Non-coding Regulatory Elements Research Articles

Functional Characterization of Insulation Effect for Synthetic Gene Circuits in Mammalian Cells.

Insulators are noncoding gene regulatory elements in eukaryotic genome, which function as chromatin partitioning boundaries, and block interference across different chromatin domains. To facilitate modular construction of synthetic gene circuit that is usually composed of multiple transcription cassettes, unwanted cross-regulations between different cassettes should be avoided. Here, we developed a quantitative method to characterize the functional effect of three insulators on the cross-regulations of six promoters in mammalian cells. We showed that the unwanted cross-regulations displayed a threshold-like effect, and the threshold position varied along with the context of promoters and insulators. We tested the function of insulators in both cascade and sensory switch circuits assembled in episomal plasmid vectors, and showed that the insulation effect was mainly revealed on the first regulatory layer of the cascade circuit. A deviation on the response curve of the sensory switch circuit with or without insulators was observed, but response intensity of some sensory switch circuits were not affected. Therefore, our results provided a general guide on the selection of insulators with varying promoters in episomal synthetic gene circuits in mammalian cells, which may be useful to reduce the effect of the unwanted cross-regulations.

Regulation of a muralytic enzyme-encoding gene by two non-coding RNAs

ABSTRACTNon-coding regulatory RNAs fine-tune gene expression post-transcriptionally. In the streptomycetes, rpfA – encoding a muralytic enzyme required for establishing and exiting dormancy – is flanked by non-coding regulatory RNA elements both upstream (riboswitch) and downstream [antisense small RNA (sRNA)]. In Streptomyces coelicolor, the upstream riboswitch decreases rpfA transcript abundance in response to the second messenger cyclic di-AMP, itself involved in cell wall metabolism and dormancy. There is, however, no obvious expression platform associated with this riboswitch and consequently, its mechanism of action is entirely unknown. Using in vitro transcription assays, we discovered that the rpfA riboswitch promoted premature transcription termination in response to cyclic di-AMP. Through an extensive mutational analysis, we determined that attenuation required ligand binding and involved an unusual extended stem-loop region unique to a subset of rpfA riboswitches in the actinobacteria. At the other end of the rpfA gene, an antisense sRNA, termed Scr3097, is expressed opposite the predicted rpfA terminator. Using northern blotting, we found that Scr3097 accumulation mirrored that of the rpfA mRNA. In liquid culture, we detected Scr3097 exclusively in exponential-phase cells, and in plate-grown culture, we observed the sRNA primarily in differentiating cultures. Using mutational analyses, we found that the sRNA increased rpfA mRNA abundance in cells. Taken together, our work revealed multiple regulatory RNAs controlling rpfA expression in the streptomycetes.

Noncoding copy-number variations are associated with congenital limb malformation.

PurposeCopy-number variants (CNVs) are generally interpreted by linking the effects of gene dosage with phenotypes. The clinical interpretation of noncoding CNVs remains challenging. We investigated the percentage of disease-associated CNVs in patients with congenital limb malformations that affect noncoding cis-regulatory sequences versus genes sensitive to gene dosage effects.MethodsWe applied high-resolution copy-number analysis to 340 unrelated individuals with isolated limb malformation. To investigate novel candidate CNVs, we re-engineered human CNVs in mice using clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing.ResultsOf the individuals studied, 10% harbored CNVs segregating with the phenotype in the affected families. We identified 31 CNVs previously associated with congenital limb malformations and four novel candidate CNVs. Most of the disease-associated CNVs (57%) affected the noncoding cis-regulatory genome, while only 43% included a known disease gene and were likely to result from gene dosage effects. In transgenic mice harboring four novel candidate CNVs, we observed altered gene expression in all cases, indicating that the CNVs had a regulatory effect either by changing the enhancer dosage or altering the topological associating domain architecture of the genome.ConclusionOur findings suggest that CNVs affecting noncoding regulatory elements are a major cause of congenital limb malformations.

A variant associated with sagittal nonsyndromic craniosynostosis alters the regulatory function of a non-coding element.

Craniosynostosis presents either as a nonsyndromic congenital anomaly or as a finding in nearly 200 genetic syndromes. Our previous genome-wide association study of sagittal nonsyndromic craniosynostosis identified associations with variants downstream from BMP2 and intronic in BBS9. Because no coding variants in BMP2 were identified, we hypothesized that conserved non-coding regulatory elements may alter BMP2 expression. In order to identify and characterize noncoding regulatory elements near BMP2, two conserved noncoding regions near the associated region on chromosome 20 were tested for regulatory activity with a Renilla luciferase assay. For a 711 base pair noncoding fragment encompassing the most strongly associated variant, rs1884302, the luciferase assay showed that the risk allele (C) of rs1884302 drives higher expression of the reporter than the common allele (T). When this same DNA fragment was tested in zebrafish transgenesis studies, a strikingly different expression pattern of the green fluorescent reporter was observed depending on whether the transgenic fish had the risk (C) or the common (T) allele at rs1884302. The in vitro results suggest that altered BMP2 regulatory function at rs1884302 may contribute to the etiology of sagittal nonsyndromic craniosynostosis. The in vivo results indicate that differences in regulatory activity depend on the presence of a C or T allele at rs1884302.

Combining molecular information on chromatin organisation with eQTLs and evolutionary conservation provides strong candidates for the evolution of gene regulation in mammalian brains

Many non-coding regulatory elements conserved in vertebrates regulate the expression of genes involved in development and play an important role in the evolution of morphology through the rewiring of developmental gene networks. Available biological datasets allow the identification of non-coding regulatory elements with high confidence; furthermore, chromatin conformation data can be used to confirm enhancer-promoter interactions in specific tissue types and developmental stages. We have devised an analysis pipeline that integrates datasets about gene expression, enhancer activity, chromatin accessibility, epigenetic marks, and Hi-C contact frequencies in various brain tissues and developmental stages, leading to the identification of eight non-coding elements that might regulate the expression of three genes with important roles in brain development in vertebrates. We have then performed comparative sequence and microsynteny analyses in order to reconstruct the evolutionary history of the regulatory landscape around these genes; we observe a general pattern of ancient regulatory elements conserved across most vertebrate lineages, together with younger elements that appear to be mammal and primate innovations.

An erythroid-specific ATP2B4 enhancer mediates red blood cell hydration and malaria susceptibility.

The lack of mechanistic explanations for many genotype-phenotype associations identified by GWAS precludes thorough assessment of their impact on human health. Here, we conducted an expression quantitative trait locus (eQTL) mapping analysis in erythroblasts and found erythroid-specific eQTLs for ATP2B4, the main calcium ATPase of red blood cells (rbc). The same SNPs were previously associated with mean corpuscular hemoglobin concentration (MCHC) and susceptibility to severe malaria infection. We showed that Atp2b4-/- mice demonstrate increased MCHC, confirming ATP2B4 as the causal gene at this GWAS locus. Using CRISPR-Cas9, we fine mapped the genetic signal to an erythroid-specific enhancer of ATP2B4. Erythroid cells with a deletion of the ATP2B4 enhancer had abnormally high intracellular calcium levels. These results illustrate the power of combined transcriptomic, epigenomic, and genome-editing approaches in characterizing noncoding regulatory elements in phenotype-relevant cells. Our study supports ATP2B4 as a potential target for modulating rbc hydration in erythroid disorders and malaria infection.

Connected Gene Communities Underlie Transcriptional Changes in Cornelia de Lange Syndrome.

Cornelia de Lange syndrome (CdLS) is a complex multisystem developmental disorder caused by mutations in cohesin subunits and regulators. While its precise molecular mechanisms are not well defined, they point toward a global deregulation of the transcriptional gene expression program. Cohesin is associated with the boundaries of chromosome domains and with enhancer and promoter regions connecting the three-dimensional genome organization with transcriptional regulation. Here, we show that connected gene communities, structures emerging from the interactions of noncoding regulatory elements and genes in the three-dimensional chromosomal space, provide a molecular explanation for the pathoetiology of CdLS associated with mutations in the cohesin-loading factor NIPBL and the cohesin subunit SMC1A NIPBL and cohesin are important constituents of connected gene communities that are centrally positioned at noncoding regulatory elements. Accordingly, genes deregulated in CdLS are positioned within reach of NIPBL- and cohesin-occupied regions through promoter-promoter interactions. Our findings suggest a dynamic model where NIPBL loads cohesin to connect genes in communities, offering an explanation for the gene expression deregulation in the CdLS.

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

Inherited determinants of early recurrent somatic mutations in prostate cancer

Prostate cancer is a highly heritable molecularly and clinically heterogeneous disease. To discover germline events involved in prostate cancer predisposition, we develop a computational approach to nominate heritable facilitators of somatic genomic events in the context of the androgen receptor signaling. Here, we use a ranking score and benign prostate transcriptomes to identify a non-coding polymorphic regulatory element at 7p14.3 that associates with DNA repair and hormone-regulated transcript levels and with an early recurrent prostate cancer-specific somatic mutation in the Speckle-Type POZ protein (SPOP) gene. The locus shows allele-specific activity that is concomitantly modulated by androgen receptor and by CCAAT/enhancer-binding protein (C/EBP) beta (CEBPB). Deletion of this locus via CRISPR-Cas9 leads to deregulation of the genes predicted to interact with the 7p14.3 locus by Hi-C chromosome conformation capture data. This study suggests that a polymorphism at 7p14.3 may predispose to SPOP mutant prostate cancer subclass through a hormone-dependent DNA damage response.

Whole exome sequencing of sporadic patients with Currarino Syndrome: A report of three trios

Currarino Syndrome is a rare congenital malformation syndrome described as a triad of anorectal, sacral and presacral anomalies. Currarino Syndrome is reported to be both familial and sporadic. Familial CS is today known as an autosomal dominant disorder caused by mutations in the transcription factor MNX1. The aim of this study was to look for genetic causes of Currarino Syndrome in sporadic patients after ruling out other causes, like chromosome aberrations, disease-causing variants in possible MNX1 cooperating transcription factors and aberrant methylation in the promoter of the MNX1 gene. The hypothesis was that MNX1 was affected through interactions with other transcription factors or through other regulatory elements and thereby possibly leading to abnormal function of the gene. We performed whole exome sequencing with an additional 6Mb custom made region on chromosome 7 (GRCh37/hg19, chr7:153.138.664–159.138.663) to detect regulatory elements in non-coding regions around the MNX1 gene. We did not find any variants in genes of interest shared between the patients. However, after analyzing the whole exome sequencing data with Filtus, the in-house SNV filtration program, we did find some interesting variants in possibly relevant genes that could be explaining these patients` phenotypes. The most promising genes were ETV3L, ARID5A and NCAPD3. To our knowledge this is the first report of whole exome sequencing in sporadic CS patients.

Super-Enhancer-Driven Transcriptional Dependencies in Cancer

Transcriptional deregulation is one of the core tenets of cancer biology and is underpinned by alterations in both protein-coding genes and noncoding regulatory elements. Large regulatory elements, so-called super-enhancers (SEs), are central to the maintenance of cancer cell identity and promote oncogenic transcription to which cancer cells become highly addicted. Such dependence on SE-driven transcription for proliferation and survival offers an Achilles heel for the therapeutic targeting of cancer cells. Indeed, inhibition of the cellular machinery required for the assembly and maintenance of SEs dampens oncogenic transcription and inhibits tumor growth. In this article, we review the organization, function, and regulation of oncogenic SEs and their contribution to the cancer cell state.

Genetic variants alter T-bet binding and gene expression in mucosal inflammatory disease.

The polarization of CD4+ T cells into distinct T helper cell lineages is essential for protective immunity against infection, but aberrant T cell polarization can cause autoimmunity. The transcription factor T-bet (TBX21) specifies the Th1 lineage and represses alternative T cell fates. Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) that may be causative for autoimmune diseases. The majority of these polymorphisms are located within non-coding distal regulatory elements. It is considered that these genetic variants contribute to disease by altering the binding of regulatory proteins and thus gene expression, but whether these variants alter the binding of lineage-specifying transcription factors has not been determined. Here, we show that SNPs associated with the mucosal inflammatory diseases Crohn’s disease, ulcerative colitis (UC) and celiac disease, but not rheumatoid arthritis or psoriasis, are enriched at T-bet binding sites. Furthermore, we identify disease-associated variants that alter T-bet binding in vitro and in vivo. ChIP-seq for T-bet in individuals heterozygous for the celiac disease-associated SNPs rs1465321 and rs2058622 and the IBD-associated SNPs rs1551398 and rs1551399, reveals decreased binding to the minor disease-associated alleles. Furthermore, we show that rs1465321 is an expression quantitative trait locus (eQTL) for the neighboring gene IL18RAP, with decreased T-bet binding associated with decreased expression of this gene. These results suggest that genetic polymorphisms may predispose individuals to mucosal autoimmune disease through alterations in T-bet binding. Other disease-associated variants may similarly act by modulating the binding of lineage-specifying transcription factors in a tissue-selective and disease-specific manner.

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.

Dense sgRNA Library Construction Using a Molecular Chipper Approach.

Genetic screens using single-guide-RNA (sgRNA) libraries and CRISPR technology have been powerful to identify genetic regulators for both coding and noncoding regions of the genome. Interrogating functional elements in noncoding regions requires sgRNA libraries that are densely covering, and ideally inexpensive, easy to implement and flexible for customization. We present a Molecular Chipper protocol for generating dense sgRNA libraries from genomic regions of interest. This approach utilizes a combination of random fragmentation and a Type III restriction enzyme to derive a dense coverage of sgRNA library from input DNA.

Genetic Marker Discovery in Complex Traits: A Field Example on Fat Content and Composition in Pigs.

Among the large number of attributes that define pork quality, fat content and composition have attracted the attention of breeders in the recent years due to their interaction with human health and technological and sensorial properties of meat. In livestock species, fat accumulates in different depots following a temporal pattern that is also recognized in humans. Intramuscular fat deposition rate and fatty acid composition change with life. Despite indication that it might be possible to select for intramuscular fat without affecting other fat depots, to date only one depot-specific genetic marker (PCK1 c.2456C>A) has been reported. In contrast, identification of polymorphisms related to fat composition has been more successful. For instance, our group has described a variant in the stearoyl-coA desaturase (SCD) gene that improves the desaturation index of fat without affecting overall fatness or growth. Identification of mutations in candidate genes can be a tedious and costly process. Genome-wide association studies can help in narrowing down the number of candidate genes by highlighting those which contribute most to the genetic variation of the trait. Results from our group and others indicate that fat content and composition are highly polygenic and that very few genes explain more than 5% of the variance of the trait. Moreover, as the complexity of the genome emerges, the role of non-coding genes and regulatory elements cannot be disregarded. Prediction of breeding values from genomic data is discussed in comparison with conventional best linear predictors of breeding values. An example based on real data is given, and the implications in phenotype prediction are discussed in detail. The benefits and limitations of using large SNP sets versus a few very informative markers as predictors of genetic merit of breeding candidates are evaluated using field data as an example.

Systematic analysis of chromatin interactions at disease associated loci links novel candidate genes to inflammatory bowel disease.

BackgroundGenome-wide association studies (GWAS) have revealed many susceptibility loci for complex genetic diseases. For most loci, the causal genes have not been identified. Currently, the identification of candidate genes is predominantly based on genes that localize close to or within identified loci. We have recently shown that 92 of the 163 inflammatory bowel disease (IBD)-loci co-localize with non-coding DNA regulatory elements (DREs). Mutations in DREs can contribute to IBD pathogenesis through dysregulation of gene expression. Consequently, genes that are regulated by these 92 DREs are to be considered as candidate genes. This study uses circular chromosome conformation capture-sequencing (4C-seq) to systematically analyze chromatin-interactions at IBD susceptibility loci that localize to regulatory DNA.ResultsUsing 4C-seq, we identify genomic regions that physically interact with the 92 DRE that were found at IBD susceptibility loci. Since the activity of regulatory elements is cell-type specific, 4C-seq was performed in monocytes, lymphocytes, and intestinal epithelial cells. Altogether, we identified 902 novel IBD candidate genes. These include genes specific for IBD-subtypes and many noteworthy genes including ATG9A and IL10RA. We show that expression of many novel candidate genes is genotype-dependent and that these genes are upregulated during intestinal inflammation in IBD. Furthermore, we identify HNF4α as a potential key upstream regulator of IBD candidate genes.ConclusionsWe reveal many novel and relevant IBD candidate genes, pathways, and regulators. Our approach complements classical candidate gene identification, links novel genes to IBD and can be applied to any existing GWAS data.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1100-3) contains supplementary material, which is available to authorized users.

Chromosome conformation elucidates regulatory relationships in developing human brain.

Three-dimensional physical interactions within chromosomes dynamically regulate gene expression in a tissue-specific manner. However, the 3D organization of chromosomes during human brain development and its role in regulating gene networks dysregulated in neurodevelopmental disorders, such as autism or schizophrenia, are unknown. Here we generate high-resolution 3D maps of chromatin contacts during human corticogenesis, permitting large-scale annotation of previously uncharacterized regulatory relationships relevant to the evolution of human cognition and disease. Our analyses identify hundreds of genes that physically interact with enhancers gained on the human lineage, many of which are under purifying selection and associated with human cognitive function. We integrate chromatin contacts with non-coding variants identified in schizophrenia genome-wide association studies (GWAS), highlighting multiple candidate schizophrenia risk genes and pathways, including transcription factors involved in neurogenesis, and cholinergic signalling molecules, several of which are supported by independent expression quantitative trait loci and gene expression analyses. Genome editing in human neural progenitors suggests that one of these distal schizophrenia GWAS loci regulates FOXG1 expression, supporting its potential role as a schizophrenia risk gene. This work provides a framework for understanding the effect of non-coding regulatory elements on human brain development and the evolution of cognition, and highlights novel mechanisms underlying neuropsychiatric disorders.

Transcriptional Dynamics at Brain Enhancers: from Functional Specialization to Neurodegeneration.

Over the last decade, the noncoding part of the genome has been shown to harbour thousands of cis-regulatory elements, such as enhancers, that activate well-defined gene expression programs. Driven by the development of numerous techniques, many of these elements are now identified in multiple tissues and cell types, and their characteristics as well as importance in development and disease are becoming increasingly clear. Here, we provide an overview of the insights that were gained from the analysis of noncoding gene regulatory elements in the brain and describe their potential contribution to cell type specialization, brain function and neurodegenerative disease.

Applying CRISPR-Cas9 tools to identify and characterize transcriptional enhancers.

The development of the CRISPR-Cas9 system triggered a revolution in the field of genome engineering. Initially, the use of this system was focused on the study of protein-coding genes but, recently, a number of CRISPR-Cas9-based tools have been developed to study non-coding transcriptional regulatory elements. These technological advances offer unprecedented opportunities for elucidating the functions of enhancers in their endogenous context. Here, we discuss the application, current limitations and future development of CRISPR-Cas9 systems to identify and characterize enhancer elements in a high-throughput manner.

QuIN: A Web Server for Querying and Visualizing Chromatin Interaction Networks.

Recent studies of the human genome have indicated that regulatory elements (e.g. promoters and enhancers) at distal genomic locations can interact with each other via chromatin folding and affect gene expression levels. Genomic technologies for mapping interactions between DNA regions, e.g., ChIA-PET and HiC, can generate genome-wide maps of interactions between regulatory elements. These interaction datasets are important resources to infer distal gene targets of non-coding regulatory elements and to facilitate prioritization of critical loci for important cellular functions. With the increasing diversity and complexity of genomic information and public ontologies, making sense of these datasets demands integrative and easy-to-use software tools. Moreover, network representation of chromatin interaction maps enables effective data visualization, integration, and mining. Currently, there is no software that can take full advantage of network theory approaches for the analysis of chromatin interaction datasets. To fill this gap, we developed a web-based application, QuIN, which enables: 1) building and visualizing chromatin interaction networks, 2) annotating networks with user-provided private and publicly available functional genomics and interaction datasets, 3) querying network components based on gene name or chromosome location, and 4) utilizing network based measures to identify and prioritize critical regulatory targets and their direct and indirect interactions. AVAILABILITY: QuIN’s web server is available at http://quin.jax.org QuIN is developed in Java and JavaScript, utilizing an Apache Tomcat web server and MySQL database and the source code is available under the GPLV3 license available on GitHub: https://github.com/UcarLab/QuIN/.