Motif Positions Research Articles

ACVPred: Enhanced prediction of anti-coronavirus peptides by transfer learning combined with data augmentation

Anti-coronavirus peptides (ACVPs) have garnered significant attention in COVID-19 therapeutic research due to their precise targeting, low risk of drug resistance, flexible synthesis, and effectiveness against viral mutations. Although some in-silico methods have been developed to predict ACVPs, they suffer from challenges such as limited datasets and a lack of interpretability. Hence, this study introduces ACVPred, an algorithm for ACVP prediction, based on two few-shot learning strategies: transfer learning and data augmentation strategies. Our experiments demonstrate that data augmentation can significantly enhance model performance, while transfer learning can effectively prevent overfitting and strengthen generalizability. Compared to existing methods, ACVPred exhibits superior performance and robust generalization both in training and independent test datasets. Moreover, the interpretability study of the model reveals that its transformer-based core can effectively capture key motifs on ACVP sequences, demonstrating strong feature learning capabilities. Additionally, the findings suggest that the sequence feature weights and key motif positions tend to be distributed towards the N-terminal end of ACVP sequences, providing vital clues for the design of ACVPs. In summary, ACVPred is not only a practical and valuable tool for aiding in the design of ACVPs, but its algorithmic concept also serves as an important reference for research on other small sample prediction problems.

Response of Rhododendron simsii and Rhododendron delavayi Superoxide Dismutase Family Genes to High-Temperature Stress

Superoxide dismutases (SODs) are the first line of defense in the antioxidant defense system, and they play an essential role in various adversity stress adaptations in Rhododendron. In this study, 9 Rhododendron simsii SODs (RsSODs) and 11 Rhododendron delavayi SODs (RdSODs) genes were identified in the genomes of R. simsii and R. delavayi. Phylogenetic relationship analysis classified SOD proteins from two Rhododendron species and other related species into three subfamilies. The results of gene structure and conserved motif analysis show that SOD proteins are strongly evolutionarily conserved, and SODs of the same subfamily have similar motif distributions, positions, and lengths. Twenty-two light-responsive elements, eight phytohormone regulatory elements, five adversity stress-related elements, and three growth and development regulatory elements were detected in the RsSOD and RdSOD promoters. Quantitative real-time fluorescence polymerase chain reaction analysis showed that among the 20 candidate genes, except for RdCSD5, the other SODs were expressed in at least one of four tissues, and all of these gene family members had high expression levels in the leaves. We then investigated the response of the RsSOD and RdSOD gene families to high-temperature stress in combination with the following specific stressors: abscisic acid, ethephon, and hydrogen peroxide treatments, followed by high-temperature stress. Different degrees of upregulated expression of the detected SOD gene family members were found for exogenous reagent treatments and different times of high-temperature stress. Thus, we provide a basis for the further functional characterization of SOD genes in R. simsii and R. delavayi in the future.

Enhancers display constrained sequence flexibility and context-specific modulation of motif function.

The information about when and where each gene is to be expressed is mainly encoded in the DNA sequence of enhancers, sequence elements that comprise binding sites (motifs) for different transcription factors (TFs). Most of the research on enhancer sequences has been focused on TF motif presence, whereas the enhancer syntax, that is, the flexibility of important motif positions and how the sequence context modulates the activity of TF motifs, remains poorly understood. Here, we explore the rules of enhancer syntax by a two-pronged approach in Drosophila melanogaster S2 cells: we (1) replace important TF motifs by all possible 65,536 eight-nucleotide-long sequences and (2) paste eight important TF motif types into 763 positions within 496 enhancers. These complementary strategies reveal that enhancers display constrained sequence flexibility and the context-specific modulation of motif function. Important motifs can be functionally replaced by hundreds of sequences constituting several distinct motif types, but these are only a fraction of all possible sequences and motif types. Moreover, TF motifs contribute with different intrinsic strengths that are strongly modulated by the enhancer sequence context (the flanking sequence, the presence and diversity of other motif types, and the distance between motifs), such that not all motif types can work in all positions. The context-specific modulation of motif function is also a hallmark of human enhancers, as we demonstrate experimentally. Overall, these two general principles of enhancer sequences are important to understand and predict enhancer function during development, evolution, and in disease.

Electrical DNA Sequence Mapping Using Oligodeoxynucleotide Labels and Nanopores.

Identifying DNA species is crucial for diagnostics. For DNA identification, single-molecule DNA sequence mapping is an alternative to DNA sequencing toward fast point-of-care testing, which traditionally relies on targeting and labeling DNA sequences with fluorescent labels and readout using optical imaging methods. A nanopore is a promising sensor as a complement to optical mapping with advantages of electric measurement suitable for portable devices and potential for high resolution. Here, we demonstrate a high-resolution nanopore-based DNA sequence mapping by labeling specific short sequence motifs with oligodeoxynucleotides (ODNs) using DNA methyltransferase (MTase) and detecting them using nanopores. We successfully detected ODNs down to the size of 11 nucleotides without introducing extra reporters and resolved neighboring sites with a distance of 141 bp (∼48 nm) on a single DNA molecule. To accurately locate the sequence motif positions on DNA, a nanopore data analysis method is proposed by considering DNA velocity change through nanopores and using ensemble statistics to translate the time-dependent signals to the location information. Our platform enables high-resolution detection of small labels on DNA and high-accuracy localization of them for DNA species identification in an all-electrical format. The method presents an alternative to optical techniques relying on fluorescent labels and is promising for miniature-scale integration for diagnostic applications.

Predicting and clustering plant CLE genes with a new method developed specifically for short amino acid sequences

BackgroundThe CLV3/ESR-RELATED (CLE) gene family encodes small secreted peptides (SSPs) and plays vital roles in plant growth and development by promoting cell-to-cell communication. The prediction and classification of CLE genes is challenging because of their low sequence similarity.ResultsWe developed a machine learning-aided method for predicting CLE genes by using a CLE motif-specific residual score matrix and a novel clustering method based on the Euclidean distance of 12 amino acid residues from the CLE motif in a site-weight dependent manner. In total, 2156 CLE candidates—including 627 novel candidates—were predicted from 69 plant species. The results from our CLE motif-based clustering are consistent with previous reports using the entire pre-propeptide. Characterization of CLE candidates provided systematic statistics on protein lengths, signal peptides, relative motif positions, amino acid compositions of different parts of the CLE precursor proteins, and decisive factors of CLE prediction. The approach taken here provides information on the evolution of the CLE gene family and provides evidence that the CLE and IDA/IDL genes share a common ancestor.ConclusionsOur new approach is applicable to SSPs or other proteins with short conserved domains and hence, provides a useful tool for gene prediction, classification and evolutionary analysis.

Engaging the nucleosome

Structural Biology Cell identity is defined by gene expression patterns that are established through the binding of specific transcription factors. However, nucleosomal units limit access of transcription factors to specific DNA motifs within the mammalian genome. To study how transcription factors bind such chromatinized, nucleosome-embedded motifs, Michael et al. focused on the pluripotency factors OCT4 and SOX2. They systematically quantified the relative affinities of these factors at different motif positions throughout the nucleosome, enabling structure determination of OCT4-SOX2–bound nucleosomes by cryo–electron microscopy. OCT4 and SOX2 bound cooperatively to strengthen DNA-binding affinity and resulted in DNA distortions that destabilized the nucleosome. This analysis reveals position-dependent binding modes that were validated in vivo, providing insights on how transcription factors read out chromatinized motifs. Science , this issue p. [1460][1] [1]: /lookup/doi/10.1126/science.abb0074

Mechanisms of OCT4-SOX2 motif readout on nucleosomes.

Transcription factors (TFs) regulate gene expression through chromatin where nucleosomes restrict DNA access. To study how TFs bind nucleosome-occupied motifs, we focused on the reprogramming factors OCT4 and SOX2 in mouse embryonic stem cells. We determined TF engagement throughout a nucleosome at base-pair resolution in vitro, enabling structure determination by cryo-electron microscopy at two preferred positions. Depending on motif location, OCT4 and SOX2 differentially distort nucleosomal DNA. At one position, OCT4-SOX2 removes DNA from histone H2A and histone H3; however, at an inverted motif, the TFs only induce local DNA distortions. OCT4 uses one of its two DNA-binding domains to engage DNA in both structures, reading out a partial motif. These findings explain site-specific nucleosome engagement by the pluripotency factors OCT4 and SOX2, and they reveal how TFs distort nucleosomes to access chromatinized motifs.

Nonreciprocal and Conditional Cooperativity Directs the Pioneer Activity of Pluripotency Transcription Factors

SUMMARYCooperative binding of transcription factors (TFs) to chromatin orchestrates gene expression programming and cell fate specification. However, the biophysical principles of TF cooperativity remain incompletely understood. Here we use single-molecule fluorescence microscopy to study the partnership between Sox2 and Oct4, two core members of the pluripotency gene regulatory network. We find that the ability of Sox2 to target DNA inside nucleosomes is strongly affected by the translational and rotational positioning of its binding motif. In contrast, Oct4 can access nucleosomal sites with equal capacities. Furthermore, the Sox2-Oct4 pair displays nonreciprocal cooperativity, with Oct4 modulating interaction of Sox2 with the nucleosome but not vice versa. Such cooperativity is conditional upon the composite motif’s residing at specific nucleosomal locations. These results reveal that pioneer factors possess distinct chromatin-binding properties and suggest that the same set of TFs can differentially regulate gene activities on the basis of their motif positions in the nucleosomal context.

Identification and Expression Analyses of SBP-Box Genes Reveal Their Involvement in Abiotic Stress and Hormone Response in Tea Plant (Camellia sinensis).

The SQUAMOSA promoter binding protein (SBP)-box gene family is a plant-specific transcription factor family. This family plays a crucial role in plant growth and development. In this study, 20 SBP-box genes were identified in the tea plant genome and classified into six groups. The genes in each group shared similar exon-intron structures and motif positions. Expression pattern analyses in five different tissues demonstrated that expression in the buds and leaves was higher than that in other tissues. The cis-elements and expression patterns of the CsSBP genes suggested that the CsSBP genes play active roles in abiotic stress responses; these responses may depend on the abscisic acid (ABA), gibberellic acid (GA), and methyl jasmonate (MeJA) signaling pathways. Our work provides a comprehensive understanding of the CsSBP family and will aid in genetically improving tea plants.

Increasing the brightness of red fluorescent proteins by saturation mutagenesis

Red fluorescent proteins (RFPs) are genetically-encoded fluorophores that are widely used for in vivo imaging. For all applications of RFPs, brighter variants are desired. Previously, we improved the brightness of mRojoA, a red-shifted mutant of the widely-used RFP mCherry, by designing a triple-decker motif of aromatic rings around its chromophore. This yielded the brighter variant mRojo-VHSV, which contains a triple-decker motif consisting of His and Tyr side chains that pack against the chromophore. This improved chromophore packing resulted in an approximately 3-fold brightness increase at physiological pH. However, the His side chain in the triple-decker motif of mRojo-VHSV adopted a perpendicular arrangement to the other two, which may result in a suboptimal packing arrangement. To further improve chromophore packing in mRojo-VHSV, we performed saturation mutagenesis of residues surrounding its triple-decker motif (positions 62, 97, 165, and 199). Using a microplate fluorescence screening assay, a total of 376 colonies were screened for improved brightness. The brightest mutant found, L199M, was expressed and purified, and its spectral properties were characterized in detail. We found that the quantum yield of this variant was improved by two-fold, resulting in a two-fold brightness increase compared to mRojo-VHSV as well as a 5.3-fold increase in brightness compared to mRojoA. The L199M improved variant is the basis for continued engineering with the goal of further improving the spectral properties of this family of RFPs.

Feeding environment and other traits shape species' roles in marine food webs.

Food webs and meso-scale motifs allow us to understand the structure of ecological communities and define species' roles within them. This species-level perspective on networks permits tests for relationships between species' traits and their patterns of direct and indirect interactions. Such relationships could allow us to predict food-web structure based on more easily obtained trait information. Here, we calculated the roles of species (as vectors of motif position frequencies) in six well-resolved marine food webs and identified the motif positions associated with the greatest variation in species' roles. We then tested whether the frequencies of these positions varied with species' traits. Despite the coarse-grained traits we used, our approach identified several strong associations between traits and motifs. Feeding environment was a key trait in our models and may shape species' roles by affecting encounter probabilities. Incorporating environment into future food-web models may improve predictions of an unknown network structure.

Novel structural features drive DNA binding properties of Cmr, a CRP family protein in TB complex mycobacteria.

Mycobacterium tuberculosis (Mtb) encodes two CRP/FNR family transcription factors (TF) that contribute to virulence, Cmr (Rv1675c) and CRPMt (Rv3676). Prior studies identified distinct chromosomal binding profiles for each TF despite their recognizing overlapping DNA motifs. The present study shows that Cmr binding specificity is determined by discriminator nucleotides at motif positions 4 and 13. X-ray crystallography and targeted mutational analyses identified an arginine-rich loop that expands Cmr’s DNA interactions beyond the classical helix-turn-helix contacts common to all CRP/FNR family members and facilitates binding to imperfect DNA sequences. Cmr binding to DNA results in a pronounced asymmetric bending of the DNA and its high level of cooperativity is consistent with DNA-facilitated dimerization. A unique N-terminal extension inserts between the DNA binding and dimerization domains, partially occluding the site where the canonical cAMP binding pocket is found. However, an unstructured region of this N-terminus may help modulate Cmr activity in response to cellular signals. Cmr’s multiple levels of DNA interaction likely enhance its ability to integrate diverse gene regulatory signals, while its novel structural features establish Cmr as an atypical CRP/FNR family member.

A two-helix motif positions the lysophosphatidic acid acyltransferase active site for catalysis within the membrane bilayer.

Phosphatidic acid (PA), the central intermediate in membrane phospholipid synthesis, is generated by two acyltransferases in a pathway conserved in all life forms. The second step in this pathway is catalyzed by 1-acyl-sn-glycerol-3-phosphate acyltransferase, called PlsC in bacteria. Here we present the crystal structure of PlsC from Thermotoga maritima, revealing an unusual hydrophobic/aromatic N-terminal two-helix motif linked to an acyltransferase αβ-domain that contains the catalytic HX4D motif. PlsC dictates the acyl chain composition of the 2-position of phospholipids, and the acyl chain selectivity 'ruler' is an appropriately placed and closed hydrophobic tunnel. We confirmed this by site-directed mutagenesis and membrane composition analysis of Escherichia coli cells that expressed mutant PlsC. Molecular dynamics (MD) simulations showed that the two-helix motif represents a novel substructure that firmly anchors the protein to one leaflet of the membrane. This binding mode allows the PlsC active site to acylate lysophospholipids within the membrane bilayer by using soluble acyl donors.

Individual position diversity in dependence socioeconomic networks increases economic output

The availability of big data recorded from massively multiplayer online role-playing games (MMORPGs) allows us to gain a deeper understanding of the potential connection between individuals’ network positions and their economic outputs. We use a statistical filtering method to construct dependence networks from weighted friendship networks of individuals. We investigate the 30 distinct motif positions in the 13 directed triadic motifs which represent microscopic dependences among individuals. Based on the structural similarity of motif positions, we further classify individuals into different groups. The node position diversity of individuals is found to be positively correlated with their economic outputs. We also find that the economic outputs of leaf nodes are significantly lower than that of the other nodes in the same motif. Our findings shed light on understanding the influence of network structure on economic activities and outputs in socioeconomic systems.

Role of the recognition helix of response regulator WalR from Bacillus anthracis in DNA binding and specificity

WalRK two-component system of Bacillus anthracis potentially regulates multiple genes spanning diverse cellular functions. Its constituent response regulator (RR), WalR belongs to the OmpR/PhoB family which possesses a winged helix-turn-helix motif for DNA binding. An in silico knowledge based model of WalR C-terminal DNA binding domain in complex with its ftsE promoter region binding motif was used to identify specific residues of the recognition helix important for DNA binding. The model was validated by mutagenesis in conjunction with in vitro DNA binding analysis. The ftsE promoter region DNA binding motif was also varied. Optimal binding of WalR to DNA required the presence of both half-sites in its binding motif. Substitution of invariant bases of WalR DNA binding motif abrogated the binding whereas changes at variable motif positions governed affinity. D199 was not in direct contact with the DNA but its substitution modified the WalR-DNA specificity indicating the importance of contact avoidance by this residue for DNA specificity. This represents the first in-depth study of RR-DNA interaction from B. anthracis.

Parametric bootstrapping for biological sequence motifs.

BackgroundBiological sequence motifs drive the specific interactions of proteins and nucleic acids. Accordingly, the effective computational discovery and analysis of such motifs is a central theme in bioinformatics. Many practical questions about the properties of motifs can be recast as random sampling problems. In this light, the task is to determine for a given motif whether a certain feature of interest is statistically unusual among relevantly similar alternatives. Despite the generality of this framework, its use has been frustrated by the difficulties of defining an appropriate reference class of motifs for comparison and of sampling from it effectively.ResultsWe define two distributions over the space of all motifs of given dimension. The first is the maximum entropy distribution subject to mean information content, and the second is the truncated uniform distribution over all motifs having information content within a given interval. We derive exact sampling algorithms for each. As a proof of concept, we employ these sampling methods to analyze a broad collection of prokaryotic and eukaryotic transcription factor binding site motifs. In addition to positional information content, we consider the informational Gini coefficient of the motif, a measure of the degree to which information is evenly distributed throughout a motif’s positions. We find that both prokaryotic and eukaryotic motifs tend to exhibit higher informational Gini coefficients (IGC) than would be expected by chance under either reference distribution. As a second application, we apply maximum entropy sampling to the motif p-value problem and use it to give elementary derivations of two new estimators.ConclusionsDespite the historical centrality of biological sequence motif analysis, this study constitutes to our knowledge the first use of principled null hypotheses for sequence motifs given information content. Through their use, we are able to characterize for the first time differerences in global motif statistics between biological motifs and their null distributions. In particular, we observe that biological sequence motifs show an unusual distribution of IGC, presumably due to biochemical constraints on the mechanisms of direct read-out.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1246-8) contains supplementary material, which is available to authorized users.

Region 4 of Rhizobium etli Primary Sigma Factor (SigA) Confers Transcriptional Laxity in Escherichia coli.

Sigma factors are RNA polymerase subunits engaged in promoter recognition and DNA strand separation during transcription initiation in bacteria. Primary sigma factors are responsible for the expression of housekeeping genes and are essential for survival. RpoD, the primary sigma factor of Escherichia coli, a γ-proteobacteria, recognizes consensus promoter sequences highly similar to those of some α-proteobacteria species. Despite this resemblance, RpoD is unable to sustain transcription from most of the α-proteobacterial promoters tested so far. In contrast, we have found that SigA, the primary sigma factor of Rhizobium etli, an α-proteobacteria, is able to transcribe E. coli promoters, although it exhibits only 48% identity (98% coverage) to RpoD. We have called this the transcriptional laxity phenomenon. Here, we show that SigA partially complements the thermo-sensitive deficiency of RpoD285 from E. coli strain UQ285 and that the SigA region σ4 is responsible for this phenotype. Sixteen out of 74 residues (21.6%) within region σ4 are variable between RpoD and SigA. Mutating these residues significantly improves SigA ability to complement E. coli UQ285. Only six of these residues fall into positions already known to interact with promoter DNA and to comprise a helix-turn-helix motif. The remaining variable positions are located on previously unexplored sites inside region σ4, specifically into the first two α-helices of the region. Neither of the variable positions confined to these helices seem to interact directly with promoter sequence; instead, we adduce that these residues participate allosterically by contributing to correct region folding and/or positioning of the HTH motif. We propose that transcriptional laxity is a mechanism for ensuring transcription in spite of naturally occurring mutations from endogenous promoters and/or horizontally transferred DNA sequences, allowing survival and fast environmental adaptation of α-proteobacteria.

Identification of cancer-related genes and motifs in the human gene regulatory network.

The authors investigated the regulatory network motifs and corresponding motif positions of cancer-related genes. First, they mapped disease-related genes to a transcription factor regulatory network. Next, they calculated statistically significant motifs and subsequently identified positions within these motifs that were enriched in cancer-related genes. Potential mechanisms of these motifs and positions are discussed. These results could be used to identify other disease- and cancer-related genes and could also suggest mechanisms for how these genes relate to co-occurring diseases.

The Brucella TIR domain containing proteins BtpA and BtpB have a structural WxxxE motif important for protection against microtubule depolymerisation

BackgroundThe TIR domain-containing proteins BtpA/Btp1/TcpB and BtpB are translocated into host cells by the facultative intracellular bacterial pathogen Brucella. Here, they interfere with Toll like receptor signalling to temper the host inflammatory response. BtpA has also been found to modulate microtubule dynamics. In both proteins we identified a WxxxE motif, previously shown to be an essential structural component in a family of bacterial type III secretion system effectors that modulate host actin dynamics by functioning as guanine nucleotide exchange factors of host GTPases. We analysed a role for the WxxxE motif in association of BtpA and BtpB with the cytoskeleton.ResultsUnlike BtpA, ectopically expressed BtpB did not show a tubular localisation, but was found ubiquitously in the cytoplasm and the nucleus, and often appeared in discrete punctae in HeLa cells. BtpB was able to protect microtubules from drug-induced destabilisation similar to BtpA. The WxxxE motif was important for the ability of BtpA and BtpB to protect microtubules against destabilising drugs. Surprisingly, ectopic expression of BtpA, although not BtpB, in HeLa cells induced the formation of filopodia. This process was invariably dependent of the WxxxE motif. Our recent resolution of the crystal structure of the BtpA TIR domain reveals that the motif positions a glycine residue that has previously been shown to be essential for interaction of BtpA with microtubules.ConclusionsOur results suggest a structural role for the WxxxE motif in the association of BtpA and BtpB with microtubules, as with the WxxxE GEF family proteins where the motif positions an adjacent catalytic loop important for interaction with specific Rho GTPases. In addition, the ability of ectopically expressed BtpA to induce filopodia in a WxxxE-dependent manner suggests a novel property for BtpA. A conserved WxxxE motif is found in most bacterial and several eukaryotic TIR domain proteins. Despite the similarity between ectopically expressed BtpA and WxxxE GEFs to modulate host actin dynamics, our results suggest that BtpA is not part of this WxxxE GEF family. The WxxxE motif may therefore be a more common structural motif than thus far described. BtpA may provide clues to cross-talk between the TLR and GTPase signalling pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-014-0053-y) contains supplementary material, which is available to authorized users.

Subtle Changes in Motif Positioning Cause Tissue-Specific Effects on Robustness of an Enhancer's Activity

Deciphering the specific contribution of individual motifs within cis-regulatory modules (CRMs) is crucial to understanding how gene expression is regulated and how this process is affected by sequence variation. But despite vast improvements in the ability to identify where transcription factors (TFs) bind throughout the genome, we are limited in our ability to relate information on motif occupancy to function from sequence alone. Here, we engineered 63 synthetic CRMs to systematically assess the relationship between variation in the content and spacing of motifs within CRMs to CRM activity during development using Drosophila transgenic embryos. In over half the cases, very simple elements containing only one or two types of TF binding motifs were capable of driving specific spatio-temporal patterns during development. Different motif organizations provide different degrees of robustness to enhancer activity, ranging from binary on-off responses to more subtle effects including embryo-to-embryo and within-embryo variation. By quantifying the effects of subtle changes in motif organization, we were able to model biophysical rules that explain CRM behavior and may contribute to the spatial positioning of CRM activity in vivo. For the same enhancer, the effects of small differences in motif positions varied in developmentally related tissues, suggesting that gene expression may be more susceptible to sequence variation in one tissue compared to another. This result has important implications for human eQTL studies in which many associated mutations are found in cis-regulatory regions, though the mechanism for how they affect tissue-specific gene expression is often not understood.