Enhancer RNAs (eRNAs) are a pivotal class of enhancer-derived non-coding RNAs that drive gene expression. Here we identify the SNAI1 enhancer RNA (SNAI1e; SCREEM2) as a key activator of SNAI1 expression and a potent enforcer of transforming growth factor-β (TGF-β)/SMAD signaling in cancer cells. SNAI1e depletion impairs TGF-β-induced epithelial-mesenchymal transition (EMT), migration, in vivo extravasation, stemness, and chemotherapy resistance in breast cancer cells. SNAI1e functions as an eRNA to cis-regulate SNAI1 enhancer activity by binding to and strengthening the enrichment of the transcriptional co-activator bromodomain containing protein 4 (BRD4) at the local enhancer. SNAI1e selectively promotes the expression of SNAI1, which encodes the EMT transcription factor SNAI1. Furthermore, we reveal that SNAI1 interacts with and anchors the inhibitory SMAD7 in the nucleus, and thereby prevents TGF-β type I receptor (TβRI) polyubiquitination and proteasomal degradation. Our findings establish SNAI1e as a critical driver of SNAI1 expression and TGF-β-induced cell plasticity. Enhancer RNAs (eRNAs) regulate gene expression in cancer. Here, the authors identify the eRNA SNAI1e as a key activator of SNAI1 expression, promoting TGF-β-induced EMT, migration, and chemotherapy resistance in breast cancer by enhancing BRD4 enrichment at the SNAI1 enhancer.

The human genome contains regulatory DNA elements, known as enhancers, that can activate gene transcription over long chromosomal distances. Here, we showed that enhancer distance can be critical for gene silencing. We demonstrated that linear recruitment of the normally distal strong HBB enhancer to developmentally silenced embryonic HBE or fetal HBG promoters through deletion or inversion of intervening DNA sequences led to strongly reactivated expression in adult erythroid cells and ex vivo differentiated hematopoietic stem and progenitor cells. A similar observation was made for the HBA locus in which deletion-to-recruit of the distal enhancer strongly reactivated embryonic HBZ expression. Overall, our work assigned function to seemingly nonregulatory genomic segments; by providing linear separation, they may support genes to autonomously control their transcriptional response to distal enhancers.

Cell-type-specific gene activation is regulated by enhancers, sometimes located at large genomic distances from target gene promoters. Whether distal enhancers require specific factors to orchestrate gene regulation remains unclear. Here, we used enhancer distance-controlled reporter screens to find candidate factors. We depleted them and employed activity-by-contact predictions to genome-wide classify genes based on enhancer distance. Predicted distal enhancers typically control tissue-restricted genes and often are strong enhancers. We find cohesin, but also mediator, most specifically required for long-range activation, with cohesin repressing short-range gene activation and prioritizing distal over proximal HBB genes competing for shared enhancers. Long-range controlled genes are also most sensitive to perturbations of other regulatory proteins and to BET inhibitor JQ1, this being more a consequence of their distinct enhancer features than distance. Our work predicts that lengthening of intervening sequences can help limit the expression of target genes to specialized cells with optimal trans-factor environments.

Cohesin (SMC1-SMC3-RAD21) constantly extrudes DNA loops to organize chromosomes into structural domains, pausing and anchoring at specific DNA-bound CTCF molecules. To study the detailed consequences of cohesin loop extrusion, we developed TArgeted Cohesin Loader (TACL) for controlled pan-cellular activation of chromatin loop formation at defined genomic locations in living cells. With TACL, we show that highly complex looping networks can exist, with extruding cohesin complexes that block each other, drive cohesin queuing and induce loop anchoring at nearly all CTCF-bound sites. TACL loops extend upon acute depletion of STAG2, PDS5A or WAPL. Activated cohesin loop extrusion hinders local gene transcription and can alter chromatin accessibility and H3K27ac distribution. TACL shows that the loading/extrusion complex NIPBL-MAU2 can be transported by cohesin to CTCF sites but, together with SMC1, to enhancers in a RAD21-independent manner. TACL thus enables studying the consequences of activated loop extrusion at defined genomic locations.

Enhancers are tissue-specific regulatory DNA elements that can activate transcription of genes over distance. Their target genes most often are located in the same contact domain—chromosomal entities formed by cohesin DNA loop extrusion and typically flanked by CTCF-bound boundaries. Enhancers shared by multiple unrelated genes are underexplored but may be more common than anticipated. Here, we analyzed the interplay between an enhancer and two distal functionally unrelated genes residing at opposite domain boundaries. The enhancer strongly activated their expression and supported their frequent interactions. Cohesin structured the domain and supported their transcription, but the genes did not rely on each other's transcription or show gene competition. Deleting either domain boundary not only extended the contact domain but led to reduced contacts within the original domain and reduction in the expression of both genes. Conversely, by isolating either gene with the enhancer in shorter domains, through insertion of new CTCF boundaries, intradomain contact frequencies increased, and the gene isolated with the enhancer was upregulated. Collectively, this shows that an enhancer can independently activate unrelated distal genes and that long-range gene regulation benefits from operating in small contact domains.

The human genome contains regulatory DNA elements, enhancers, that can activate gene transcription over long chromosomal distances. Here, we show that enhancer distance can be critical for gene silencing. We demonstrate that linear recruitment of the normally distal HBB super-enhancer to developmentally silenced HBG promoters, through deletion or inversion of intervening DNA sequences, results in strongly reactivated HBG expression in adult erythroid cells and ex vivo differentiated hematopoietic stem and progenitor cells. A similar observation is made in the HBA locus, where deletion-to-recruit of the distal enhancer strongly reactivates embryonic HBZ expression. Overall, our work assigns function to seemingly non-regulatory genomic segments: by providing linear separation they may support genes to autonomously control their transcriptional response to distal enhancers. ### Competing Interest Statement Patent applications PCT/EP2022/053341 (WdL) and PCT/EP2023/070536 (WdL, A-K.F and PHLK) describing the Del2Rec methodology were filed.

Single-nucleotide polymorphisms (SNPs) near the ERAP2 gene are associated with various autoimmune conditions, as well as protection against lethal infections. Due to high linkage disequilibrium, numerous trait-associated SNPs are correlated with ERAP2 expression; however, their functional mechanisms remain unidentified. We show by reciprocal allelic replacement that ERAP2 expression is directly controlled by the splice region variant rs2248374. However, disease-associated variants in the downstream LNPEP gene promoter are independently associated with ERAP2 expression. Allele-specific conformation capture assays revealed long-range chromatin contacts between the gene promoters of LNPEP and ERAP2 and showed that interactions were stronger in patients carrying the alleles that increase susceptibility to autoimmune diseases. Replacing the SNPs in the LNPEP promoter by reference sequences lowered ERAP2 expression. These findings show that multiple SNPs act in concert to regulate ERAP2 expression and that disease-associated variants can convert a gene promoter region into a potent enhancer of a distal gene.

The cohesin complex (SMC1-SMC3-RAD21) shapes chromosomes by DNA loop extrusion, but individual extrusion trajectories were so far unappreciable in vivo. Here, we site-specifically induced dozens of extrusion trajectories anchored at artificial loading sites in living cells. Extruding cohesin transports loading proteins MAU2-NIPBL over megabase DNA distances to blocking CTCF sites that then loop back to the loading sequences, showing that CTCF-CTCF interactions are unnecessary for stabilized contacts between loop extrusion obstacles. When stalled, cohesin can block other extruding cohesin from either direction. Without RAD21, MAU2-NIPBL exclusively accumulate at loading sites, here genome-wide defined as enhancers. SMC1 now also selectively accumulates here, suggesting that cohesin may load modularly on chromatin. Genes inside high cohesin extrusion trajectories are collectively hindered in transcription. This work characterizes the impact, entry and exit sites of individual cohesin loop extrusion trajectories.

AbstractSingle nucleotide polymorphisms (SNP) near theERAP2gene are associated with autoimmune conditions such asCrohn’s disease, andbirdshot chorioretinopathy, as well as protection against lethal infections, including theBlack Death. Due to high linkage disequilibrium (LD), a great number of trait-associated SNPs are correlated withERAP2expression, however their functional mechanisms remain unidentified. We used genome editing and functional genomics to identify causal variants that remain obscured by LD. We demonstrate by reciprocal allelic replacement thatERAP2expression is directly controlled by the genotype of splice region SNP rs2248374. However, we demonstrate that autoimmune disease-risk SNPs located near the downstreamLNPEPgene promoter are independently associated withERAP2expression. Allele-specific conformation capture assays revealed long-range chromatin contacts between theLNPEPpromoter region and theERAP2promoter and showed that interactions were stronger in patients carrying the alleles that increase susceptibility to autoimmune diseases. Replacing the disease-associated SNPs in theLNPEPpromoter by reference sequences loweredERAP2expression. These findings show that clustered GWAS signals associated with diverse autoimmune conditions and lethal infections act in concert to control ERAP2 expression and that disease-associated variants can convert a gene promoter region into a potent enhancer of a distal gene.

Monoallelic inactivation of CCCTC-binding factor (CTCF) in human cancer drives altered methylated genomic states, altered CTCF occupancy at promoter and enhancer regions, and deregulated global gene expression. In patients with T cell acute lymphoblastic leukemia (T-ALL), we find that acquired monoallelic CTCF-inactivating events drive subtle and local genomic effects in nearly half of t(5; 14) (q35; q32.2) rearranged patients, especially when CTCF-binding sites are preserved in between the BCL11B enhancer and the TLX3 oncogene. These solitary intervening sites insulate TLX3 from the enhancer by inducing competitive looping to multiple binding sites near the TLX3 promoter. Reduced CTCF levels or deletion of the intervening CTCF site abrogates enhancer insulation by weakening competitive looping while favoring TLX3 promoter to BCL11B enhancer looping, which elevates oncogene expression levels and leukemia burden.

During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitutions, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Polϵ) for PCNA binding. However, CAF-1 does not affect the activity of the lagging strand DNA polymerase Delta (Polδ). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand these processes may be coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication.

Developmental gene expression is often controlled by distal regulatory DNA elements called enhancers. Distant enhancer action is restricted to structural chromosomal domains that are flanked by CTCF-associated boundaries and formed through cohesin chromatin loop extrusion. To better understand how enhancers, genes and CTCF boundaries together form structural domains and control expression, we used a bottom-up approach, building series of active regulatory landscapes in inactive chromatin. We demonstrate here that gene transcription levels and activity over time reduce with increased enhancer distance. The enhancer recruits cohesin to stimulate domain formation and engage flanking CTCF sites in loop formation. It requires cohesin exclusively for the activation of distant genes, not of proximal genes, with nearby CTCF boundaries supporting efficient long-range enhancer action. Our work supports a dual activity model for enhancers: its classic role of stimulating transcription initiation and elongation from target gene promoters and a role of recruiting cohesin for the creation of chromosomal domains, the engagement of CTCF sites in chromatin looping and the activation of distal target genes.

Bovine leukemia virus (BLV)-induced tumoral development is a multifactorial phenomenon that remains incompletely understood. Here, we highlight the critical role of the cellular CCCTC-binding factor (CTCF) both in the regulation of BLV transcriptional activities and in the deregulation of the three-dimensional (3D) chromatin architecture surrounding the BLV integration site. We demonstrated the in vivo recruitment of CTCF to three conserved CTCF binding motifs along the provirus. Next, we showed that CTCF localized to regions of transitions in the histone modifications profile along the BLV genome and that it is implicated in the repression of the 5 ' Long Terminal Repeat (LTR) promoter activity, thereby contributing to viral latency, while favoring the 3 ' LTR promoter activity. Finally, we demonstrated that BLV integration deregulated the host cellular 3D chromatin organization through the formation of viral/host chromatin loops. Altogether, our results highlight CTCF as a new critical effector of BLV transcriptional regulation and BLV-induced physiopathology.

Enhancers play a central role in the spatiotemporal control of gene expression and tend to work in a cell-type-specific manner. In addition, they are suggested to be major contributors to phenotypic variation, evolution and disease. There is growing evidence that enhancer dysfunction due to genetic, structural or epigenetic mechanisms contributes to a broad range of human diseases referred to as enhanceropathies. Such mechanisms often underlie the susceptibility to common diseases, but can also play a direct causal role in cancer or Mendelian diseases. Despite the recent gain of insights into enhancer biology and function, we still have a limited ability to predict how enhancer dysfunction impacts gene expression. Here we discuss the major challenges that need to be overcome when studying the role of enhancers in disease etiology and highlight opportunities and directions for future studies, aiming to disentangle the molecular basis of enhanceropathies.

Recent years have seen a vast expansion of knowledge on three-dimensional (3D) genome organization. The majority of studies on chromosome topology consists of pairwise interaction data of bulk populations of cells and therefore conceals heterogenic and more complex folding patterns. Here, we discuss novel methodologies to study the variation in genome topologies between different cells and techniques that allow analysis of complex, multi-way interactions. These technologies will aid the interpretation of genome-wide chromosome conformation data and provide strategies to further dissect the interplay between genome architecture and transcription regulation.

In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens.

STRIP is a start-to-end streamlined and automated procedure for COVID-19 testing, centering on a single Tecan Fluent liquid-handling robot that can process over 14,000 samples per day. Key features of the customized Tecan Fluent robot are (1) on-board 1D and 2D barcode scanners, (2) an automated tube decapper, (3) two robotic arms for simultaneous processing of different procedural steps, (4) a newly-designed spin vessel to keep magnetic beads in solution and immediately transferable to 384-well plates, (5) a built-in magnetic deck and a 384-channel pipetting head for rapid RNA isolation, (6) a heating device for fast drying of RNA prior to elution, (7) a built-in plate sealer and (8) a plate storage system to allow processing of multiple sample plates in a single run (See Materials). Here we describe the RNA extraction and RT-qPCR protocol as currently applied in STRIP.

To understand how chromatin domains coordinate gene expression, we dissected select genetic elements organizing topology and transcription around the Prdm14 super enhancer in mouse embryonic stem cells. Taking advantage of allelic polymorphisms, we developed methods to sensitively analyze changes in chromatin topology, gene expression, and protein recruitment. We show that enhancer insulation does not rely strictly on loop formation between its flanking boundaries, that the enhancer activates the Slco5a1 gene beyond its prominent domain boundary, and that it recruits cohesin for loop extrusion. Upon boundary inversion, we find that oppositely oriented CTCF terminates extrusion trajectories but does not stall cohesin, while deleted or mutated CTCF sites allow cohesin to extend its trajectory. Enhancer-mediated gene activation occurs independent of paused loop extrusion near the gene promoter. We expand upon the loop extrusion model to propose that cohesin loading and extrusion trajectories originating at an enhancer contribute to gene activation.

Recent years have seen a vast expansion of knowledge on three-dimensional (3D) genome organization. The majority of studies on chromosome topology consists of pairwise interaction data of bulk populations of cells and therefore conceals heterogenic and more complex folding patterns. Here, we discuss novel methodologies to study the variation in genome topologies between different cells and techniques that allow analysis of complex, multi-way interactions. These technologies will aid the interpretation of genome-wide chromosome conformation data and provide strategies to further dissect the interplay between genome architecture and transcription regulation.

An in-house multiplex RT-qPCR , targeting SARS-CoV-2and PDV as internal control [1][2], developed on QuantStudio 7 Pro Real-Time PCR Systems using Life Technologies Taqman FastVirus 1-step mastermix with E-gene primers and probe as described by Corman et al. and N1 primers and probes as described by the CDC[3, 4]. 1.Clancy, A. eta al., The development of a qualitative real-time RT-PCR assay for the detection of hepatitis C virus European Journal Microbial Infectious Diseases, 2008. 276(12): p.1177. 2.Wolters, F., et al., Multi-center evaluation of cepheid xpert® xpress SARS-CoV-2 point-of-care test during the SARS-CoV-2 pandemic. Journal of Clinical Virology, 2020. 128: p. 104426 3.Corman, V.M., et al., Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill, 2020. 25(3). 4.Lu, X., et al., US CDC Real-Time Reverse Transcription PCR Panel for Detection of Severe Acute Respiratory Syndrome Coronavirus 2.Emerging Infectious Disease journal, 2020. 26(8): p. 1654.