Promoter Enhancer Research Articles

CT-FOCS: a novel method for inferring cell type-specific enhancer-promoter maps.

Spatiotemporal gene expression patterns are governed to a large extent by the activity of enhancer elements, which engage in physical contacts with their target genes. Identification of enhancer–promoter (EP) links that are functional only in a specific subset of cell types is a key challenge in understanding gene regulation. We introduce CT-FOCS (cell type FOCS), a statistical inference method that uses linear mixed effect models to infer EP links that show marked activity only in a single or a small subset of cell types out of a large panel of probed cell types. Analyzing 808 samples from FANTOM5, covering 472 cell lines, primary cells and tissues, CT-FOCS inferred such EP links more accurately than recent state-of-the-art methods. Furthermore, we show that strictly cell type-specific EP links are very uncommon in the human genome.

How subtle changes in 3D structure can create large changes in transcription.

Animal genomes are organized into topologically associated domains (TADs). TADs are thought to contribute to gene regulation by facilitating enhancer-promoter (E-P) contacts within a TAD and preventing these contacts across TAD borders. However, the absolute difference in contact frequency across TAD boundaries is usually less than 2-fold, even though disruptions of TAD borders can change gene expression by 10-fold. Existing models fail to explain this hypersensitive response. Here, we propose a futile cycle model of enhancer-mediated regulation that can exhibit hypersensitivity through bistability and hysteresis. Consistent with recent experiments, this regulation does not exhibit strong correlation between E-P contact and promoter activity, even though regulation occurs through contact. Through mathematical analysis and stochastic simulation, we show that this system can create an illusion of E-P biochemical specificity and explain the importance of weak TAD boundaries. It also offers a mechanism to reconcile apparently contradictory results from recent global TAD disruption with local TAD boundary deletion experiments. Together, these analyses advance our understanding of cis-regulatory contacts in controlling gene expression and suggest new experimental directions.

Exploring the role of eRNA in regulating gene expression.

eRNAs as the products of enhancers can regulate gene expression via various possible ways, but which regulation way is more reasonable is debatable in biology, and in particular, how eRNAs impact gene expression remains unclear. Here we introduce a mechanistic model of gene expression to address these issues. This model considers three possible regulation ways of eRNA: Type-I by which eRNA regulates transcriptional activity by facilitating the formation of enhancer-promoter (E-P) loop, Type-II by which eRNA directly promotes the mRNA production rate, and mixed regulation (i.e., the combination of Type-I and Type-II). We show that with the increase of the E-P loop length, mRNA distribution can transition from unimodality to bimodality or vice versa in all the three regulation cases. However, in contrast to the other two regulations, Type-II regulation can lead to the highest mean mRNA level and the lowest mRNA noise, independent of the E-P loop length. These results would not only reveal the essential mechanism of how eRNA regulates gene expression, but also imply a new mechanism for phenotypic switching, namely the E-P loop can induce phenotypic switching.

High-Resolution Mapping of Multiway Enhancer-Promoter Interactions Regulating Pathogen Detection

Eukaryotic gene expression regulation involves thousands of distal regulatory elements. Understanding the quantitative contribution of individual enhancers to gene expression is critical for assessing the role of disease-associated genetic risk variants. Yet, we lack the ability to accurately link genes with their distal regulatory elements. To address this, we used 3D enhancer-promoter (E-P) associations identified using split-pool recognition of interactions by tag extension (SPRITE) to build a predictive model of gene expression. Our model dramatically outperforms models using genomic proximity and can be used to determine the quantitative impact of enhancer loss on gene expression in different genetic backgrounds. We show that genes that form stable E-P hubs have less cell-to-cell variability in gene expression. Finally, we identified transcription factors that regulate stimulation-dependent E-P interactions. Together, our results provide a framework for understanding quantitative contributions of E-P interactions and associated genetic variants to gene expression.

Robust Hi-C Maps of Enhancer-Promoter Interactions Reveal the Function of Non-coding Genome in Neural Development and Diseases

Genome-wide mapping of chromatin interactions at high resolution remains experimentally and computationally challenging. Here we used a low-input "easy Hi-C" protocol to map the 3D genome architecture in human neurogenesis and brain tissues and also demonstrated that a rigorous Hi-C bias-correction pipeline (HiCorr) can significantly improve the sensitivity and robustness of Hi-C loop identification at sub-TAD level, especially the enhancer-promoter (E-P) interactions. We used HiCorr to compare the high-resolution maps of chromatin interactions from 10 tissue or cell types with a focus on neurogenesis and brain tissues. We found that dynamic chromatin loops are better hallmarks for cellular differentiation than compartment switching. HiCorr allowed direct observation of cell-type- and differentiation-specific E-P aggregates spanning large neighborhoods, suggesting a mechanism that stabilizes enhancer contacts during development. Interestingly, we concluded that Hi-C loop outperforms eQTL in explaining neurological GWAS results, revealing a unique value of high-resolution 3D genome maps in elucidating the disease etiology.

Functionally Related Genes Cluster into Genomic Regions That Coordinate Transcription at a Distance in Saccharomyces cerevisiae.

Balancing gene expression is a fundamental challenge of all cell types. To properly regulate transcription on a genome-wide level, there are myriad mechanisms employed by the cell. One layer to this regulation is through spatial positioning, with particular chromosomal loci exerting an influence on transcription throughout a region. Many coregulated gene families utilize spatial positioning to coordinate transcription, with functionally related genes clustering together which can allow coordinated expression via adjacent gene coregulation. The mechanisms underlying this process have not been elucidated, though there are many coregulated gene families that exhibit this genomic distribution. In the present study, we tested for a role for the enhancer-promoter (EP) hypothesis, which demonstrates that regulatory elements can exert transcriptional effects over a broad distance, in coordinating transcriptional coregulation using budding yeast, Saccharomyces cerevisiae We empirically validated the EP model, finding that the genomic distance a promoter can affect varies by locus, which can profoundly affect levels of transcription, phenotype, and the extent of transcriptional disruption throughout a genomic region. Using the nitrogen metabolism, ribosomal protein, toxin response, and heat shock gene families as our test case, we report functionally clustered genes localize to genomic loci that are more conducive to transcriptional regulation at a distance compared to the unpaired members of the same families. Furthermore, we report that the coregulation of functional clusters is dependent, in part, on chromatin maintenance and remodeling, providing one mechanism underlying adjacent gene coregulation.IMPORTANCE The two-dimensional, physical positioning of genes along a chromosome can impact proper transcriptional regulation throughout a genomic region. The transcription of neighboring genes is correlated in a genome-wide manner, which is a characteristic of eukaryotes. Many coregulated gene families can be found clustered with another member of the same set-which can result in adjacent gene coregulation of the pair. Due to the myriad gene families that exhibit a nonrandom genomic distribution, there are likely multiple mechanisms working in concert to properly regulate transcriptional coordination of functionally clustered genes. In this study, we utilized budding yeast in an attempt to elucidate mechanisms that underlie this coregulation: testing and empirically validating the enhancer-promoter hypothesis in this species and reporting that functionally related genes cluster to genomic regions that are more conducive to transcriptional regulation at a distance. These clusters rely, in part, on chromatin maintenance and remodelers to maintain proper transcriptional coordination. Our work provides insight into the mechanisms underlying adjacent gene coregulation.

Positive and Negative Control of Enhancer-Promoter Interactions by Other DNA Loops Generates Specificity and Tunability

Enhancers are ubiquitous and critical gene-regulatory elements. However, quantitative understanding of the role of DNA looping in the regulation of enhancer action and specificity is limited. We used the Escherichia coli NtrC enhancer-σ54 promoter system as an invivo model, finding that NtrC activation is highly sensitive to the enhancer-promoter (E-P) distance in the 300-6,000bp range. DNA loops formed by Lac repressor were able to strongly regulate enhancer action either positively or negatively, recapitulating promoter targeting and insulation. A single LacI loop combining targeting and insulation produced a strong shift in specificity for enhancer choice between two σ54 promoters. A combined kinetic-thermodynamic model was used to quantify the effect of DNA-looping interactions on promoter activity and revealed that sensitivity to E-P distance and to control by other loops is itself dependent on enhancer and promoter parameters that may be subject to regulation.

Control of Enhancer-Promoter Contact by Alternative DNA Loops

Enhancers are ubiquitous and critical gene regulatory elements. However, quantitative understanding of the role of DNA looping in the regulation of enhancer action and specificity is limited. We used the Escherichia coli NtrC enhancer-σ54 promoter system as a model in vivo system, showing that activation is highly sensitive to the enhancer-promoter (E-P) distance in the 300-6000 bp range, and can be regulated positively or negatively by assisting or insulating loops formed by Lac repressor. Two σ54 promoters controlled by the same enhancer compete weakly, and a single LacI loop can produce a 15-fold shift in promoter choice by the enhancer. A combined kinetic-thermodynamic model quantifies loop interactions and reveals that the response to changes in the E-P tether by insulators and other loops is highly tunable. While the NtrC system is sensitive to E-P distance and looping control, other enhancers and promoters may resist such manipulation.

Abstract 2280: Systematic analysis of causal noncoding mutations in pediatric B-cell acute lymphoblastic leukemia

Abstract B cell acute lymphoblastic leukemia (B-ALL) is the most prevalent childhood hematological malignancy and the leading cause of childhood cancer-related mortality. To date, the molecular pathogenesis of B-ALL has not been completely defined, limiting our ability to develop targeted therapies. Despite great advances in identifying mutations in cancer patients, the functional consequences of noncoding mutations are still difficult to interpret. Transcriptional enhancers are key determinants of tissue-specific gene expression. Several recent studies have reported novel mutations in cancer genome that disrupt enhance function and thus expression of their target genes. However, systematic detection of noncoding mutations that perturb either enhancer sequence or enhancer-promoter (EP) interaction is still challenging. Such analysis can help to identify novel driver mutations and improve our understanding of the disease. Here we introduce a novel algorithm for identifying causal mutations that disrupt enhancer function and target gene expression in B-ALL. The algorithm takes as input whole genome sequencing (WGS) and RNA-Seq data to predict causal noncoding mutations. We apply the algorithm to analyze data from 163 B-ALL patients and identify 332 noncoding mutations that disrupt EP interactions and gene expression. Our analysis recapitulate previously known translocations such as t(14;X) that leads to CRLF2 overexpression, as well as novel causal translocations that leads to deregulation of TMEM106A and MIR663B. Systematic analysis of the identified mutations indicated that SNVs in enhancer region have a higher variant allele frequency (VAF), suggesting enhancer mutations likely occur earlier than coding mutations during oncogenesis. In addition, pathway analysis show that the majority (57%) of mutations perturbing metabolic pathways are noncoding mutations. Further analysis and experimental follow-up of the predicted noncoding mutations may reveal novel insights into the pathogenesis of B-cell ALL. Citation Format: Bing He, Peng Gao, Yang Ding, Chia-Hui Chen, Hannah Kim, Stephen P. Hunger, Sarah K. Tasian, Kai Tan. Systematic analysis of causal noncoding mutations in pediatric B-cell acute lymphoblastic leukemia [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 2280.

Current Advances on the Important Roles of Enhancer RNAs in Gene Regulation and Cancer

Revealing the gene regulation networks governing cancer initiation and development is necessary while it remains uncompleted. In recent years, enhancers have been reported to be widely transcribed, resulting in the generation of enhancer RNAs (eRNAs). Previous studies have reported that eRNAs are a subclass of long noncoding RNAs (lncRNAs), which play a critical role in gene regulation and cancer development. These eRNAs can promote enhancer-promoter (E-P) looping formation by binding to other protein factors or propel expression of downstream protein-coding gene. In this review, we have focused on the characteristics of eRNAs and illustrated the biological function and potential mechanism of eRNAs in regulating gene expression and cancer development.

Enhancer Sharing Promotes Neighborhoods of Transcriptional Regulation Across Eukaryotes.

Enhancers physically interact with transcriptional promoters, looping over distances that can span multiple regulatory elements. Given that enhancer–promoter (EP) interactions generally occur via common protein complexes, it is unclear whether EP pairing is predominantly deterministic or proximity guided. Here, we present cross-organismic evidence suggesting that most EP pairs are compatible, largely determined by physical proximity rather than specific interactions. By reanalyzing transcriptome datasets, we find that the transcription of gene neighbors is correlated over distances that scale with genome size. We experimentally show that nonspecific EP interactions can explain such correlation, and that EP distance acts as a scaling factor for the transcriptional influence of an enhancer. We propose that enhancer sharing is commonplace among eukaryotes, and that EP distance is an important layer of information in gene regulation.

Global view of enhancer-promoter interactome in human cells.

Enhancer mapping has been greatly facilitated by various genomic marks associated with it. However, little is available in our toolbox to link enhancers with their target promoters, hampering mechanistic understanding of enhancer-promoter (EP) interaction. We develop and characterize multiple genomic features for distinguishing true EP pairs from noninteracting pairs. We integrate these features into a probabilistic predictor for EP interactions. Multiple validation experiments demonstrate a significant improvement over state-of-the-art approaches. Systematic analyses of EP interactions across 12 cell types reveal several global features of EP interactions: (i) a larger fraction of EP interactions are cell type specific than enhancers; (ii) promoters controlled by multiple enhancers have higher tissue specificity, but the regulating enhancers are less conserved; (iii) cohesin plays a role in mediating tissue-specific EP interactions via chromatin looping in a CTCF-independent manner. Our approach presents a systematic and effective strategy to decipher the mechanisms underlying EP communication.

Arouser Reveals a Role for Synapse Number in the Regulation of Ethanol Sensitivity

A reduced sensitivity to the sedating effects of alcohol is a characteristic associated with alcohol use disorders (AUDs). A genetic screen for ethanol sedation mutants in Drosophila identified arouser (aru), which functions in developing neurons to reduce ethanol sensitivity. Genetic evidence suggests that aru regulates ethanol sensitivity through its activation by Egfr/Erk signaling and its inhibition by PI3K/Akt signaling. The aru mutant also has an increased number of synaptic terminals in the larva and adult fly. Both the increased ethanol sensitivity and synapse number of the aru mutant are restored upon adult social isolation, suggesting a causal relationship between synapse number and ethanol sensitivity. We thus show that a developmental abnormality affecting synapse number and ethanol sensitivity is not permanent and can be reversed by manipulating the environment of the adult fly.

Sequence Independent Phylogenetic Footprinting Analysis Identifies Novel Transcription Regulation for Orexin Receptors

The orexin receptors (OX1/2R) are G‐protein coupled receptors involved in the regulation of energy metabolism (Tsujino et al. 2009). Current evidence suggests that changes in expression of the OX1/2R are implicated in the development of obesity (Teske et al. 2006). However, there is limited information about the transcriptional regulation of these receptors. A sequence‐independent phylogenetic footprinting bioinformatic analysis of the OXR promoters from six species (human, rat, mouse, chimpanzee, dog and cow) identified several transcription factor binding sites (TFBS). Our preliminary results indicate the transcription factors (TFs) SP1, NFATc2, EGR1 and PPARγ are involved in the transcriptional regulation of OX1/2R. These TFs have been implicated in several rodent models of obesity, yet their specific role in the brain is undefined (Plagemann et al. 2009, van Beekum et al. 2009, T. C. Yang et al. 2006). This study is ongoing and we are currently analyzing TFBS co‐occurrences and OXR promoter's enhancer structures. Simultaneously, we have started working towards validation of the TFBS predictions in set of new immortalized rat hypothalamic cell lines, which either contain or do not contain both OXR. These findings will provide novel insight into the transcriptional regulation of the OX1/2R.Funding: The Department of Veterans Affairs and the National Institute of Health, NIDDK, DK078985‐02S2.

A Gain-of-Function Suppressor Screen for Genes Involved in Dorsal–Ventral Boundary Formation in the Drosophila Wing

The Drosophila wing primordium is subdivided into a dorsal (D) and a ventral (V) compartment by the activity of the LIM-homeodomain protein Apterous in D cells. Cell interactions between D and V cells induce the activation of Notch at the DV boundary. Notch is required for the maintenance of the compartment boundary and the growth of the wing primordium. Beadex, a gain-of-function allele of dLMO, results in increased levels of dLMO protein, which interferes with the activity of Apterous and results in defects in DV axis formation. We performed a gain-of-function enhancer-promoter (EP) screen to search for suppressors of Beadex when overexpressed in D cells. We identified 53 lines corresponding to 35 genes. Loci encoding for micro-RNAs and proteins involved in chromatin organization, transcriptional control, and vesicle trafficking were characterized in the context of dLMO activity and DV boundary formation. Our results indicate that a gain-of-function genetic screen in a sensitized background, as opposed to classical loss-of-function-based screenings, is a very efficient way to identify redundant genes involved in a developmental process.

Antagonistic Interactions between Ikaros and the Chromatin Remodeler Mi-2β Determine Silencer Activity and Cd4 Gene Expression

Lineage commitment is induced by changes in gene expression dictated by the intimate interaction between transcription factors and chromatin regulators. Here, we revealed the antagonistic interplay between Ikaros and its associate the chromatin remodeler Mi-2beta during T cell development, as exemplified by the regulation of Cd4 expression. Loss of Ikaros or Mi-2beta led to activation or repression, respectively, of the Cd4 locus at inappropriate stages of development. Their combined mutation reverted to normal CD4 expression. In double-negative thymocytes, Ikaros binding to the Cd4 silencer contributed to its repressive activity. In double-positive thymocytes, concomitant binding of Mi-2beta with Ikaros to the Cd4 silencer caused silencer inactivation, thereby allowing for CD4 expression. Mi-2beta facilitated recruitment of histone acetyl transferases to the silencer. This recruitment possibly antagonized Ikaros and associated repressive activities. Thus, concomitant interactions between functionally opposing chromatin-regulating machineries are an important mode of gene regulation during lineage determination.

The hypoxia-induced paralogs Scylla and Charybdis inhibit growth by down-regulating S6K activity upstream of TSC in Drosophila

Diverse extrinsic and intrinsic cues must be integrated within a developing organism to ensure appropriate growth at the cellular and organismal level. In Drosophila, the insulin receptor/TOR/S6K signaling network plays a fundamental role in the control of metabolism and cell growth. Here we show that scylla and charybdis, two homologous genes identified as growth suppressors in an EP (enhancer/promoter) overexpression screen, act as negative regulators of growth. The simultaneous loss of both genes generates flies that are more susceptible to reduced oxygen concentrations (hypoxia) and that show mild overgrowth phenotypes. Conversely, scylla or charybdis overactivation reduces growth. Growth inhibition is associated with a reduction in S6K but not PKB/Akt activity. Together, genetic and biochemical analysis places Scylla/Charybdis downstream of PKB and upstream of TSC. Furthermore, we show that scylla and charybdis are induced under hypoxic conditions and that scylla is a target of Drosophila HIF-1 (hypoxia-inducible factor-1) like its mammalian counterpart RTP801/REDD1, thus establishing a potential cross-talk between growth and oxygen sensing.

Osa modulates the expression of Apterous target genes in the Drosophila wing

The establishment of the dorsal–ventral axis of the Drosophila wing depends on the activity of the LIM-homeodomain protein Apterous. Apterous activity depends on the formation of a higher order complex with its cofactor Chip to induce the expression of its target genes. Apterous activity levels are modulated during development by dLMO. Expression of dLMO in the Drosophila wing is regulated by two distinct Chip dependent mechanisms. Early in development, Chip bridges two molecules of Apterous to induce expression of dLMO in the dorsal compartment. Later in development, Chip, independently of Apterous, is required for expression of dLMO in the wing pouch. We have conducted a modular P-element based EP (enhancer/promoter) misexpression screen to look for genes involved in Apterous activity. We have found Osa, a member of the Brahma chromatin-remodeling complex, as a positive modulator of Apterous activity in the Drosophila wing. Osa mediates activation of some Apterous target genes and repression of others, including dLMO. Osa has been shown to bind Chip. We propose that Chip recruits Osa to the Apterous target genes, thus mediating activation or repression of their expression.

An EP overexpression screen for genetic modifiers of Notch pathway function in Drosophila melanogaster

The Notch pathway comprises a signal transduction cascade required for the proper formation of multiple tissues during metazoan development. Originally described in Drosophila for its role in nervous system formation, the pathway has attracted much wider interest owing to its fundamental roles in a range of developmental and disease-related processes. Despite extensive analysis, Notch signaling is not completely understood and it appears that additional components of the pathway remain to be identified and characterized. Here, we describe a novel genetic strategy to screen for additional Notch pathway genes. The strategy combines partial loss of function for pathway activity with Enhancer-promoter (EP)-induced overexpression of random loci across the dorsoventral wing margin. Mastermind (Mam) is a nuclear component of the Notch signaling cascade. Using a GAL4-UAS-driven dominant-negative form of Mam, we created a genotype that exhibits a completely penetrant dominant wing-nicking phenotype. This phenotype was assayed for enhancement or suppression after outcrossing to several thousand EP lines. The screen identified known components or modifiers of Notch pathway function, as well as several potential new components. Our results suggest that a genetic screen that combines partial loss of function with random gene overexpression might be a useful strategy in the analysis of developmental pathways.

Osa modulates the expression of Apterous target genes in the Drosophila wing

The establishment of the dorsal–ventral axis of the Drosophila wing depends on the activity of the LIM-homeodomain protein Apterous. Apterous activity depends on the formation of a higher order complex with its cofactor Chip to induce the expression of its target genes. Apterous activity levels are modulated during development by dLMO. Expression of dLMO in the Drosophila wing is regulated by two distinct Chip dependent mechanisms. Early in development, Chip bridges two molecules of Apterous to induce expression of dLMO in the dorsal compartment. Later in development, Chip, independently of Apterous, is required for expression of dLMO in the wing pouch. We have conducted a modular P-element based EP (enhancer/promoter) misexpression screen to look for genes involved in Apterous activity. We have found Osa, a member of the Brahma chromatin-remodeling complex, as a positive modulator of Apterous activity in the Drosophila wing. Osa mediates activation of some Apterous target genes and repression of others, including dLMO. Osa has been shown to bind Chip. We propose that Chip recruits Osa to the Apterous target genes, thus mediating activation or repression of their expression.