ABSTRACT
Enhancers are distal DNA elements believed to loop and contact promoters to control gene expression. Recently, we found diffraction-sized transcriptional condensates at genes controlled by clusters of enhancers (super-enhancers). However, a direct function of endogenous condensates in controlling gene expression remains elusive. Here, we develop live-cell super-resolution and multi-color 3D-imaging approaches to investigate putative roles of endogenous condensates in the regulation of super-enhancer controlled gene Sox2. In contrast to enhancer distance, we find instead that the condensate's positional dynamics are a better predictor of gene expression. A basal gene bursting occurs when the condensate is far (>1 µm), but burst size and frequency are enhanced when the condensate moves in proximity (<1 µm). Perturbations of cohesin and local DNA elements do not prevent basal bursting but affect the condensate and its burst enhancement. We propose a three-way kissing model whereby the condensate interacts transiently with gene locus and regulatory DNA elements to control gene bursting.
Subject(s)
Gene Expression Regulation , SOXB1 Transcription Factors , Super Enhancers , Transcription, Genetic , DNA/genetics , Enhancer Elements, Genetic , SOXB1 Transcription Factors/genetics , Animals , Mice , Embryonic Stem Cells/metabolism , Microscopy/methodsABSTRACT
Super-enhancers are compound regulatory elements that control expression of key cell identity genes. They recruit high levels of tissue-specific transcription factors and co-activators such as the Mediator complex and contact target gene promoters with high frequency. Most super-enhancers contain multiple constituent regulatory elements, but it is unclear whether these elements have distinct roles in activating target gene expression. Here, by rebuilding the endogenous multipartite α-globin super-enhancer, we show that it contains bioinformatically equivalent but functionally distinct element types: classical enhancers and facilitator elements. Facilitators have no intrinsic enhancer activity, yet in their absence, classical enhancers are unable to fully upregulate their target genes. Without facilitators, classical enhancers exhibit reduced Mediator recruitment, enhancer RNA transcription, and enhancer-promoter interactions. Facilitators are interchangeable but display functional hierarchy based on their position within a multipartite enhancer. Facilitators thus play an important role in potentiating the activity of classical enhancers and ensuring robust activation of target genes.
Subject(s)
Gene Expression Regulation , Super Enhancers , Transcription, Genetic , alpha-Globins , Enhancer Elements, Genetic , Promoter Regions, Genetic , Transcription Factors/metabolism , alpha-Globins/geneticsABSTRACT
The recent development of spatial omics methods has enabled single-cell profiling of the transcriptome and 3D genome organization with high spatial resolution. Expanding the repertoire of spatial omics tools, a spatially resolved single-cell epigenomics method will accelerate understanding of the spatial regulation of cell and tissue functions. Here, we report a method for spatially resolved epigenomic profiling of single cells using in situ tagmentation and transcription followed by multiplexed imaging. We demonstrated the ability to profile histone modifications marking active promoters, putative enhancers, and silent promoters in individual cells, and generated high-resolution spatial atlas of hundreds of active promoters and putative enhancers in embryonic and adult mouse brains. Our results suggested putative promoter-enhancer pairs and enhancer hubs regulating developmentally important genes. We envision this approach will be generally applicable to spatial profiling of epigenetic modifications and DNA-binding proteins, advancing our understanding of how gene expression is spatiotemporally regulated by the epigenome.
Subject(s)
Epigenomics , Histone Code , Mice , Animals , Promoter Regions, Genetic , Epigenesis, Genetic , Transcriptome , Enhancer Elements, Genetic , ChromatinABSTRACT
Regulatory landscapes drive complex developmental gene expression, but it remains unclear how their integrity is maintained when incorporating novel genes and functions during evolution. Here, we investigated how a placental mammal-specific gene, Zfp42, emerged in an ancient vertebrate topologically associated domain (TAD) without adopting or disrupting the conserved expression of its gene, Fat1. In ESCs, physical TAD partitioning separates Zfp42 and Fat1 with distinct local enhancers that drive their independent expression. This separation is driven by chromatin activity and not CTCF/cohesin. In contrast, in embryonic limbs, inactive Zfp42 shares Fat1's intact TAD without responding to active Fat1 enhancers. However, neither Fat1 enhancer-incompatibility nor nuclear envelope-attachment account for Zfp42's unresponsiveness. Rather, Zfp42's promoter is rendered inert to enhancers by context-dependent DNA methylation. Thus, diverse mechanisms enabled the integration of independent Zfp42 regulation in the Fat1 locus. Critically, such regulatory complexity appears common in evolution as, genome wide, most TADs contain multiple independently expressed genes.
Subject(s)
Chromatin , Placenta , Animals , CCCTC-Binding Factor/metabolism , Chromatin Assembly and Disassembly , Enhancer Elements, Genetic , Evolution, Molecular , Female , Genome , Mammals/metabolism , Placenta/metabolism , Pregnancy , Promoter Regions, Genetic , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The primary regulators of metazoan gene expression are enhancers, originally functionally defined as DNA sequences that can activate transcription at promoters in an orientation-independent and distance-independent manner. Despite being crucial for gene regulation in animals, what mechanisms underlie enhancer selectivity for promoters, and more fundamentally, how enhancers interact with promoters and activate transcription, remain poorly understood. In this Review, we first discuss current models of enhancer-promoter interactions in space and time and how enhancers affect transcription activation. Next, we discuss different mechanisms that mediate enhancer selectivity, including repression, biochemical compatibility and regulation of 3D genome structure. Through 3D polymer simulations, we illustrate how the ability of 3D genome folding mechanisms to mediate enhancer selectivity strongly varies for different enhancer-promoter interaction mechanisms. Finally, we discuss how recent technical advances may provide new insights into mechanisms of enhancer-promoter interactions and how technical biases in methods such as Hi-C and Micro-C and imaging techniques may affect their interpretation.
Subject(s)
Enhancer Elements, Genetic , Promoter Regions, Genetic , Enhancer Elements, Genetic/genetics , Promoter Regions, Genetic/genetics , Animals , Humans , Transcriptional Activation/genetics , Gene Expression Regulation/genetics , Chromatin/metabolism , Chromatin/geneticsABSTRACT
Elucidating the regulatory mechanisms of human brain evolution is essential to understanding human cognition and mental disorders. We generated multi-omics profiles and constructed a high-resolution map of 3D genome architecture of rhesus macaque during corticogenesis. By comparing the 3D genomes of human, macaque, and mouse brains, we identified many human-specific chromatin structure changes, including 499 topologically associating domains (TADs) and 1,266 chromatin loops. The human-specific loops are significantly enriched in enhancer-enhancer interactions, and the regulated genes show human-specific expression changes in the subplate, a transient zone of the developing brain critical for neural circuit formation and plasticity. Notably, many human-specific sequence changes are located in the human-specific TAD boundaries and loop anchors, which may generate new transcription factor binding sites and chromatin structures in human. Collectively, the presented data highlight the value of comparative 3D genome analyses in dissecting the regulatory mechanisms of brain development and evolution.
Subject(s)
Brain/embryology , Evolution, Molecular , Fetus/embryology , Genome , Organogenesis/genetics , Animals , Base Sequence , Chromatin/metabolism , DNA Transposable Elements/genetics , Enhancer Elements, Genetic/genetics , Gene Expression Regulation, Developmental , Humans , Macaca mulatta , Mice , Species Specificity , Synteny/genetics , Transcription Factors/metabolismABSTRACT
Cell-type- and condition-specific profiles of gene expression require coordination between protein-coding gene promoters and cis-regulatory sequences called enhancers. Enhancers can stimulate gene activity at great genomic distances from their targets, raising questions about how enhancers communicate with specific gene promoters and what molecular mechanisms underlie enhancer function. Characterization of enhancer loci has identified the molecular features of active enhancers that accompany the binding of transcription factors and local opening of chromatin. These characteristics include coactivator recruitment, histone modifications, and noncoding RNA transcription. However, it remains unclear which of these features functionally contribute to enhancer activity. Here, we discuss what is known about how enhancers regulate their target genes and how enhancers and promoters communicate. Further, we describe recent data demonstrating many similarities between enhancers and the gene promoters they control, and we highlight unanswered questions in the field, such as the potential roles of transcription at enhancers.
Subject(s)
Enhancer Elements, Genetic , Gene Expression Regulation , Genome , Promoter Regions, Genetic , RNA Polymerase II/genetics , Transcription, Genetic , Animals , Chromatin/chemistry , Chromatin/metabolism , DNA/genetics , DNA/metabolism , Eukaryotic Cells/metabolism , Genetic Loci , Histone Code , Histones/genetics , Histones/metabolism , Humans , RNA Polymerase II/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Enhancers switch genes on and off in response to a variety of intrinsic and external cellular signals. They are the cornerstone of gene regulation and the most pervasive constituents of the regulatory genome. Sequence polymorphisms in enhancer DNAs are a major source of population diversity and predilection to disease. To view this SnapShot, open or download the PDF.
Subject(s)
Enhancer Elements, Genetic/genetics , Gene Expression Regulation/genetics , Animals , Heterochromatin/genetics , Heterochromatin/metabolism , Humans , Models, Spatial Interaction , Polycomb-Group Proteins/metabolismABSTRACT
Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here, we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal lead to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knockin mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.
Subject(s)
Enhancer Elements, Genetic/genetics , High-Throughput Screening Assays/methods , Polydactyly/genetics , Animals , Enhancer Elements, Genetic/physiology , Gene Expression Regulation, Developmental/genetics , Gene Knock-In Techniques/methods , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Humans , Mice , Mutation , Phenotype , Polydactyly/metabolism , RNA, Untranslated/geneticsABSTRACT
Cell differentiation and function are regulated across multiple layers of gene regulation, including modulation of gene expression by changes in chromatin accessibility. However, differentiation is an asynchronous process precluding a temporal understanding of regulatory events leading to cell fate commitment. Here we developed simultaneous high-throughput ATAC and RNA expression with sequencing (SHARE-seq), a highly scalable approach for measurement of chromatin accessibility and gene expression in the same single cell, applicable to different tissues. Using 34,774 joint profiles from mouse skin, we develop a computational strategy to identify cis-regulatory interactions and define domains of regulatory chromatin (DORCs) that significantly overlap with super-enhancers. During lineage commitment, chromatin accessibility at DORCs precedes gene expression, suggesting that changes in chromatin accessibility may prime cells for lineage commitment. We computationally infer chromatin potential as a quantitative measure of chromatin lineage-priming and use it to predict cell fate outcomes. SHARE-seq is an extensible platform to study regulatory circuitry across diverse cells in tissues.
Subject(s)
Chromatin/metabolism , Gene Expression Profiling , RNA/genetics , Single-Cell Analysis , Animals , Cell Differentiation/genetics , Cell Line , Cell Lineage/genetics , Enhancer Elements, Genetic/genetics , Female , Gene Expression Regulation , Histones/metabolism , Mice, Inbred C57BL , Protein Processing, Post-Translational , RNA/metabolismABSTRACT
Transcriptome studies reveal pervasive transcription of complex genomes, such as those of mammals. Despite popular arguments for functionality of most, if not all, of these transcripts, genome-wide analysis of selective constraints indicates that most of the produced RNA are junk. However, junk is not garbage. On the contrary, junk transcripts provide the raw material for the evolution of diverse long non-coding (lnc) RNAs by non-adaptive mechanisms, such as constructive neutral evolution. The generation of many novel functional entities, such as lncRNAs, that fuels organismal complexity does not seem to be driven by strong positive selection. Rather, the weak selection regime that dominates the evolution of most multicellular eukaryotes provides ample material for functional innovation with relatively little adaptation involved.
Subject(s)
RNA, Long Noncoding/genetics , RNA, Messenger/genetics , Animals , DNA, Intergenic/genetics , Enhancer Elements, Genetic/genetics , Evolution, Molecular , Humans , RNA, Long Noncoding/metabolism , RNA, Messenger/metabolism , Transcription, GeneticABSTRACT
To discover regulatory elements driving the specificity of gene expression in different cell types and regions of the developing human brain, we generated an atlas of open chromatin from nine dissected regions of the mid-gestation human telencephalon, as well as microdissected upper and deep layers of the prefrontal cortex. We identified a subset of open chromatin regions (OCRs), termed predicted regulatory elements (pREs), that are likely to function as developmental brain enhancers. pREs showed temporal, regional, and laminar differences in chromatin accessibility and were correlated with gene expression differences across regions and gestational ages. We identified two functional de novo variants in a pRE for autism risk gene SLC6A1, and using CRISPRa, demonstrated that this pRE regulates SCL6A1. Additionally, mouse transgenic experiments validated enhancer activity for pREs proximal to FEZF2 and BCL11A. Thus, this atlas serves as a resource for decoding neurodevelopmental gene regulation in health and disease.
Subject(s)
Chromatin/genetics , Chromatin/metabolism , Enhancer Elements, Genetic , Gene Expression Regulation, Developmental/genetics , Prefrontal Cortex/embryology , Telencephalon/embryology , Animals , Autistic Disorder/genetics , Cell Line , Chromatin Immunoprecipitation Sequencing , Euchromatin/genetics , GABA Plasma Membrane Transport Proteins/genetics , Gene Ontology , Genetic Predisposition to Disease , Gestational Age , Humans , Mice , Mice, Transgenic , Nucleotide Motifs , Point Mutation , Prefrontal Cortex/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Spatio-Temporal Analysis , Telencephalon/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The high mobility group (HMG) transcription factor TCF-1 is essential for early T cell development. Although in vitro biochemical assays suggest that HMG proteins can serve as architectural elements in the assembly of higher-order nuclear organization, the contribution of TCF-1 on the control of three-dimensional (3D) genome structures during T cell development remains unknown. Here, we investigated the role of TCF-1 in 3D genome reconfiguration. Using gain- and loss-of-function experiments, we discovered that the co-occupancy of TCF-1 and the architectural protein CTCF altered the structure of topologically associating domains in T cell progenitors, leading to interactions between previously insulated regulatory elements and target genes at late stages of T cell development. The TCF-1-dependent gain in long-range interactions was linked to deposition of active enhancer mark H3K27ac and recruitment of the cohesin-loading factor NIPBL at active enhancers. These data indicate that TCF-1 has a role in controlling global genome organization during T cell development.
Subject(s)
Chromatin , Enhancer Elements, Genetic , CCCTC-Binding Factor/genetics , CCCTC-Binding Factor/metabolism , Cell Cycle Proteins/metabolism , Enhancer Elements, Genetic/genetics , Gene Expression Regulation , T-Lymphocytes/metabolismABSTRACT
Over one million candidate regulatory elements have been identified across the human genome, but nearly all are unvalidated and their target genes uncertain. Approaches based on human genetics are limited in scope to common variants and in resolution by linkage disequilibrium. We present a multiplex, expression quantitative trait locus (eQTL)-inspired framework for mapping enhancer-gene pairs by introducing random combinations of CRISPR/Cas9-mediated perturbations to each of many cells, followed by single-cell RNA sequencing (RNA-seq). Across two experiments, we used dCas9-KRAB to perturb 5,920 candidate enhancers with no strong a priori hypothesis as to their target gene(s), measuring effects by profiling 254,974 single-cell transcriptomes. We identified 664 (470 high-confidence) cis enhancer-gene pairs, which were enriched for specific transcription factors, non-housekeeping status, and genomic and 3D conformational proximity to their target genes. This framework will facilitate the large-scale mapping of enhancer-gene regulatory interactions, a critical yet largely uncharted component of the cis-regulatory landscape of the human genome.
Subject(s)
Chromosome Mapping/methods , Enhancer Elements, Genetic/genetics , Gene Expression Regulation/genetics , CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Gene Expression Profiling , Gene Regulatory Networks/genetics , Genome, Human , Genome-Wide Association Study , Genomics , Humans , Quantitative Trait Loci , Transcription Factors/geneticsABSTRACT
A complete chart of cis-regulatory elements and their dynamic activity is necessary to understand the transcriptional basis of differentiation and function of an organ system. We generated matched epigenome and transcriptome measurements in 86 primary cell types that span the mouse immune system and its differentiation cascades. This breadth of data enable variance components analysis that suggests that genes fall into two distinct classes, controlled by either enhancer- or promoter-driven logic, and multiple regression that connects genes to the enhancers that regulate them. Relating transcription factor (TF) expression to the genome-wide accessibility of their binding motifs classifies them as predominantly openers or closers of local chromatin accessibility, pinpointing specific cis-regulatory elements where binding of given TFs is likely functionally relevant, validated by chromatin immunoprecipitation sequencing (ChIP-seq). Overall, this cis-regulatory atlas provides a trove of information on transcriptional regulation through immune differentiation and a foundational scaffold to define key regulatory events throughout the immunological genome.
Subject(s)
Immune System/immunology , Immune System/metabolism , Regulatory Elements, Transcriptional/genetics , Animals , Binding Sites/genetics , Chromatin , Chromatin Immunoprecipitation/methods , Enhancer Elements, Genetic/genetics , Epigenomics/methods , Gene Expression Regulation/genetics , Mice , Mice, Inbred C57BL , Promoter Regions, Genetic/genetics , Protein Binding/genetics , Transcription Factors/metabolism , Transcriptome/geneticsABSTRACT
The temporal order of DNA replication (replication timing [RT]) is highly coupled with genome architecture, but cis-elements regulating either remain elusive. We created a series of CRISPR-mediated deletions and inversions of a pluripotency-associated topologically associating domain (TAD) in mouse ESCs. CTCF-associated domain boundaries were dispensable for RT. CTCF protein depletion weakened most TAD boundaries but had no effect on RT or A/B compartmentalization genome-wide. By contrast, deletion of three intra-TAD CTCF-independent 3D contact sites caused a domain-wide early-to-late RT shift, an A-to-B compartment switch, weakening of TAD architecture, and loss of transcription. The dispensability of TAD boundaries and the necessity of these "early replication control elements" (ERCEs) was validated by deletions and inversions at additional domains. Our results demonstrate that discrete cis-regulatory elements orchestrate domain-wide RT, A/B compartmentalization, TAD architecture, and transcription, revealing fundamental principles linking genome structure and function.
Subject(s)
DNA Replication Timing/physiology , DNA Replication/genetics , DNA Replication/physiology , Animals , CCCTC-Binding Factor/genetics , CCCTC-Binding Factor/metabolism , Chromatin , DNA/genetics , DNA Replication Timing/genetics , Embryonic Stem Cells , Enhancer Elements, Genetic/genetics , Mammals/genetics , Mammals/metabolism , Mice , Repressor Proteins/metabolism , Spatio-Temporal AnalysisABSTRACT
Mammalian switch/sucrose non-fermentable (mSWI/SNF) complexes are multi-component machines that remodel chromatin architecture. Dissection of the subunit- and domain-specific contributions to complex activities is needed to advance mechanistic understanding. Here, we examine the molecular, structural, and genome-wide regulatory consequences of recurrent, single-residue mutations in the putative coiled-coil C-terminal domain (CTD) of the SMARCB1 (BAF47) subunit, which cause the intellectual disability disorder Coffin-Siris syndrome (CSS), and are recurrently found in cancers. We find that the SMARCB1 CTD contains a basic α helix that binds directly to the nucleosome acidic patch and that all CSS-associated mutations disrupt this binding. Furthermore, these mutations abrogate mSWI/SNF-mediated nucleosome remodeling activity and enhancer DNA accessibility without changes in genome-wide complex localization. Finally, heterozygous CSS-associated SMARCB1 mutations result in dominant gene regulatory and morphologic changes during iPSC-neuronal differentiation. These studies unmask an evolutionarily conserved structural role for the SMARCB1 CTD that is perturbed in human disease.
Subject(s)
Chromatin Assembly and Disassembly/genetics , Chromosomal Proteins, Non-Histone/metabolism , Mutation/genetics , Nucleosomes/metabolism , SMARCB1 Protein/genetics , Transcription Factors/metabolism , Amino Acid Sequence , Enhancer Elements, Genetic/genetics , Female , Genome, Human , HEK293 Cells , HeLa Cells , Heterozygote , Humans , Male , Models, Molecular , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Protein Binding , Protein Domains , SMARCB1 Protein/chemistry , SMARCB1 Protein/metabolismABSTRACT
While Mediator plays a key role in eukaryotic transcription, little is known about its mechanism of action. This study combines CRISPR-Cas9 genetic screens, degron assays, Hi-C, and cryoelectron microscopy (cryo-EM) to dissect the function and structure of mammalian Mediator (mMED). Deletion analyses in B, T, and embryonic stem cells (ESC) identified a core of essential subunits required for Pol II recruitment genome-wide. Conversely, loss of non-essential subunits mostly affects promoters linked to multiple enhancers. Contrary to current models, however, mMED and Pol II are dispensable to physically tether regulatory DNA, a topological activity requiring architectural proteins. Cryo-EM analysis revealed a conserved core, with non-essential subunits increasing structural complexity of the tail module, a primary transcription factor target. Changes in tail structure markedly increase Pol II and kinase module interactions. We propose that Mediator's structural pliability enables it to integrate and transmit regulatory signals and act as a functional, rather than an architectural bridge, between promoters and enhancers.
Subject(s)
Mediator Complex/metabolism , RNA Polymerase II/metabolism , Animals , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/metabolism , CRISPR-Cas Systems/genetics , Cell Cycle Proteins/metabolism , Cells, Cultured , Chromosomal Proteins, Non-Histone/metabolism , Cryoelectron Microscopy , Enhancer Elements, Genetic , Gene Editing , Humans , Male , Mediator Complex/chemistry , Mediator Complex/genetics , Mice , Mice, Inbred C57BL , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/metabolism , Promoter Regions, Genetic , Protein Structure, Quaternary , RNA Polymerase II/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , CohesinsABSTRACT
Stochastic activation of clustered Protocadherin (Pcdh) α, ß, and γ genes generates a cell-surface identity code in individual neurons that functions in neural circuit assembly. Here, we show that Pcdhα gene choice involves the activation of an antisense promoter located in the first exon of each Pcdhα alternate gene. Transcription of an antisense long noncoding RNA (lncRNA) from this antisense promoter extends through the sense promoter, leading to DNA demethylation of the CTCF binding sites proximal to each promoter. Demethylation-dependent CTCF binding to both promoters facilitates cohesin-mediated DNA looping with a distal enhancer (HS5-1), locking in the transcriptional state of the chosen Pcdhα gene. Uncoupling DNA demethylation from antisense transcription by Tet3 overexpression in mouse olfactory neurons promotes CTCF binding to all Pcdhα promoters, resulting in proximity-biased DNA looping of the HS5-1 enhancer. Thus, antisense transcription-mediated promoter demethylation functions as a mechanism for distance-independent enhancer/promoter DNA looping to ensure stochastic Pcdhα promoter choice.
Subject(s)
Cadherins/genetics , DNA Demethylation , RNA, Antisense/metabolism , RNA, Long Noncoding/genetics , Animals , Binding Sites , CCCTC-Binding Factor/chemistry , CCCTC-Binding Factor/metabolism , Cadherins/metabolism , Cell Line , Enhancer Elements, Genetic , Exons , Female , Humans , Mice , Mice, Transgenic , Multigene Family , Neurons/cytology , Neurons/metabolism , Promoter Regions, Genetic , RNA Polymerase II/metabolism , RNA, Antisense/genetics , Transcription, GeneticABSTRACT
Non-coding regions amplified beyond oncogene borders have largely been ignored. Using a computational approach, we find signatures of significant co-amplification of non-coding DNA beyond the boundaries of amplified oncogenes across five cancer types. In glioblastoma, EGFR is preferentially co-amplified with its two endogenous enhancer elements active in the cell type of origin. These regulatory elements, their contacts, and their contribution to cell fitness are preserved on high-level circular extrachromosomal DNA amplifications. Interrogating the locus with a CRISPR interference screening approach reveals a diversity of additional elements that impact cell fitness. The pattern of fitness dependencies mirrors the rearrangement of regulatory elements and accompanying rewiring of the chromatin topology on the extrachromosomal amplicon. Our studies indicate that oncogene amplifications are shaped by regulatory dependencies in the non-coding genome.