Identification of Highly Repetitive Enhancers with Long-range Regulation Potential in Barley via STARR-seq.

Enhancers are DNA sequences that can strengthen transcription initiation. However, the global identification of plant enhancers is complicated due to uncertainty in the distance and orientation of enhancers, especially in species with large genomes. In this study, we performed self-transcribing active regulatory region sequencing (STARR-seq) for the first time to identify enhancers across the barley genome. A total of 7323 enhancers were successfully identified, and among 45 randomly selected enhancers, over 75% were effective as validated by a dual-luciferase reporter assay system in the lower epidermis of tobacco leaves. Interestingly, up to 53.5% of the barley enhancers were repetitive sequences, especially transposable elements (TEs), thus reinforcing the vital role of repetitive enhancers in gene expression. Both the common active mark H3K4me3 and repressive mark H3K27me3 were abundant among the barley STARR-seq enhancers. In addition, the functional range of barley STARR-seq enhancers seemed much broader than that of rice or maize and extended to ±100 kb of the gene body, and this finding was consistent with the high expression levels of genes in the genome. This study specifically depicts the unique features of barley enhancers and provides available barley enhancers for further utilization.

Comprehensive network modeling approaches unravel dynamic enhancer-promoter interactions across neural differentiation.

BACKGROUND: Increasing evidence suggests that a substantial proportion of disease-associated mutations occur in enhancers, regions of non-coding DNA essential to gene regulation. Understanding the structures and mechanisms of the regulatory programs this variation affects can shed light on the apparatuses of human diseases. RESULTS: We collect epigenetic and gene expression datasets from seven early time points during neural differentiation. Focusing on this model system, we construct networks of enhancer-promoter interactions, each at an individual stage of neural induction. These networks serve as the base for a rich series of analyses, through which we demonstrate their temporal dynamics and enrichment for various disease-associated variants. We apply the Girvan-Newman clustering algorithm to these networks to reveal biologically relevant substructures of regulation. Additionally, we demonstrate methods to validate predicted enhancer-promoter interactions using transcription factor overexpression and massively parallel reporter assays. CONCLUSIONS: Our findings suggest a generalizable framework for exploring gene regulatory programs and their dynamics across developmental processes; this includes a comprehensive approach to studying the effects of disease-associated variation on transcriptional networks. The techniques applied to our networks have been published alongside our findings as a computational tool, E-P-INAnalyzer. Our procedure can be utilized across different cellular contexts and disorders.

Principle and application of self-transcribing active regulatory region sequencing in enhancer discovery research.

Self-transcribing active regulatory region sequencing (STARR-seq) is a high-throughput sequencing method capable of simultaneously discovering and validating all enhancers within the genome. In this method, candidate sequences are inserted into plasmid vectors and electroporated into cells. Acting as both enhancers and target genes, the self-transcription of these sequences will also be enhanced by themselves. By sequencing the transcriptome and comparing the results with the non-inserted control, the locations and activity of enhancers can be determined. In traditional enhancer discovery strategies, the chromatin open regions and transcription active regions were sequenced and predicted as enhancers. However, the activity of these putative enhancers could only be validated one by one without a high-throughput method. STARR-seq solved this limitation, allowing simultaneous enhancers discovery and activity validation in a high-throughput manner. Since the introduction of STARR-seq, it has been widely used to discover enhancers and validate enhancer activity in a number of organisms and cells. In this review, we present the traditional enhancer prediction methods and the basic principles, development history, specific applications of STARR-seq, and its future prospects, aiming to provide a reference for researchers in related fields conducting enhancer studies.

A RORE-dependent Intronic Enhancer in the IL-7 Receptor-α Locus Controls Glucose Metabolism via Vγ4+ γδT17 Cells.

The IL-7R regulates the homeostasis, activation, and distribution of T cells in peripheral tissues. Although several transcriptional enhancers that regulate IL-7Rα expression in αß T cells have been identified, enhancers active in γδ T cells remain unknown. In this article, we discovered an evolutionarily conserved noncoding sequence (CNS) in intron 2 of the IL-7Rα-chain (IL-7Rα) locus and named this region CNS9. CNS9 contained a conserved retinoic acid receptor-related orphan receptor (ROR)-responsive element (RORE) and exerted RORγt-dependent enhancer activity in vitro. Mice harboring point mutations in the RORE in CNS9 (CNS9-RORmut) showed reduced IL-7Rα expression in IL-17-producing Vγ4+ γδ T cells. In addition, the cell number and IL-17A production of Vγ4+ γδ T cells were reduced in the adipose tissue of CNS9-RORmut mice. Consistent with the reduction in IL-17A, CNS9-RORmut mice exhibited decreased IL-33 expression in the adipose tissue, resulting in fewer regulatory T cells and glucose intolerance. The CNS9-ROR motif was partially responsible for IL-7Rα expression in RORγt+ regulatory T cells, whereas IL-7Rα expression was unaffected in RORγt-expressing Vγ2+ γδ T cells, Th17 cells, type 3 innate lymphoid cells, and invariant NKT cells. Our results indicate that CNS9 is a RORΕ-dependent, Vγ4+ γδ T cell-specific IL-7Rα enhancer that plays a critical role in adipose tissue homeostasis via regulatory T cells, suggesting that the evolutionarily conserved RORΕ in IL-7Rα intron 2 may influence the incidence of type 2 diabetes.

Comprehensive mapping and modelling of the rice regulome landscape unveils the regulatory architecture underlying complex traits.

Unraveling the regulatory mechanisms that govern complex traits is pivotal for advancing crop improvement. Here we present a comprehensive regulome atlas for rice (Oryza sativa), charting the chromatin accessibility across 23 distinct tissues from three representative varieties. Our study uncovers 117,176 unique open chromatin regions (OCRs), accounting for ~15% of the rice genome, a notably higher proportion compared to previous reports in plants. Integrating RNA-seq data from matched tissues, we confidently predict 59,075 OCR-to-gene links, with enhancers constituting 69.54% of these associations, including many known enhancer-to-gene links. Leveraging this resource, we re-evaluate genome-wide association study results and discover a previously unknown function of OsbZIP06 in seed germination, which we subsequently confirm through experimental validation. We optimize deep learning models to decode regulatory grammar, achieving robust modeling of tissue-specific chromatin accessibility. This approach allows to predict cross-variety regulatory dynamics from genomic sequences, shedding light on the genetic underpinnings of cis-regulatory divergence and morphological disparities between varieties. Overall, our study establishes a foundational resource for rice functional genomics and precision molecular breeding, providing valuable insights into regulatory mechanisms governing complex traits.

A pattern emerges in chromatin aging: AP-1 steals the show.

During aging, transcriptional programs of cell identity are partially eroded, reducing cellular fitness and resilience. Patrick et al.1 unveil a general mechanism for this process that consists of the progressive loss of transcription factor AP-1 from cell identity enhancers and its relocation by competition to stress-response elements.

DKK1-SE recruits AP1 to activate the target gene DKK1 thereby promoting pancreatic cancer progression.

Super-enhancers are a class of DNA cis-regulatory elements that can regulate cell identity, cell fate, stem cell pluripotency, and even tumorigenesis. Increasing evidence shows that epigenetic modifications play an important role in the pathogenesis of various types of cancer. However, the current research is far from enough to reveal the complex mechanism behind it. This study found a super-enhancer enriched with abnormally active histone modifications in pancreatic ductal adenocarcinoma (PDAC), called DKK1-super-enhancer (DKK1-SE). The major active component of DKK1-SE is component enhancer e1. Mechanistically, AP1 induces chromatin remodeling in component enhancer e1 and activates the transcriptional activity of DKK1. Moreover, DKK1 was closely related to the malignant clinical features of PDAC. Deletion or knockdown of DKK1-SE significantly inhibited the proliferation, colony formation, motility, migration, and invasion of PDAC cells in vitro, and these phenomena were partly mitigated upon rescuing DKK1 expression. In vivo, DKK1-SE deficiency not only inhibited tumor proliferation but also reduced the complexity of the tumor microenvironment. This study identifies that DKK1-SE drives DKK1 expression by recruiting AP1 transcription factors, exerting oncogenic effects in PDAC, and enhancing the complexity of the tumor microenvironment.

DNA-binding factor footprints and enhancer RNAs identify functional non-coding genetic variants.

BACKGROUND: Genome-wide association studies (GWAS) have revealed a multitude of candidate genetic variants affecting the risk of developing complex traits and diseases. However, the highlighted regions are typically in the non-coding genome, and uncovering the functional causative single nucleotide variants (SNVs) is challenging. Prioritization of variants is commonly based on genomic annotation with markers of active regulatory elements, but current approaches still poorly predict functional variants. To address this, we systematically analyze six markers of active regulatory elements for their ability to identify functional variants. RESULTS: We benchmark against molecular quantitative trait loci (molQTL) from assays of regulatory element activity that identify allelic effects on DNA-binding factor occupancy, reporter assay expression, and chromatin accessibility. We identify the combination of DNase footprints and divergent enhancer RNA (eRNA) as markers for functional variants. This signature provides high precision, but with a trade-off of low recall, thus substantially reducing candidate variant sets to prioritize variants for functional validation. We present this as a framework called FINDER-Functional SNV IdeNtification using DNase footprints and eRNA. CONCLUSIONS: We demonstrate the utility to prioritize variants using leukocyte count trait and analyze variants in linkage disequilibrium with a lead variant to predict a functional variant in asthma. Our findings have implications for prioritizing variants from GWAS, in development of predictive scoring algorithms, and for functionally informed fine mapping approaches.

Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells.

Sequencing-based mapping of ensemble pairwise interactions among regulatory elements support the existence of topological assemblies known as promoter-enhancer hubs or cliques in cancer. Yet, prevalence, regulators, and functions of promoter-enhancer hubs in individual cancer cells remain unclear. Here, we systematically integrated functional genomics, transcription factor screening, and optical mapping of promoter-enhancer interactions to identify key promoter-enhancer hubs, examine heterogeneity of their assembly, determine their regulators, and elucidate their role in gene expression control in individual triple negative breast cancer (TNBC) cells. Optical mapping of individual SOX9 and MYC alleles revealed the existence of frequent multiway interactions among promoters and enhancers within spatial hubs. Our single-allele studies further demonstrated that lineage-determining SOX9 and signaling-dependent NOTCH1 transcription factors compact MYC and SOX9 hubs. Together, our findings suggest that promoter-enhancer hubs are dynamic and heterogeneous topological assemblies, which are controlled by oncogenic transcription factors and facilitate subtype-restricted gene expression in cancer.

Potential Transcriptional Enhancers in Coronaviruses: From Infectious Bronchitis Virus to SARS-CoV-2.

Coronaviruses constitute a global threat to human and animal health. It is essential to investigate the long-distance RNA-RNA interactions that approximate remote regulatory elements in strategies, including genome circularization, discontinuous transcription, and transcriptional enhancers, aimed at the rapid replication of their large genomes, pathogenicity, and immune evasion. Based on the primary sequences and modeled RNA-RNA interactions of two experimentally defined coronaviral enhancers, we detected via an in silico primary and secondary structural analysis potential enhancers in various coronaviruses, from the phylogenetically ancient avian infectious bronchitis virus (IBV) to the recently emerged SARS-CoV-2. These potential enhancers possess a core duplex-forming region that could transition between closed and open states, as molecular switches directed by viral or host factors. The duplex open state would pair with remote sequences in the viral genome and modulate the expression of downstream crucial genes involved in viral replication and host immune evasion. Consistently, variations in the predicted IBV enhancer region or its distant targets coincide with cases of viral attenuation, possibly driven by decreased open reading frame (ORF)3a immune evasion protein expression. If validated experimentally, the annotated enhancer sequences could inform structural prediction tools and antiviral interventions.

Specific multivalent molecules boost CRISPR-mediated transcriptional activation.

CRISPR/Cas-based transcriptional activators can be enhanced by intrinsically disordered regions (IDRs). However, the underlying mechanisms are still debatable. Here, we examine 12 well-known IDRs by fusing them to the dCas9-VP64 activator, of which only seven can augment activation, albeit independently of their phase separation capabilities. Moreover, modular domains (MDs), another class of multivalent molecules, though ineffective in enhancing dCas9-VP64 activity on their own, show substantial enhancement in transcriptional activation when combined with dCas9-VP64-IDR. By varying the number of gRNA binding sites and fusing dCas9-VP64 with different IDRs/MDs, we uncover that optimal, rather than maximal, cis-trans cooperativity enables the most robust activation. Finally, targeting promoter-enhancer pairs yields synergistic effects, which can be further amplified via enhancing chromatin interactions. Overall, our study develops a versatile platform for efficient gene activation and sheds important insights into CRIPSR-based transcriptional activators enhanced with multivalent molecules.

Transcriptional bursting: from fundamentals to novel insights.

Transcription occurs as irregular bursts in a very wide range of systems, including numerous different species and many genes within these. In this review, we examine the underlying theories, discuss how these relate to experimental measurements, and explore some of the discrepancies that have emerged among various studies. Finally, we consider more recent works that integrate novel concepts, such as the involvement of biomolecular condensates in enhancer-promoter interactions and their effects on the dynamics of transcriptional bursting.

An i-motif-regulated enhancer, eRNA and adjacent lncRNA affect Lhb expression through distinct mechanisms in a sex-specific context.

Genome-wide studies have demonstrated regulatory roles for diverse non-coding elements, but their precise and interrelated functions have often remained enigmatic. Addressing the need for mechanistic insight, we studied their roles in expression of Lhb which encodes the pituitary gonadotropic hormone that controls reproduction. We identified a bi-directional enhancer in gonadotrope-specific open chromatin, whose functional eRNA (eRNA2) supports permissive chromatin at the Lhb locus. The central untranscribed region of the enhancer contains an iMotif (iM), and is bound by Hmgb2 which stabilizes the iM and directs transcription specifically towards the functional eRNA2. A distinct downstream lncRNA, associated with an inducible G-quadruplex (G4) and iM, also facilitates Lhb expression, following its splicing in situ. GnRH activates Lhb transcription and increased levels of all three RNAs, eRNA2 showing the highest response, while estradiol, which inhibits Lhb, repressed levels of eRNA2 and the lncRNA. The levels of these regulatory RNAs and Lhb mRNA correlate highly in female mice, though strikingly not in males, suggesting a female-specific function. Our findings, which shed new light on the workings of non-coding elements and non-canonical DNA structures, reveal novel mechanisms regulating transcription which have implications not only in the central control of reproduction but also for other inducible genes.

A transcriptional enhancer regulates cardiac maturation.

Cardiomyocyte maturation is crucial for generating adult cardiomyocytes and the application of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). However, regulation at the cis-regulatory element level and its role in heart disease remain unclear. Alpha-actinin 2 (ACTN2) levels increase during CM maturation. In this study, we investigated a clinically relevant, conserved ACTN2 enhancer's effects on CM maturation using hPSC and mouse models. Heterozygous ACTN2 enhancer deletion led to abnormal CM morphology, reduced function and mitochondrial respiration. Transcriptomic analyses in vitro and in vivo showed disrupted CM maturation and upregulated anabolic mammalian target for rapamycin (mTOR) signaling, promoting senescence and hindering maturation. As confirmation, ACTN2 enhancer deletion induced heat shock protein 90A expression, a chaperone mediating mTOR activation. Conversely, targeting the ACTN2 enhancer via enhancer CRISPR activation (enCRISPRa) promoted hPSC-CM maturation. Our studies reveal the transcriptional enhancer's role in cardiac maturation and disease, offering insights into potentially fine-tuning gene expression to modulate cardiomyocyte physiology.

Quantitative analysis of cis-regulatory elements in transcription with KAS-ATAC-seq.

Cis-regulatory elements (CREs) are pivotal in orchestrating gene expression throughout diverse biological systems. Accurate identification and in-depth characterization of functional CREs are crucial for decoding gene regulation networks during cellular processes. In this study, we develop Kethoxal-Assisted Single-stranded DNA Assay for Transposase-Accessible Chromatin with Sequencing (KAS-ATAC-seq) to quantitatively analyze the transcriptional activity of CREs. A main advantage of KAS-ATAC-seq lies in its precise measurement of ssDNA levels within both proximal and distal ATAC-seq peaks, enabling the identification of transcriptional regulatory sequences. This feature is particularly adept at defining Single-Stranded Transcribing Enhancers (SSTEs). SSTEs are highly enriched with nascent RNAs and specific transcription factors (TFs) binding sites that define cellular identity. Moreover, KAS-ATAC-seq provides a detailed characterization and functional implications of various SSTE subtypes. Our analysis of CREs during mouse neural differentiation demonstrates that KAS-ATAC-seq can effectively identify immediate-early activated CREs in response to retinoic acid (RA) treatment. Our findings indicate that KAS-ATAC-seq provides more precise annotation of functional CREs in transcription. Future applications of KAS-ATAC-seq would help elucidate the intricate dynamics of gene regulation in diverse biological processes.

Super-enhancer omics in stem cell.

The hallmarks of stem cells, such as proliferation, self-renewal, development, differentiation, and regeneration, are critical to maintain stem cell identity which is sustained by genetic and epigenetic factors. Super-enhancers (SEs), which consist of clusters of active enhancers, play a central role in maintaining stemness hallmarks by specifically transcriptional model. The SE-navigated transcriptional complex, including SEs, non-coding RNAs, master transcriptional factors, Mediators and other co-activators, forms phase-separated condensates, which offers a toggle for directing diverse stem cell fate. With the burgeoning technologies of multiple-omics applied to examine different aspects of SE, we firstly raise the concept of "super-enhancer omics", inextricably linking to Pan-omics. In the review, we discuss the spatiotemporal organization and concepts of SEs, and describe links between SE-navigated transcriptional complex and stem cell features, such as stem cell identity, self-renewal, pluripotency, differentiation and development. We also elucidate the mechanism of stemness and oncogenic SEs modulating cancer stem cells via genomic and epigenetic alterations hijack in cancer stem cell. Additionally, we discuss the potential of targeting components of the SE complex using small molecule compounds, genome editing, and antisense oligonucleotides to treat SE-associated organ dysfunction and diseases, including cancer. This review also provides insights into the future of stem cell research through the paradigm of SEs.

Artificially inserted strong promoter containing multiple G-quadruplexes induces long-range chromatin modification.

Although the role of G-quadruplex (G4) DNA structures has been suggested in chromosomal looping this was not tested directly. Here, to test causal function, an array of G4s, or control sequence that does not form G4s, were inserted within chromatin in cells. In vivo G4 formation of the inserted G4 sequence array, and not the control sequence, was confirmed using G4-selective antibody. Compared to the control insert, we observed a remarkable increase in the number of 3D chromatin looping interactions from the inserted G4 array. This was evident within the immediate topologically associated domain (TAD) and throughout the genome. Locally, recruitment of enhancer histone marks and the transcriptional coactivator p300/Acetylated-p300 increased in the G4-array, but not in the control insertion. Resulting promoter-enhancer interactions and gene activation were clear up to 5 Mb away from the insertion site. Together, these show the causal role of G4s in enhancer function and long-range chromatin interactions. Mechanisms of 3D topology are primarily based on DNA-bound architectural proteins that induce/stabilize long-range interactions. Involvement of the underlying intrinsic DNA sequence/structure in 3D looping shown here therefore throws new light on how long-range chromosomal interactions might be induced or maintained.

Cooperative insulation of regulatory domains by CTCF-dependent physical insulation and promoter competition.

The specificity of gene expression during development requires the insulation of regulatory domains to avoid inappropriate enhancer-gene interactions. In vertebrates, this insulator function is mostly attributed to clusters of CTCF sites located at topologically associating domain (TAD) boundaries. However, TAD boundaries allow some physical crosstalk across regulatory domains, which is at odds with the specific and precise expression of developmental genes. Here we show that developmental genes and nearby clusters of CTCF sites cooperatively foster the robust insulation of regulatory domains. By genetically dissecting a couple of representative loci in mouse embryonic stem cells, we show that CTCF sites prevent undesirable enhancer-gene contacts (i.e. physical insulation), while developmental genes preferentially contribute to regulatory insulation through non-structural mechanisms involving promoter competition rather than enhancer blocking. Overall, our work provides important insights into the insulation of regulatory domains, which in turn might help interpreting the pathological consequences of certain structural variants.

Multi-omics profiling of retinal pigment epithelium reveals enhancer-driven activation of RANK-NFATc1 signaling in traumatic proliferative vitreoretinopathy.

During the progression of proliferative vitreoretinopathy (PVR) following ocular trauma, previously quiescent retinal pigment epithelial (RPE) cells transition into a state of rapid proliferation, migration, and secretion. The elusive molecular mechanisms behind these changes have hindered the development of effective pharmacological treatments, presenting a pressing clinical challenge. In this study, by monitoring the dynamic changes in chromatin accessibility and various histone modifications, we chart the comprehensive epigenetic landscape of RPE cells in male mice subjected to traumatic PVR. Coupled with transcriptomic analysis, we reveal a robust correlation between enhancer activation and the upregulation of the PVR-associated gene programs. Furthermore, by constructing transcription factor regulatory networks, we identify the aberrant activation of enhancer-driven RANK-NFATc1 pathway as PVR advanced. Importantly, we demonstrate that intraocular interventions, including nanomedicines inhibiting enhancer activity, gene therapies targeting NFATc1 and antibody therapeutics against RANK pathway, effectively mitigate PVR progression. Together, our findings elucidate the epigenetic basis underlying the activation of PVR-associated genes during RPE cell fate transitions and offer promising therapeutic avenues targeting epigenetic modulation and the RANK-NFATc1 axis for PVR management.