Distal and proximal cis-regulatory elements sense X chromosome dosage and developmental state at the Xist locus.

Developmental genes such as Xist, which initiates X chromosome inactivation, are controlled by complex cis-regulatory landscapes, which decode multiple signals to establish specific spatiotemporal expression patterns. Xist integrates information on X chromosome dosage and developmental stage to trigger X inactivation in the epiblast specifically in female embryos. Through a pooled CRISPR screen in differentiating mouse embryonic stem cells, we identify functional enhancer elements of Xist at the onset of random X inactivation. Chromatin profiling reveals that X-dosage controls the promoter-proximal region, while differentiation cues activate several distal enhancers. The strongest distal element lies in an enhancer cluster associated with a previously unannotated Xist-enhancing regulatory transcript, which we named Xert. Developmental cues and X-dosage are thus decoded by distinct regulatory regions, which cooperate to ensure female-specific Xist upregulation at the correct developmental time. With this study, we start to disentangle how multiple, functionally distinct regulatory elements interact to generate complex expression patterns in mammals.

Xrn2 substrate mapping identifies torpedo loading sites and extensive premature termination of RNA pol II transcription.

The exonuclease torpedo Xrn2 loads onto nascent RNA 5'-PO4 ends and chases down pol II to promote termination downstream from polyA sites. We report that Xrn2 is recruited to preinitiation complexes and "travels" to 3' ends of genes. Mapping of 5'-PO4 ends in nascent RNA identified Xrn2 loading sites stabilized by an active site mutant, Xrn2(D235A). Xrn2 loading sites are approximately two to 20 bases downstream from where CPSF73 cleaves at polyA sites and histone 3' ends. We propose that processing of all mRNA 3' ends comprises cleavage and limited 5'-3' trimming by CPSF73, followed by handoff to Xrn2. A similar handoff occurs at tRNA 3' ends, where cotranscriptional RNase Z cleavage generates novel Xrn2 substrates. Exonuclease-dead Xrn2 increased transcription in 3' flanking regions by inhibiting polyA site-dependent termination. Surprisingly, the mutant Xrn2 also rescued transcription in promoter-proximal regions to the same extent as in 3' flanking regions. eNET-seq revealed Xrn2-mediated degradation of sense and antisense nascent RNA within a few bases of the TSS, where 5'-PO4 ends may be generated by decapping or endonucleolytic cleavage. These results suggest that a major fraction of pol II complexes terminates prematurely close to the start site under normal conditions by an Xrn2-mediated torpedo mechanism.

Hypoxia-driven deSUMOylation of EXOSC10 promotes adaptive changes in the transcriptome profile.

Reduced oxygen availability (hypoxia) triggers adaptive cellular responses via hypoxia-inducible factor (HIF)-dependent transcriptional activation. Adaptation to hypoxia also involves transcription-independent processes like post-translational modifications; however, these mechanisms are poorly characterized. Investigating the involvement of protein SUMOylation in response to hypoxia, we discovered that hypoxia strongly decreases the SUMOylation of Exosome subunit 10 (EXOSC10), the catalytic subunit of the RNA exosome, in an HIF-independent manner. EXOSC10 is a multifunctional exoribonuclease enriched in the nucleolus that mediates the processing and degradation of various RNA species. We demonstrate that the ubiquitin-specific protease 36 (USP36) SUMOylates EXOSC10 and we reveal SUMO1/sentrin-specific peptidase 3 (SENP3) as the enzyme-mediating deSUMOylation of EXOSC10. Under hypoxia, EXOSC10 dissociates from USP36 and translocates from the nucleolus to the nucleoplasm concomitant with its deSUMOylation. Loss of EXOSC10 SUMOylation does not detectably affect rRNA maturation but affects the mRNA transcriptome by modulating the expression levels of hypoxia-related genes. Our data suggest that dynamic modulation of EXOSC10 SUMOylation and localization under hypoxia regulates the RNA degradation machinery to facilitate cellular adaptation to low oxygen conditions.

Single-cell RNA sequencing uncovers the nuclear decoy lincRNA PIRAT as a regulator of systemic monocyte immunity during COVID-19.

The systemic immune response to viral infection is shaped by master transcription factors, such as NF-κB, STAT1, or PU.1. Although long noncoding RNAs (lncRNAs) have been suggested as important regulators of transcription factor activity, their contributions to the systemic immunopathologies observed during SARS-CoV-2 infection have remained unknown. Here, we employed a targeted single-cell RNA sequencing approach to reveal lncRNAs differentially expressed in blood leukocytes during severe COVID-19. Our results uncover the lncRNA PIRAT (PU.1-induced regulator of alarmin transcription) as a major PU.1 feedback-regulator in monocytes, governing the production of the alarmins S100A8/A9, key drivers of COVID-19 pathogenesis. Knockout and transgene expression, combined with chromatin-occupancy profiling, characterized PIRAT as a nuclear decoy RNA, keeping PU.1 from binding to alarmin promoters and promoting its binding to pseudogenes in naïve monocytes. NF-κB-dependent PIRAT down-regulation during COVID-19 consequently releases a transcriptional brake, fueling alarmin production. Alarmin expression is additionally enhanced by the up-regulation of the lncRNA LUCAT1, which promotes NF-κB-dependent gene expression at the expense of targets of the JAK-STAT pathway. Our results suggest a major role of nuclear noncoding RNA networks in systemic antiviral responses to SARS-CoV-2 in humans.

Determination of primary microRNA processing in clinical samples by targeted pri-miR-sequencing.

MicroRNA expression is important for gene regulation and deregulated microRNA expression is often observed in diseases such as cancer. The processing of primary microRNA transcripts is an important regulatory step in microRNA biogenesis. Due to low expression level and association with chromatin, primary microRNAs are challenging to study in clinical samples where input material is limited. Here, we present a high-sensitivity targeted method to determine processing efficiency of several hundred primary microRNAs from total RNA that requires relatively few RNA sequencing reads. We validate the method using RNA from HeLa cells and show the applicability to clinical samples by analyzing RNA from normal liver and hepatocellular carcinoma. We identify 24 primary microRNAs with significant changes in processing efficiency from normal liver to hepatocellular carcinoma, among those the highly expressed miRNA-122 and miRNA-21, demonstrating that differential processing of primary microRNAs is occurring and could be involved in disease. With our method presented here we provide means to study pri-miRNA processing in disease from clinical samples.

An atlas of active enhancers across human cell types and tissues.

Enhancers control the correct temporal and cell-type-specific activation of gene expression in multicellular eukaryotes. Knowing their properties, regulatory activity and targets is crucial to understand the regulation of differentiation and homeostasis. Here we use the FANTOM5 panel of samples, covering the majority of human tissues and cell types, to produce an atlas of active, in vivo-transcribed enhancers. We show that enhancers share properties with CpG-poor messenger RNA promoters but produce bidirectional, exosome-sensitive, relatively short unspliced RNAs, the generation of which is strongly related to enhancer activity. The atlas is used to compare regulatory programs between different cells at unprecedented depth, to identify disease-associated regulatory single nucleotide polymorphisms, and to classify cell-type-specific and ubiquitous enhancers. We further explore the utility of enhancer redundancy, which explains gene expression strength rather than expression patterns. The online FANTOM5 enhancer atlas represents a unique resource for studies on cell-type-specific enhancers and gene regulation.

Identifying lncRNA-mediated regulatory modules via ChIA-PET network analysis.

BACKGROUND: Although several studies have provided insights into the role of long non-coding RNAs (lncRNAs), the majority of them have unknown function. Recent evidence has shown the importance of both lncRNAs and chromatin interactions in transcriptional regulation. Although network-based methods, mainly exploiting gene-lncRNA co-expression, have been applied to characterize lncRNA of unknown function by means of 'guilt-by-association', no strategy exists so far which identifies mRNA-lncRNA functional modules based on the 3D chromatin interaction graph. RESULTS: To better understand the function of chromatin interactions in the context of lncRNA-mediated gene regulation, we have developed a multi-step graph analysis approach to examine the RNA polymerase II ChIA-PET chromatin interaction network in the K562 human cell line. We have annotated the network with gene and lncRNA coordinates, and chromatin states from the ENCODE project. We used centrality measures, as well as an adaptation of our previously developed Markov State Models (MSM) clustering method, to gain a better understanding of lncRNAs in transcriptional regulation. The novelty of our approach resides in the detection of fuzzy regulatory modules based on network properties and their optimization based on co-expression analysis between genes and gene-lncRNA pairs. This results in our method returning more bona fide regulatory modules than other state-of-the art approaches for clustering on graphs. CONCLUSIONS: Interestingly, we find that lncRNA network hubs tend to be significantly enriched in evolutionary conserved lncRNAs and enhancer-like functions. We validated regulatory functions for well known lncRNAs, such as MALAT1 and the enhancer-like lncRNA FALEC. In addition, by investigating the modular structure of bigger components we mine putative regulatory functions for uncharacterized lncRNAs.

Microprocessor dynamics shows co- and post-transcriptional processing of pri-miRNAs.

miRNAs are small regulatory RNAs involved in the regulation of translation of target transcripts. miRNA biogenesis is a multistep process starting with the cleavage of the primary miRNA transcript in the nucleus by the Microprocessor complex. Endogenous processing of pri-miRNAs is challenging to study and the in vivo kinetics of this process is not known. Here, we present a method for determining the processing kinetics of pri-miRNAs within intact cells over time, using a pulse-chase approach to label transcribed RNA during 15 min, and follow the processing within a 1-hour window after labeling with bromouridine. We show that pri-miRNAs exhibit different processing kinetics ranging from fast over intermediate to slow processing, and we provide evidence that pri-miRNA processing can occur both cotranscriptionally and post-transcriptionally.

Genome-wide measurement of RNA dissociation from chromatin classifies transcripts by their dynamics and reveals rapid dissociation of enhancer lncRNAs.

Long non-coding RNAs (lncRNAs) are involved in gene expression regulation in cis. Although enriched in the cell chromatin fraction, to what degree this defines their regulatory potential remains unclear. Furthermore, the factors underlying lncRNA chromatin tethering, as well as the molecular basis of efficient lncRNA chromatin dissociation and its impact on enhancer activity and target gene expression, remain to be resolved. Here, we developed chrTT-seq, which combines the pulse-chase metabolic labeling of nascent RNA with chromatin fractionation and transient transcriptome sequencing to follow nascent RNA transcripts from their transcription on chromatin to release and allows the quantification of dissociation dynamics. By incorporating genomic, transcriptomic, and epigenetic metrics, as well as RNA-binding protein propensities, in machine learning models, we identify features that define transcript groups of different chromatin dissociation dynamics. Notably, lncRNAs transcribed from enhancers display reduced chromatin retention, suggesting that, in addition to splicing, their chromatin dissociation may shape enhancer activity.

Second order regulator Collier directly controls intercalary-specific segment polarity gene expression.

In Drosophila, trunk metamerization is established by a cascade of segmentation gene activities: the gap genes, the pair rule genes, and the segment polarity genes. In the anterior head, metamerization requires also gap-like genes and segment polarity genes. However, because the pair rule genes are not active in this part of the embryo, the question on which gene activities are fulfilling the role of the second order regulator genes still remains to be solved. Here we provide first molecular evidence that the Helix-Loop-Helix-COE transcription factor Collier fulfills this role by directly activating the expression of the segment polarity gene hedgehog in the posterior part of the intercalary segment. Collier thereby occupies a newly identified binding site within an intercalary-specific cis-regulatory element. Moreover, we identified a direct physical association between Collier and the basic-leucine-zipper transcription factor Cap'n'collar B, which seems to restrict the activating input of Collier to the posterior part of the intercalary segment and to lead to the attenuation of hedgehog expression in the intercalary lobes at later stages.

Unique establishment of procephalic head segments is supported by the identification of cis-regulatory elements driving segment-specific segment polarity gene expression in Drosophila.

Anterior head segmentation is governed by different regulatory mechanisms than those that control trunk segmentation in Drosophila. For segment polarity genes, both initial mode of activation as well as cross-regulatory interactions among them differ from the typical genetic circuitry in the trunk and are unique for each of the procephalic segments. In order to better understand the segment-specific gene network responsible for the procephalic expression of the earliest active segment polarity genes wingless and hedgehog, we started to identify and analyze cis-regulatory DNA elements of these genes. For hedgehog, we could identify a cis-regulatory element, ic-CRE, that mediates expression specifically in the posterior part of the intercalary segment and requires promoter-specific interaction for its function. The intercalary stripe is the last part of the metameric hedgehog expression pattern that appears during embryonic development, which probably reflects the late and distinct establishment of this segment. The identification of a cis-regulatory element that is specific for one head segment supports the mutant-based observation that the expression of segment polarity genes is governed by a unique gene network in each of the procephalic segments. This provides further indication that the anterior-most head segments represent primary segments, which are set up independently, in contrast to the secondary segments of the trunk, which resemble true repetitive units.

Targeting Polyadenylation for Retention of RNA at Chromatin.

The various steps of RNA polymerase II transcription, including transcription initiation, splicing, and termination, are interlinked and tightly coordinated. Efficient 3'end processing is defined by sequence motifs emerging in the nascent-transcribed RNA strand and the cotranscriptional binding of regulatory proteins. The processing of a mature 3'end consists of cleavage and polyadenylation and is coupled with RNA polymerase II transcription termination and the dissociation of the nascent RNA transcript from the chromatin-associated transcriptional template. The subcellular and subnuclear topological specificity of the various RNA species is important for their functions. For instance, the formation of RNA-binding protein interactions, critical for the final outcome of gene expression, may require the nucleoplasmic fully spliced and polyadenylated form of an RNA transcript. Thus, interfering with the critical step of transcription termination and 3'end formation provides a means for assaying the functional potential of a given RNA of interest.In this protocol, we describe a method for blocking 3'end processing of the nascent RNA transcript, by using RNase H-inactive antisense oligonucleotides targeting cleavage and polyadenylation, delivered via transient transfection in cell culture.

Functional impacts of non-coding RNA processing on enhancer activity and target gene expression.

Tight regulation of gene expression is orchestrated by enhancers. Through recent research advancements, it is becoming clear that enhancers are not solely distal regulatory elements harboring transcription factor binding sites and decorated with specific histone marks, but they rather display signatures of active transcription, showing distinct degrees of transcription unit organization. Thereby, a substantial fraction of enhancers give rise to different species of non-coding RNA transcripts with an unprecedented range of potential functions. In this review, we bring together data from recent studies indicating that non-coding RNA transcription from active enhancers, as well as enhancer-produced long non-coding RNA transcripts, may modulate or define the functional regulatory potential of the cognate enhancer. In addition, we summarize supporting evidence that RNA processing of the enhancer-associated long non-coding RNA transcripts may constitute an additional layer of regulation of enhancer activity, which contributes to the control and final outcome of enhancer-targeted gene expression.

Transient N-6-Methyladenosine Transcriptome Sequencing Reveals a Regulatory Role of m6A in Splicing Efficiency.

Splicing efficiency varies among transcripts, and tight control of splicing kinetics is crucial for coordinated gene expression. N-6-methyladenosine (m6A) is the most abundant RNA modification and is involved in regulation of RNA biogenesis and function. The impact of m6A on regulation of RNA splicing kinetics is unknown. Here, we provide a time-resolved high-resolution assessment of m6A on nascent RNA transcripts and unveil its importance for the control of RNA splicing kinetics. We find that early co-transcriptional m6A deposition near splice junctions promotes fast splicing, while m6A modifications in introns are associated with long, slowly processed introns and alternative splicing events. In conclusion, we show that early m6A deposition specifies the fate of transcripts regarding splicing kinetics and alternative splicing.

Long ncRNA A-ROD activates its target gene DKK1 at its release from chromatin.

Long ncRNAs are often enriched in the nucleus and at chromatin, but whether their dissociation from chromatin is important for their role in transcription regulation is unclear. Here, we group long ncRNAs using epigenetic marks, expression and strength of chromosomal interactions; we find that long ncRNAs transcribed from loci engaged in strong long-range chromosomal interactions are less abundant at chromatin, suggesting the release from chromatin as a crucial functional aspect of long ncRNAs in transcription regulation of their target genes. To gain mechanistic insight into this, we functionally validate the long ncRNA A-ROD, which enhances DKK1 transcription via its nascent spliced released form. Our data provide evidence that the regulatory interaction requires dissociation of A-ROD from chromatin, with target specificity ensured within the pre-established chromosomal proximity. We propose that the post-transcriptional release of a subset of long ncRNAs from the chromatin-associated template plays an important role in their function as transcription regulators.

LincRNA H19 protects from dietary obesity by constraining expression of monoallelic genes in brown fat.

Increasing brown adipose tissue (BAT) thermogenesis in mice and humans improves metabolic health and understanding BAT function is of interest for novel approaches to counteract obesity. The role of long noncoding RNAs (lncRNAs) in these processes remains elusive. We observed maternally expressed, imprinted lncRNA H19 increased upon cold-activation and decreased in obesity in BAT. Inverse correlations of H19 with BMI were also observed in humans. H19 overexpression promoted, while silencing of H19 impaired adipogenesis, oxidative metabolism and mitochondrial respiration in brown but not white adipocytes. In vivo, H19 overexpression protected against DIO, improved insulin sensitivity and mitochondrial biogenesis, whereas fat H19 loss sensitized towards HFD weight gains. Strikingly, paternally expressed genes (PEG) were largely absent from BAT and we demonstrated that H19 recruits PEG-inactivating H19-MBD1 complexes and acts as BAT-selective PEG gatekeeper. This has implications for our understanding how monoallelic gene expression affects metabolism in rodents and, potentially, humans.

Long ncRNA expression associates with tissue-specific enhancers.

Long non-coding RNAs (ncRNA) have recently been demonstrated to be expressed from a subset of enhancers and to be required for the distant regulation of gene expression. Several approaches to predict enhancers have been developed based on various chromatin marks and occupancy of enhancer-binding proteins. Despite the rapid advances in the field, no consensus how to define tissue specific enhancers yet exists. Here, we identify 2,695 long ncRNAs annotated by ENCODE (corresponding to 28% of all ENCODE annotated long ncRNAs) that overlap tissue-specific enhancers. We use a recently developed algorithm to predict tissue-specific enhancers, PreSTIGE, that is based on the H3K4me1 mark and tissue specific expression of mRNAs. The expression of the long ncRNAs overlapping enhancers is significantly higher when the enhancer is predicted as active in a specific cell line, suggesting a general interdependency of active enhancers and expression of long ncRNAs. This dependency is not identified using previous enhancer prediction algorithms that do not account for expression of their downstream targets. The predicted enhancers that overlap annotated long ncRNAs generally have a lower ratio of H3K4me1 to H3K4me3, suggesting that enhancers expressing long ncRNAs might be associated with specific epigenetic marks. In conclusion, we demonstrate the tissue-specific predictive power of PreSTIGE and provide evidence for thousands of long ncRNAs that are expressed from active tissue-specific enhancers, suggesting a particularly important functional relationship between long ncRNAs and enhancer activity in determining tissue-specific gene expression.

Polyadenylation site-induced decay of upstream transcripts enforces promoter directionality.

Active human promoters produce promoter-upstream transcripts (PROMPTs). Why these RNAs are coupled to decay, whereas their neighboring promoter-downstream mRNAs are not, is unknown. Here high-throughput sequencing demonstrates that PROMPTs generally initiate in the antisense direction closely upstream of the transcription start sites (TSSs) of their associated genes. PROMPT TSSs share features with mRNA-producing TSSs, including stalled RNA polymerase II (RNAPII) and the production of small TSS-associated RNAs. Notably, motif analyses around PROMPT 3' ends reveal polyadenylation (pA)-like signals. Mutagenesis studies demonstrate that PROMPT pA signals are functional but linked to RNA degradation. Moreover, pA signals are under-represented in promoter-downstream versus promoter-upstream regions, thus allowing for more efficient RNAPII progress in the sense direction from gene promoters. We conclude that asymmetric sequence distribution around human gene promoters serves to provide a directional RNA output from an otherwise bidirectional transcription process.

The human cap-binding complex is functionally connected to the nuclear RNA exosome.

Nuclear processing and quality control of eukaryotic RNA is mediated by the RNA exosome, which is regulated by accessory factors. However, the mechanism of exosome recruitment to its ribonucleoprotein (RNP) targets remains poorly understood. Here we report a physical link between the human exosome and the cap-binding complex (CBC). The CBC associates with the ARS2 protein to form CBC-ARS2 (CBCA) and then further connects, together with the ZC3H18 protein, to the nuclear exosome targeting (NEXT) complex, thus forming CBC-NEXT (CBCN). RNA immunoprecipitation using CBCN factors as well as the analysis of combinatorial depletion of CBCN and exosome components underscore the functional relevance of CBC-exosome bridging at the level of target RNA. Specifically, CBCA suppresses read-through products of several RNA families by promoting their transcriptional termination. We suggest that the RNP 5' cap links transcription termination to exosomal RNA degradation through CBCN.