Super enhancer lncRNAs: a novel hallmark in cancer.

Super enhancers (SEs) consist of clusters of enhancers, harboring an unusually high density of transcription factors, mediator coactivators and epigenetic modifications. SEs play a crucial role in the maintenance of cancer cell identity and promoting oncogenic transcription. Super enhancer lncRNAs (SE-lncRNAs) refer to either transcript from SEs locus or interact with SEs, whose transcriptional activity is highly dependent on SEs. Moreover, these SE-lncRNAs can interact with their associated enhancer regions in cis and modulate the expression of oncogenes or key signal pathways in cancers. Inhibition of SEs would be a promising therapy for cancer. In this review, we summarize the research of SE-lncRNAs in different kinds of cancers so far and decode the mechanism of SE-lncRNAs in carcinogenesis to provide novel ideas for the cancer therapy.

Identification of the Efficient Enhancer Elements in FVIII-Padua for Gene Therapy Study of Hemophilia A.

Hemophilia A (HA) is a common X-linked recessive hereditary bleeding disorder. Coagulation factor VIII (FVIII) is insufficient in patients with HA due to the mutations in the F8 gene. The restoration of plasma levels of FVIII via both recombinant B-domain-deleted FVIII (BDD-FVIII) and B-domain-deleted F8 (BDDF8) transgenes was proven to be helpful. FVIII-Padua is a 23.4 kb tandem repeat mutation in the F8 associated with a high F8 gene expression and thrombogenesis. Here we screened a core enhancer element in FVIII-Padua for improving the F8 expression. In detail, we identified a 400 bp efficient enhancer element, C400, in FVIII-Padua for the first time. The core enhancer C400 extensively improved the transcription of BDDF8 driven by human elongation factor-1 alpha in HepG2, HeLa, HEK-293T and induced pluripotent stem cells (iPSCs) with different genetic backgrounds, as well as iPSCs-derived endothelial progenitor cells (iEPCs) and iPSCs-derived mesenchymal stem cells (iMSCs). The expression of FVIII protein was increased by C400, especially in iEPCs. Our research provides a novel molecular target to enhance expression of FVIII protein, which has scientific value and application prospects in both viral and nonviral HA gene therapy strategies.

Enhancer-promoter interactions become more instructive in the transition from cell-fate specification to tissue differentiation.

To regulate expression, enhancers must come in proximity to their target gene. However, the relationship between the timing of enhancer-promoter (E-P) proximity and activity remains unclear, with examples of uncoupled, anticorrelated and correlated interactions. To assess this, we selected 600 characterized enhancers or promoters with tissue-specific activity in Drosophila embryos and performed Capture-C in FACS-purified myogenic or neurogenic cells during specification and tissue differentiation. This enabled direct comparison between E-P proximity and activity transitioning from OFF-to-ON and ON-to-OFF states across developmental conditions. This showed remarkably similar E-P topologies between specified muscle and neuronal cells, which are uncoupled from activity. During tissue differentiation, many new distal interactions emerge where changes in E-P proximity reflect changes in activity. The mode of E-P regulation therefore appears to change as embryogenesis proceeds, from largely permissive topologies during cell-fate specification to more instructive regulation during terminal tissue differentiation, when E-P proximity is coupled to activation.

Dissection of a CTCF topological boundary uncovers principles of enhancer-oncogene regulation.

Enhancer-gene communication is dependent on topologically associating domains (TADs) and boundaries enforced by the CCCTC-binding factor (CTCF) insulator, but the underlying structures and mechanisms remain controversial. Here, we investigate a boundary that typically insulates fibroblast growth factor (FGF) oncogenes but is disrupted by DNA hypermethylation in gastrointestinal stromal tumors (GISTs). The boundary contains an array of CTCF sites that enforce adjacent TADs, one containing FGF genes and the other containing ANO1 and its putative enhancers, which are specifically active in GIST and its likely cell of origin. We show that coordinate disruption of four CTCF motifs in the boundary fuses the adjacent TADs, allows the ANO1 enhancer to contact FGF3, and causes its robust induction. High-resolution micro-C maps reveal specific contact between transcription initiation sites in the ANO1 enhancer and FGF3 promoter that quantitatively scales with FGF3 induction such that modest changes in contact frequency result in strong changes in expression, consistent with a causal relationship.

MYC activity at enhancers drives prognostic transcriptional programs through an epigenetic switch.

The transcription factor MYC is overexpressed in most cancers, where it drives multiple hallmarks of cancer progression. MYC is known to promote oncogenic transcription by binding to active promoters. In addition, MYC has also been shown to invade distal enhancers when expressed at oncogenic levels, but this enhancer binding has been proposed to have low gene-regulatory potential. Here, we demonstrate that MYC directly regulates enhancer activity to promote cancer type-specific gene programs predictive of poor patient prognosis. MYC induces transcription of enhancer RNA through recruitment of RNA polymerase II (RNAPII), rather than regulating RNAPII pause-release, as is the case at promoters. This process is mediated by MYC-induced H3K9 demethylation and acetylation by GCN5, leading to enhancer-specific BRD4 recruitment through its bromodomains, which facilitates RNAPII recruitment. We propose that MYC drives prognostic cancer type-specific gene programs through induction of an enhancer-specific epigenetic switch, which can be targeted by BET and GCN5 inhibitors.

The immunoglobulin heavy chain super enhancer controls class switch recombination in developing B cells.

Class switch recombination (CSR) plays an important role in adaptive immune response by enabling mature B cells to replace the initial IgM by another antibody class (IgG, IgE or IgA). CSR is preceded by transcription of the IgH constant genes and is controlled by the super-enhancer 3' regulatory region (3'RR) in an activation-specific manner. The 3'RR is composed of four enhancers (hs3a, hs1-2, hs3b and hs4). In mature B cells, 3'RR activity correlates with transcription of its enhancers. CSR can also occur in primary developing B cells though at low frequency, but in contrast to mature B cells, the transcriptional elements that regulate the process in developing B cells are ill-known. In particular, the role of the 3'RR in the control of constant genes' transcription and CSR has not been addressed. Here, by using a mouse line devoid of the 3'RR and a culture system that highly enriches in pro-B cells, we show that the 3'RR activity is indeed required for switch transcription and CSR, though its effect varies in an isotype-specific manner and correlates with transcription of hs4 enhancer only.

Super-Enhancers and Their Parts: From Prediction Efforts to Pathognomonic Status.

Super-enhancers (SEs) are regions of the genome that play a crucial regulatory role in gene expression by promoting large-scale transcriptional responses in various cell types and tissues. Recent research suggests that alterations in super-enhancer activity can contribute to the development and progression of various disorders. The aim of this research is to explore the multifaceted roles of super-enhancers in gene regulation and their significant implications for understanding and treating complex diseases. Here, we study and summarise the classification of super-enhancer constituents, their possible modes of interaction, and cross-regulation, including super-enhancer RNAs (seRNAs). We try to investigate the opportunity of SE dynamics prediction based on the hierarchy of enhancer single elements (enhancers) and their aggregated action. To further our understanding, we conducted an in silico experiment to compare and differentiate between super-enhancers and locus-control regions (LCRs), shedding light on the enigmatic relationship between LCRs and SEs within the human genome. Particular attention is paid to the classification of specific mechanisms and their diversity, exemplified by various oncological, cardiovascular, and immunological diseases, as well as an overview of several anti-SE therapies. Overall, the work presents a comprehensive analysis of super-enhancers across different diseases, aiming to provide insights into their regulatory roles and may act as a rationale for future clinical interventions targeting these regulatory elements.

An Emerging Role for Enhancer RNAs in Brain Disorders.

Noncoding DNA undergoes widespread context-dependent transcription to produce noncoding RNAs. In recent decades, tremendous advances in genomics and transcriptomics have revealed important regulatory roles for noncoding DNA elements and the RNAs that they produce. Enhancers are one such element that are well-established drivers of gene expression changes in response to a variety of factors such as external stimuli, cellular responses, developmental cues, and disease states. They are known to act at long distances, interact with multiple target gene loci simultaneously, synergize with other enhancers, and associate with dynamic chromatin architectures to form a complex regulatory network. Recent advances in enhancer biology have revealed that upon activation, enhancers transcribe long noncoding RNAs, known as enhancer RNAs (eRNAs), that have been shown to play important roles in enhancer-mediated gene regulation and chromatin-modifying activities. In the brain, enhancer dysregulation and eRNA transcription has been reported in numerous disorders from acute injuries to chronic neurodegeneration. Because this is an emerging area, a comprehensive understanding of eRNA function has not yet been achieved in brain disorders; however, the findings to date have illuminated a role for eRNAs in activity-driven gene expression and phenotypic outcomes. In this review, we highlight the breadth of the current literature on eRNA biology in brain health and disease and discuss the challenges as well as focus areas and strategies for future in-depth research on eRNAs in brain health and disease.

Decoding chromatin states by proteomic profiling of nucleosome readers.

DNA and histone modifications combine into characteristic patterns that demarcate functional regions of the genome1,2. While many 'readers' of individual modifications have been described3-5, how chromatin states comprising composite modification signatures, histone variants and internucleosomal linker DNA are interpreted is a major open question. Here we use a multidimensional proteomics strategy to systematically examine the interaction of around 2,000 nuclear proteins with over 80 modified dinucleosomes representing promoter, enhancer and heterochromatin states. By deconvoluting complex nucleosome-binding profiles into networks of co-regulated proteins and distinct nucleosomal features driving protein recruitment or exclusion, we show comprehensively how chromatin states are decoded by chromatin readers. We find highly distinctive binding responses to different features, many factors that recognize multiple features, and that nucleosomal modifications and linker DNA operate largely independently in regulating protein binding to chromatin. Our online resource, the Modification Atlas of Regulation by Chromatin States (MARCS), provides in-depth analysis tools to engage with our results and advance the discovery of fundamental principles of genome regulation by chromatin states.

Enhancer Arrays Regulating Developmental Genes: Sox2 Enhancers as a Paradigm.

Enhancers are the primary regulatory DNA sequences in eukaryotes and are mostly located in the non-coding sequences of genes, namely, intergenic regions and introns. The essential characteristic of an enhancer is the ability to activate proximal genes, e.g., a reporter gene in a reporter assay, regardless of orientation, relative position, and distance from the gene. These characteristics are ascribed to the interaction (spatial proximity) of the enhancer sequence and the gene promoter via DNA looping, discussed in the latter part of this chapter.Developmentally regulated genes are associated with multiple enhancers carrying distinct cell and developmental stage specificities, which form arrays on the genome. We discuss the array of enhancers regulating the Sox2 gene as a paradigm. Sox2 enhancers are the best studied enhancers of a single gene in developmental regulation. In addition, the Sox2 gene is located in a genomic region with a very sparse gene distribution (no other protein-coding genes in ~1.6 Mb in the mouse genome), termed a "gene desert," which means that most identified enhancers in the region are associated with Sox2 regulation. Furthermore, the importance of the Sox2 gene in stem cell regulation and neural development justifies focusing on Sox2-associated enhancers.

Enhancer Activation by Transcription Factors and Underlying Mechanisms.

Enhancers are classified into two classes based on various criteria. Class I enhancers participate primarily in finely tuned cell-specific regulation, as exemplified by the neural enhancers discussed in Chap. 9 . They are activated by simultaneous binding of transcription factors (TFs) to adjacent sites in the core sequence and are marked by moderate levels of H3K27ac modification. Class II enhancers are activated by the reiterated binding of the same TFs at multiple sites and are marked by high levels of H3K27ac modification. Class II enhancers are exemplified by enhancers in the SCR downstream of the Sox2 gene, as also discussed in Chap. 9 . Both classes of enhancers activate transcription similarly with low selectivity toward the promoters.The genomic loci broadly covered by high-level H3K27ac modification were once dubbed "Super-enhancers," implying that they are densely packed enhancers with superpowers in gene regulation. However, marking with H3K27ac modification does not predict the enhancer activity of a sequence; a "Super enhancer" region includes a few ordinary Class II enhancers. Currently, the most reliable criterion for enhancer prediction is cross-species sequence conservation.The mechanism by which transcription factors find and stay on the target enhancer site remains elusive. Results from two approaches, single-molecule live imaging and kinetic analysis using FRAP, are discussed.

Endothelial gene regulatory elements associated with cardiopharyngeal lineage differentiation.

Endothelial cells (EC) differentiate from multiple sources, including the cardiopharyngeal mesoderm, which gives rise also to cardiac and branchiomeric muscles. The enhancers activated during endothelial differentiation within the cardiopharyngeal mesoderm are not completely known. Here, we use a cardiogenic mesoderm differentiation model that activates an endothelial transcription program to identify endothelial regulatory elements activated in early cardiogenic mesoderm. Integrating chromatin remodeling and gene expression data with available single-cell RNA-seq data from mouse embryos, we identify 101 putative regulatory elements of EC genes. We then apply a machine-learning strategy, trained on validated enhancers, to predict enhancers. Using this computational assay, we determine that 50% of these sequences are likely enhancers, some of which are already reported. We also identify a smaller set of regulatory elements of well-known EC genes and validate them using genetic and epigenetic perturbation. Finally, we integrate multiple data sources and computational tools to search for transcriptional factor binding motifs. In conclusion, we show EC regulatory sequences with a high likelihood to be enhancers, and we validate a subset of them using computational and cell culture models. Motif analyses show that the core EC transcription factors GATA/ETS/FOS is a likely driver of EC regulation in cardiopharyngeal mesoderm.

Enhancer-MDLF: a novel deep learning framework for identifying cell-specific enhancers.

Enhancers, noncoding DNA fragments, play a pivotal role in gene regulation, facilitating gene transcription. Identifying enhancers is crucial for understanding genomic regulatory mechanisms, pinpointing key elements and investigating networks governing gene expression and disease-related mechanisms. Existing enhancer identification methods exhibit limitations, prompting the development of our novel multi-input deep learning framework, termed Enhancer-MDLF. Experimental results illustrate that Enhancer-MDLF outperforms the previous method, Enhancer-IF, across eight distinct human cell lines and exhibits superior performance on generic enhancer datasets and enhancer-promoter datasets, affirming the robustness of Enhancer-MDLF. Additionally, we introduce transfer learning to provide an effective and potential solution to address the prediction challenges posed by enhancer specificity. Furthermore, we utilize model interpretation to identify transcription factor binding site motifs that may be associated with enhancer regions, with important implications for facilitating the study of enhancer regulatory mechanisms. The source code is openly accessible at https://github.com/HaoWuLab-Bioinformatics/Enhancer-MDLF.

From sequence to consequence: Deciphering the complex cisregulatory landscape.

Cell type-specific expression of genes plays a pivotal role in the development and evolution of multicellular organisms over millions of years. The majority of regulatory control resides within the non-coding regions of the genome, referred to as 'dark matter', which contains cis-regulatory modules. These cis-regulatory modules function collectively and can impact gene expression even when located far from the target gene, exhibiting context-specific behaviour. Consequently, the cis-regulatory code governing gene expression patterns is intricate, in contrast to the universally understood genetic code. This overview centres on the current knowledge regarding cis-regulatory elements, primarily enhancers and their role in governing the spatiotemporal gene expression patterns, and how they have evolved and adapted across different species.

Atlas of cardiac endothelial cell enhancer elements linking the mineralocorticoid receptor to pathological gene expression.

Endothelial cells play crucial roles in physiology and are increasingly recognized as therapeutic targets in cardiovascular disease. Here, we analyzed the regulatory landscape of cardiac endothelial cells by assessing chromatin accessibility, histone modifications, and 3D chromatin organization and confirmed the functional relevance of enhancer-promoter interactions by CRISPRi-mediated enhancer silencing. We used this dataset to explore mechanisms of transcriptional regulation in cardiovascular disease and compared six different experimental models of heart failure, hypertension, or diabetes. Enhancers that regulate gene expression in diseased endothelial cells were enriched with binding sites for a distinct set of transcription factors, including the mineralocorticoid receptor (MR), a known drug target in heart failure and hypertension. For proof of concept, we applied endothelial cell-specific MR deletion in mice to confirm MR-dependent gene expression and predicted direct MR target genes. Overall, we have compiled here a comprehensive atlas of cardiac endothelial cell enhancer elements that provides insight into the role of transcription factors in cardiovascular disease.

Enhancer-promoter interactions are reconfigured through the formation of long-range multiway hubs as mouse ES cells exit pluripotency.

Enhancers bind transcription factors, chromatin regulators, and non-coding transcripts to modulate the expression of target genes. Here, we report 3D genome structures of single mouse ES cells as they are induced to exit pluripotency and transition through a formative stage prior to undergoing neuroectodermal differentiation. We find that there is a remarkable reorganization of 3D genome structure where inter-chromosomal intermingling increases dramatically in the formative state. This intermingling is associated with the formation of a large number of multiway hubs that bring together enhancers and promoters with similar chromatin states from typically 5-8 distant chromosomal sites that are often separated by many Mb from each other. In the formative state, genes important for pluripotency exit establish contacts with emerging enhancers within these multiway hubs, suggesting that the structural changes we have observed may play an important role in modulating transcription and establishing new cell identities.

Increased enhancer-promoter interactions during developmental enhancer activation in mammals.

Remote enhancers are thought to interact with their target promoters via physical proximity, yet the importance of this proximity for enhancer function remains unclear. Here we investigate the three-dimensional (3D) conformation of enhancers during mammalian development by generating high-resolution tissue-resolved contact maps for nearly a thousand enhancers with characterized in vivo activities in ten murine embryonic tissues. Sixty-one percent of developmental enhancers bypass their neighboring genes, which are often marked by promoter CpG methylation. The majority of enhancers display tissue-specific 3D conformations, and both enhancer-promoter and enhancer-enhancer interactions are moderately but consistently increased upon enhancer activation in vivo. Less than 14% of enhancer-promoter interactions form stably across tissues; however, these invariant interactions form in the absence of the enhancer and are likely mediated by adjacent CTCF binding. Our results highlight the general importance of enhancer-promoter physical proximity for developmental gene activation in mammals.

The impact of genomic distance on enhancer-promoter interactions at the CFTR locus.

We identified and characterized multiple cell-type selective enhancers of the CFTR gene promoter in previous work and demonstrated active looping of these elements to the promoter. Here we address the impact of genomic spacing on these enhancer:promoter interactions and on CFTR gene expression. Using CRISPR/Cas9, we generated clonal cell lines with deletions between the -35 kb airway enhancer and the CFTR promoter in the 16HBE14o- airway cell line, or between the intron 1 (185 + 10 kb) intestinal enhancer and the promoter in the Caco2 intestinal cell line. The effect of these deletions on CFTR transcript abundance, as well as the 3D looping structure of the locus was investigated in triplicate clones of each modification. Our results indicate that both small and larger deletions upstream of the promoter can perturb CFTR expression and -35 kb enhancer:promoter interactions in the airway cells, though the larger deletions are more impactful. In contrast, the small intronic deletions have no effect on CFTR expression and intron 1 enhancer:promoter interactions in the intestinal cells, whereas larger deletions do. Clonal variation following a specific CFTR modification is a confounding factor particularly in 16HBE14o- cells.