Human gene regulatory evolution is driven by the divergence of regulatory element function in both cis and trans.

Gene regulatory divergence between species can result from cis-acting local changes to regulatory element DNA sequences or global trans-acting changes to the regulatory environment. Understanding how these mechanisms drive regulatory evolution has been limited by challenges in identifying trans-acting changes. We present a comprehensive approach to directly identify cis- and trans-divergent regulatory elements between human and rhesus macaque lymphoblastoid cells using assay for transposase-accessible chromatin coupled to self-transcribing active regulatory region (ATAC-STARR) sequencing. In addition to thousands of cis changes, we discover an unexpected number (∼10,000) of trans changes and show that cis and trans elements exhibit distinct patterns of sequence divergence and function. We further identify differentially expressed transcription factors that underlie ∼37% of trans differences and trace how cis changes can produce cascades of trans changes. Overall, we find that most divergent elements (67%) experienced changes in both cis and trans, revealing a substantial role for trans divergence-alone and together with cis changes-in regulatory differences between species.

Uncovering dark matter in cancer by identifying epigenetic drivers.

The complex relationship between chromatin accessibility, transcriptional regulation, and cancer transitions presents a daunting puzzle. Terekhanova et al. created a pan-cancer epigenetic and transcriptomic atlas at single-cell resolution, yielding important insights into the underlying chromatin architecture of cancer transitions and novel discoveries with the potential to advance precision medicine.

Systematic identification of genotype-dependent enhancer variants in eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a rare atopic disorder associated with esophageal dysfunction, including difficulty swallowing, food impaction, and inflammation, that develops in a small subset of people with food allergies. Genome-wide association studies (GWASs) have identified 9 independent EoE risk loci reaching genome-wide significance (p < 5 × 10-8) and 27 additional loci of suggestive significance (5 × 10-8 < p < 1 × 10-5). In the current study, we perform linkage disequilibrium (LD) expansion of these loci to nominate a set of 531 variants that are potentially causal. To systematically interrogate the gene regulatory activity of these variants, we designed a massively parallel reporter assay (MPRA) containing the alleles of each variant within their genomic sequence context cloned into a GFP reporter library. Analysis of reporter gene expression in TE-7, HaCaT, and Jurkat cells revealed cell-type-specific gene regulation. We identify 32 allelic enhancer variants, representing 6 genome-wide significant EoE loci and 7 suggestive EoE loci, that regulate reporter gene expression in a genotype-dependent manner in at least one cellular context. By annotating these variants with expression quantitative trait loci (eQTL) and chromatin looping data in related tissues and cell types, we identify putative target genes affected by genetic variation in individuals with EoE. Transcription factor enrichment analyses reveal possible roles for cell-type-specific regulators, including GATA3. Our approach reduces the large set of EoE-associated variants to a set of 32 with allelic regulatory activity, providing functional insights into the effects of genetic variation in this disease.

High-throughput functional dissection of noncoding SNPs with biased allelic enhancer activity for insulin resistance-relevant phenotypes.

Most of the single-nucleotide polymorphisms (SNPs) associated with insulin resistance (IR)-relevant phenotypes by genome-wide association studies (GWASs) are located in noncoding regions, complicating their functional interpretation. Here, we utilized an adapted STARR-seq to evaluate the regulatory activities of 5,987 noncoding SNPs associated with IR-relevant phenotypes. We identified 876 SNPs with biased allelic enhancer activity effects (baaSNPs) across 133 loci in three IR-relevant cell lines (HepG2, preadipocyte, and A673), which showed pervasive cell specificity and significant enrichment for cell-specific open chromatin regions or enhancer-indicative markers (H3K4me1, H3K27ac). Further functional characterization suggested several transcription factors (TFs) with preferential allelic binding to baaSNPs. We also incorporated multi-omics data to prioritize 102 candidate regulatory target genes for baaSNPs and revealed prevalent long-range regulatory effects and cell-specific IR-relevant biological functional enrichment on them. Specifically, we experimentally verified the distal regulatory mechanism at IRS1 locus, in which rs952227-A reinforces IRS1 expression by long-range chromatin interaction and preferential binding to the transcription factor HOXC6 to augment the enhancer activity. Finally, based on our STARR-seq screening data, we predicted the enhancer activity of 227,343 noncoding SNPs associated with IR-relevant phenotypes (fasting insulin adjusted for BMI, HDL cholesterol, and triglycerides) from the largest available GWAS summary statistics. We further provided an open resource (http://www.bigc.online/fnSNP-IR) for better understanding genetic regulatory mechanisms of IR-relevant phenotypes.

Epigenomics of conventional type-I dendritic cells depicted preferential control of TLR9 versus TLR3 response by NCoR1 through differential IRF3 activation.

Tight control of gene regulation in dendritic cells (DCs) is important to mount pathogen specific immune responses. Apart from transcription factor binding, dynamic regulation of enhancer activity through global transcriptional repressors like Nuclear Receptor Co-repressor 1 (NCoR1) plays a major role in fine-tuning of DC responses. However, how NCoR1 regulates enhancer activity and gene expression in individual or multiple Toll-like receptor (TLR) activation in DCs is largely unknown. In this study, we did a comprehensive epigenomic analysis of murine conventional type-I DCs (cDC1) across different TLR ligation conditions. We profiled gene expression changes along with H3K27ac active enhancers and NCoR1 binding in the TLR9, TLR3 and combined TLR9 + TLR3 activated cDC1. We observed spatio-temporal activity of TLR9 and TLR3 specific enhancers regulating signal specific target genes. Interestingly, we found that NCoR1 differentially controls the TLR9 and TLR3-specific responses. NCoR1 depletion specifically enhanced TLR9 responses as evident from increased enhancer activity as well as TLR9-specific gene expression, whereas TLR3-mediated antiviral response genes were negatively regulated. We validated that NCoR1 KD cDC1 showed significantly decreased TLR3 specific antiviral responses through decreased IRF3 activation. In addition, decreased IRF3 binding was observed at selected ISGs leading to their decreased expression upon NCoR1 depletion. Consequently, the NCoR1 depleted cDC1 showed reduced Sendai Virus (SeV) clearance and cytotoxic potential of CD8+ T cells upon TLR3 activation. NCoR1 directly controls the majority of these TLR specific enhancer activity and the gene expression. Overall, for the first time, we revealed NCoR1 mediates transcriptional control towards TLR9 as compared to TLR3 in cDC1.

Analysis of Enhancers and Transcriptional Networks in Thermogenic Adipocytes.

Transcription factor (TF) networks orchestrate the regulation of gene programs in mammalian cells, including white and brown adipocytes. In this protocol, we outline how genomics and transcriptomics data can be integrated to infer causal TFs of a given cellular response or cell type using "Integrated analysis of Motif Activity and Gene Expression changes of transcription factors" (IMAGE). Here, we show how key regulatory TFs controlling white and brown adipocyte gene programs can be predicted from chromatin accessibility and RNA-seq data. Furthermore, we demonstrate how information about target sites and target genes of the predicted key regulators can be integrated to propose testable hypotheses regarding the role and mechanisms of TFs.

FoxA-dependent demethylation of DNA initiates epigenetic memory of cellular identity.

Tissue-specific DNA methylation patterns are created by transcription factors that recruit methylation and demethylation enzymes to cis-regulatory elements. To date, it is not known whether transcription factors are needed to continuously maintain methylation profiles in development and mature tissues or whether they only establish these marks during organ development. We queried the role of the pioneer factor FoxA in generating hypomethylated DNA at liver enhancers. We discovered a set of FoxA-binding sites that undergo regional, FoxA-dependent demethylation during organ development. Conditional ablation of FoxA genes in the adult liver demonstrated that continued FoxA presence was not required to maintain the hypomethylated state, even when massive cell proliferation was induced. This study provides strong evidence for the stable, epigenetic nature of tissue-specific DNA methylation patterns directed by lineage-determining transcription factors during organ development.

Acute perturbation strategies in interrogating RNA polymerase II elongation factor function in gene expression.

The regulation of gene expression catalyzed by RNA polymerase II (Pol II) requires a host of accessory factors to ensure cell growth, differentiation, and survival under environmental stress. Here, using the auxin-inducible degradation (AID) system to study transcriptional activities of the bromodomain and extraterminal domain (BET) and super elongation complex (SEC) families, we found that the CDK9-containing BRD4 complex is required for the release of Pol II from promoter-proximal pausing for most genes, while the CDK9-containing SEC is required for activated transcription in the heat shock response. By using both the proteolysis targeting chimera (PROTAC) dBET6 and the AID system, we found that dBET6 treatment results in two major effects: increased pausing due to BRD4 loss, and reduced enhancer activity attributable to BRD2 loss. In the heat shock response, while auxin-mediated depletion of the AFF4 subunit of the SEC has a more severe defect than AFF1 depletion, simultaneous depletion of AFF1 and AFF4 leads to a stronger attenuation of the heat shock response, similar to treatment with the SEC inhibitor KL-1, suggesting a possible redundancy among SEC family members. This study highlights the usefulness of orthogonal acute depletion/inhibition strategies to identify distinct and redundant biological functions among Pol II elongation factor paralogs.

Analysis of Regulatory DNA Sequences by Dual-Luciferase Reporter Assays in Ewing Sarcoma.

Reporter gene assays allow for examining the influence of regulatory DNA sequences on the transcription of target genes. In Ewing sarcoma, the study of these DNA sequences is especially paramount for its main driver mutation is a fusion transcription factor that binds different motifs than its wild-type constituents. Here, we describe the process of analyzing the enhancer activity of regulatory DNA sequences using transfection-based dual-luciferase reporter assays in Ewing sarcoma cell lines. To this end, we provide a protocol for cloning sequences of interest from genomic DNA into a firefly luciferase-containing plasmid, transfecting Ewing sarcoma cells with plasmids and measuring luciferase expression by luminescence. The entire procedure can be completed in 14 days.

Cis-acting lnc-eRNA SEELA directly binds histone H4 to promote histone recognition and leukemia progression.

BACKGROUND: Long noncoding enhancer RNAs (lnc-eRNAs) are a subset of stable eRNAs identified from annotated lncRNAs. They might act as enhancer activity-related therapeutic targets in cancer. However, the underlying mechanism of epigenetic activation and their function in cancer initiation and progression remain largely unknown. RESULTS: We identify a set of lncRNAs as lnc-eRNAs according to the epigenetic signatures of enhancers. We show that these lnc-eRNAs are broadly activated in MLL-rearranged leukemia (MLL leukemia), an aggressive leukemia caused by a chromosomal translocation, through a mechanism by which the HOXA cluster initiates enhancer activity, and the epigenetic reader BRD4 cooperates with the coregulator MLL fusion oncoprotein to induce transcriptional activation. To demonstrate the functional roles of lnc-eRNAs, two newly identified lnc-eRNAs transcribed from the SEELA eRNA cluster (SEELA), SEELA1 and SEELA2, are chosen for further studies. The results show that SEELA mediated cis-activated transcription of the nearby oncogene Serine incorporate 2 (SERINC2) by directly binding to the K31 amino acid (aa) of histone H4. Chromatin-bound SEELA strengthens the interaction between chromatin and histone modifiers to promote histone recognition and oncogene transcription. Further studies show that the SEELA-SERINC2 axis regulated aspects of cancer metabolism, such as sphingolipid synthesis, to affect leukemia progression. CONCLUSIONS: This study shows that lnc-eRNAs are epigenetically activated by cancer-initiating oncoproteins and uncovers a cis-activating mechanism of oncogene transcription control based on lnc-eRNA-mediated epigenetic regulation of enhancer activity, providing insights into the critical roles of lnc-eRNAs in cancer initiation and progression.

ASXL3 bridges BRD4 to BAP1 complex and governs enhancer activity in small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) is a more aggressive subtype of lung cancer that often results in rapid tumor growth, early metastasis, and acquired therapeutic resistance. Consequently, such phenotypical characteristics of SCLC set limitations on viable procedural options, making it difficult to develop both screenings and effective treatments. In this study, we examine a novel mechanistic insight in SCLC cells that could potentially provide a more sensitive therapeutic alternative for SCLC patients. METHODS: Biochemistry studies, including size exclusion chromatography, mass spectrometry, and western blot analysis, were conducted to determine the protein-protein interaction between additional sex combs-like protein 3 (ASXL3) and bromodomain-containing protein 4 (BRD4). Genomic studies, including chromatin immunoprecipitation sequencing (ChIP-seq), RNA sequencing, and genome-wide analysis, were performed in both human and mouse SCLC cells to determine the dynamic relationship between BRD4/ASXL3/BAP1 epigenetic axis in chromatin binding and its effects on transcriptional activity. RESULTS: We report a critical link between BAP1 complex and BRD4, which is bridged by the physical interaction between ASXL3 and BRD4 in an SCLC subtype (SCLC-A), which expresses a high level of ASCL1. We further showed that ASXL3 functions as an adaptor protein, which directly interacts with BRD4's extra-terminal (ET) domain via a novel BRD4 binding motif (BBM), and maintains chromatin occupancy of BRD4 to active enhancers. Genetic depletion of ASXL3 results in a genome-wide reduction of histone H3K27Ac levels and BRD4-dependent gene expression in SCLC. Pharmacologically induced inhibition with BET-specific chemical degrader (dBET6) selectively inhibits cell proliferation of a subtype of SCLC that is characterized with high expression of ASXL3. CONCLUSIONS: Collectively, this study provides a mechanistic insight into the oncogenic function of BRD4/ASXL3/BAP1 epigenetic axis at active chromatin enhancers in SCLC-A subtype, as well as a potential new therapeutic option that could become more effective in treating SCLC patients with a biomarker of ASXL3-highly expressed SCLC cells.

Enhancer RNAs Mediate Estrogen-Induced Decommissioning of Selective Enhancers by Recruiting ERα and Its Cofactor.

The function of enhancer RNAs (eRNAs) in transcriptional regulation remains obscure. By analyzing the genome-wide nascent transcript profiles in breast cancer cells, we identify a special group of eRNAs that are essential for estrogen-induced transcriptional repression. Using eRNAs of TM4SF1 and EFEMP1 as the paradigms, we find that these RNA molecules not only stabilize promoter-enhancer interactions but also recruit liganded estrogen receptor α (ERα) to particular enhancer regions, facilitate the formation of a functional transcriptional complex, and cause gene silencing. Interestingly, ERα is shown to directly bind with eRNAs by its DNA-binding domain. These eRNAs help with the formation of a specific ERα-centered transcriptional complex and promote the association of the histone demethylase KDM2A, which dismisses RNA polymerase II from designated enhancers and suppresses the transcription of target genes. Our work demonstrates a complete mechanism underlying the action of eRNAs in modulating and refining the locus-specific transcriptional program.

The Role of Chromatin Accessibility in cis-Regulatory Evolution.

Transcription factor (TF) binding is determined by sequence as well as chromatin accessibility. Although the role of accessibility in shaping TF-binding landscapes is well recorded, its role in evolutionary divergence of TF binding, which in turn can alter cis-regulatory activities, is not well understood. In this work, we studied the evolution of genome-wide binding landscapes of five major TFs in the core network of mesoderm specification, between Drosophila melanogaster and Drosophila virilis, and examined its relationship to accessibility and sequence-level changes. We generated chromatin accessibility data from three important stages of embryogenesis in both Drosophila melanogaster and Drosophila virilis and recorded conservation and divergence patterns. We then used multivariable models to correlate accessibility and sequence changes to TF-binding divergence. We found that accessibility changes can in some cases, for example, for the master regulator Twist and for earlier developmental stages, more accurately predict binding change than is possible using TF-binding motif changes between orthologous enhancers. Accessibility changes also explain a significant portion of the codivergence of TF pairs. We noted that accessibility and motif changes offer complementary views of the evolution of TF binding and developed a combined model that captures the evolutionary data much more accurately than either view alone. Finally, we trained machine learning models to predict enhancer activity from TF binding and used these functional models to argue that motif and accessibility-based predictors of TF-binding change can substitute for experimentally measured binding change, for the purpose of predicting evolutionary changes in enhancer activity.

Development of enhancer-trapping and -detection vectors mediated by the Tol2 transposon in zebrafish.

Enhancers are key transcriptional drivers of gene expression. The identification of enhancers in the genome is central for understanding gene-expression programs. Although transposon-mediated enhancer trapping (ET) is a powerful approach to the identification of enhancers in zebrafish, its efficiency varies considerably. To improve the ET efficiency, we constructed Tol2-mediated ET vectors with a reporter gene (mCherry) expression box driven by four minimal promoters (Gata, Myc, Krt4 and Oct4), respectively. The ET efficiency and expression background were compared among the four promoters by zebrafish embryo injection at the one-cell stage. The results showed that the Gata minimal promoter yielded the lowest basic expression and the second-highest trapping efficiency (44.6% at 12 hpf (hour post-fertilization) and 23.1% at 72 hpf, n = 305 and n = 307). The Krt4 promoter had the highest trapping efficiency (64% at 12 hpf and 67.1% at 72 hpf, n = 302 and n = 301) and the strongest basic expression. To detect enhancer activity, chicken 5'HS4 double insulators were cloned into the two ET vectors with the Gata or Krt4 minimal promoter, flanking the mCherry expression box. The resulting detection vectors were injected into zebrafish embryos. mCherry expression driven by the Gata promoter (about 5%, n = 301) was decreased significantly compared with that observed for embryos injected with the ET vectors (23% at 72 hpf, n = 308). These results suggest that the insulators block the genome-position effects and that this vector is fit for enhancer-activity evaluation. To assess the compatibility between the enhancers and the minimal promoters, four enhancers (CNS1, Z48, Hand2 and Hs769) were cloned upstream of the Gata or Beta-globin minimal promoter in the enhancer-activity-detection vectors. The resulting recombinant vectors were assayed by zebrafish embryo injection. We found that Z48 and CNS1 responded to the Gata minimal promoter, and that Hand2 only responded to the Beta-globin minimal promoter. In contrast, Hs769 did not respond to either the Gata or Beta-globin minimal promoters. These results suggest the existence of compatibility between enhancers and minimal promoters. This study represents a systematic approach to the discovery of optional ET and enhancer-detection vectors. We are eager to provide a superior tool for understanding functional genomics.

Causal Transcription Regulatory Network Inference Using Enhancer Activity as a Causal Anchor.

Transcription control plays a crucial role in establishing a unique gene expression signature for each of the hundreds of mammalian cell types. Though gene expression data have been widely used to infer cellular regulatory networks, existing methods mainly infer correlations rather than causality. We developed statistical models and likelihood-ratio tests to infer causal gene regulatory networks using enhancer RNA (eRNA) expression information as a causal anchor and applied the framework to eRNA and transcript expression data from the FANTOM Consortium. Predicted causal targets of transcription factors (TFs) in mouse embryonic stem cells, macrophages and erythroblastic leukaemia overlapped significantly with experimentally-validated targets from ChIP-seq and perturbation data. We further improved the model by taking into account that some TFs might act in a quantitative, dosage-dependent manner, whereas others might act predominantly in a binary on/off fashion. We predicted TF targets from concerted variation of eRNA and TF and target promoter expression levels within a single cell type, as well as across multiple cell types. Importantly, TFs with high-confidence predictions were largely different between these two analyses, demonstrating that variability within a cell type is highly relevant for target prediction of cell type-specific factors. Finally, we generated a compendium of high-confidence TF targets across diverse human cell and tissue types.

Enhancer activities of amphioxus Brachyury genes in embryos of the ascidian, Ciona intestinalis.

The notochord and somites are distinctive chordate structures. The T-box transcription factor gene, Brachyury, is expressed in notochord and plays a pivotal role in its formation. In the cephalochordate, Branchiostoma floridae, Brachyury is duplicated into BfBra1 and BfBra2, which are expressed in the somite-formation region as well. In a series of experiments to elucidate the regulatory machinery of chordate Brachyury expression, we carried out a lacZ reporter assay of BfBra in embryos of the urochordate, Ciona intestinalis. Vista analyses suggest the presence of conserved non-coding sequences, not only in the 5'-upstream, but also in the 3'-downstream and in introns of BfBra. We found that: (1) 5'-upstream sequences of both BfBra1 and BfBra2 promote lacZ expression in muscle cells, (2) 3'-downstream sequences have enhancer activity that promotes lacZ expression in notochord cells, and (3) introns of BfBra2 and BfBra1 exhibit lacZ expression preferentially in muscle and notochord cells. These results suggest shared cephalochordate Brachyury enhancer machinery that also works in urochordates. We discuss the results in relation to evolutionary modification of Brachyury expression in formation of chordate-specific organs characteristic of each lineage.

Non-coding Transcripts from Enhancers: New Insights into Enhancer Activity and Gene Expression Regulation.

Long non-coding RNAs (lncRNAs) have gained widespread interest in the past decade owing to their enormous amount and surprising functions implicated in a variety of biological processes. Some lncRNAs exert function as enhancers, i.e., activating gene transcription by serving as the cis-regulatory molecules. Furthermore, recent studies have demonstrated that many enhancer elements can be transcribed and produce RNA molecules, which are termed as enhancer RNAs (eRNAs). The eRNAs are not merely the by-product of the enhancer transcription. In fact, many of them directly exert or regulate enhancer activity in gene activation through diverse mechanisms. Here, we provide an overview of enhancer activity, transcription of enhancer itself, characteristics of eRNAs, as well as their roles in regulating enhancer activity and gene expression.

Genes expressed in mouse cortical progenitors are enriched in Pax, Lhx, and Sox transcription factor putative binding sites.

Considerable progress has been made in the understanding of molecular and cellular mechanisms controlling the development of the mammalian cortex. The proliferative and neurogenic properties of cortical progenitors located in the ventricular germinal zone start being understood. Little is known however on the cis-regulatory control that finely tunes gene expression in these progenitors. Here, we undertook an in silico-based approach to address this question, followed by some functional validation. Using the Eurexpress database, we established a list of 30 genes specifically expressed in the cortical germinal zone, we selected mouse/human conserved non-coding elements (CNEs) around these genes and we performed motif-enrichment search in these CNEs. We found an over-representation of motifs corresponding to binding sites for Pax, Sox, and Lhx transcription factors, often found as pairs and located within 100bp windows. A small subset of CNEs (n=7) was tested for enhancer activity, by ex-vivo and in utero electroporation assays. Two showed strong enhancer activity in the germinal zone progenitors. Mutagenesis experiments on a selected CNE showed the functional importance of the Pax, Sox, and Lhx TFBS for conferring enhancer activity to the CNE. Overall, from a cis-regulatory viewpoint, our data suggest an input from Pax, Sox and Lhx transcription factors to orchestrate corticogenesis. These results are discussed with regards to the known functional roles of Pax6, Sox2 and Lhx2 in cortical development.

Computational schemes for the prediction and annotation of enhancers from epigenomic assays.

Identifying and annotating distal regulatory enhancers is critical to understand the mechanisms that control gene expression and cell-type-specific activities. Next-generation sequencing techniques have provided us an exciting toolkit of genome-wide assays that can be used to predict and annotate enhancers. However, each assay comes with its own specific set of analytical needs if enhancer prediction is to be optimal. Furthermore, integration of multiple genome-wide assays allows for different genomic features to be combined, and can improve predictive performance. Herein, we review the genome-wide assays and analysis schemes that are used to predict and annotate enhancers. In particular, we focus on three key computational topics: predicting enhancer locations, determining the cell-type-specific activity of enhancers, and linking enhancers to their target genes.