance of 5-hydroxymethylcytosine (5hmC), a mark of active demethylation and gene activation, nor did study evaluate 5hmC in cell-free DNA (cfDNA) as a non-invasive “liquid biopsy” mainly due to technological constraints.Methods: In a proof-of-concept study, we used the nano-hmC-Seal, a highly sensitive chemical labeling technique, and the next-generation sequencing to profile 5hmC in plasma cfDNA samples from 19 newly diagnosed, treatment-naïve European American patients with MM (n=9) and its premalignant precursor conditions - monoclonal gammopathy of undetermined significance (MGUS, n=5) and smoldering multiple myeloma (SMM; n=5) from the University of Chicago between 2011-12. Three of the 9 MM patients developed refractory disease or relapsed within 12 months after diagnosis. We profiled 5hmC with 1-2 ng of DNA extracted from 1ml of plasma for library construction. We obtained ~25 million reads per sample, providing a depth of coverage ~600X in terms of gene bodies. We normalized raw fragment counts using DESeq2.Results: We found that in cfDNA, 5hmC loci were enriched within gene bodies, while depleted in CpG islands. Our analysis, based on cis-regulatory elements from the ENCODE (Encyclopedia of DNA Elements), showed substantial overlap of 5hmC reads with histone modifications marking active transcription.We identified 183 genes containing differential 5hmC in gene bodies at 5% false discovery rate (p<0.001) between MGUS, SMM, and MM patients. Furthermore, 5hmC features in cfDNA showed the potential of separating MM patients at the time of diagnosis according to relapse status (39 genes, p<0.001). Many of these genes were previously implicated in MM, including those found to be differentially methylated (e.g., SYTL1, PSG1, GDF2, DNMT3A, GRP84) in a previous study of CD138+ cells from premalignant MGUS, early, and advanced stages of MM, and healthy control (Heuck CJ, 2013). We are profiling additional ~200 plasma samples from MM patients to 1) validate these preliminary findings, and 2) evaluate independent prognostic value of 5hmC in cfDNA controlling for established prognostic indices. These results will be presented at the ASH meeting.Conclusion: These preliminary findings suggest that 5hmC signatures in cfDNA differ 1) between MM and its two precursor conditions (i.e., MGUS and SMM), and 2) between MM patients by relapse status. [Display omitted] DisclosuresZimmerman:Abbvie: Employment.

CLLU1 Expression Is Not Confined to Chronic Lymphocytic Leukemia Cells; Putative Role of Gene-Body Hypomethylation in Regulating Splice Variant-Specific Transcriptional Dynamics

Background: Alternative splicing of the CLLU1 gene generates 6 transcript variants (accession numbers from the National Center for Biotechnology Information: AJ845162-AJ845167), two of which comprise a putative coding DNA sequence (AJ845165 and AJ845166, CDS transcripts). Previous studies showed that CLLU1 overexpression is highly confined to chronic lymphocytic leukemia (CLL) cells and that only the AJ845162 is expressed in both high- and low-risk CLL patients, while expression of the CDS-transcripts is confined to the high-risk subgroup.Pair-wise linear regression analysis revealed a powerful linear relation between the expression levels of all the splice variants, thereby it was deduced that CLLU1 transcription is orchestrated by a common promoter, which is located upstream of CLLU1 exon 1 (Buhl et al., Leukemia 2009). We recently documented (Papamichos et al., Haematologica 2016; s1) that this promoter was evolutionarily derived by an endogenous retrovirus sequence.Herein we argue that CLLU1 expression is not restricted to CLL cells while also we highlight that transcription of CDS transcripts is regulated by an alternative intronic promoter.Methods:CLLU1 high-throughput sequencing of polyadenylated RNA (RNA-Seq) data across 51 human tissues and 2 human cell lines, generated by the Genotype-Tissue Expression (GTEx) project of the US National Institutes of Health Common Fund (GTEx Analysis Release V6p), were analyzed via GTEx Portal. CLLU1 GeneChip expression data (AJ845164 transcript-specific probe set 1558185_at from Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array), generated by Genevestigator project, were downloaded from Genevisible portal.CLLU1 chromatin immunoprecipitation coupled with high throughput sequencing (ChIP-Seq) on histone H3 protein subunit modified at lysine position 4 with trimethylation (H3K4me3) as well as transcription factor (TF) ChIP-Seq data were downloaded from the Encyclopedia of DNA Elements (ENCODE) Regulation supertrack, which is available in UCSC Genome Browser Database. Data on CLLU1 gene-body methylation status in 63 cell lines from ENCODE were downloaded from the HAIB Methyl450 ENCODE track.Results:CLLU1 GeneChip expression data show that the AJ845164 transcript (corresponding to the RP11-693J15.4 long non-coding RNA) is promiscuously expressed in various normal B-cell subpopulations andin mature B-cell neoplasms other than CLL, especially in mantle cell lymphoma.RNA-Seq data analysis reveals that AJ845162 transcript is expressed in normal somatic tissues such as skeletal muscle, salivary gland, and esophageal mucosa. Vice versa, expression of CLLU1 CDS transcripts is confined to transformed lymphocytes.Structure interrogation of CDS transcripts indicates that these variants initiate from an alternative transcriptional start site which is located 2429 nucleotides downstream from the canonical one. H3K4me3 ChIP-Seq data signify the presence of an alternative intronic promoter in this genomic segment. TF Chip-Seq data reveal that the alternative promoter region comprises high affinity binding sites for signal transducer and activator of transcription 3 (STAT3) and Activator protein 1 (AP-1). Analysis of CLLU1 -body methylation status in five lymphoblastoid cell lines reveals that the alternative promoter remains heavily methylated in these cell types. This finding is in accordance with previous studies reporting that CLLU1 gene body is hypermethylated in B-cells from healthy individuals and in IgVH-mutated (IgVH-M) CLL cases while appears hypomethylated in IgVH-unmutated (IgVH-UM) CLL cells.Conclusion: Not all CLLU1 transcripts are equal in discriminating CLL from other mature B-cell malignancies.Whilst of retroviral origin, CLLU1 upstream promoter is not subjected to stringent epigenetic repression but remains active in somatic cells, a tantalizing finding that warrants further investigation.The level of intragenic DNA methylation represents a decisive regulator of alternative promoter usage (Neri et al., Nature 2017). It would be tantalizing to speculate that CLLU1 -body hypomethylation, occuring exclusively in IgVH-unmutated CLL, is mechanistically linked to the epigenetic derepression of the alternative intronic CLLU1 promoter and the subsequent high expression of CLLU1 CDS transcripts in high-risk CLL. DisclosuresNo relevant conflicts of interest to declare.

Endogenous Retrovirus Derepression Drives Ectopic UGT2B17 Overexpression in Multiple Myeloma Cells: Molecular Sequelae and Pathophysiological Implications

Introduction: UGT2B17 is a phase II detoxification enzyme which catalyzes the glucuronidation of C19 steroid hormones and lipid-soluble drugs. UGT2B17 gene is ubiquitously expressed in hepatic tissues as well as in steroid target tissues including breast, uterus, and prostate. UGT2B17 mRNA levels directly correlate with functional glucuronidation activity toward vorinostat (Gruber et al., Blood 2013), which is used against a variety of cancers including multiple myeloma (MM).Progression from monoclonal gammopathy of undetermined significance to MM is associated with increased genomic hypomethylation. Importantly it is transposable elements (TEs), remaining stochastically methylated in normal cells, that lend themselves predominantly susceptible to hypomethylation during myelomatogenesis. Hypomethylated TEs could subsequently acquire the potential to exert a significant impact on host gene expression. Herein we present evidence that an aberrantly activated long terminal repeat (LTR) of the ERV1 family of endogenous retroviruses (ERVs) drives the lineage-inordinate expression of UGT2B17 in MM cells.Methods:UGT2B17 high-throughput sequencing of polyadenylated RNA (RNA-Seq) data across 27 normal tissues from 95 human individuals, generated from the PRJEB4337 BioProject, were analyzed via the National Center for Biotechnology Information (NCBI) portal. Publicly available UGT2B17 RNA-Seq data coupled with ribosome profiling (Ribo-Seq) data across 17 cell types were downloaded from GWIPS-viz ribo-seq genome browser. UGT2B17 Transcription factor (TF) chromatin immunoprecipitation coupled with high throughput sequencing (ChIP-seq) data as well as ChIP-Seq data on histone H3 protein subunit modified at lysine position 4 with trimethylation (H3K4me3) were downloaded from the Encyclopedia of DNA Elements (ENCODE) Regulation supertrack, publicly available in UCSC Genome Browser Database. RepeatMasker software was employed to reveal the presence of integrated TEs in the genomic segment comprising UGT2B17 on chromosome 4.Results: Data analysis from the PRJEB4337 BioProject reveals low-level ectopic UGT2B17 transcription initiation in 5 of 27 normal human tissues (stomach, colon, appendix, spleen, and white blood cells) from a novel exon (E0) which is located ~7500 nucleotides (nts) upstream of the canonical UGT2B17 first exon (E1). Notably, H3K4me3 ChIP-Seq data indicate the presence of a highly potent promoter sequence immediately upstream of E0. RepeatMasker scan reveals that the genomic segment that comprises UGT2B17 E0 was evolutionary derived from a human-specific Harlequin-int LTR of the ERV1 family.Data from the GWIPS-viz genome browser show that the LTR-driven UGT2B17 transcription is minimal in 15 cell types, including normal somatic cells, cancer cells, and embryonic stem cells. In stark contrast, specifically in EBV-immortalized lymphocytes and the MM.1 myeloma cell line, UGT2B17 E0 presents higher transcription rate and also higher ribosome coverage compared to the UGT2B17 E1.TF ChIP-seq data from ENCODE reveal that UGT2B17 E0 comprises a binding site for RNA polymerase II subunit A (POLR2A) which, amongst several cell types examined, is functional specifically in B-lineage cells. Furthermore the intragenic segment that is located between E0 and E1 comprises a high-affinity binding site for RELA proto-oncogene. Of note, adjacent to the POLR2A and RELA binding sites, located are two binding sites for the CCCTC-binding factor (CTCF), which is known to act as a transcriptional insulator restricting activation of ectopic promoters.Conclusion: A retroviral-derived promoter that exhibits only basal activity in most normal or malignant somatic cells has the potential to initiate transcription of the adjacent UGT2B17 locus specifically in EBV-immortalized lymphocytes and MM cells. It is highly likely that ectopic UGT2B17 expression, restricted in B-lineage transformed and cancer cells, is induced by a lineage-specific aberration of CTCF function. Furthermore, the RELA binding site may serve as a potent enhancer of UGT2B17 inordinate transcription.The UGT2B17 LTR-driven misexpression in B-lineage transformed and cancer cell types may impact the effectiveness of vorinostat-based interventions in MM patients, a tantalizing scenario warranting further interrogation in primary MM samples. DisclosuresNo relevant conflicts of interest to declare.

Epigenetic Regulation of the N-Terminal Truncated Isoform of Matrix Metalloproteinase-2 (NTT-MMP-2) and Its Presence in Renal and Cardiac Diseases.

Several clinical and experimental studies have documented a compelling and critical role for the full-length matrix metalloproteinase-2 (FL-MMP-2) in ischemic renal injury, progressive renal fibrosis, and diabetic nephropathy. A novel N-terminal truncated isoform of MMP-2 (NTT-MMP-2) was recently discovered, which is induced by hypoxia and oxidative stress by the activation of a latent promoter located in the first intron of the MMP2 gene. This NTT-MMP-2 isoform is enzymatically active but remains intracellular in or near the mitochondria. In this perspective article, we first present the findings about the discovery of the NTT-MMP-2 isoform, and its functional and structural differences as compared with the FL-MMP-2 isoform. Based on publicly available epigenomics data from the Encyclopedia of DNA Elements (ENCODE) project, we provide insights into the epigenetic regulation of the latent promoter located in the first intron of the MMP2 gene, which support the activation of the NTT-MMP-2 isoform. We then focus on its functional assessment by covering the alterations found in the kidney of transgenic mice expressing the NTT-MMP-2 isoform. Next, we highlight recent findings regarding the presence of the NTT-MMP-2 isoform in renal dysfunction, in kidney and cardiac diseases, including damage observed in aging, acute ischemia-reperfusion injury (IRI), chronic kidney disease, diabetic nephropathy, and human renal transplants with delayed graft function. Finally, we briefly discuss how our insights may guide further experimental and clinical studies that are needed to elucidate the underlying mechanisms and the role of the NTT-MMP-2 isoform in renal dysfunction, which may help to establish it as a potential therapeutic target in kidney diseases.

Integrative analysis of liver-specific non-coding regulatory SNPs associated with the risk of coronary artery disease

Genetic factors underlying coronary artery disease (CAD) have been widely studied using genome-wide association studies (GWASs). However, the functional understanding of the CAD loci has been limited by the fact that a majority of GWAS variants are located within non-coding regions with no functional role. High cholesterol and dysregulation of the liver metabolism such as non-alcoholic fatty liver disease confer an increased risk of CAD. Here, we studied the function of non-coding single-nucleotide polymorphisms in CAD GWAS loci located within liver-specific enhancer elements by identifying their potential target genes using liver cis-eQTL analysis and promoter Capture Hi-C in HepG2 cells. Altogether, 734 target genes were identified of which 121 exhibited correlations to liver-related traits. To identify potentially causal regulatory SNPs, the allele-specific enhancer activity was analyzed by (1) sequence-based computational predictions, (2) quantification of allele-specific transcription factor binding, and (3) STARR-seq massively parallel reporter assay. Altogether, our analysis identified 1,277 unique SNPs that display allele-specific regulatory activity. Among these, susceptibility enhancers near important cholesterol homeostasis genes (APOB, APOC1, APOE, and LIPA) were identified, suggesting that altered gene regulatory activity could represent another way by which genetic variation regulates serum lipoprotein levels. Using CRISPR-based perturbation, we demonstrate how the deletion/activation of a single enhancer leads to changes in the expression of many target genes located in a shared chromatin interaction domain. Our integrative genomics approach represents a comprehensive effort in identifying putative causal regulatory regions and target genes that could predispose to clinical manifestation of CAD by affecting liver function.

Low Quantity Single Strand CAGE (LQ-ssCAGE) Maps Regulatory Enhancers and Promoters.

The Cap Analysis of Gene Expression (CAGE) is a powerful method to identify Transcription Start Sites (TSSs) of capped RNAs while simultaneously measuring transcripts expression level. CAGE allows mapping at single nucleotide resolution at all active promoters and enhancers. Large CAGE datasets have been produced over the years from individual laboratories and consortia, including the Encyclopedia of DNA Elements (ENCODE) and Functional Annotation of the Mammalian Genome (FANTOM) consortia. These datasets constitute open resource for TSS annotations and gene expression analysis. Here, we provide an experimental protocol for the most recent CAGE method called Low Quantity (LQ) single strand (ss) CAGE "LQ-ssCAGE", which enables cost-effective profiling of low quantity RNA samples. LQ-ssCAGE is especially useful for samples derived from cells cultured in small volumes, cellular compartments such as nuclear RNAs or for samples from developmental stages. We demonstrate the reproducibility and effectiveness of the method by constructing 240 LQ-ssCAGE libraries from 50ng of THP-1 cell extracted RNAs and discover lowly expressed novel enhancer and promoter-derived lncRNAs.

Both EZH2 and JMJD6 regulate cell cycle genes in breast cancer

BackgroundStrong evidences support the critical role of Jumonji domain containing 6 (JMJD6) in progression of breast cancer. Here we explore potential partners that coregulate gene expression, to understand additional pathways that are activated by higher amounts of JMJD6.MethodsWe used Gene Set Enrichment Analysis (GSEA) data to identify factors that display gene expression similar to cells treated with JMJD6 siRNA. Using chromatin immunoprecipitations (ChIP) against genomic regions that bind JMJD6 identified by in house and public database Encyclopaedia of DNA Elements (ENCODE), we confirmed JMJD6 occupancy by ChIP PCR. We tested the association of co-regulated genes with patient prognosis using The Cancer Genome Atlas (TCGA) datasets.ResultsJMJD6 profiles overlapped with those of Enhancer of Zeste homolog 2 (EZH2) and together they appear to co-regulate a unique cassette of genes in both ER+ and ER- cells. 496 genes including aurora kinases, which are currently being tested as novel therapeutic targets in breast cancer were co-regulated in MDA MB 231 cells. JMJD6 and EZH2 neither inter-regulated nor physically interacted with one another. Since both proteins are chromatin modulators, we performed ChIP linked PCR analysis and show that JMJD6 bound in the neighbourhood of co-regulated genes, though EZH2 data did not show any peaks within 100 kb of these sites. Alignment of binding site sequences suggested that atleast two types of binding partners could offer their DNA binding properties to enrich JMJD6 at regulatory sites. In clinical samples, JMJD6 and EZH2 expression significantly correlated in both normal and tumor samples, however the strongest correlation was observed in triple-negative breast cancer (TNBC) subtype. Co-expression of JMJD6 and EZH2 imposed poorer prognosis in breast cancer.ConclusionsJMJD6 and EZH2 regulate the same crucial cell cycle regulatory and therapeutic targets but their mechanisms appear to be independent of each other. Blocking of a single molecule may not axe cell proliferation completely and blocking both JMJD6 and EZH2 simultaneously may be more effective in breast cancer patients.

Revealing Epigenetic Factors of circRNA Expression by Machine Learning in Various Cellular Contexts.

SummaryCircular RNAs (circRNAs) have been identified as naturally occurring RNAs that are highly represented in the eukaryotic transcriptome. Although a large number of circRNAs have been reported, the underlying regulatory mechanism of circRNAs biogenesis remains largely unknown. Here, we integrated in-depth multi-omics data including epigenome, transcriptome, and non-coding RNA and identified candidate circRNAs in six cellular contexts. Next, circRNAs were divided into two classes (high versus low) with different expression levels. Machine learning models were constructed that predicted circRNA expression levels based on 11 different histone modifications and host gene expression. We found that the models achieve great accuracy in predicting high versus low expressed circRNAs. Furthermore, the expression levels of host genes of circRNAs, H3k36me3, H3k79me2, and H4k20me1 contributed greatly to the classification models in six cellular contexts. In summary, all these results suggest that epigenetic modifications, particularly histone modifications, can effectively predict expression levels of circRNAs.

How Retroactivity Affects the Behavior of Incoherent Feedforward Loops.

SummaryAn incoherent feedforward loop (IFFL) is a network motif known for its ability to accelerate responses and generate pulses. It remains an open question to understand the behavior of IFFLs in contexts with high levels of retroactivity, where an upstream transcription factor binds to numerous downstream binding sites. Here we study the behavior of IFFLs by simulating and comparing ODE models with different levels of retroactivity. We find that increasing retroactivity in an IFFL can increase, decrease, or keep the network's response time and pulse amplitude constant. This suggests that increasing retroactivity, traditionally considered an impediment to designing robust synthetic systems, could be exploited to improve the performance of IFFLs. In contrast, we find that increasing retroactivity in a negative autoregulated circuit can only slow the response. The ability of an IFFL to flexibly handle retroactivity may have contributed to its significant abundance in both bacterial and eukaryotic regulatory networks.

Novel Functional Variants in the Notch Pathway and Survival of Chinese Colorectal Cancer

Background: Notch signaling pathway plays a crucial role in progression of colorectal cancer (CRC).Genetic variants in Notch pathway genes may impact the prognosis in CRC. Method: The associations genotypes of single-nucleotide polymorphisms (SNPs) from Notch pathway genes and overall survival (OS) in 1116 Chinese eastern CRC were investigated. The ChIP sequencing data from the encyclopedia of DNA Elements (ENCODE), Expression quantitative trait loci (eQTL) analysis and Dual luciferase assays were used to evaluated the association of SNPs genotype and candidate gene expression. The function of candidate gene was explored in The Cancer Genome Atlas (TCGA) CRC data and elucidated in vitro experiments. Findings: We identified MINAR1 rs72430409 was significantly associated with a greater death risk (HR=1.98, 95% CI=1.55–2.54, P=6.8×10-8). The analysis of ENCODE data showed that rs72430409 were potential cis-regulatory elements for the MINAR1 promoter, eQTL analysis revealed that rs72430409 A allele were correlated with increased MINAR1 expression. Dual luciferase assays revealed that rs72430409 A allele increased MINAR1 promoter activity. The expression of MINAR1 in TCGA CRC samples were significantly higher than that in normal colorectal tissue and high expression of MINAR1 was associated with a shortened OS, likely via activating the epithelial mesenchymal transition (EMT) pathway. RNAi mediated silencing of MINAR1 led to decreased migration and proliferation in CRC cells, and MINAR1 silencing could downregulate the expression of key effector genes in EMT and glycolysis. Interpretation:Our study identified novel functional genetic variants which could independently predicted CRC survival, the gene harbouring this loci functioned as a putative oncogene in CRC by regulating EMT and glycolysis. Funding Statement: This research was supported by Natural Science Foundation of China and Shang Shanghai Natural Science Foundation. Declaration of Interests: The authors have no conflicts of interest to declare. Ethics Approval Statement: The research proposal was approved by the FUSCC institutional review board.

NIMBus: a negative binomial regression based Integrative Method for mutation Burden Analysis

BackgroundIdentifying frequently mutated regions is a key approach to discover DNA elements influencing cancer progression.However, it is challenging to identify these burdened regions due to mutation rate heterogeneity across the genome and across different individuals. Moreover, it is known that this heterogeneity partially stems from genomic confounding factors, such as replication timing and chromatin organization. The increasing availability of cancer whole genome sequences and functional genomics data from the Encyclopedia of DNA Elements (ENCODE) may help address these issues.ResultsWe developed a negative binomial regression-based Integrative Method for mutation Burden analysiS (NIMBus). Our approach addresses the over-dispersion of mutation count statistics by (1) using a Gamma–Poisson mixture model to capture the mutation-rate heterogeneity across different individuals and (2) estimating regional background mutation rates by regressing the varying local mutation counts against genomic features extracted from ENCODE. We applied NIMBus to whole-genome cancer sequences from the PanCancer Analysis of Whole Genomes project (PCAWG) and other cohorts. It successfully identified well-known coding and noncoding drivers, such as TP53 and the TERT promoter. To further characterize the burdening of non-coding regions, we used NIMBus to screen transcription factor binding sites in promoter regions that intersect DNase I hypersensitive sites (DHSs). This analysis identified mutational hotspots that potentially disrupt gene regulatory networks in cancer. We also compare this method to other mutation burden analysis methods.ConclusionNIMBus is a powerful tool to identify mutational hotspots. The NIMBus software and results are available as an online resource at github.gersteinlab.org/nimbus.

Abstract 4687: Oncogenic role of the transcription factor YY1 and its target Survivin in non-Hodgkin's lymphoma

Abstract Objective Yin Yang 1 (YY1) is a transcription factor with a dual role in cancer genesis. Hematological malignancies, including non-Hodgkin lymphomas (NHL), are often characterized by a frequent development of resistance to therapy and a high rate of tumor recurrence. Our previous observations showed that YY1 is overexpressed in hematological tumors. The aim of this study was to investigate the functional role of YY1 in anti-cancer therapy induced cell death and to identify YY1 downstream resistant factors. Experimental Designs The NHL Raji cell line was used as a cellular model. The expression of YY1 was assessed by q-PCR and WB. YY1 knock-down (KD) was achieved using retroviral shRNA. Viability was measured by both the MTT assay and the Trypan Blue live/dead cell count. Cell death was induced following Vincristine and Doxorubicin treatments. The following analyses were used in this study: the JASPAR database, the Encyclopedia of DNA Elements (ENCODE) and the Cancer Genome Atlas (TCGA) datasets relative to NHL patients have been analyzed to assess the expression levels of YY1 and apoptotic genes, according to tumor stages and patients' clinical-pathological features. Results Silencing of YY1 in Raji cells resulted in increased sensitivity to both Vincristine and Doxorubicin treatments and augmented apoptosis. Jaspar analysis identified several potential YY1 transcriptional targets involved in cellular death regulation. As a result of q-PCR screening and validation, Survivin has been identified as a potential YY1 target, as it was significantly downregulated following YY1 KD. Bioinformatic analysis of deposited Chip-Seq databases in ENCODE revealed that YY1 is able to target Survivin transcriptional regulatory regions in different cancer cell lines, as well as in normal B lymphocytes. The TCGA-deposited datasets identified specific clusters of YY1 expression significantly correlated with Survivin. Conclusions Our findings demonstrated that (1) YY1 silencing resulted in the sensitization of Raji tumor cells to drug-induced apoptosis (2) q-PCR-mediated screening of putative YY1 targets led to the identification of Survivin as a potential YY1 target, as when YY1 was KD, Survivin levels were strongly downregulated (3) YY1 might be positively regulating Survivin expression (4) Survivin could be a potential target of YY1 in NHL and (5) YY1 and Survivin in NHL may represent two novel diagnostic and prognostic biomarkers to assess patients' response to chemotherapy. Citation Format: Silvia Vivarelli, Luca Falzone, Saverio Candido, Benjamin Bonavida, Massimo Libra. Oncogenic role of the transcription factor YY1 and its target Survivin in non-Hodgkin's lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4687.

Abstract 29: Integrating computational epigenetic and statistical approaches to investigate how genome-wide transcription factor (TF)-DNA bindings affect breast cancer risk

Abstract Background: Fine-mapping and functional genomic studies of risk loci identified by genome wide association studies (GWAS) of breast cancer suggest that risk-associated regulatory variants may disrupt DNA binding affinities of transcription factors (TFs), such as FOXA1 and ESR1, thus altering gene expression and affecting breast cancer risk. To date, however, no studies have directly investigated how genome-wide TF-DNA bindings in a tissue-specific context affect breast cancer risk. Methods: We recently developed an analytic framework using computational epigenetic approaches and generalized mixed models to identify breast cancer risk associated cis-regulatory elements that are occupied by TFs. Summary statistics derived from GWAS of imputed data for approximately 11 million genetic variants were obtained from the Breast Cancer Association Consortium (BCAC). A total of 113 ChIP-seq datasets for TFs and chromatin features were annotated from the Encyclopedia of DNA Elements (ENCODE) and Roadmap projects and the Cistrome database (http://cistrome.org/) in multiple breast cancer cell lines..Chi-square values reported in the BCAC dataset were used to measure breast cancer risk associated with a genetic variant. To investigate TF-DNA bindings for breast cancer risk, we constructed generalized mixed models to evaluate the associations between the Chi-square values and binding sites of single TF or co-occupied by multiple TFs, given linkage disequilibrium (LD) blocks of variants to handle their dependence. To define approximately independent LD blocks similar to other studies, we defined LD blocks using non-overlapping segments of 100k bps. In addition, we investigated the effect of putative cis-regulatory elements characterized by TF-DNA bindings and chromatin features on breast cancer risk. Results: We identified a total of 22 TFs where their TF-DNA bindings were significantly associated with breast cancer risk at P &amp;lt; 1 × 10−5, with the top five TFs being FOXA1, SIN3AK, ESR1, TCF7l2, and AR (P &amp;lt; 1 × 10−10). We further observed stronger associations of breast cancer risk with co-occupied TF-TF DNA bindings, with the top five TF pairs (P &amp;lt; 1 × 10−16) being FOXA1+E2F1, FOXA1+NR2F2, FOXA1+ESR1, FOXA1+SIN3, and FOXA1+TCF12. The interactions of TF-TF DNA bindings for these five TF pairs were highly significant (P &amp;lt; 1 × 10−5). In addition, our results showed a significant interaction (P &amp;lt; 1 × 10−5) between chromatin features and the TF-DNA bindings. As compared with quiescent/low chromatin features, more TF-DNA bindings in enhancers, strong or weak transcription, or heterochromatin were associated with lower breast cancer risk. Conclusions: Our findings suggest that putative regulatory variants, which alter the TF-DNA binding affinities, particularly those located in TF-TF co-occupied sites, and TF-DNA binding colocalized with chromatin features, play an important role in breast cancer risk. Our approaches can be generalized to investigate the effect of genome-wide TF-DNA bindings on the risk of any human cancers that have comprehensive ChIP-seq and large-scale GWAS data. Citation Format: Wanqing Wen, Zhishan Chen, Quan Long, Wei Zheng, Xingyi Guo. Integrating computational epigenetic and statistical approaches to investigate how genome-wide transcription factor (TF)-DNA bindings affect breast cancer risk [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 29.

Abstract 4616: Functional genomic analyses of 21q22.3 locus identify potential functional variants and candidate gene YBEY for breast cancer risk

Abstract We have previously identified SNP rs3541811, located in the 21q22.3 locus, to be associated with breast cancer risk in Asians. However, the underlying causal functional variants and gene(s) responsible for this association are unknown. In this study, we performed functional genomic analyses to identify potential functional variants and target genes that may mediate this association. Functional annotation for SNPs highly correlated with rs3541881, using epigenetic data from the Encyclopedia of DNA Elements (ENCODE) and Roadmap projects, showed evidence of promoter and/or enhancer activities of five putative functional SNPs, including rs35418111, rs2078203, rs8134832, rs57385578, and rs8126917. These SNPs were assessed for interactions with nuclear proteins by EMSA assay. Our results showed that compared to the reference allele, alternate allele rs2078203 (A &amp;gt; G) significantly increased DNA-protein interactions, while an opposite trend was observed for rs35418111 (G &amp;gt; A). Cis-expression quantitative loci (eQTL) analysis, using data from the Genotype-Tissue Expression (GTEx) project, The Cancer Genome Atlas (TCGA), Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), and the Shanghai Breast Cancer Study, indicated that the risk allele rs35418111 is associated with a decreased expression of the YBEY (C21or57) gene, indicating its putative oncogenic function that may contribute to breast cancer risk. The gene YBEY is a relatively uncharacterized endoribonuclease in humans, which is thought to function in rRNA cleavage and maturation, similar to its bacterial homologues. We further investigated whether YBEY may have any biological effect in human breast cancers by knocking-down YBEY gene expression using siRNA in MCF-7, T47D, and MDA-MB-231 cell lines. Transient knockdown of YBEY gene expression in three breast cancer cell lines consistently affected cell proliferation, colony formation, and migration/invasion, regardless of hormone receptor status. Our study provides support for a significant role for human YBEY gene in breast cancer pathogenesis and the association between the rs35418111/21q22.3 locus and breast cancer risk, which may be mediated through its correlated potential functional SNPs that regulate expression of the YBEY gene. Citation Format: Chris Shidal, Xiang Shu, Jie Wu, Jifeng Wang, Shuya Huang, Joshua Bauer, Xingyi Guo, Wei Zheng, Xiao-Ou Shu, Qiuyin Cai. Functional genomic analyses of 21q22.3 locus identify potential functional variants and candidate gene YBEY for breast cancer risk [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4616.

Computational Prediction of Drug Responses in Cancer Cell Lines From Cancer Omics and Detection of Drug Effectiveness Related Methylation Sites

Accurately predicting the response of a cancer patient to a therapeutic agent remains an important challenge in precision medicine. With the rise of data science, researchers have applied computational models to study the drug inhibition effects on cancers based on cancer genomics and transcriptomics. Moreover, a common epigenetic modification, DNA methylation, has been related to the occurrence and development of cancer, as well as drug effectiveness. Therefore, it is helpful for improvement of drug response prediction through exploring the relationship between DNA methylation and drug effectiveness. Here, we proposed a computational model to predict drug responses in cancers through integration of cancer genomics, transcriptomics, epigenomics, and compound chemical properties. Meanwhile, we applied a regularized regression model (Least Absolute Shrinkage and Selection Operator, lasso) to detect the methylation sites that were closely related to drug effectiveness. The prediction models were trained on a well-known pharmacogenomics data resource, Genomics of Drug Sensitivity in Cancer (GDSC). The cross-validation indicates that the performance of the prediction model using DNA methylation is comparable to that of using other cancer omics, including oncogene mutation and gene expression data. It indicates the important role of DNA methylation in prediction of drug responses. Encyclopedia of DNA Elements (ENCODE) and Transcriptional Regulatory Relationships Unraveled by Sentence-based Text mining (TRRUST2) database analyses suggest that the methylation sites associated with drug effectiveness are mainly located in the transcription factor (TF) binding region. Therefore, we hypothesized that the sensitivity of cancer cells to drugs could be regulated by changing the methylation modification of TF binding region. In conclusion, we confirmed the important role of DNA methylation in prediction of drug responses, and provided some methylation sites that closely related to the drug effectiveness, which may be a great regulatory target for improvement of drug treatment effects on cancer patients.

An atlas of dynamic chromatin landscapes in mouse fetal development

The Encyclopedia of DNA Elements (ENCODE) project has established a genomic resource for mammalian development, profiling a diverse panel of mouse tissues at 8 developmental stages from 10.5 days after conception until birth, including transcriptomes, methylomes and chromatin states. Here we systematically examined the state and accessibility of chromatin in the developing mouse fetus. In total we performed 1,128 chromatin immunoprecipitation with sequencing (ChIP–seq) assays for histone modifications and 132 assay for transposase-accessible chromatin using sequencing (ATAC–seq) assays for chromatin accessibility across 72 distinct tissue-stages. We used integrative analysis to develop a unified set of chromatin state annotations, infer the identities of dynamic enhancers and key transcriptional regulators, and characterize the relationship between chromatin state and accessibility during developmental gene regulation. We also leveraged these data to link enhancers to putative target genes and demonstrate tissue-specific enrichments of sequence variants associated with disease in humans. The mouse ENCODE data sets provide a compendium of resources for biomedical researchers and achieve, to our knowledge, the most comprehensive view of chromatin dynamics during mammalian fetal development to date.

Expanded encyclopaedias of DNA elements in the human and mouse genomes

The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE1 and Roadmap Epigenomics2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.