Regulatory Sequence Elements Research Articles

Interpreting deep neural networks for the prediction of translation rates

BackgroundThe 5’ untranslated region of mRNA strongly impacts the rate of translation initiation. A recent convolutional neural network (CNN) model accurately quantifies the relationship between massively parallel synthetic 5’ untranslated regions (5’UTRs) and translation levels. However, the underlying biological features, which drive model predictions, remain elusive. Uncovering sequence determinants predictive of translation output may allow us to develop a more detailed understanding of translation regulation at the 5’UTR.ResultsApplying model interpretation, we extract representations of regulatory logic from CNNs trained on synthetic and human 5’UTR reporter data. We reveal a complex interplay of regulatory sequence elements, such as initiation context and upstream open reading frames (uORFs) to influence model predictions. We show that models trained on synthetic data alone do not sufficiently explain translation regulation via the 5’UTR due to differences in the frequency of regulatory motifs compared to natural 5’UTRs.ConclusionsOur study demonstrates the significance of model interpretation in understanding model behavior, properties of experimental data and ultimately mRNA translation. By combining synthetic and human 5’UTR reporter data, we develop a model (OptMRL) which better captures the characteristics of human translation regulation. This approach provides a general strategy for building more successful sequence-based models of gene regulation, as it combines global sampling of random sequences with the subspace of naturally occurring sequences. Ultimately, this will enhance our understanding of 5’UTR sequences in disease and our ability to engineer translation output.

Deep learning the cis-regulatory code for gene expression in selected model plants

Elucidating the relationship between non-coding regulatory element sequences and gene expression is crucial for understanding gene regulation and genetic variation. We explored this link with the training of interpretable deep learning models predicting gene expression profiles from gene flanking regions of the plant species Arabidopsis thaliana, Solanum lycopersicum, Sorghum bicolor, and Zea mays. With over 80% accuracy, our models enabled predictive feature selection, highlighting e.g. the significant role of UTR regions in determining gene expression levels. The models demonstrated remarkable cross-species performance, effectively identifying both conserved and species-specific regulatory sequence features and their predictive power for gene expression. We illustrated the application of our approach by revealing causal links between genetic variation and gene expression changes across fourteen tomato genomes. Lastly, our models efficiently predicted genotype-specific expression of key functional gene groups, exemplified by underscoring known phenotypic and metabolic differences between Solanum lycopersicum and its wild, drought-resistant relative, Solanum pennellii.

Exploring the Function of Gene Promoter Regulatory Elements Using CRISPR Tools.

Gene promoters serve as pivotal regulators of transcription, orchestrating the initiation, rate, and specificity of gene expression, resulting in cellular diversity found among distinct cell types within multicellular organisms. Identification of the sequence and function of promoters' regulatory elements and their complex interaction with transcription factors, enhancers, silencers, and insulators is fundamental to coordinated transcriptional processes within cells. Identifying these regulatory elements and scrutinizing their functions and interactions through the use of synthetic promoters can pave the way for researchers in various fields ranging from uncovering the origins of diseases associated with promoter mutations to harnessing these regulatory components in biotechnological applications.In this chapter, we describe the manipulation of regulatory elements within promoters, with a specific focus on the use of CRISPR technology on enhancers and silencer elements of the Ovalbumin gene promoter. We explain and discuss processes for the deletion of/interference with regulatory elements within the promoter, employing CRISPR-based approaches. Furthermore, we demonstrate that a CRISPR/Cas-manipulated promoter can activate gene transcription in cell types where it is normally inactive. This confirms that CRISPR technology can be effectively used to engineer synthetic promoters with desired characteristics, such as inducibility, tissue-specificity, or enhanced transcriptional strength. Such an approach provides valuable insights into the mechanisms and dynamics of gene expression, thereby offering new opportunities in the fields of biotechnology and medicine.

Toward Identification of Functional Sequences and Variants in Noncoding DNA.

Understanding the noncoding part of the genome, which encodes gene regulation, is necessary to identify genetic mechanisms of disease and translate findings from genome-wide association studies into actionable results for treatments and personalized care. Here we provide an overview of the computational analysis of noncoding regions, starting from gene-regulatory mechanisms and their representation in data. Deep learning methods, when applied to these data, highlight important regulatory sequence elements and predict the functional effects of genetic variants. These and other algorithms are used to predict damaging sequence variants. Finally, we introduce rare-variant association tests that incorporate functional annotations and predictions in order to increase interpretability and statistical power.

Genetic risk variants for multiple sclerosis are linked to differences in alternative pre-mRNA splicing.

Multiple sclerosis (MS) is a chronic immune-mediated disease of the central nervous system to which a genetic predisposition contributes. Over 200 genetic regions have been associated with increased disease risk, but the disease-causing variants and their functional impact at the molecular level are mostly poorly defined. We hypothesized that single-nucleotide polymorphisms (SNPs) have an impact on pre-mRNA splicing in MS. Our study focused on 10 bioinformatically prioritized SNP-gene pairs, in which the SNP has a high potential to alter alternative splicing events (ASEs). We tested for differential gene expression and differential alternative splicing in B cells from MS patients and healthy controls. We further examined the impact of the SNP genotypes on ASEs and on splice isoform expression levels. Novel genotype-dependent effects on splicing were verified with splicing reporter minigene assays. We were able to confirm previously described findings regarding the relation of MS-associated SNPs with the ASEs of the pre-mRNAs from GSDMB and SP140. We also observed an increased IL7R exon 6 skipping when comparing relapsing and progressive MS patients to healthy subjects. Moreover, we found evidence that the MS risk alleles of the SNPs rs3851808 (EFCAB13), rs1131123 (HLA-C), rs10783847 (TSFM), and rs2014886 (TSFM) may contribute to a differential splicing pattern. Of particular interest is the genotype-dependent exon skipping of TSFM due to the SNP rs2014886. The minor allele T creates a donor splice site, resulting in the expression of the exon 3 and 4 of a short TSFM transcript isoform, whereas in the presence of the MS risk allele C, this donor site is absent, and thus the short transcript isoform is not expressed. In summary, we found that genetic variants from MS risk loci affect pre-mRNA splicing. Our findings substantiate the role of ASEs with respect to the genetics of MS. Further studies on how disease-causing genetic variants may modify the interactions between splicing regulatory sequence elements and RNA-binding proteins can help to deepen our understanding of the genetic susceptibility to MS.

Evolutionary Changes in the Chromatin Landscape Contribute to Reorganization of a Developmental Gene Network During Rapid Life History Evolution in Sea Urchins.

Chromatin configuration is highly dynamic during embryonic development in animals, exerting an important point of control in transcriptional regulation. Yet there exists remarkably little information about the role of evolutionary changes in chromatin configuration to the evolution of gene expression and organismal traits. Genome-wide assays of chromatin configuration, coupled with whole-genome alignments, can help address this gap in knowledge in several ways. In this study we present a comparative analysis of regulatory element sequences and accessibility throughout embryogenesis in three sea urchin species with divergent life histories: a lecithotroph Heliocidaris erythrogramma, a closely related planktotroph H. tuberculata, and a distantly related planktotroph Lytechinus variegatus. We identified distinct epigenetic and mutational signatures of evolutionary modifications to the function of putative cis-regulatory elements in H. erythrogramma that have accumulated nonuniformly throughout the genome, suggesting selection, rather than drift, underlies many modifications associated with the derived life history. Specifically, regulatory elements composing the sea urchin developmental gene regulatory network are enriched for signatures of positive selection and accessibility changes which may function to alter binding affinity and access of developmental transcription factors to these sites. Furthermore, regulatory element changes often correlate with divergent expression patterns of genes involved in cell type specification, morphogenesis, and development of other derived traits, suggesting these evolutionary modifications have been consequential for phenotypic evolution in H. erythrogramma. Collectively, our results demonstrate that selective pressures imposed by changes in developmental life history rapidly reshape the cis-regulatory landscape of core developmental genes to generate novel traits and embryonic programs.

An oligonucleotide/oligosaccharide-binding-fold protein enhances the alternative splicing event producing thylakoid membrane-bound ascorbate peroxidase in Nicotiana tabacum.

The stromal and thylakoid membrane-bound ascorbate peroxidase isoforms are produced by the alternative splicing event of the 3′-terminal region of the APXII gene in spinach (Spinacia oleracea) and tobacco (Nicotiana tabacum), but not in Arabidopsis (Arabidopsis thaliana). However, all alternative splicing variants were detected in APXII gene-transformed Arabidopsis, indicating the occurrence of its regulatory mechanisms in Arabidopsis. The efficiency of this alternative splicing event in producing thylakoid membrane-bound ascorbate peroxidase mRNA is regulated by a splicing regulatory cis element, but trans splicing regulatory factor(s) for alternative splicing remain unclear. To identify this factor, we conducted a forward genetic screen using Arabidopsis in combination with a luciferase reporter system to evaluate the alternative splicing efficiency of thylakoid membrane-bound ascorbate peroxidase mRNA production. We isolated 9 mutant lines that showed low efficiency of the AS in producing thylakoid membrane-bound ascorbate peroxidase mRNA compared with that in the control plants. From one mutant [APXII alternative splicing inhibition (apsi1)], the causal gene responsible for the phenotype, AT5G38890 (oligonucleotide/oligosaccharide-binding-fold protein, APSI1), was identified. The levels of thylakoid membrane-bound ascorbate peroxidase mRNA from the transformed APXII gene decreased and increased in APSI1 knockout and APSI1-overexpressing plants, respectively. APSI1 was localized to the nucleus and specifically bound to the splicing regulatory cis element sequence. Tobacco plants that disrupted the closest homologs of APSI1 showed low levels of endogenous thylakoid membrane-bound ascorbate peroxidase mRNA. These results indicate that APSI1 is an enhancing component of the alternative splicing event of APXII.

Tumor-suppressive circRHOBTB3 is excreted out of cells via exosome to sustain colorectal cancer cell fitness

Background & AimsTo clarify the biological roles, circularization process and secretion pathway of circRHOBTB3 in colorectal cancer (CRC) progression.MethodsWe performed a comprehensive analysis of circRNA levels in serum exosomes from multiple types of cancer patients in public databases and verified the higher level of circRHOBTB3 in CRC sera versus healthy donors by RT-qPCR. Then, the function of circRHOBTB3 in CRC was investigated in vitro and in vivo. RNA-seq and RNA pull-down assays together with mass spectrometry identified the downstream signals and the binding proteins of circRHOBTB3. Finally, Antisense oligonucleotides (ASOs) were designed to target circularization and secretion elements of circRHOBTB3 for CRC therapy.ResultscircRHOBTB3 levels were increased in the sera but was downregulated in tissue samples in CRC, and the downregulation was associated with poor prognosis. Furthermore, circRHOBTB3 acts a tumor-suppressive circRNA by repressing metabolic pathways, intracellular ROS production in CRC. Several key elements were discovered to regulate circRHOBTB3 circularization and exosomal secretion. Moreover, SNF8 was identified that sorts circRHOBTB3 into exosomes. Interestingly, we found that CRC cells could actively secrete more circRHOBTB3 than normal cells. According to the sequence of regulatory elements for circularization and exosomal secretion, we designed and synthesized ASOs, which increased circRHOBTB3 expression and blocked circRHOBTB3 exosomal secretion. More importantly, ASOs could inhibit CRC growth and metastasis in vitro and in vivo.ConclusionscircRHOBTB3 plays a tumor-suppressive role in CRC and has to be excreted out of cells to sustain cancer cell fitness. ASOs targeting regulatory elements for circularization and exosomal secretion will become a novel antitumor strategy.

Alternative Splicing in Human Biology and Disease.

Alternative pre-mRNA splicing allows for the production of multiple mRNAs from an individual gene, which not only expands the protein-coding potential of the genome but also enables complex mechanisms for the post-transcriptional control of gene expression. Regulation of alternative splicing entails a combinatorial interplay between an abundance of trans-acting splicing factors, cis-acting regulatory sequence elements and their concerted effects on the core splicing machinery. Given the extent and biological significance of alternative splicing in humans, it is not surprising that aberrant splicing patterns can cause or contribute to a wide range of diseases. In this introductory chapter, we outline the mechanisms that govern alternative pre-mRNA splicing and its regulation and discuss how dysregulated splicing contributes to human diseases affecting the motor system and the brain.

Long-range promoter-enhancer contacts are conserved during evolution and contribute to gene expression robustness.

Gene expression is regulated through complex molecular interactions, involving cis-acting elements that can be situated far away from their target genes. Data on long-range contacts between promoters and regulatory elements are rapidly accumulating. However, it remains unclear how these regulatory relationships evolve and how they contribute to the establishment of robust gene expression profiles. Here, we address these questions by comparing genome-wide maps of promoter-centered chromatin contacts in mouse and human. We show that there is significant evolutionary conservation of cis-regulatory landscapes, indicating that selective pressures act to preserve not only regulatory element sequences but also their chromatin contacts with target genes. The extent of evolutionary conservation is remarkable for long-range promoter–enhancer contacts, illustrating how the structure of regulatory landscapes constrains large-scale genome evolution. We show that the evolution of cis-regulatory landscapes, measured in terms of distal element sequences, synteny, or contacts with target genes, is significantly associated with gene expression evolution.

Improved Gene Therapy for Metachromatic Leukodystrophy

Metachromatic Leukodystrophy (MLD) is an autosomal recessive lysosomal storage disease (LSD) characterized by a decreased Arylsulfatatse A (ARSA) enzymatic activity. The most common form, late infantile MLD, universally results in rapid loss of neurologic function in early childhood. Ex-vivo hematopoietic stem cell (HSC) gene therapy using a lentiviral vector (LV) can improve clinical outcomes by supplying a functional copy of the ARSA cDNA (Biffi A, et al, Science 2013). Unfortunately, this approach is only successful in pre- and minimally symptomatic individuals and only a small subset of individuals are diagnosed during the limited therapeutic window. As such, the development of additional approaches targeting early symptomatic individuals are critically needed.The only clinical vector (CV) approved to treat MLD patients, PawMut6, includes the human ARSA cDNA gene under the control of the human Phosphoglycerate Kinase (PGK) promoter and includes, in the integrating transcriptional unit, the viral sequences Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) sequence to increase titer and mRNA translation (Biffi A, et al, Science 2013). To increase expression of ARSA cDNA at single integration level, we generated several LVs that include the ARSA gene with a variety of insulators to optimize ARSA expression and enhance safety in transduced cell lines. We placed the ARSA cDNA under the control of the human Elongation Factor 1 alpha (EF1-alpha) promoter, which has been shown to promote higher transcription rates in different cell lines, compared to human PGK as shown by Jane Yuxia Qin, PLos One 2010. Our constructs carry versions of the ARSA gene with and without the 5' and 3' untranslated regions (UTR+ or UTR-) and a Traceable Codon Optimized (TCO) modified sequence to distinguish the transgene from the endogenous ARSA. An ankyrin or foamy insulator have been incorporated to minimize genotoxicity caused by integration events. The WPRE has been proven to enhance the performance of viral vectors. However, to prevent WPRE integration in the host genome, we placed it directly after the 3'-self inactivating LTR (SIN-LTR) together with a strong bovine growth hormone polyA signal (for sequence termination) (BGHpA), as shown by Breda L. et al, Mol Ther 2021.We compared the ARSA activity (normalized to vector copy number (VCN)) of our constructs to that of PawMut6, the LV currently used in clinical trial, on MLD primary patient fibroblast cultures. Our top performing vectors, TCO-EAAWP-UTR +, TCO-EAFWP-UTR - and TCO-AEAFWP-UTR - showed 2X, 10X and 4X more ARSA activity, respectively, compared to that generated using PawMut6. We also detected a superior ability of our vectors to secrete functional ARSA enzyme into the culture media of transduced primary MLD patient fibroblast cells, which is a critical modality for transfer of functional ARSA from microglia to oligodendrocytes. Extracellular vesicle isolation, purification, and immunoblot analysis has demonstrated small vesicle secretion is the primary modality by which ARSA is secreted, having significant implication for how we approach treatment of MLD. In parallel experiments on murine HSC, the TCO-AEAFWP-UTR - vector reproduced similar results, with about 4x more ARSA activity. To exclude potential toxicity, we performed bone marrow transplants on WT animals with HSCs transduced at up to 13 copies per genome. Mice transplanted with high VCN transduced bone marrow did not show signs of bone marrow failure or distress; more extensive evaluation of these animal models is ongoing. Clonogenic assays and secondary transplants are in progress. Upon completion of the in-vivo studies in WT mice, at least two of our best vector candidates will be utilized on a MLD mouse model (ARSA-KO) that we generated using CRISPR-Cas9. Analysis will include pathological sections of the CNS, brain lysate collection and sulfatase activity assays. Our studies are currently focused on completing in-vivo validation and toxicity assays to move our best vector to the pre-clinical and IND application. The accumulated data on our novel vectors imply new mechanistic considerations for treatment of MLD and demonstrate utility as a strong approach for treating early symptomatic patients. DisclosuresVanderver: Homology: Research Funding; Takeda: Research Funding; Ionis and Illumina Inc: Research Funding; Biogen: Research Funding; Eli Lily and Company: Research Funding; Orchard Therapeutics: Research Funding; Gilead Sciences Inc: Research Funding. Adang: MEGMA: Consultancy; Orchard Therapeutics: Consultancy; Takeda Pharmaceuticals: Consultancy. Rivella: Keros Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Forma Theraputics: Consultancy; MeiraGTx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Consultancy.

LncRNAs in development and differentiation: from sequence motifs to functional characterization

The number of long noncoding RNAs (lncRNAs) with characterized developmental and cellular functions continues to increase, but our understanding of the molecular mechanisms underlying lncRNA functions, and how they are dictated by RNA sequences, remains limited. Relatively short, conserved sequence motifs embedded in lncRNA transcripts are often important determinants of lncRNA localization, stability and interactions. Identifying such RNA motifs remains challenging due to the substantial length of lncRNA transcripts and the rapid evolutionary turnover of lncRNA sequences. Nevertheless, the recent discovery of specific RNA elements, together with their experimental interrogation, has enabled the first step in classifying heterogeneous lncRNAs into sub-groups with similar molecular mechanisms and functions. In this Review, we focus on lncRNAs with roles in development, cell differentiation and normal physiology in vertebrates, and we discuss the sequence elements defining their functions. We also summarize progress on the discovery of regulatory RNA sequence elements, as well as their molecular functions and interaction partners.

Epstein-Barr virus-positive gastric cancer involves enhancer activation through activating transcription factor 3.

Epstein‐Barr virus (EBV) is associated with particular forms of gastric cancer (GC). We previously showed that EBV infection into gastric epithelial cells induced aberrant DNA hypermethylation in promoter regions and silencing of tumor suppressor genes. We here undertook integrated analyses of transcriptome and epigenome alteration during EBV infection in gastric cells, to investigate activation of enhancer regions and related transcription factors (TFs) that could contribute to tumorigenesis. Formaldehyde‐assisted isolation of regulatory elements (FAIRE) sequencing (‐seq) data revealed 19 992 open chromatin regions in putative H3K4me1+ H3K4me3− enhancers in EBV‐infected MKN7 cells (MKN7_EB), with 10 260 regions showing increase of H3K27ac. Motif analysis showed candidate TFs, eg activating transcription factor 3 (ATF3), to possibly bind to these activated enhancers. ATF3 was considerably upregulated in MKN7_EB due to EBV factors including EBV‐determined nuclear antigen 1 (EBNA1), EBV‐encoded RNA 1, and latent membrane protein 2A. Expression of mutant EBNA1 decreased copy number of the EBV genome, resulting in relative downregulation of ATF3 expression. Epstein‐Barr virus was also infected into normal gastric epithelial cells, GES1, confirming upregulation of ATF3. Chromatin immunoprecipitation‐seq analysis on ATF3 binding sites and RNA‐seq analysis on ATF3 knocked‐down MKN7_EB revealed 96 genes targeted by ATF3‐activating enhancers, which are related with cancer hallmarks, eg evading growth suppressors. These 96 ATF3 target genes were significantly upregulated in MKN7_EB compared with MKN7 and significantly downregulated when ATF3 was knocked down in EBV‐positive GC cells SNU719 and NCC24. Knockdown of ATF3 in EBV‐infected MKN7, SNU719, and NCC24 cells all led to significant decrease of cellular growth through an increase of apoptotic cells. These indicate that enhancer activation though ATF3 might contribute to tumorigenesis of EBV‐positive GC.

Cationic porphyrins as destabilizer of a G-quadruplex located at the promoter of human MYH7 β gene

G-quadruplex (GQ) architecture is adopted by guanine rich sequences, present throughout the eukaryotic genome including promoter locations and telomeric ends. The in vivo presence indicates their involvement and role in various biological processes. Various small ligands have been developed to interact and stabilize/destabilize G-quadruplex structures. Cationic porphyrins are among the most studied ligands, reported to bind and stabilize G-quadruplexes. Herein, we report the recognition and destabilization of a parallel G-quadruplex by porphyrins (TMPyP3 and TMPyP4). This G-quadruplex forming 23-nt G-rich sequence is in the promoter region of Human Myosin Heavy Chain β gene (MYH7β). Presence of various putative regulatory sequence elements (TATA Box, CCAAT, SP-1) located in the vicinity of this quadruplex motif, highlight its regulatory implications. Biophysical methods as Circular Dichroism Spectroscopy, UV-Absorption Spectroscopy, UV-Thermal Denaturation and Fluorescence Spectroscopy (steady as well as Time Resolved) have been used for studying the interaction and binding parameters. It is proposed that porphyrins have a destabilizing effect on the G-quadruplexes with parallel topology and a stronger binding specifically via intercalation mode is needed to cause destabilization. The study deals with better understanding and insights of DNA-Drug interactions in biological systems.Communicated by Ramaswamy H. Sarma

Abstract AP11: IDENTIFYING FUNCTIONAL NONCODING SOMATIC MUTATIONS IN OVARIAN CANCER

Abstract Epithelial ovarian cancer (OC) is a heterogeneous disease that stratifies into different histologic subtypes including high-grade serous (HGSOC), clear cell (CCOC), endometrioid (EnOC) and mucinous (MOC) ovarian cancer. HGSOC is the most common, and each histotype is characterized by largely distinct germline genetics, somatic alterations and clinical biomarkers. Recently, whole genome sequencing (WGS) studies have catalogued genome-wide somatic variation for most OC histotypes. These data demonstrate that OC histotypes harbor thousands of noncoding somatic mutations and our next major challenge is to distinguish the few important noncoding somatic drivers from the thousands of passenger mutations. We hypothesized that driver noncoding somatic mutations impact disease development and progression through altering the sequence of regulatory elements (REs), such as enhancers and promoters, resulting in perturbed expression of genes involved in neoplastic development of OC. To systematically address this hypothesis, we established genome-wide H3K27ac epigenomic profiles, annotating active REs for the different ovarian cancer histotypes using chromatin immunoprecipitation sequencing (ChIP-seq) in 20 fresh frozen primary OC tissue samples—five tumors for each major histotype. In parallel, we performed transcriptional profiling using RNA sequencing (RNA-seq). Together, these two datasets enabled us to evaluate epigenetic alterations and the transcriptome. We identified histotype-specific active REs, and common active REs across all histotypes. First, we used the RNA-seq data to assess the effect of the histotype-specific REs in gene expression, and to find target genes of cis-REs and novel histotype-specific biomarkers. Next, we integrated these unique profiles with WGS data from 232 OCs (169 HGSOCs, 35 CCOCs and 28 EnOCs). The number of somatic single nucleotide variants per sample range from 481 to 40,764 (mean=7199, sd = 5751). Of these, 9.4% were noncoding and overlapped active REs in OC. Using a Poisson binomial distribution, we tested the significance of the observed number of mutated samples for any given active RE. We identified several frequently mutated REs in HGSOC, CCOC and EnOC, including promoters (POLR3E and CHCHD6 for HGSOC, CA5BP1 and C16orf87 for EnOC, DKC1 and SLIT3 for CCOC+EnOC), enhancers and super enhancers (KLF6 for HGSOC). We developed a novel approach to identifying target genes associated with a collection of frequently mutated enhancers by correlating enhancer activity (H3K27ac ChIP-Seq signal) vs. gene expression (RNA-seq signal), identifying HOXD cluster genes and C19orf44 as targets of mutated REs in OC. In conclusion, we have used a multi-omics method to identify functional, driver noncoding somatic mutations for ovarian cancer based on their interaction with disease-specific regulatory elements. This represents a powerful way to distinguish important noncoding somatic drivers from a much larger number of passenger mutations that accumulate during tumor development. Citation Format: Rosario I. Corona, Ji-Heui Seo, Dennis J. Hazelett, Xianzhi Lin, Paulette Y. Mhawech-Fauceglia, Jenny Lester, Sohrab P. Shah, David G. Huntsman, Beth Y. Karlan, Alexander Gusev, Benjamin P. Berman, Matthew L. Freedman, Simon A. Gayther, Kate Lawrenson. IDENTIFYING FUNCTIONAL NONCODING SOMATIC MUTATIONS IN OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP11.

Atypical chromatin structure of immune-related genes expressed in chicken erythrocytes.

The major biological role of red blood cells is to carry oxygen to the tissues in the body. However, another role of the erythroid cell is to participate in the immune response. Mature erythrocytes from chickens express Toll-like receptors and several cytokines in response to stimulation of the immune system. We previously reported the application of a biochemical fractionation protocol to isolate highly enriched transcribed DNA from polychromatic erythrocytes from chickens. In conjunction with next-generation DNA, RNA sequencing, chromatin immunoprecipitation-DNA sequencing, and formaldehyde-assisted isolation of regulatory elements (FAIRE) sequencing, we identified the active chromosomal compartments and determined their structural signatures in relation to expression levels. Here, we present the detailed chromatin characteristics of erythroid genes participating in the innate immune response. Our studies revealed an atypical chromatin structure for several genes coding for Toll-like receptors, interleukins, and interferon regulatory factors. The body of these genes had nucleosome-free regions intermingled with nucleosomes modified with H3K4me3 and H3K27ac, suggesting a dynamic unstable chromatin structure. We further show that human genes involved in cell identity have gene bodies with the same chromatin-instability features as the chicken polychromatic erythrocyte genes participating in the innate immune response.

Using secondary structure to predict the effects of genetic variants on alternative splicing.

Accurate interpretation of genomic variants that alter RNA splicing is critical to precision medicine. We present a computational framework, Prediction of variant Effect on Percent Spliced In (PEPSI), that predicts the splicing impact of coding and noncoding variants for the Fifth Critical Assessment of Genome Interpretation (CAGI5) "Vex-seq" challenge. PEPSI is a random forest regression model trained on multiple layers of features associated with sequence conservation and regulatory sequence elements. Compared to other splicing defect prediction tools from the literature, our framework integrates secondary structure information in predicting variants that disrupt splicing regulatory elements (SREs). We applied our model to classify splice-disrupting variants among 2,094 single-nucleotide polymorphisms from the Exome Aggregation Consortium using model-predicted changes in percent spliced in (ΔPSI) associated with tested variants. Benchmarking our model against widely used state-of-the-art tools, we demonstrate that PEPSI achieves comparable performance in terms of sensitivity and precision. Moreover, we also show that using secondary structure context can help resolve several cases where changes in the counts of SREs do not correspond with the directionality of ΔPSI measured for tested variants.

Differential repression of the Ve R-genes in tomato

Resistance to Verticillium wilt disease has been associated with the tomato Ve R-gene locus that comprises two genes, Ve1 and Ve2. Previous transcript analyses revealed that the tomato Ve genes are expressed differentially and that Ve1 induction by pathogen colonization mitigates the Ve2-induced defense/stress cascade. Using a promoter-reporter assay, we have sought to map regulatory sequence elements that may be responsible for Ve gene regulation. Systematic deletion mutations of the shared upstream intergenic region between Ve1 and Ve2 were fused to an EYFP reporter sequence and plant transformants expressing these constructs were selected. We showed that both functional Ve1 and Ve2 promoter regions have similar transcriptional activity, comparable to endogenous Ve2 transcript levels, suggesting that more distant regulatory elements or chromatin mechanism normally repress Ve1 promoter activity and/or influence encoded endogenous mRNA levels.

Abstract 395: Identifying the functional drivers of noncoding somatic mutations in ovarian cancer

Abstract Epithelial ovarian cancer (OC) is a heterogeneous disease that has been stratified into different histologic subtypes: high-grade serous (HGSOC), clear cell (CCOC), endometrioid (EnOC) and mucinous (MOC). HGSOC is the most common, but every histotype is characterized by largely distinct germline genetics, somatic alterations and clinical biomarkers. Recently, whole-genome sequencing (WGS) studies have catalogued genome-wide somatic variation for most OC histotypes. These data demonstrate that OC histotypes harbor thousands of noncoding somatic mutations and our next major challenge is to distinguish the few important noncoding somatic drivers from the thousands of passenger mutations. We hypothesized that driver noncoding somatic mutations impact disease development and progression through altering the sequence of regulatory elements (REs), such as enhancers and promoters, eventually resulting in perturbation of the expression of target genes. To systematically address this hypothesis, we established genome-wide H3K27ac epigenomic profiles, annotating active REs for the different ovarian cancer histotypes using chromatin immunoprecipitation sequencing (ChIP-seq) in 20 fresh frozen primary OC tissue samples—five tumors for each major histotype. In parallel, we performed transcriptional profiling using RNA sequencing (RNA-seq). Together, these two datasets enabled us to evaluate epigenetic alterations and the transcriptome. We identified histotype-specific active REs, and common active REs across all histotypes. We used the RNA-seq data to assess the effect of the histotype-specific REs in gene expression, and to find target genes of cis-REs and novel histotype-specific biomarkers. Next, we integrated these unique profiles with WGS data from 232 OCs (169 HGSOCs, 35 CCOCs and 28 EnOCs). The number of somatic single-nucleotide variants per sample range from 481 to 40,764 (mean=7199, sd = 5751). Of these, 9.4% were noncoding and overlapped active REs in OC. Using a Poisson binomial distribution, we tested the significance of the observed number of mutated samples for any given active RE. Importantly, the method adjusted for interpatient mutation rate heterogeneity. Using the Benjamini-Hochberg procedure for p-value correction, we identified several significantly mutated active REs, including the promoters of POLR3E and WDR74. In conclusion, we have used an integrative method to identify functional, driver noncoding somatic mutations for ovarian cancer based on their interaction with disease-specific regulatory elements, and their putative target genes. This represents a powerful way to distinguish important noncoding somatic drivers from a much larger number of passenger mutations that accumulate during tumor development. The approach can be applied to other cancer types if sufficient depth and quality of genetic, transcriptomic and epigenomic datasets are available. Citation Format: Rosario I. Corona, Ji-Heui Seo, Dennis J. Hazelett, Xianzhi Lin, Paulette Y. Mhawech-Fauceglia, Jenny Lester, Sohrab Shah, David G. Huntsman, Beth Y. Karlan, Benjamin P. Berman, Matthew L. Freedman, Simon A. Gayther, Kate Lawrenson. Identifying the functional drivers of noncoding somatic mutations in ovarian cancer [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 395.

In vivo response of the human epigenome to vitamin D: A Proof-of-principle study

In vitro cell culture studies showed that the hormonal form of vitamin D3, 1α,25-dihydroxyvitamin D3, significantly (p < 0.05) affects the human epigenome at thousands of genomic loci. Phase II of the VitDbol vitamin D intervention trial (NCT02063334) involved a proof-of-principle study of one individual, who was exposed three times every 28 days to an oral bolus (2000 μg) of vitamin D3. Blood samples were taken directly before each supplementation as well as one and two days after, chromatin was isolated from peripheral blood mononuclear cells without any further in vitro culture and at all nine time points epigenome-wide chromatin accessibility was assessed by applying FAIRE-seq (formaldehyde-assisted isolation of regulatory elements sequencing). The vitamin D3 bolus resulted in an average raise in 25-hydroxyvitamin D3 (25(OH)D3) serum concentration of 11.9 and 19.4 nM within one and two days, respectively. Consistently accessible chromatin was detected at 5205 genomic loci, the 853 most prominent of which a self-organizing map algorithm classified into early, delayed and non-responding genomic regions: 70 loci showed already after one day and 361 sites after two days significant (p < 0.0001) chromatin opening or closing. Interestingly, more than half of these genomic regions overlap with transcription start sites, but the change of chromatin accessibility at these sites has no direct effect on the transcriptome. Some of the vitamin D responsive chromatin sites cluster at specific loci within the human genome, the most prominent of which is the human leukocyte antigen region in chromosome 6. In conclusion, this study demonstrates that under in vivo conditions a rather minor rise in 25(OH)D3 serum levels is sufficient to result in significant changes at hundreds of sites within the epigenome of human leukocytes.