Search

Vascular endothelial growth factors (VEGFs) are best known as key regulators of angiogenesis and lymphangiogenesis. Although VEGFs have been promising therapeutic targets for various cardiovascular diseases, their regulatory landscape in endothelial cells remains elusive. Several studies have highlighted the involvement of noncoding RNAs (ncRNAs) in the modulation of VEGF expression. In this study, we investigated the role of two classes of ncRNAs, long ncRNAs (lncRNAs) and enhancer RNAs (eRNAs), in the transcriptional regulation of VEGFA and VEGFC . By integrating genome-wide global run-on sequencing (GRO-Seq) and chromosome conformation capture (Hi-C) data, we identified putative lncRNAs and eRNAs associated with VEGFA and VEGFC genes in endothelial cells. A subset of the identified putative enhancers demonstrated regulatory activity in a reporter assay. Importantly, we demonstrate that deletion of enhancers and lncRNAs by CRISPR/Cas9 promoted significant changes in VEGFA and VEGFC expression. Transcriptome sequencing (RNA-Seq) data from lncRNA deletions showed downstream factors implicated in VEGFA- and VEGFC -linked pathways, such as angiogenesis and lymphangiogenesis, suggesting functional roles for these lncRNAs. Our study uncovers novel lncRNAs and eRNAs regulating VEGFA and VEGFC that can be targeted to modulate the expression of these important molecules in endothelial cells.

Background: Planar equilibrium radionuclide angiocardiography (ERNA) has been used as the gold standard for assessment of left ventricular (LV) function for over three decades. However, this imaging modality has recently gained less favor due to growing concerns about radiation exposure. We developed a novel approach that involves integrating short axis slices of gated bloodpool SPECT for quantification of LV function with improved signal-to-noise ratio and reduced radioactive dose while maintaining image quality and quantitative precision., Methods: Twenty patients referred for ERNA underwent standard in vitro 99m Tc-labeling of red blood cells (RBC), and were initially imaged following a low-dose (~ 8 mCi) injection using a dedicated cardiac SPECT camera, and then had planar imaging following a high-dose (~ 25 mCi) injection. Four different quantification methods were utilized to assess the LV function and were compared for quantitative precision and inter-observer reproducibility of the quantitative assessments., Results: The Yale method resulted in the most consistent assessment of LV function compared with the gold standard high-dose ERNA method, along with excellent inter-observer reproducibility., Conclusions: The new low-dose 99m Tc-RBC imaging method provides precise quantification of LV function with a greater than 67% reduction in dose and may potentially improve assessment of regional function.

Enhancer RNAs (eRNAs) participate in tumor growth and immune regulation through complex signaling pathways. However, the immune-related function of the eRNA-mRNA axis in lung adenocarcinoma (LUAD) is unclear. Data on the expression of eRNAs and mRNAs were downloaded from The Cancer Genome Atlas, GEO, and UCSC Xena, including LUAD, and pan-cancer clinical data and mutational information. Immune gene files were obtained from ImmLnc and ImmPort databases. Survival indices, including relapse-free and overall survival, were analyzed using the Kaplan-Meier and log-rank methods. The level of immune cell infiltration, degree of tumor hypoxia, and tumor cell stemness characteristics were quantified using the single-sample gene set enrichment analysis algorithm. The immune infiltration score and infiltration degree were evaluated using the ESTIMATE and CIBERSORT algorithms. The tumor mutation burden and microsatellite instability were examined using the Spearman test. The LUAD-associated immune-related LINC00987/A2M axis was down-regulated in most cancer types, indicating poor survival and cancer progression. Immune cell infiltration was closely related to abnormal expression of the LINC00987/A2M axis, linking its expression to a possible evaluation of sensitivity to checkpoint inhibitors and response to chemotherapy. Abnormal expression of the LINC00987/A2M axis was characterized by heterogeneity in the degree of tumor hypoxia and stemness characteristics. The abnormal distribution of immune cells in LUAD was also verified through pan-cancer analysis. Comprehensive bioinformatic analysis showed that the LINC00987/A2M axis is a functional and effective tumor suppressor and biomarker for assessing the immune microenvironment and prognostic and therapeutic evaluations of LUAD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ma, Lin, Wang, Min, Wang and Tang.)

Recent studies have reported widespread stimulus-dependent transcription of mammalian enhancers into noncoding enhancer RNAs (eRNAs), some of which have central roles in the enhancer-mediated induction of target genes and modulation of phenotypic outcomes during development and disease. In cerebral ischemia, the expression and functions of eRNAs are virtually unknown. Here, we applied genome-wide H3K27ac ChIP-seq and genome-wide RNA-seq to identify enhancer elements and stroke-induced eRNAs, respectively, in the mouse cerebral cortex during transient focal ischemia. Following a 1-h middle cerebral artery occlusion (MCAO) and 6 h of reperfusion, we identified 77 eRNAs that were significantly upregulated in stroke as compared to sham, of which 55 were exclusively expressed in stroke. The knockdown of two stroke-induced eRNAs in the mouse brain resulted in significantly larger infarct volumes as compared to controls, suggesting that these eRNAs are involved in the post-stroke neuroprotective response. A preliminary comparison of eRNA expression in the male versus female cortices revealed sex-dependent patterns that may underlie the physiological differences in response to stroke between the two sexes. Together, this study is the first to illuminate the eRNA landscape in the post-stroke cortex and demonstrate the significance of an eRNA in modulating post-stroke cortical brain damage.

Enhancers are well established as critical regulators of gene expression, but the mechanisms underlying the molecular basis of their specificity and activity are only partly understood. One of the most exciting recent observations is the discovery of enhancer RNA (eRNA), a class of noncoding RNAs derived from enhancer regions. Transcription of developmentally regulated enhancers has been observed to be associated with their active state. The nature of transcripts (eRNA) and their functional attributes are diverse and context dependent. The majority of eRNA are nonpolyadenylated and present in low abundance owing to their low stability, and may represent transcriptional noise. However, some eRNAs have been reported to be reasonably long and stable, are enriched in nuclei, exhibit tissue-specific expression and may contribute to enhancer function. Transcription of enhancers has been postulated to mediate enhancer function through either the act of transcription or via the transcribed RNA per se and is a useful feature to be analysed to understand mechanisms underlying enhancer activity. Enhancer Eβ at the murine TCRβ locus has been reported to exhibit enhanced occupancy of RNA polymerase II in developing thymocytes. Here, we investigated the transcriptional potential of Eβ in developing thymocytes and detected overlapping bidirectional transcripts at Eβ ranging between 0.7 and 1.7 kb. These noncoding transcripts are capped, polyadenylated, nuclear and expressed specifically in thymocytes. Delineation of these characteristics is important to further investigate their functional roles in mediating enhancer activity., (© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Enhancer RNAs, a type of long non-coding RNAs (lncRNAs), play a critical role in the occurrence and development of glioma. RNA-seq data from 161 glioblastoma multiforme (GBM) samples were acquired from The Cancer Genome Atlas database. Then, 70 eRNAs were identified as prognosis-related genes, which had significant relations with overall survival (log-rank test, p < 0.05). AC003092.1 was demonstrated as an immune-related eRNA by functional enrichment analysis. We divided samples into two groups based on AC003092.1 expression: AC003092.1 High (AC003092.1&#95;H) and AC003092.1 Low (AC003092.1&#95;L) and systematically analyzed the influence of AC003092.1 on the immune microenvironment by single-sample gene-set enrichment analysis and CIBERSORTx. We quantified AC003092.1 and TFPI2 levels in 11 high-grade gliomas, 5 low-grade gliomas, and 7 GBM cell lines. Our study indicates that AC003092.1 is related to glioma-immunosuppressive microenvironment, and these results offer innovative sights into GBM immune therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Guo, Zhong, Wang, Xie, Duan, Zhang, Zhang, Liang, Cui, Hu, Lu, Wu, Dong, He and Mou.)

Effective synthesis of mammalian messenger (m)RNAs depends on many factors that together direct RNA polymerase II (pol II) through the different stages of the transcription cycle and ensure efficient cotranscriptional processing of mRNAs. In addition to the many proteins involved in transcription initiation, elongation, and termination, several noncoding (nc)RNAs also function as global transcriptional regulators. Understanding the mode of action of these non-protein regulators has been an intense area of research in recent years. Here, we describe how these ncRNAs influence key regulatory steps of the transcription process, to affect large numbers of genes. Through direct association with pol II or by modulating the activity of transcription or RNA processing factors, these regulatory RNAs perform critical roles in gene expression., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Responses to developmental and environmental cues depend on precise spatiotemporal control of gene transcription. Enhancers, which comprise DNA elements bound by regulatory proteins, can activate target genes in response to these external signals. Recent studies have shown that enhancers are transcribed to produce enhancer RNAs (eRNAs). Do eRNAs play a functional role in activating gene expression or are they non-functional byproducts of nearby transcription machinery? The unstable nature of eRNAs and over-reliance on knockdown approaches have made elucidating the possible functions of eRNAs challenging. We focus here on studies using cloned eRNAs to study their function as transcripts, revealing roles for eRNAs in enhancer-promoter looping, recruiting transcriptional machinery, and facilitating RNA polymerase pause-release to regulate gene expression., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Enhancers are transcribed by RNA polymerase II (Pol II). In order to study the regulation of enhancer transcription and its function in target gene control, methods are required that track genome transcription with high precision in vivo. Here, we provide step-by-step guidance for performing native elongating transcript sequencing (NET-Seq) in mammalian cells. NET-Seq allows quantitative measurements of transcription genome-wide, including enhancer transcription, with single-nucleotide and DNA strand resolution. The approach consists of capturing and efficiently converting the 3'-ends of the nascent RNA into a sequencing library followed by next-generation sequencing and computational data analysis. The protocol includes quality control measurements to monitor the success of the main steps. Following this protocol, a NET-Seq library is obtained within 5 days., (© 2021. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The Cap Analysis of Gene Expression (CAGE) is a powerful method to identify Transcription Start Sites (TSSs) of capped RNAs while simultaneously measuring transcripts expression level. CAGE allows mapping at single nucleotide resolution at all active promoters and enhancers. Large CAGE datasets have been produced over the years from individual laboratories and consortia, including the Encyclopedia of DNA Elements (ENCODE) and Functional Annotation of the Mammalian Genome (FANTOM) consortia. These datasets constitute open resource for TSS annotations and gene expression analysis. Here, we provide an experimental protocol for the most recent CAGE method called Low Quantity (LQ) single strand (ss) CAGE "LQ-ssCAGE", which enables cost-effective profiling of low quantity RNA samples. LQ-ssCAGE is especially useful for samples derived from cells cultured in small volumes, cellular compartments such as nuclear RNAs or for samples from developmental stages. We demonstrate the reproducibility and effectiveness of the method by constructing 240 LQ-ssCAGE libraries from 50 ng of THP-1 cell extracted RNAs and discover lowly expressed novel enhancer and promoter-derived lncRNAs., (© 2021. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The discovery of new classes of non-coding RNAs has always been preceded or accompanied by technological breakthroughs, and these outstanding progresses in transcriptomics approaches enabled to regularly add new members to the list. From the first detection of tRNAs, through the revolution of miRNAs discovery, to the recent identification of eRNAs or the identification of new functions for already known ncRNAs, this introductive review provides a very concise historical and functional overview of most prominent small regulatory non-coding RNA families.

Chronic vascular inflammation plays a key role in the pathogenesis of atherosclerosis. Long non-coding RNAs (lncRNAs) have emerged as essential inflammation regulators. We identify a novel lncRNA termed lncRNA-MAP3K4 that is enriched in the vessel wall and regulates vascular inflammation. In the aortic intima, lncRNA-MAP3K4 expression was reduced by 50% during the progression of atherosclerosis (chronic inflammation) and 70% during endotoxemia (acute inflammation). lncRNA-MAP3K4 knockdown reduced the expression of key inflammatory factors (eg, ICAM-1, E-selectin, MCP-1) in endothelial cells or vascular smooth muscle cells and decreased monocytes adhesion to endothelium, as well as reducing TNF-α, IL-1β, COX2 expression in macrophages. Mechanistically, lncRNA-MAP3K4 regulates inflammation through the p38 MAPK signaling pathway. lncRNA-MAP3K4 shares a bidirectional promoter with MAP3K4, an upstream regulator of the MAPK signaling pathway, and regulates its transcription in cis. lncRNA-MAP3K4 and MAP3K4 show coordinated expression in response to inflammation in vivo and in vitro. Similar to lncRNA-MAP3K4, MAP3K4 knockdown reduced the expression of inflammatory factors in several different vascular cells. Furthermore, lncRNA-MAP3K4 and MAP3K4 knockdown showed cooperativity in reducing inflammation in endothelial cells. Collectively, these findings unveil the role of a novel lncRNA in vascular inflammation by cis-regulating MAP3K4 via a p38 MAPK pathway., (© 2020 Federation of American Societies for Experimental Biology.)

Enhancers are noncoding DNA elements that are present upstream or downstream of a gene to control its spatial and temporal expression. Specific histone modifications, such as monomethylation on histone H3 lysine 4 (H3K4me1) and H3K27ac, have been widely used to assign enhancer regions in mammalian genomes. In recent years, emerging evidence suggests that active enhancers are bidirectionally transcribed to produce enhancer RNAs (eRNAs). This finding not only adds a new reliable feature to define enhancers but also raises a fundamental question of how eRNAs function to activate transcription. Although some believe that eRNAs are merely transcriptional byproducts, many studies have demonstrated that eRNAs execute crucial tasks in regulating chromatin conformation and transcription activation. In this review, we summarize the current understanding of eRNAs from their biogenesis, functions, and regulation to their pathological significance. Additionally, we discuss the challenges and possible mechanisms of eRNAs in regulated transcription., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

It is now evident that transcriptional gene regulation usually requires the re-organization of chromatin architecture. Increasing evidence suggested various kinds of RNAs are involved in this process. Especially the nascent RNAs retained at their site of transcription can serve as a scaffold for organizing transcriptionally either favorable or unfavorable chromatin structures. An emerging concept of phase separation explains how these chromatin structures can be maintained as physically discrete subcompartments within membrane-less nucleoplasm. Evidences that support the crucial role of nascent RNAs in the formation of phase-separated condensates are now rapidly growing.

Since the discovery that enhancers can support transcription, the roles of enhancer RNAs have remained largely elusive. We identified that enhancer RNAs interact with and augment bromodomain engagement with acetylated chromatin. Here, we discuss our recent findings and the potential mechanisms underlying the regulation and functions of enhancer RNA-bromodomain associations.

Transcriptional responses to external stimuli remain poorly understood. Using global nuclear run-on followed by sequencing (GRO-seq) and precision nuclear run-on sequencing (PRO-seq), we show that CDK8 kinase activity promotes RNA polymerase II pause release in response to interferon-γ (IFN-γ), a universal cytokine involved in immunity and tumor surveillance. The Mediator kinase module contains CDK8 or CDK19, which are presumed to be functionally redundant. We implemented cortistatin A, chemical genetics, transcriptomics, and other methods to decouple their function while assessing enzymatic versus structural roles. Unexpectedly, CDK8 and CDK19 regulated different gene sets via distinct mechanisms. CDK8-dependent regulation required its kinase activity, whereas CDK19 governed IFN-γ responses through its scaffolding function (i.e., it was kinase independent). Accordingly, CDK8, not CDK19, phosphorylates the STAT1 transcription factor (TF) during IFN-γ stimulation, and CDK8 kinase inhibition blocked activation of JAK-STAT pathway TFs. Cytokines such as IFN-γ rapidly mobilize TFs to "reprogram" cellular transcription; our results implicate CDK8 and CDK19 as essential for this transcriptional reprogramming. • Mediator kinases CDK8 and CDK19 are distinct transcription regulators in the IFN-γ pathway • CDK8 acts as a kinase while CDK19 functions as a scaffold in driving IFN-γ gene profiles • CDK8 and CDK19 activate distinct gene sets • Mediator kinase CDK8 promotes polymerase II pause release during the IFN-γ response Steinparzer et al. discover that the Mediator kinases CDK8 and CDK19 are functionally and mechanistically distinct transcription regulators in the IFN-γ antiviral program: CDK8 and CDK19 activate distinct gene sets, and they do so in kinase-dependent (CDK8) and kinase-independent (CDK19) ways. Further, CDK8 kinase activity promotes RNAPII pause release. [ABSTRACT FROM AUTHOR]

Long noncoding RNAs (lncRNAs) are characterized as a group of endogenous RNAs that are more than 200 nucleotides in length and have no protein-encoding function. More and more evidence indicates that lncRNAs play vital roles in various human diseases, especially in tumorigenesis. Focally amplified lncRNA on chromosome 1 (FAL1), a novel lncRNA with enhancer-like activity, has been identified as an oncogene in multiple cancers and high expression level of FAL1 is usually associated with poor prognosis. Dysregulation of FAL1 has been shown to promote the proliferation and metastasis of cancer cells. In the present review, we summarized and illustrated the functions and underlying molecular mechanisms of FAL1 in the occurrence and development of different cancers and other diseases. FAL1 has the potential to appear as a feasible diagnostic and prognostic tool and new therapeutic target for cancer patients though further investigation is needed so as to accelerate clinical application. [ABSTRACT FROM AUTHOR]

Systemic RNAi, initiated by double-stranded RNA (dsRNA) ingestion, has been reported in diverse invertebrates, including honey bees, demonstrating environmental RNA uptake that undermines homologous gene expression. However, the question why any organism would take up RNA from the environment has remained largely unanswered. Here, we report on horizontal RNA flow among honey bees mediated by secretion and ingestion of worker and royal jelly diets. We demonstrate that transmission of jelly-secreted dsRNA to larvae is biologically active and triggers gene knockdown that lasts into adulthood. Worker and royal jellies harbor differential naturally occurring RNA populations. Jelly RNAs corresponded to honey bee protein-coding genes, transposable elements, and non-coding RNA, as well as bacteria, fungi, and viruses. These results reveal an inherent property of honey bees to share RNA among individuals and generations. Our findings suggest a transmissible RNA pathway, playing a role in social immunity and signaling between members of the hive. • Ingested dsRNA spreads via hemolymph and is secreted in worker and royal jellies • Functional dsRNA flows horizontally among honey bees by jelly consumption • Worker and royal jellies harbor differential natural ssRNA and dsRNA populations • Diverse pathogenic RNA fragments naturally occur in worker and royal jellies RNA mobility among cells has been documented in plants and animals. Maori et al. show that RNA spreads further in honey bees and is horizontally transferred between individuals and across generations. Their findings demonstrate a transmissible RNA pathway with potential roles in social immunity and epigenetic communication between honey bees. [ABSTRACT FROM AUTHOR]

Transcription of the proto-oncogene SPHK1 is regulated by KHPS1 , an antisense RNA that activates SPHK1 expression by forming a triple-helical RNA-DNA-DNA structure at the SPHK1 enhancer. Triplex-mediated tethering of KHPS1 to its target gene is required for recruitment of E2F1 and p300 and transcription of the RNA derived from the SPHK1 enhancer (eRNA-Sphk1). eRNA-Sphk1 evicts CTCF, which insulates the enhancer from the SPHK1 promoter, thus facilitating SPHK1 expression. Genomic deletion of the triplex-forming sequence attenuates SPHK1 expression, leading to decreased cell migration and invasion. Replacement of the triplex-forming region (TFR) of KHPS1 by the TFR of the lncRNA MEG3 tethers KHPS1 to the MEG3 target gene TGFBR1 , underscoring the interchangeability and anchoring function of sequences involved in triplex formation. Altogether, the results reveal a triplex-driven feedforward mechanism involving lncRNA-dependent induction of eRNA, which enhances expression of specific target genes. • RNA-DNA triplex formation is required for transcription activation of the proto-oncogene SPHK1 • lncRNA bound to the SPHK1 induces the synthesis of eRNA-Sphk1 and SPHK1 mRNA • eRNA-Sphk1 evicts CTCF, which insulates the enhancer from the SPHK1 promoter • Deletion of the triplex-forming region impairs SPHK1 expression and cell viability Blank-Giwojna et al. demonstrate that the antisense RNA KHPS1 forms an RNA-DNA triplex at the SPHK1 enhancer. Tethering KHPS1 to the enhancer is required for recruitment of E2F1 and p300, transcription of enhancer-derived RNA, and activation of SPHK1 expression. The results uncover a triplex-driven feedforward mechanism of transcriptional regulation. [ABSTRACT FROM AUTHOR]

Cap analysis of gene expression (CAGE) is an approach to identify and monitor the activity (transcription initiation frequency) of transcription start sites (TSSs) at single base-pair resolution across the genome. It has been effectively used to identify active promoter and enhancer regions in cancer cells, with potential utility to identify key factors to immunotherapy. Here, we overview a series of CAGE protocols and describe detailed experimental steps of the latest protocol based on the Illumina sequencing platform; both experimental steps (see Subheadings 3.1-3.11) and computational processing steps (see Subheadings 3.12-3.20) are described.

The DNA-binding protein PU.1 is a myeloid lineage-determining and pioneering transcription factor due to its ability to bind "closed" genomic sites and maintain "open" chromatin state for myeloid lineage-specific genes. The precise mechanism of PU.1 in cell type-specific programming is yet to be elucidated. The melanoma cell line B16BL6, although it is nonmyeloid lineage, expressed Toll-like receptors and activated the transcription factor NF-κB upon stimulation by the bacterial cell wall component lipopolysaccharide. However, it did not produce cytokines, such as IL-1β mRNA. Ectopic PU.1 expression induced remodeling of a novel distal enhancer (located ∼10 kbp upstream of the IL-1β transcription start site), marked by nucleosome depletion, enhancer-promoter looping, and histone H3 lysine 27 acetylation (H3K27ac). PU.1 induced enhancer-promoter looping and H3K27ac through two distinct PU.1 regions. These PU.1-dependent events were independently required for subsequent signal-dependent and co-dependent events: NF-κB recruitment and further H3K27ac, both of which were required for enhancer RNA (eRNA) transcription. In murine macrophage RAW264.7 cells, these PU.1-dependent events were constitutively established and readily expressed eRNA and subsequently IL-1β mRNA by lipopolysaccharide stimulation. In summary, this study showed a sequence of epigenetic events in programming IL-1β transcription by the distal enhancer priming and eRNA production mediated by PU.1 and the signal-dependent transcription factor NF-κB., (© 2019 Ha et al.)

Although accumulating evidence has demonstrated the key roles of enhancers in gene expression regulation, the contribution of genome-wide enhancer-enhancer interactions to developmental decisions remains unclear. Here we explored the cooperative regulation patterns among enhancers to understand their regulatory mechanism. We first filtered robust enhancers in embryonic stem cells (ESCs) through integrating bidirectional transcription, genomic location, and epigenetic modification. Genome-wide enhancer-enhancer interactions were then identified based on enhancer-promoter relationships that were derived from Hi-C data. We further explored the interacting principles of the identified enhancer-enhancer interactions. The results revealed that the observed cooperativity occurred mainly between enhancers distributed within a 1-kb to 10-Mb distance across the genome. In addition, enhancer-enhancer pairs had higher expression correlations than non-interacting pairs. Finally, we identified robust enhancers during human cardiac commitment, and we found that enhancers exhibited strong stage-specific expression patterns. We further inferred the enhancer-enhancer interactions based on RNA sequencing (RNA-seq) data in heart development, according to the regulatory principles characterized from Hi-C data. The identified enhancer-enhancer interaction networks (EEINs) presented highly dynamic linkages. Moreover, enhancers cooperatively targeted many marker genes in each developmental stage to regulate stage-specific functions, which contribute to the organization of cell identity in heart development. Our work will increase the understanding of enhancer regulation in human heart development., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Biological roles for extracellular RNA (eRNA) have become apparent. For example, eRNA can induce contact activation in blood via activation of the plasma proteases factor XII (FXII) and factor XI (FXI). We sought to reveal the biological role of the secretory enzyme ribonuclease 1 (RNase 1) in an organismal context by generating and analyzing RNase 1 knockout ( Rnase1 -/- ) mice. We found that these mice are viable, healthy, and fertile, though larger than Rnase1 +/+ mice. Rnase1 -/- plasma contains more RNA than does the plasma of Rnase1 mice, and is consistent with the known effects of eRNA on FXII activation. The apparent activity of FXI in the plasma of +/+ mice was 1000-fold higher when measured in an assay triggered by a low concentration of tissue factor than in assays based on recalcification, consistent with eRNA enhancing FXI activation by thrombin. These findings suggest that one of the physiological functions of RNase 1 is to degrade eRNA in blood plasma. Loss of this function facilitates FXII and FXI activation, which could have effects on inflammation and blood coagulation. We anticipate that Rnase1 mice will be a useful tool for evaluating other hypotheses about the functions of RNase 1 and of eRNA in vivo.-/- mice clots more rapidly than does wild-type plasma. This phenotype appeared to be due to increased levels of the active form of FXII (FXIIa) in the plasma of Rnase1 -/- mice compared to Rnase1 +/+ mice, and is consistent with the known effects of eRNA on FXII activation. The apparent activity of FXI in the plasma of Rnase1 -/- mice was 1000-fold higher when measured in an assay triggered by a low concentration of tissue factor than in assays based on recalcification, consistent with eRNA enhancing FXI activation by thrombin. These findings suggest that one of the physiological functions of RNase 1 is to degrade eRNA in blood plasma. Loss of this function facilitates FXII and FXI activation, which could have effects on inflammation and blood coagulation. We anticipate that Rnase1 -/- mice will be a useful tool for evaluating other hypotheses about the functions of RNase 1 and of eRNA in vivo., (© 2019 Garnett et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Enhancers are non-coding DNA elements that function in cis to regulate transcription from nearby genes. Through direct interactions with gene promoters, enhancers give rise to spatially and temporally precise gene expression profiles in distinct cell or tissue types. In the brain, the accurate regulation of these intricate expression programs across different neuronal classes gives rise to an incredible cellular and functional diversity. Newly developed technologies have recently allowed more accurate enhancer mapping and more sophisticated enhancer manipulation, producing rapid progress in our understanding of enhancer biology. Furthermore, identification of disease-linked genetic variation in enhancer regions has highlighted the potential influence of enhancers in brain health and disease. This review outlines the key role of enhancers as transcriptional regulators, reviews the current understanding of enhancer regulation in neuronal development, function and dysfunction and provides our thoughts on how enhancers can be targeted for technological and therapeutic goals. [ABSTRACT FROM AUTHOR]

Summary Extensive tracts of the mammalian genome that lack protein-coding function are still transcribed into long noncoding RNA. While these lncRNAs are generally short lived, length restricted, and non-polyadenylated, how their expression is distinguished from protein-coding genes remains enigmatic. Surprisingly, depletion of the ubiquitous Pol-II-associated transcription elongation factor SPT6 promotes a redistribution of H3K36me3 histone marks from active protein coding to lncRNA genes, which correlates with increased lncRNA transcription. SPT6 knockdown also impairs the recruitment of the Integrator complex to chromatin, which results in a transcriptional termination defect for lncRNA genes. This leads to the formation of extended, polyadenylated lncRNAs that are both chromatin restricted and form increased levels of RNA:DNA hybrid (R-loops) that are associated with DNA damage. Additionally, these deregulated lncRNAs overlap with DNA replication origins leading to localized DNA replication stress and a cellular senescence phenotype. Overall, our results underline the importance of restricting lncRNA expression. Graphical Abstract Highlights • SPT6 promotes the selective distribution of H3K36me3 over protein-coding genes • SPT6 loss leads to formation of extended lncRNAs that are prone to R-loop formation • Deregulated Pol II collides with DNA replisomes on lncRNA genes • Collision between Pol II and DNA replisome leads to cellular senescence Nojima and colleagues demonstrate that loss of human SPT6 protein elevates levels of noncoding transcription and perturbs its termination. The deregulated noncoding transcription increases R-loop formation with consequent DNA damage, transcription-replication collision, and cellular senescence. This implies that noncoding transcription is restricted to avoid such conflicts in proliferating cells. [ABSTRACT FROM AUTHOR]

Summary Monomethylation of histone H3 at lysine 4 (H3K4me1) and acetylation of histone H3 at lysine 27 (H3K27ac) are correlated with transcriptionally engaged enhancer elements, but the functional impact of these modifications on enhancer activity is not well understood. Here we used CRISPR/Cas9 genome editing to separate catalytic activity-dependent and independent functions of Mll3 (Kmt2c) and Mll4 (Kmt2d, Mll2), the major enhancer H3K4 monomethyltransferases. Loss of H3K4me1 from enhancers in Mll3/4 catalytically deficient cells causes partial reduction of H3K27ac, but has surprisingly minor effects on transcription from either enhancers or promoters. In contrast, loss of Mll3/4 proteins leads to strong depletion of enhancer Pol II occupancy and eRNA synthesis, concomitant with downregulation of target genes. Interestingly, downregulated genes exhibit reduced polymerase levels in gene bodies, but not at promoters, suggestive of pause-release defects. Altogether, our results suggest that enhancer H3K4me1 provides only a minor contribution to the long-range coactivator function of Mll3/4. [ABSTRACT FROM AUTHOR]

MyoD upstream noncoding RNA (MUNC) initiates in the distal regulatory region (DRR) enhancer of MYOD and is formally classified as an enhancer RNA (DRR eRNA ). MUNC is required for optimal myogenic differentiation, induces specific myogenic transcripts in trans ( MYOD , MYOGENIN , and MYH3 ), and has a functional human homolog. The vast majority of eRNAs are believed to act in cis primarily on their neighboring genes (1, 2), making it likely that MUNC action is dependent on the induction of MYOD RNA. Surprisingly, MUNC overexpression in MYOD C2C12 cells induces many myogenic transcripts in the complete absence of MyoD protein. Genomewide analysis showed that, while many genes are regulated by MUNC in a MyoD-dependent manner, there is a set of genes that are regulated by MUNC, both upward and downward, independently of MyoD. MUNC and MyoD even appear to act antagonistically on certain transcripts. Deletion mutagenesis showed that there are at least two independent functional sites on the MUNC long noncoding RNA (lncRNA), with exon 1 more active than exon 2 and with very little activity from the intron. Thus, although MUNC is an eRNA of -/- C2C12 cells induces many myogenic transcripts in the complete absence of MyoD protein. Genomewide analysis showed that, while many genes are regulated by MUNC in a MyoD-dependent manner, there is a set of genes that are regulated by MUNC, both upward and downward, independently of MyoD. MUNC and MyoD even appear to act antagonistically on certain transcripts. Deletion mutagenesis showed that there are at least two independent functional sites on the MUNC long noncoding RNA (lncRNA), with exon 1 more active than exon 2 and with very little activity from the intron. Thus, although MUNC is an eRNA of MYOD , it is also a trans -acting lncRNA whose sequence, structure, and cooperating factors, which include but are not limited to MyoD, determine the regulation of many myogenic genes., (Copyright © 2018 American Society for Microbiology.)

Nuclear receptors induce both transcriptional activation and repression programs responsible for development, homeostasis, and disease. Here, we report a previously overlooked enhancer decommissioning strategy underlying a large estrogen receptor alpha (ERα)-dependent transcriptional repression program. The unexpected signature for this E 2 -induced program resides in indirect recruitment of ERα to a large cohort of pioneer factor basally active FOXA1-bound enhancers that lack cognate ERα DNA-binding elements. Surprisingly, these basally active estrogen-repressed (BAER) enhancers are decommissioned by ERα-dependent recruitment of the histone demethylase KDM2A, functioning independently of its demethylase activity. Rather, KDM2A tethers the E3 ubiquitin-protein ligase NEDD4 to ubiquitylate/dismiss Pol II to abrogate eRNA transcription, with consequent target gene downregulation. Thus, our data reveal that Pol II ubiquitylation/dismissal may serve as a potentially broad strategy utilized by indirectly bound nuclear receptors to abrogate large programs of pioneer factor-mediated, eRNA-producing enhancers., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Enhancers can act as cis-regulatory elements (CREs) to control development and cellular function by regulating gene expression in a tissue-specific and ubiquitous manner. However, the regulatory network and characteristic of different types of enhancers (e.g., transcribed/non-transcribed enhancers, tissue-specific/ubiquitous enhancers) across multiple tissues are still unclear. Results: Here, a total of 53,924 active enhancers and 10,307 enhancer-associated RNAs (eRNAs) in 10 tissues (adrenal, brain, breast, heart, liver, lung, ovary, placenta, skeletal muscle and kidney) were identified through the integration of histone modifications (H3K4me1, H3K27ac and H3K4me3) and DNase I hypersensitive sites (DHSs) data. Moreover, 40,101 tissue-specific enhancers (TS-Enh), 1,241 ubiquitously expressed enhancers (UE-Enh) as well as transcribed enhancers (T-Enh), including 7,727 unidirectionally transcribed enhancers (1D-Enh) and 1,215 bidirectionally transcribed enhancers (2D-Enh) were defined in 10 tissues. The results show that enhancers exhibited high GC content, genomic variants and transcription factor binding sites (TFBS) enrichment in all tissues. These characteristics were significantly different between TS-Enh and UE-Enh, T-Enh and NT-Enh, 2D-Enh and 1D-Enh. Furt hermore, the results showed that enhancers obviously upregulate the expression of adjacent target genes which were remarkably correlated with the functions of corresponding tissues. Finally, a free user-friendly tissue-specific enhancer database, TiED (http://lcbb.swjtu.edu.cn/TiED), has been built to store, visualize, and confer these results. Conclusion: Genome-wide analysis of the regulatory network and characteristic of various types of enhancers showed that enhancers associated with TFs, eRNAs and target genes appeared in tissue specificity and function across different tissues., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Enhancers are thought to activate transcription by physically contacting promoters via looping. However, direct assays demonstrating these contacts are required to mechanistically verify such cellular determinants of enhancer function. Here, we present versatile cell-free assays to further determine the role of enhancer-promoter contacts (EPCs). We demonstrate that EPC is linked to mutually stimulatory transcription at the enhancer and promoter in vitro. SRC-3 was identified as a critical looping determinant for the estradiol-(E2)-regulated GREB1 locus. Surprisingly, the GREB1 enhancer and promoter contact two internal gene body SRC-3 binding sites, GBS1 and GBS2, which stimulate their transcription. Utilizing time-course 3C assays, we uncovered SRC-3-dependent dynamic chromatin interactions involving the enhancer, promoter, GBS1, and GBS2. Collectively, these data suggest that the enhancer and promoter remain "poised" for transcription via their contacts with GBS1 and GBS2. Upon E2 induction, GBS1 and GBS2 disengage from the enhancer, allowing direct EPC for active transcription., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Summary CBP/p300 are transcription co-activators whose binding is a signature of enhancers, cis -regulatory elements that control patterns of gene expression in multicellular organisms. Active enhancers produce bi-directional enhancer RNAs (eRNAs) and display CBP/p300-dependent histone acetylation. Here, we demonstrate that CBP binds directly to RNAs in vivo and in vitro. RNAs bound to CBP in vivo include a large number of eRNAs. Using steady-state histone acetyltransferase (HAT) assays, we show that an RNA binding region in the HAT domain of CBP—a regulatory motif unique to CBP/p300—allows RNA to stimulate CBP’s HAT activity. At enhancers where CBP interacts with eRNAs, stimulation manifests in RNA-dependent changes in the histone acetylation mediated by CBP, such as H3K27ac, and by corresponding changes in gene expression. By interacting directly with CBP, eRNAs contribute to the unique chromatin structure at active enhancers, which, in turn, is required for regulation of target genes. [ABSTRACT FROM AUTHOR]

Following the discovery of widespread enhancer transcription, enhancers and promoters have been found to be far more similar than previously thought. In this issue of Genes & Development , two studies (Henriques and colleagues [pp. 26-41] and Mikhaylichenko and colleagues [pp. 42-57]) shine new light on the transcriptional nature of promoters and enhancers in Drosophila Together, these studies support recent work in mammalian cells that indicates that most active enhancers drive local transcription using factors and mechanisms similar to those of promoters. Intriguingly, enhancer transcription is shown to be coordinated by SPT5- and P-TEFb-mediated pause-release, but the pause half-life is shorter, and termination is more rapid at enhancers than at promoters. Moreover, bidirectional transcription from promoters is associated with enhancer activity, lending further credence to models in which regulatory elements exist along a spectrum of promoter-ness and enhancer-ness. We propose a general unified model to explain possible functions of transcription at enhancers., (© 2018 Tippens et al.; Published by Cold Spring Harbor Laboratory Press.)

Gene expression is regulated by promoters, which initiate transcription, and enhancers, which control their temporal and spatial activity. However, the discovery that mammalian enhancers also initiate transcription questions the inherent differences between enhancers and promoters. Here, we investigate the transcriptional properties of enhancers during Drosophila embryogenesis using characterized developmental enhancers. We show that while the timing of enhancer transcription is generally correlated with enhancer activity, the levels and directionality of transcription are highly varied among active enhancers. To assess how this impacts function, we developed a dual transgenic assay to simultaneously measure enhancer and promoter activities from a single element in the same embryo. Extensive transgenic analysis revealed a relationship between the direction of endogenous transcription and the ability to function as an enhancer or promoter in vivo, although enhancer RNA (eRNA) production and activity are not always strictly coupled. Some enhancers (mainly bidirectional) can act as weak promoters, producing overlapping spatio-temporal expression. Conversely, bidirectional promoters often act as strong enhancers, while unidirectional promoters generally cannot. The balance between enhancer and promoter activity is generally reflected in the levels and directionality of eRNA transcription and is likely an inherent sequence property of the elements themselves., (© 2018 Mikhaylichenko et al.; Published by Cold Spring Harbor Laboratory Press.)

Sequencing environmental DNA (eDNA) is increasingly being used as an alternative to traditional morphological-based identification to characterize biological assemblages and monitor anthropogenic impacts in marine environments. Most studies only assess eDNA which, compared to eRNA, can persist longer in the environment after cell death. Therefore, eRNA may provide a more immediate census of the environment due to its relatively weaker stability, leading some researchers to advocate for the use of eRNA as an additional, or perhaps superior proxy for portraying ecological changes. A variety of pre-treatment techniques for screening eDNA and eRNA derived operational taxonomic units (OTUs) have been employed prior to statistical analyses, including removing singleton taxa (i.e., OTUs found only once) and discarding those not present in both eDNA and eRNA datasets. In this study, we used bacterial (16S ribosomal RNA gene) and eukaryotic (18S ribosomal RNA gene) eDNA- and eRNA-derived data from benthic communities collected at increasing distances along a transect from an oil production platform (Taranaki, New Zealand). Macro-infauna (visual classification of benthic invertebrates) and physico-chemical data were analyzed in parallel. We tested the effect of removing singleton taxa, and removing taxa not present in the eDNA and eRNA libraries from the same environmental sample (trimmed by shared OTUs), by comparing the impact of the oil production platform on alpha- and beta-diversity of the eDNA/eRNA-based biological assemblages, and by correlating these to the morphologically identified macro-faunal communities and the physico-chemical data. When trimmed by singletons, presence/absence information from eRNA data represented the best proxy to detect changes on species diversity for both bacteria and eukaryotes. However, assessment of quantitative beta-diversity from read abundance information of bacteria eRNA did not, contrary to eDNA, reveal any impact from the oil production activity. Overall, the data appeared more robust when trimmed by shared OTUs, showing a greater effect of the platform on alpha- and beta-diversity. Trimming by shared OTUs likely removes taxa derived from legacy DNA and technical artefacts introduced through reverse transcriptase, polymerase-chain-reaction and sequencing. Findings from our scoping study suggest that metabarcoding-based biomonitoring surveys should, if funds, time and expertise allow, be assessed using both eDNA and eRNA products., Competing Interests: Dr. Xavier Pochon is an Academic Editor for PeerJ.

Recent work has shown that RNA polymerase II-mediated transcription at distal cis-regulatory elements serves as a mark of highly active enhancers. Production of noncoding RNAs at enhancers, termed eRNAs, correlates with higher expression of genes that the enhancer interacts with; hence, eRNAs provide a new tool to model gene activity in normal and disease tissues. Moreover, this unique class of noncoding RNA has diverse roles in transcriptional regulation. Transcribed enhancers can be identified by a common signature of epigenetic marks by overlaying a series of genome-wide chromatin immunoprecipitation and RNA sequencing datasets. A computational approach to filter non-enhancer elements and other classes of noncoding RNAs is essential to not cloud downstream analysis. Here we present a protocol that combines wet and dry bench methods to accurately identify transcribed enhancers genome-wide as well as an experimental procedure to validate these datasets.

Enhancers are principal regulators that allow spatiotemporal tissue-specific control of gene expression. While mounting evidence suggests that enhancer-derived long noncoding RNAs (long ncRNAs), including enhancer RNAs (eRNAs), are an important component of enhancer function, their expression has not been broadly analyzed at a single cell level via imaging techniques. This protocol describes a method to image eRNA in single cells by in situ hybridization followed by tyramide signal amplification (TSA). The procedure can be multiplexed to simultaneously visualize both eRNA and protein-coding transcript at the site of transcriptional elongation, thereby permitting analysis of dynamics between the two transcript species in single cells. Our approach is not limited to eRNAs, but can be implemented on other transcripts.