Transcriptional Elements Research Articles

Modular engineering of Shiraia bambusicola for hypocrellin production through an efficient CRISPR system

Shiraia bambusicola exhibits an excellent capability to produce high-value pharmacological drugs, such as hypocrellin. However, less effective molecular tools hamper the processes to discover or exploit these metabolites. To address this issue, the more effective CRISPR/Cas9 system was constructed by optimizing the sgRNA transcription elements and disrupting the endogenous non-homologous end-joining pathway. These tactics prompted the gene-targeting frequency of 100% and simultaneously multiplex genome editing in S. bambusicola. This optimal CRISPR system encouraged us to rewire the entire hypocrellin flux and improve the yield by orchestrating the substrate pool supply, the central hypocrellin pathway, and the antioxidant system. Thus, 8632 mg/L hypocrellin was obtained, resulting in a 12-fold increase than that of the wild-type strain. This engineered S. bambusicola can still endure oxidative stresses from higher target metabolites and sustain an excellent biological activity. This study provides a whole conception to establish the more efficient genome-editing system. Higher conserved transcription elements for sgRNA expressions inspire us to adopt this system for gene modifications of other filamentous fungi. The rational and global biosystems outline will offer guidance to modulate metabolite productivity in other filamentous fungi.

Hemocyanin and hexamerinsexpression in response to hypoxia in stoneflies (Plecoptera, Insecta).

Many freshwater ecosystems worldwide undergo hypoxia events that can trigger physiological, behavioral, and molecular responses in many organisms. Among such molecular responses, the regulation of the hemocyanin (Hc) protein expression which plays a major role in oxygen transportation within aquatic insects remains poorly understood. The stoneflies (Plecoptera) are aquatic insects that possess a functional Hc in the hemolymph similar to crustacean that co-occurs with a nonfunctional Hc protein, hexamerins (Hx). However, the role of both proteins during hypoxia remains undetermined. Here, we evaluated the effect of hypoxia on the expression of Hc and Hx proteins via a comparison between hypoxia and normoxia amino acid sequence variation and protein expression pattern within 23 stonefly species. We induced short-term hypoxia in wild-caught stoneflies species, sequenced the target region of Hc and Hx by complementary DNA synthesis, characterized the protein biochemistry using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ultrafiltration, and polarographic fluorometric method, and amplified the genome region of the hypoxia-inducible factor (HIF) transcriptional response element that regulated Hc using genome walking library approach. We found a lack of Hc expression in all examined species during hypoxia conditions, despite recognition of the HIF gene region as a possible regulatory factor of Hc, suggesting that compensatory responses as metabolic changes or behavioral tracheal movements to enhance respiratory efficiency could be possible mechanics to compensate for hypoxia. A short Hc-like novel isoform was detected instead in these 23 species, possibly due to either protein degradation or alternative splicing mechanisms, suggesting that the protein could be performing a different function other than oxygen transportation. Hx during hypoxia was expressed and exhibited species-level amino acid changes, highlighting a possible role during hypoxia. Our results demonstrate that hypoxia could enable a similar potential adaptive response of multiple species regarding specific physiological requirements, thereby shedding light on community behavior in stress environments that may help us to improve conservation practices and biomonitoring.

A predictable conserved DNA base composition signature defines human core DNA replication origins

DNA replication initiates from multiple genomic locations called replication origins. In metazoa, DNA sequence elements involved in origin specification remain elusive. Here, we examine pluripotent, primary, differentiating, and immortalized human cells, and demonstrate that a class of origins, termed core origins, is shared by different cell types and host ~80% of all DNA replication initiation events in any cell population. We detect a shared G-rich DNA sequence signature that coincides with most core origins in both human and mouse genomes. Transcription and G-rich elements can independently associate with replication origin activity. Computational algorithms show that core origins can be predicted, based solely on DNA sequence patterns but not on consensus motifs. Our results demonstrate that, despite an attributed stochasticity, core origins are chosen from a limited pool of genomic regions. Immortalization through oncogenic gene expression, but not normal cellular differentiation, results in increased stochastic firing from heterochromatin and decreased origin density at TAD borders.

PKR kinase directly regulates tau expression and Alzheimer's disease-related tau phosphorylation.

Deposition of extensively hyperphosphorylated tau in specific brain cells is a clear pathological hallmark in Alzheimer's disease and a number of other neurodegenerative disorders, collectively termed the tauopathies. Furthermore, hyperphosphorylation of tau prevents it from fulfilling its physiological role as a microtubule‐stabilizing protein and leaves it increasingly vulnerable to self‐assembly, suggestive of a central underlying role of hyperphosphorylation as a contributing factor in the etiology of these diseases. Via in vitro phosphorylation and regulation of kinase activity within cells and acute brain tissue, we reveal that the inflammation associated kinase, protein kinase R (PKR), directly phosphorylates numerous abnormal and disease‐modifying residues within tau including Thr181, Ser199/202, Thr231, Ser262, Ser396, Ser404 and Ser409. Similar to disease processes, these PKR‐mediated phosphorylations actively displace tau from microtubules in cells. In addition, PKR overexpression and knockdown, respectively, increase and decrease tau protein and mRNA levels in cells. This regulation occurs independent of noncoding transcriptional elements, suggesting an underlying mechanism involving intra‐exonic regulation of the tau‐encoding microtubule‐associated protein tau (MAPT) gene. Finally, acute encephalopathy in wild type mice, induced by intracranial Langat virus infection, results in robust inflammation and PKR upregulation accompanied by abnormally phosphorylated full‐length‐ and truncated tau. These findings indicate that PKR, independent of other kinases and upon acute brain inflammation, is capable of triggering pathological modulation of tau, which, in turn, might form the initial pathologic seed in several tauopathies such as Alzheimer's disease and Chronic traumatic encephalopathy where inflammation is severe.

Early effect of laser irradiation in signaling pathways of diabetic rat submandibular salivary glands

Low-power laser irradiation (LPLI) is clinically used to modulate inflammation, proliferation and apoptosis. However, its molecular mechanisms are still not fully understood. This study aimed to describe the effects of LPLI upon inflammatory, apoptotic and proliferation markers in submandibular salivary glands (SMGs) in an experimental model of chronic disorder, 24h after one time irradiation. Diabetes was induced in rats by the injection of streptozotocin. After 29 days, these animals were treated with LPLI in the SMG area, and euthanized 24h after this irradiation. Treatment with LPLI significantly decreased diabetes-induced high mobility group box 1 (HMGB1) and tumor necrosis factor alpha (TNF-α) expression, while enhancing the activation of the transcriptional factor cAMP response element binding (CREB) protein. LPLI also reduced the expression of bax, a mitochondrial apoptotic marker, favoring the cell survival. These findings suggest that LPLI can hamper the state of chronic inflammation and favor homeostasis in diabetic rats SMGs.

Molecular biology and structure of a novel penaeid shrimp densovirus elucidate convergent parvoviral host capsid evolution

The giant tiger prawn (Penaeus monodon) is a decapod crustacean widely reared for human consumption. Currently, viruses of two distinct lineages of parvoviruses (PVs, family Parvoviridae; subfamily Hamaparvovirinae) infect penaeid shrimp. Here, a PV was isolated and cloned from Vietnamese P. monodon specimens, designated Penaeus monodon metallodensovirus (PmMDV). This is the first member of a third divergent lineage shown to infect penaeid decapods. PmMDV has a transcription strategy unique among invertebrate PVs, using extensive alternative splicing and incorporating transcription elements characteristic of vertebrate-infecting PVs. The PmMDV proteins have no significant sequence similarity with other PVs, except for an SF3 helicase domain in its nonstructural protein. Its capsid structure, determined by cryoelectron microscopy to 3-Å resolution, has a similar surface morphology to Penaeus stylirostris densovirus, despite the lack of significant capsid viral protein (VP) sequence similarity. Unlike other PVs, PmMDV folds its VP without incorporating a βA strand and displayed unique multimer interactions, including the incorporation of a Ca2+ cation, attaching the N termini under the icosahedral fivefold symmetry axis, and forming a basket-like pentamer helix bundle. While the PmMDV VP sequence lacks a canonical phospholipase A2 domain, the structure of an EDTA-treated capsid, determined to 2.8-Å resolution, suggests an alternative membrane-penetrating cation-dependent mechanism in its N-terminal region. PmMDV is an observed example of convergent evolution among invertebrate PVs with respect to host-driven capsid structure and unique as a PV showing a cation-sensitive/dependent basket structure for an alternative endosomal egress.

Combinatorial ETS1-dependent control of oncogenic NOTCH1 enhancers in T-cell leukemia.

Notch activation is highly prevalent among cancers, in particular T-cell acute lymphoblastic leukemia (T-ALL). However, the use of pan-Notch inhibitors to treat cancers has been hampered by adverse effects, particularly intestinal toxicities. To circumvent this barrier in T-ALL, we aimed to inhibit ETS1, a developmentally important T-cell transcription factor previously shown to co-bind Notch response elements. Using complementary genetic approaches in mouse models, we show that ablation of Ets1 leads to strong Notch-mediated suppressive effects on T-cell development and leukemogenesis, but milder intestinal effects than pan-Notch inhibitors. Mechanistically, genome-wide chromatin profiling studies demonstrate that Ets1 inactivation impairs recruitment of multiple Notch-associated factors and Notch-dependent activation of transcriptional elements controlling major Notch-driven oncogenic effector pathways. These results uncover previously unrecognized hierarchical heterogeneity of Notch-controlled genes and points to Ets1-mediated enucleation of Notch-Rbpj transcriptional complexes as a target for developing specific anti-Notch therapies in T-ALL that circumvent the barriers of pan-Notch inhibition.

Ureidothiophene inhibits interaction of bacterial RNA polymerase with -10 promotor element.

Bacterial RNA polymerase is a potent target for antibiotics, which utilize a plethora of different modes of action, some of which are still not fully understood. Ureidothiophene (Urd) was found in a screen of a library of chemical compounds for ability to inhibit bacterial transcription. The mechanism of Urd action is not known. Here, we show that Urd inhibits transcription at the early stage of closed complex formation by blocking interaction of RNA polymerase with the promoter –10 element, while not affecting interactions with –35 element or steps of transcription after promoter closed complex formation. We show that mutation in the region 1.2 of initiation factor σ decreases sensitivity to Urd. The results suggest that Urd may directly target σ region 1.2, which allosterically controls the recognition of –10 element by σ region 2. Alternatively, Urd may block conformational changes of the holoenzyme required for engagement with –10 promoter element, although by a mechanism distinct from that of antibiotic fidaxomycin (lipiarmycin). The results suggest a new mode of transcription inhibition involving the regulatory domain of σ subunit, and potentially pinpoint a novel target for development of new antibacterials.

Prediction and Recognition of Gram-Negative Bacterial Promoter Sequences: An Analysis of Available Web Tools

Many computational methods aim to improve the prediction and recognition of transcription elements in prokaryotes. Despite this, the natural features of those elements make their prediction and recognition remain as an open field of research. In this paper, we compared the open-access tools BacPP, BPROM, bTSSfinder, CNNPromoter_b, iPro70-PseZNC, NNPP2, PePPer, and PromPredict. First, we listed the overall functionalities of each tool and the resources available on their web pages. Later, we carried out a comparison of prediction results using 206 intergenic regions. When evaluating the prediction using intergenic regions containing a single promoter within each, NNPP2 and BacPP obtained >90% correct predictions, with NNPP2 obtaining the highest values of match between predicted promoter location and location indicated by RegulonDB. Overall, many discrepancies were observed among the results. They may be explained by the differences in the methodologies that each tool applies for promoter prediction, not excluding the natural features of promoters as a factor as well. In any case, the results highlight the necessity to continue the efforts to improve promoter prediction, perhaps combining multiple approaches. Through said efforts, some of the challenges of the postgenomic era may be tackled as well.

A Viral Toolbox of Genetically Encoded Fluorescent Synaptic Tags.

SummaryFibronectin intrabodies generated with mRNA display (FingRs) are a recently developed tool for labeling excitatory or inhibitory synapses, with the benefit of not altering endogenous synaptic protein expression levels or synaptic transmission. Here, we generated a viral vector FingR toolbox that allows for multi-color, neuron-type-specific labeling of excitatory or inhibitory synapses in multiple brain regions. We screened various fluorophores, FingR fusion configurations, and transcriptional control regulations in adeno-associated virus (AAV) and retrovirus vector designs. We report the development of a red FingR variant and demonstrated dual labeling of excitatory and inhibitory synapses in the same cells. Furthermore, we developed cre-inducible FingR AAV variants and demonstrated their utility, finding that the density of inhibitory synapses in aspiny striatal cholinergic interneurons remained unchanged in response to dopamine depletion. Finally, we generated FingR retroviral vectors, which enabled us to track the development of excitatory and inhibitory synapses in hippocampal adult-born granule cells.

Impact of oxygen availability on heterologous geneexpression and polypeptide secretion dynamics in Yarrowia lipolytica-based protein production platforms.

Industrially relevant traits of Yarrowia lipolytica, like high growth rate, capacity to grow at high cell density or to synthesize biomolecules with high productivities, strongly rely on sufficient oxygen provision. Although the impact of oxygen availability (OA) on the physiology of Y. lipolytica has been already studied, its influence on recombinant protein (rProt) synthesis and secretion has been largely neglected to date. With the aim to fill this gap, a fluorescent reporter protein (yellow fluorescent protein [YFP]) was used herein as a proxy to follow simultaneously rProt synthesis and secretion in Y. lipolytica under different OAs. This study covers the analysis of the reporter gene expression through reverse transcription quantitative polymerase chain reaction, polypeptide synthesis and its retention-to-secretion ratio using flow cytometry and fluorymetry during shake flasks and bioreactor cultivations under different OA. The results gathered demonstrate that OA has a dramatic impact on the kinetics of intracellular and extracellular YFP accumulation. Higher rProt production and secretion were favoured under high OA, and were largely related to OA and not to cell growth. Our observations also suggest the existence of some upper limit of secretory protein accumulation inside the cells above which massive secretion is initiated. Moreover, at low OA, the first bottleneck in rProt synthesis occurs as early as at transcription level, which could results from a lower availability of transcriptional machinery elements. Finally, using flow cytometry and bioreactor cultivations, we highlighted that ovoid cells are generally more efficient in terms of rProt synthesis.

Rapid and Efficient Synthetic Assembly of Multiplex Luciferase Reporter Plasmids for the Simultaneous Monitoring of Up to Six Cellular Signaling Pathways.

High-throughput cell-based screening assays are valuable tools in the discovery of chemical probes and therapeutic agents. Such assays are designed to examine the effects of small compounds on targets, pathways, or phenotypes participating in normal and disease processes. While most cell-based assays measure single quantities, multiplexed assays seek to address these limitations by obtaining multiple simultaneous measurements. The signals from such measurements should be independently detectable and cover large dynamic ranges. Luciferases are good candidates for generation of such signals. They are genetically encoded, versatile, and cost-effective, and their output signals can be sensitively detected. We recently developed a multiplex luciferase assay that allows monitoring the activity of five experimental pathways against one control simultaneously. We used synthetic assembly cloning to assemble all six luciferase reporter units into a single vector over eight stitching rounds. Because all six reporters are on a single piece of DNA, a single vector ensures stoichiometric ratios of each transcriptional unit in each transfected cell, resulting in lower experimental variation. Our proof-of-concept multiplex hextuple luciferase assay was designed to simultaneously monitor the p53, TGF-β, NF-κβ, c-Myc, and MAPK/JNK signaling pathways. The same synthetic assembly cloning pipeline allows the stitching of numerous other cellular pathway luciferase reporters. Here we present an improved three-step synthetic assembly protocol to quickly and efficiently generate multiplex hextuple luciferase reporter plasmids for other signaling pathways of interest. This improved assembly protocol provides the opportunity to analyze any five desired pathways at once much more quickly. Protocols are provided on how to prepare DNA components and destination vector plasmids, design synthetic DNA, perform assembly cloning of new transcriptional reporter elements, implement multipartite synthetic assembly cloning of single-pathway luciferase reporters, and carry out one-step assembly of final multiplex hextuple luciferase vectors. We present protocols on how to perform multiplex hextuple luciferase in an accompanying Current Protocols in Molecular Biology article. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of DNA parts and destination vectors for synthetic assembly cloning Basic Protocol 2: DNA synthesis and assembly cloning of a typical transcriptional reporter element Alternate Protocol: DNA synthesis and assembly cloning of a challenging transcriptional reporter element Basic Protocol 3: Multipartite synthetic assembly cloning of individual pathway luciferase reporters Basic Protocol 4: One step assembly into final multiplex hextuple luciferase vectors Support Protocol: Generation of home-made chemocompetent E. coli DH10B-T1R cells.

Simultaneous Examination of Cellular Pathways using Multiplex Hextuple Luciferase Assaying.

Multiplex experimentation that can assay multiple cellular signaling pathways in the same cells requires orthogonal genetically encoded reporters that report over large dynamic ranges. Luciferases are cost-effective, versatile candidates whose output signals can be sensitively detected in a multiplex fashion. Commonly used dual luciferase reporter assays detect one luciferase that is coupled to a single cellular pathway and a second that is coupled to a control pathway for normalization purposes. We have expanded this approach to multiplex hextuple luciferase assays that can report on five cellular signaling pathways and one control, each of which is encoded by a unique luciferase. Light emission by the six luciferases can be distinguished by the use of two distinct substrates, each specific for three luciferases, followed by spectral decomposition of the light emitted by each of the three luciferase enzymes with bandpass filters. Here, we present detailed protocols on how to perform multiplex hextuple luciferase assaying to monitor pathway fluxes through transcriptional response elements for five specific signaling pathways (i.e., c-Myc, NF-κβ, TGF-β, p53, and MAPK/JNK) using the constitutive CMV promoter as normalization control. Protocols are provided for preparing reporter vector plasmids for multiplex reporter assaying, performing cell culture and multiplex luciferase reporter vector plasmid transfection, executing multiplex luciferase assays, and analyzing and interpreting data obtained by a plate reader appropriately equipped to detect the different luminescences. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of vectors for multiplex hextuple luciferase assaying Basic Protocol 2: Cell culture work for multiplex hextuple luciferase assays Basic Protocol 3: Transfection of luciferase reporter plasmids followed by drug and recombinant protein treatments Basic Protocol 4: Performing the multiplex hextuple luciferase assay.

Fur-Dam Regulatory Interplay at an Internal Promoter of the Enteroaggregative Escherichia coli Type VI Secretion sci1 Gene Cluster.

The type VI secretion system (T6SS) is a weapon for delivering effectors into target cells that is widespread in Gram-negative bacteria. The T6SS is a highly versatile machine, as it can target both eukaryotic and prokaryotic cells, and it has been proposed that T6SSs are adapted to the specific needs of each bacterium. The expression of T6SS gene clusters and the activation of the secretion apparatus are therefore tightly controlled. In enteroaggregative Escherichia coli (EAEC), the sci1 T6SS gene cluster is subject to a complex regulation involving both the ferric uptake regulator (Fur) and DNA adenine methylase (Dam)-dependent DNA methylation. In this study, an additional, internal, promoter was identified within the sci1 gene cluster using +1 transcriptional mapping. Further analyses demonstrated that this internal promoter is controlled by a mechanism strictly identical to that of the main promoter. The Fur binding box overlaps the -10 transcriptional element and a Dam methylation site, GATC-32. Hence, the expression of the distal sci1 genes is repressed and the GATC-32 site is protected from methylation in iron-rich conditions. The Fur-dependent protection of GATC-32 was confirmed by an in vitro methylation assay. In addition, the methylation of GATC-32 negatively impacted Fur binding. The expression of the sci1 internal promoter is therefore controlled by iron availability through Fur regulation, whereas Dam-dependent methylation maintains a stable ON expression in iron-limited conditions.IMPORTANCE Bacteria use weapons to deliver effectors into target cells. One of these weapons, the type VI secretion system (T6SS), assembles a contractile tail acting as a spring to propel a toxin-loaded needle. Its expression and activation therefore need to be tightly regulated. Here, we identified an internal promoter within the sci1 T6SS gene cluster in enteroaggregative E. coli We show that this internal promoter is controlled by Fur and Dam-dependent methylation. We further demonstrate that Fur and Dam compete at the -10 transcriptional element to finely tune the expression of T6SS genes. We propose that this elegant regulatory mechanism allows the optimum production of the T6SS in conditions where enteroaggregative E. coli encounters competing species.

Effect of Transgene Location, Transcriptional Control Elements and Transgene Features in Armed Oncolytic Adenoviruses.

Clinical results with oncolytic adenoviruses (OAds) used as antitumor monotherapies show limited efficacy. To increase OAd potency, transgenes have been inserted into their genome, a strategy known as “arming OAds”. Here, we review different parameters that affect the outcome of armed OAds. Recombinant adenovirus used in gene therapy and vaccination have been the basis for the design of armed OAds. Hence, early region 1 (E1) and early region 3 (E3) have been the most commonly used transgene insertion sites, along with partially or complete E3 deletions. Besides transgene location and orientation, transcriptional control elements, transgene function, either virocentric or immunocentric, and even the codons encoding it, greatly impact on transgene levels and virus fitness.

Characteristics of the Font of Small Seal Character from the Transcription Element

Characteristics of the Font of Small Seal Character from the Transcription Element

Structure, Regulation, and Function of Linear and Circular Long Non-Coding RNAs.

Long non-coding RNAs (lncRNAs), including linear lncRNAs and circular RNAs (circRNAs), exhibit a surprising range of structures. Linear lncRNAs and circRNAs are generated by different pathways. Linear lncRNAs perform functions that depend on their specific sequences, transcription, and DNA elements of their gene loci. In some cases, linear lncRNAs contain a short open reading frame encoding a peptide. circRNAs are covalently closed RNAs with tissue-specific and cell-specific expression patterns that have recently been extensively investigated. Pioneering work focusing on their biogenesis and functional characterization indicates that circRNAs regulate cell development via multiple mechanisms and play critical roles in the immune system. Furthermore, circRNAs in exosomes function on target cells. As with linear lncRNAs, specific circRNAs can also be translated. In this review, we summarize current understanding and highlight the diverse structure, regulation, and function of linear lncRNAs and circRNAs.

Multifaceted effects of soluble human CD6 in experimental cancer models

BackgroundCD6 is a lymphocyte surface co-receptor physically associated with the T-cell receptor (TCR)/CD3 complex at the center of the immunological synapse. There, CD6 assists in cell-to-cell contact stabilization and modulation...

Identification of promoter elements in the Dolichospermum circinale AWQC131C saxitoxin gene cluster and the experimental analysis of their use for heterologous expression

BackgroundDolichospermum circinale is a filamentous bloom-forming cyanobacterium responsible for biosynthesis of the paralytic shellfish toxins (PST), including saxitoxin. PSTs are neurotoxins and in their purified form are important analytical standards for monitoring the quality of water and seafood and biomedical research tools for studying neuronal sodium channels. More recently, PSTs have been recognised for their utility as local anaesthetics. Characterisation of the transcriptional elements within the saxitoxin (sxt) biosynthetic gene cluster (BGC) is a first step towards accessing these molecules for biotechnology.ResultsIn D. circinale AWQC131C the sxt BGC is transcribed from two bidirectional promoter regions encoding five individual promoters. These promoters were identified experimentally using 5′ RACE and their activity assessed via coupling to a lux reporter system in E. coli and Synechocystis sp. PCC 6803. Transcription of the predicted drug/metabolite transporter (DMT) encoded by sxtPER was found to initiate from two promoters, PsxtPER1 and PsxtPER2. In E. coli, strong expression of lux from PsxtP, PsxtD and PsxtPER1 was observed while expression from Porf24 and PsxtPER2 was remarkably weaker. In contrast, heterologous expression in Synechocystis sp. PCC 6803 showed that expression of lux from PsxtP, PsxtPER1, and Porf24 promoters was statistically higher compared to the non-promoter control, while PsxtD showed poor activity under the described conditions.ConclusionsBoth of the heterologous hosts investigated in this study exhibited high expression levels from three of the five sxt promoters. These results indicate that the majority of the native sxt promoters appear active in different heterologous hosts, simplifying initial cloning efforts. Therefore, heterologous expression of the sxt BGC in either E. coli or Synechocystis could be a viable first option for producing PSTs for industrial or biomedical purposes.

Crossroads between transposons and gene regulation.

Transposons are mobile genetic elements that have made a large contribution to genome evolution in a largely species-specific manner. A wide variety of different transposons have invaded genomes throughout evolution, acting in a first instance as 'selfish' elements, whose success was determined by their ability to self-replicate and expand within the host genome. However, their coevolution with the host has created many crossroads between transposons and the regulation of host gene expression. Transposons are an abundant source of transcriptional modulatory elements, such as gene promoters and enhancers, splicing and termination sites, and regulatory non-coding RNAs. Moreover, transposons have driven the evolution of host defence mechanisms that have been repurposed for gene regulation. However, dissecting the potential functional roles of transposons remains challenging owing to their evolutionary path, as well as their repetitive nature, which requires the development of specialized analytical tools. In this special issue, we present a collection of articles that lay out current paradigms in the field and discuss a vision for future research. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.