The generation of the post-cranial embryonic body relies on the coordinated production of spinal cord neurectoderm and presomitic mesoderm cells from neuromesodermal progenitors (NMPs). This process is orchestrated by pro-neural and pro-mesodermal transcription factors that are co-expressed in NMPs together with Hox genes, which are essential for axial allocation of NMP derivatives. NMPs reside in a posterior growth region, which is marked by the expression of Wnt, FGF and Notch signalling components. Although the importance of Wnt and FGF in influencing the induction and differentiation of NMPs is well established, the precise role of Notch remains unclear. Here, we show that the Wnt/FGF-driven induction of NMPs from human embryonic stem cells (hESCs) relies on Notch signalling. Using hESC-derived NMPs and chick embryo grafting, we demonstrate that Notch directs a pro-mesodermal character at the expense of neural fate. We show that Notch also contributes to activation of HOX gene expression in human NMPs, partly in a non-cell-autonomous manner. Finally, we provide evidence that Notch exerts its effects via the establishment of a negative-feedback loop with FGF signalling.

Expansion microscopy (ExM) is a versatile super-resolution microscopy pipeline, leveraging nanoscale biomolecular crosslinking and osmotically driven swelling of hydrogels. Currently, ExM is a laborious and skill-intensive technique, involving manual handling of the hydrogels that can compromise the integrity of the gels and capacity to track gel isotropy, hence diminishing reproducibility. We have developed a 3D-printable microplate system to contain the entire ExM workflow within each well, enabling in situ image acquisition and eliminating the need for direct handling of the hydrogels. The preservation of the gel geometry and orientation of the microplate wells enables convenient tracking of gel expansion, pre-and post-ExM image acquisition, and distortion mapping of every cell or region of interest. We demonstrate the utility of this approach with both single-color and multiplexed ExM of cultured HeLa cells and dissected pupal Drosophila melanogasterwing tissue to reveal distortion-prone structures ranging from sub-cellular organelles to micron-scale tissue regions.

The authors declare no conflicts of interest.

ABSTRACT Enhancers confer precise spatiotemporal patterns of gene expression in response to developmental and environmental stimuli. Over the last decade, the transcription of enhancer RNAs (eRNAs) – nascent RNAs transcribed from active enhancers – has emerged as a key factor regulating enhancer activity. eRNAs are relatively short-lived RNA species that are transcribed at very high rates but also quickly degraded. Nevertheless, eRNAs are deeply intertwined within enhancer regulatory networks and are implicated in a number of transcriptional control mechanisms. Enhancers show changes in function and sequence over evolutionary time, raising questions about the relationship between enhancer sequences and eRNA function. Moreover, the vast majority of single nucleotide polymorphisms associated with human complex diseases map to the non-coding genome, with causal disease variants enriched within enhancers. In this Primer, we survey the diverse roles played by eRNAs in enhancer-dependent gene expression, evaluating different models for eRNA function. We also explore questions surrounding the genetic conservation of enhancers and how this relates to eRNA function and dysfunction.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease. CAV1 and CAV2 organize membrane lipid rafts (MLRs) important for cell signaling and neuronal survival, and overexpression of CAV1 ameliorates ALS phenotypes in vivo. Genome-wide association studies localize a large proportion of ALS risk variants within the non-coding genome, but further characterization has been limited by lack of appropriate tools. By designing and applying a pipeline to identify pathogenic genetic variation within enhancer elements responsible for regulating gene expression, we identify disease-associated variation within CAV1/CAV2 enhancers, which replicate in an independent cohort. Discovered enhancer mutations reduce CAV1/CAV2 expression and disrupt MLRs in patient-derived cells, and CRISPR-Cas9 perturbation proximate to a patient mutation is sufficient to reduce CAV1/CAV2 expression in neurons. Additional enrichment of ALS-associated mutations within CAV1 exons positions CAV1 as an ALS risk gene. We propose CAV1/CAV2 overexpression as a personalized medicine target for ALS.

The master tumor suppressor p53 controls transcription of a wide-ranging gene network involved in apoptosis, cell cycle arrest, DNA damage repair, and senescence. Recent studies revealed pervasive binding of p53 to cis-regulatory elements (CREs), which are non-coding segments of DNA that spatially and temporally control transcription through the combinatorial binding of local transcription factors. Although the role of p53 as a strong trans-activator of gene expression is well known, the co-regulatory factors and local sequences acting at p53-bound CREs are comparatively understudied. We designed and executed a massively parallel reporter assay (MPRA) to investigate the effect of transcription factor binding motifs and local sequence context on p53-bound CRE activity. Our data indicate that p53-bound CREs are both positively and negatively affected by alterations in local sequence context and changes to co-regulatory TF motifs. Our data suggest p53 has the flexibility to cooperate with a variety of transcription factors in order to regulate CRE activity. By utilizing different sets of co-factors across CREs, we hypothesize that global p53 activity is guarded against loss of any one regulatory partner, allowing for dynamic and redundant control of p53-mediated transcription.

The phosphatidylinositol 3-kinase-related protein kinases (PIKKs) are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1-Ddc2 complex (ATM and ATR-ATRIP in humans) play pivotal roles in DNA replication, DNA damage signalling and repair. Here, we present the first structural insight for dimers of Mec1-Ddc2 and Tel1 using single particle electron microscopy. Both kinases reveal a head-to-head dimer with one major dimeric interface through their N-terminal HEAT repeats. Their dimeric interface is significantly distinct from the interface of mTOR Complex 1 dimer, which oligomerises through two spatially separate interfaces. We also observe different structural organisation of kinase domains of Mec1 and Tel1. The kinase domains in the Mec1-Ddc2 dimer are located in close proximity to each other. However, in the Tel1 dimer they are fully separated providing potential access of substrates to this kinase, even in its dimeric form.

The phosphatidylinositol 3-kinase-related protein kinases are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1·Ddc2 complex (ATM and ATR-ATRIP in humans) play pivotal roles in DNA replication, DNA damage signaling, and repair. Here, we present the first structural insight for dimers of Mec1·Ddc2 and Tel1 using single-particle electron microscopy. Both kinases reveal a head to head dimer with one major dimeric interface through the N-terminal HEAT (named after Huntingtin, elongation factor 3, protein phosphatase 2A, and yeast kinase TOR1) repeat. Their dimeric interface is significantly distinct from the interface of mTOR complex 1 dimer, which oligomerizes through two spatially separate interfaces. We also observe different structural organizations of kinase domains of Mec1 and Tel1. The kinase domains in the Mec1·Ddc2 dimer are located in close proximity to each other. However, in the Tel1 dimer they are fully separated, providing potential access of substrates to this kinase, even in its dimeric form.

Actin-related protein Arp8 is a component of the INO80 chromatin remodeling complex. Yeast Arp8 (yArp8) comprises two domains: a 25-KDa N-terminal domain, found only in yeast, and a 75-KDa C-terminal domain (yArp8CTD) that contains the actin fold and is conserved across other species. The crystal structure shows that yArp8CTD contains three insertions within the actin core. Using a combination of biochemistry and EM, we show that Arp8 forms a complex with nucleosomes, and that the principal interactions are via the H3 and H4 histones, mediated through one of the yArp8 insertions. We show that recombinant yArp8 exists in monomeric and dimeric states, but the dimer is the biologically relevant form required for stable interactions with histones that exploits the twofold symmetry of the nucleosome core. Taken together, these data provide unique insight into the stoichiometry, architecture, and molecular interactions between components of the INO80 remodeling complex and nucleosomes, providing a first step toward building up the structure of the complex.

Transcription, the synthesis of RNA from a DNA template, is performed by multisubunit RNA polymerases (RNAPs) in all cellular organisms. The bridge helix (BH) is a distinct feature of all multisubunit RNAPs and makes direct interactions with several active site-associated mobile features implicated in the nucleotide addition cycle and RNA and DNA binding. Because the BH has been captured in both kinked and straight conformations in different crystals structures of RNAP, recently supported by molecular dynamics studies, it has been proposed that cycling between these conformations is an integral part of the nucleotide addition cycle. To further evaluate the role of the BH, we conducted systematic alanine scanning mutagenesis of the Escherichia coli RNAP BH to determine its contributions to activities required for transcription. Combining our data with an atomic model of E. coli RNAP, we suggest that alterations in the interactions between the BH and (i) the trigger loop, (ii) fork loop 2, and (iii) switch 2 can help explain the observed changes in RNAP functionality associated with some of the BH variants. Additionally, we show that extensive defects in E. coli RNAP functionality depend upon a single previously not studied lysine residue (Lys-781) that is strictly conserved in all bacteria. It appears that direct interactions made by the BH with other conserved features of RNAP are lost in some of the E. coli alanine substitution variants, which we infer results in conformational changes in RNAP that modify RNAP functionality.

Spt4/5 in archaea and eukaryote and its bacterial homolog NusG is the only elongation factor conserved in all three domains of life and plays many key roles in cotranscriptional regulation and in recruiting other factors to the elongating RNA polymerase. Here, we present the crystal structure of Spt4/5 as well as the structure of RNA polymerase-Spt4/5 complex using cryoelectron microscopy reconstruction and single particle analysis. The Spt4/5 binds in the middle of RNA polymerase claw and encloses the DNA, reminiscent of the DNA polymerase clamp and ring helicases. The transcription elongation complex model reveals that the Spt4/5 is an upstream DNA holder and contacts the nontemplate DNA in the transcription bubble. These structures reveal that the cellular RNA polymerases also use a strategy of encircling DNA to enhance its processivity as commonly observed for many nucleic acid processing enzymes including DNA polymerases and helicases.

Foot-and-mouth disease virus (FMDV), a positive sense, single-stranded RNA virus, causes a highly contagious disease in cloven-hoofed livestock. Like other picornaviruses, FMDV has a conserved 2C protein assigned to the superfamily 3 helicases, a group of AAA+ ATPases that has a predicted N-terminal membrane-binding amphipathic helix attached to the main ATPase domain. In infected cells, 2C is involved in the formation of membrane vesicles, where it co-localizes with viral RNA replication complexes, but its precise role in virus replication has not been elucidated. We show here that deletion of the predicted N-terminal amphipathic helix enables overexpression in Escherichia coli of a highly soluble truncated protein, 2C(34-318), that has ATPase and RNA binding activity. ATPase activity was abrogated by point mutations in the Walker A (K116A) and B (D160A) motifs and Motif C (N207A) in the active site. Unliganded 2C(34-318) exhibits concentration-dependent self-association to yield oligomeric forms, the largest of which is tetrameric. Strikingly, in the presence of ATP and RNA, FMDV 2C(34-318) containing the N207A mutation, which binds but does not hydrolyze ATP, was found to oligomerize specifically into hexamers. Visualization of FMDV 2C-ATP-RNA complexes by negative stain electron microscopy revealed hexameric ring structures with 6-fold symmetry that are characteristic of AAA+ ATPases. ATPase assays performed by mixing purified active and inactive 2C(34-318) subunits revealed a coordinated mechanism of ATP hydrolysis. Our results provide new insights into the structure and mechanism of picornavirus 2C proteins that will facilitate new investigations of their roles in infection.

Gene transcription is a fundamental cellular process carried out by RNA polymerase (RNAP) enzymes and is highly regulated through the action of gene regulatory complexes. Important mechanistic insights have been gained from structural studies on multisubunit RNAP from bacteria, yeast and archaea, although the initiation process that involves the conversion of the inactive transcription complex to an active one has yet to be fully understood. RNAPs are unambiguously closely related in structure and function across all kingdoms of life and have conserved mechanisms. In bacteria, sigma (sigma) factors direct RNAP to specific promoter sites and the RNAP/sigma holoenzyme can either form a stable closed complex that is incompetent for transcription (as in the case of sigma(54)) or can spontaneously proceed to an open complex that is competent for transcription (as in the case of sigma(70)). The conversion of the RNAP/sigma(54) closed complex to an open complex requires ATP hydrolysis by enhancer-binding proteins, hence providing an ideal model system for studying the initiation process biochemically and structurally. In this review, we present recent structural studies of the two major bacterial RNAP holoenzymes and focus on mechanistic advances in the transcription initiation process via enhancer-binding proteins.

Transcription initiation involves the conversion from closed promoter complexes, comprising RNA polymerase (RNAP) and double-stranded promoter DNA, to open complexes, in which the enzyme is able to access the DNA template in a single-stranded form. The complex between bacterial RNAP and its major variant sigma factor sigma(54) remains as a closed complex until ATP hydrolysis-dependent remodeling by activator proteins occurs. This remodeling facilitates DNA melting and allows the transition to the open complex. Here we present cryoelectron microscopy reconstructions of bacterial RNAP in complex with sigma(54) alone, and of RNAP-sigma(54) with an AAA+ activator. Together with photo-crosslinking data that establish the location of promoter DNA within the complexes, we explain why the RNAP-sigma(54) closed complex is unable to access the DNA template and propose how the structural changes induced by activator binding can initiate conformational changes that ultimately result in formation of the open complex.

bEBPs (bacterial enhancer-binding proteins) are AAA+ (ATPase associated with various cellular activities) transcription activators that activate gene transcription through a specific bacterial sigma factor, sigma(54). Sigma(54)-RNAP (RNA polymerase) binds to promoter DNA sites and forms a stable closed complex, unable to proceed to transcription. The closed complex must be remodelled using energy from ATP hydrolysis provided by bEBPs to melt DNA and initiate transcription. Recently, large amounts of structural and biochemical data have produced insights into how ATP hydrolysis within the active site of bEBPs is coupled to the re-modelling of the closed complex. In the present article, we review some of the key nucleotides, mutations and techniques used and how they have contributed towards our understanding of the function of bEBPs.

Bacterial sigma (sigma) factors confer gene specificity upon the RNA polymerase, the central enzyme that catalyses gene transcription. The binding of the alternative sigma factor sigma(54) confers upon the RNA polymerase special functional and regulatory properties, making it suited for control of several major adaptive responses. Here, we summarize our current understanding of the interactions the sigma(54) factor makes with the bacterial transcription machinery.

Bacterial transcription relies on the binding of dissociable sigma (sigma) factors to RNA polymerase (RNAP) for promoter specificity. The major variant sigma factor (sigma54) forms a stable closed complex with RNAP bound to DNA that rarely spontaneously isomerises to an open complex. ATP hydrolysis by bacterial enhancer-binding proteins is used to remodel the RNAP-sigma54-DNA closed complex. Recently, a wealth of structural information on bacterial enhancer-binding proteins has enabled unprecedented insights into their mechanism. These data provide a structural basis for nucleotide binding and hydrolysis, oligomerisation and the conversion of ATPase activity into remodelling events within the RNAP-sigma54 closed complex, and represent advances towards a complete understanding of the sigma54-dependent transcription activation mechanism.