•Traditional aptamers fold into structures with established nucleic acid motifs.•Modified nucleotides expand the repertoire of known nucleic acid structural motifs.•Hydrophobic modifications make ...essential inter-molecular and intramolecular interactions.•The flexible phosphodiester bond of aptamers allows for high shape complementarity.•In most cases, aptamer binding occurs without a conformational change of the target.
Understanding the structural rules that govern specific, high-affinity binding characteristic of aptamer–protein interactions is important in view of the increasing use of aptamers across many applications. From the modest number of 16 aptamer–protein structures currently available, trends are emerging. The flexible phosphodiester backbone allows folding into precise three-dimensional structures using known nucleic acid motifs as scaffolds that orient specific functional groups for target recognition. Still, completely novel motifs essential for structure and function are found in modified aptamers with diversity-enhancing side chains. Aptamers and antibodies, two classes of macromolecules used as affinity reagents with entirely different backbones and composition, recognize protein epitopes of similar size and with comparably high shape complementarity.
Methods for analyzing chromosome conformation in mammalian cells are either low resolution or low throughput and are technically challenging. In next-generation (NG) Capture-C, we have redesigned the ...Capture-C method to achieve unprecedented levels of sensitivity and reproducibility. NG Capture-C can be used to analyze many genetic loci and samples simultaneously. High-resolution data can be produced with as few as 100,000 cells, and single-nucleotide polymorphisms can be used to generate allele-specific tracks. The method is straightforward to perform and should greatly facilitate the investigation of many questions related to gene regulation as well as the functional dissection of traits examined in genome-wide association studies.
Non-coding mutations at the far end of a large gene desert surrounding the SOX9 gene result in a human craniofacial disorder called Pierre Robin sequence (PRS). Leveraging a human stem cell ...differentiation model, we identify two clusters of enhancers within the PRS-associated region that regulate SOX9 expression during a restricted window of facial progenitor development at distances up to 1.45 Mb. Enhancers within the 1.45 Mb cluster exhibit highly synergistic activity that is dependent on the Coordinator motif. Using mouse models, we demonstrate that PRS phenotypic specificity arises from the convergence of two mechanisms: confinement of Sox9 dosage perturbation to developing facial structures through context-specific enhancer activity and heightened sensitivity of the lower jaw to Sox9 expression reduction. Overall, we characterize the longest-range human enhancers involved in congenital malformations, directly demonstrate that PRS is an enhanceropathy, and illustrate how small changes in gene expression can lead to morphological variation.
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•Extreme long-range enhancer clusters overlap PRS patient mutations at the SOX9 locus•PRS enhancers drive stage-specific SOX9 expression in the cranial neural crest•Mandible development has heightened sensitivity to perturbation of SOX9 gene dosage•Deletion of mouse EC1.45 leads to quantitative changes in mandible morphology
Non-coding mutations over a megabase from SOX9 cause the craniofacial disorder Pierre Robin sequence (PRS). Long et al. leverage a human neural crest model to demonstrate that PRS is caused by loss of extreme long-range enhancers active during a restricted developmental window and explore mechanisms underlying the specificity of disease manifestations.
•National-scale assessment of headwater stream nutrient status, relative to rivers•Greater potential for P limitation in rivers and N limitation in headwater streams•Greater potential for P and N ...co-limitation in headwater streams than rivers•Nutrient impairment of water quality was greatest in Lowland-High-Alkalinity rivers.•Nutrient Limitation Assessment could help inform the prioritisation of remediation.
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This study provides a first national-scale assessment of the nutrient status of British headwater streams within the wider river network, by joint analysis of the national Countryside Survey Headwater Stream and Harmonised River Monitoring Scheme datasets. We apply a novel Nutrient Limitation Assessment methodology to explore the extent to which nutrients may potentially limit primary production in headwater streams and rivers, by coupling ternary assessment of nitrogen (N), phosphorus (P), and carbon (C) depletion, with N:P stoichiometry, and threshold P and N concentrations. P limitation was more commonly seen in the rivers, with greater prevalence of N limitation in the headwater streams. High levels of potential P and N co-limitation were found in the headwater streams, especially the Upland-Low-Alkalinity streams. This suggests that managing both P and N inputs may be needed to minimise risks of degradation of these sensitive headwater stream environments. Although localised nutrient impairment of headwater streams can occur, there were markedly lower rates of P and N impairment of headwater streams relative to downstream rivers at the national scale. Nutrient source contributions, relative to hydrological dilution, increased with catchment scale, corresponding with increases in the extent of agricultural and urban land-use. The estimated nutrient reductions needed to achieve compliance with Water Framework Directive standards, and to reach limiting concentrations, were greatest for the Lowland-High-Alkalinity rivers and streams. Preliminary assessments suggest that reducing P concentrations in the Lowland-High-Alkalinity headwater streams, and N concentrations in the Upland-Low-Alkalinity rivers, might offer greater overall benefits for water-quality remediation at the national scale, relative to the magnitude of nutrient reductions required. This approach could help inform the prioritisation of nutrient remediation, as part of a directional approach to water quality management based on closing the gaps between current and target nutrient concentrations.
The genome is organized via CTCF-cohesin-binding sites, which partition chromosomes into 1-5 megabase (Mb) topologically associated domains (TADs), and further into smaller sub-domains (sub-TADs). ...Here we examined in vivo an ∼80 kb sub-TAD, containing the mouse α-globin gene cluster, lying within a ∼1 Mb TAD. We find that the sub-TAD is flanked by predominantly convergent CTCF-cohesin sites that are ubiquitously bound by CTCF but only interact during erythropoiesis, defining a self-interacting erythroid compartment. Whereas the α-globin regulatory elements normally act solely on promoters downstream of the enhancers, removal of a conserved upstream CTCF-cohesin boundary extends the sub-TAD to adjacent upstream CTCF-cohesin-binding sites. The α-globin enhancers now interact with the flanking chromatin, upregulating expression of genes within this extended sub-TAD. Rather than acting solely as a barrier to chromatin modification, CTCF-cohesin boundaries in this sub-TAD delimit the region of chromatin to which enhancers have access and within which they interact with receptive promoters.
Chromosome conformation capture (3C) methods are central to understanding the link between nuclear structure and function, and the physical interactions between distal regulatory elements and ...promoters. However, no one method is appropriate to address all biological questions, as each variant differs markedly in resolution, reproducibility, throughput and biases. A thorough appreciation of the strengths and weaknesses of each technique is critical when choosing the correct method for a specific application or for gauging how best to interpret different sources of data. In addition, the analysis method must be carefully considered, as this choice can profoundly affect the output. In this Review, we describe and compare the different available 3C-based approaches, with a focus on the analysis of mammalian genomes.
The promoters of mammalian genes are commonly regulated by multiple distal enhancers, which physically interact within discrete chromatin domains. How such domains form and how the regulatory ...elements within them interact in single cells is not understood. To address this we developed Tri-C, a new chromosome conformation capture (3C) approach, to characterize concurrent chromatin interactions at individual alleles. Analysis by Tri-C identifies heterogeneous patterns of single-allele interactions between CTCF boundary elements, indicating that the formation of chromatin domains likely results from a dynamic process. Within these domains, we observe specific higher-order structures that involve simultaneous interactions between multiple enhancers and promoters. Such regulatory hubs provide a structural basis for understanding how multiple cis-regulatory elements act together to establish robust regulation of gene expression.
Selection of aptamers from nucleic acid libraries by in vitro evolution represents a powerful method of identifying high-affinity ligands for a broad range of molecular targets. Nevertheless, a ...sizeable fraction of proteins remain difficult targets due to inherently limited chemical diversity of nucleic acids. We have exploited synthetic nucleotide modifications that confer protein-like diversity on a nucleic acid scaffold, resulting in a new generation of binding reagents called SOMAmers (Slow Off-rate Modified Aptamers). Here we report a unique crystal structure of a SOMAmer bound to its target, platelet-derived growth factor B (PDGF-BB). The SOMAmer folds into a compact structure and exhibits a hydrophobic binding surface that mimics the interface between PDGF-BB and its receptor, contrasting sharply with mainly polar interactions seen in traditional protein-binding aptamers. The modified nucleotides circumvent the intrinsic diversity constraints of natural nucleic acids, thereby greatly expanding the structural vocabulary of nucleic acid ligands and considerably broadening the range of accessible protein targets.
Many genes determining cell identity are regulated by clusters of Mediator-bound enhancer elements collectively referred to as super-enhancers. These super-enhancers have been proposed to manifest ...higher-order properties important in development and disease. Here we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer individually and in informative combinations, we demonstrate that each constituent enhancer seems to act independently and in an additive fashion with respect to hematological phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects. Our study highlights the importance of functional genetic analyses for the identification of new concepts in transcriptional regulation.
The mouse X-inactivation center (Xic) locus represents a powerful model for understanding the links between genome architecture and gene regulation, with the non-coding genes Xist and Tsix showing ...opposite developmental expression patterns while being organized as an overlapping sense/antisense unit. The Xic is organized into two topologically associating domains (TADs) but the role of this architecture in orchestrating cis-regulatory information remains elusive. To explore this, we generated genomic inversions that swap the Xist/Tsix transcriptional unit and place their promoters in each other's TAD. We found that this led to a switch in their expression dynamics: Xist became precociously and ectopically upregulated, both in male and female pluripotent cells, while Tsix expression aberrantly persisted during differentiation. The topological partitioning of the Xic is thus critical to ensure proper developmental timing of X inactivation. Our study illustrates how the genomic architecture of cis-regulatory landscapes can affect the regulation of mammalian developmental processes.
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