Specific Loop Research Articles

ChIAMM: A Mixture Model for Statistical Analysis of Long-Range Chromatin Interactions From ChIA-PET Experiments.

Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) is an important experimental method for detecting specific protein-mediated chromatin loops genome-wide at high resolution. Here, we proposed a new statistical approach with a mixture model, chromatin interaction analysis using mixture model (ChIAMM), to detect significant chromatin interactions from ChIA-PET data. The statistical model is cast into a Bayesian framework to consider more systematic biases: the genomic distance, local enrichment, mappability, and GC content. Using different ChIA-PET datasets, we evaluated the performance of ChIAMM and compared it with the existing methods, including ChIA-PET Tool, ChiaSig, Mango, ChIA-PET2, and ChIAPoP. The result showed that the new approach performed better than most top existing methods in detecting significant chromatin interactions in ChIA-PET experiments.

Co-crystal structures of HIV TAR RNA bound to lab-evolved proteins show key roles for arginine relevant to the design of cyclic peptide TAR inhibitors

RNA-protein interfaces control key replication events during the HIV-1 life cycle. The viral trans-activator of transcription (Tat) protein uses an archetypal arginine-rich motif (ARM) to recruit the host positive transcription elongation factor b (pTEFb) complex onto the viral trans-activation response (TAR) RNA, leading to activation of HIV transcription. Efforts to block this interaction have stimulated production of biologics designed to disrupt this essential RNA-protein interface. Here, we present four co-crystal structures of lab-evolved TAR-binding proteins (TBPs) in complex with HIV-1 TAR. Our results reveal that high-affinity binding requires a distinct sequence and spacing of arginines within a specific β2-β3 hairpin loop that arose during selection. Although loops with as many as five arginines were analyzed, only three arginines could bind simultaneously with major-groove guanines. Amino acids that promote backbone interactions within the β2-β3 loop were also observed to be important for high-affinity interactions. Based on structural and affinity analyses, we designed two cyclic peptide mimics of the TAR-binding β2-β3 loop sequences present in two high-affinity TBPs (KD values of 4.2 ± 0.3 and 3.0 ± 0.3 nm). Our efforts yielded low-molecular weight compounds that bind TAR with low micromolar affinity (KD values ranging from 3.6 to 22 μm). Significantly, one cyclic compound within this series blocked binding of the Tat-ARM peptide to TAR in solution assays, whereas its linear counterpart did not. Overall, this work provides insight into protein-mediated TAR recognition and lays the ground for the development of cyclic peptide inhibitors of a vital HIV-1 RNA-protein interaction.

A hybrid approach reveals the allosteric regulation of GTP cyclohydrolase I

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). GFRP binds to GCH1 to form inhibited or activated complexes dependent on availability of cofactor ligands, BH4 and phenylalanine, respectively. We determined high-resolution structures of human GCH1-GFRP complexes by cryoelectron microscopy (cryo-EM). Cryo-EM revealed structural flexibility of specific and relevant surface lining loops, which previously was not detected by X-ray crystallography due to crystal packing effects. Further, we studied allosteric regulation of isolated GCH1 by X-ray crystallography. Using the combined structural information, we are able to obtain a comprehensive picture of the mechanism of allosteric regulation. Local rearrangements in the allosteric pocket upon BH4 binding result in drastic changes in the quaternary structure of the enzyme, leading to a more compact, tense form of the inhibited protein, and translocate to the active site, leading to an open, more flexible structure of its surroundings. Inhibition of the enzymatic activity is not a result of hindrance of substrate binding, but rather a consequence of accelerated substrate binding kinetics as shown by saturation transfer difference NMR (STD-NMR) and site-directed mutagenesis. We propose a dissociation rate controlled mechanism of allosteric, noncompetitive inhibition.

Predicting unrecognized enhancer-mediated genome topology by an ensemble machine learning model.

Transcriptional enhancers commonly work over long genomic distances to precisely regulate spatiotemporal gene expression patterns. Dissecting the promoters physically contacted by these distal regulatory elements is essential for understanding developmental processes as well as the role of disease-associated risk variants. Modern proximity-ligation assays, like HiChIP and ChIA-PET, facilitate the accurate identification of long-range contacts between enhancers and promoters. However, these assays are technically challenging, expensive, and time-consuming, making it difficult to investigate enhancer topologies, especially in uncharacterized cell types. To overcome these shortcomings, we therefore designed LoopPredictor, an ensemble machine learning model, to predict genome topology for cell types which lack long-range contact maps. To enrich for functional enhancer-promoter loops over common structural genomic contacts, we trained LoopPredictor with both H3K27ac and YY1 HiChIP data. Moreover, the integration of several related multi-omics features facilitated identifying and annotating the predicted loops. LoopPredictor is able to efficiently identify cell type–specific enhancer-mediated loops, and promoter–promoter interactions, with a modest feature input requirement. Comparable to experimentally generated H3K27ac HiChIP data, we found that LoopPredictor was able to identify functional enhancer loops. Furthermore, to explore the cross-species prediction capability of LoopPredictor, we fed mouse multi-omics features into a model trained on human data and found that the predicted enhancer loops outputs were highly conserved. LoopPredictor enables the dissection of cell type–specific long-range gene regulation and can accelerate the identification of distal disease-associated risk variants.

Characterization of the ExoU activation mechanism using EPR and integrative modeling

ExoU, a type III secreted phospholipase effector of Pseudomonas aeruginosa, serves as a prototype to model large, dynamic, membrane-associated proteins. ExoU is synergistically activated by interactions with membrane lipids and ubiquitin. To dissect the activation mechanism, structural homology was used to identify an unstructured loop of approximately 20 residues in the ExoU amino acid sequence. Mutational analyses indicate the importance of specific loop amino acid residues in mediating catalytic activity. Engineered disulfide cross-links show that loop movement is required for activation. Site directed spin labeling EPR and DEER (double electron–electron resonance) studies of apo and holo states demonstrate local conformational changes at specific sites within the loop and a conformational shift of the loop during activation. These data are consistent with the formation of a substrate-binding pocket providing access to the catalytic site. DEER distance distributions were used as constraints in RosettaDEER to construct ensemble models of the loop in both apo and holo states, significantly extending the range for modeling a conformationally dynamic loop.

3D Genomics of Acute Meyloid Leukemia Reveals the Imbalance between DNA Methylation Canyon Interactions and Leukemic Specific Enhancer Network Interactions

Changes in 3D chromatin organization like enhancer hijacking are believed to the driver for disease development like leukemia. Here we performed high-resolution HiC assays on primary acute myeloid leukemia (AML) samples and cell lines to dissect the abnormal 3D chromatin organization in AML. Our data set covers 5 AML samples and 3 AML cell lines. This dataset includes the common genetic abnormalities in AML: MLL-rearrangement, NPM1 mutation, RUNX1 mutation, and IDH1/TET2 mutations. We have recently generated high-resolution map for normal human hematopoietic stem cells (HSC) (Zhang et al. Mole Cell. 2020). In comparison with the HSC 3D chromatin organization, we found TADs and loops are very stable in both primary leukemia samples and cell lines. Less than 5% of all TADs in HSC fuse in AML, mimicking the enhancer hijacking scenario. These fusion events do not cause the gene expression changes of genes in the fused TAD. Interestingly, in TET2 or IDH1 mutated AML blast, two-fold more TAD fusion events occurred in primary AML blast in comparison with RUNX1 and MLL-r leukemia, with a loss in the CTCF sites on the TAD fusion break point. We previously found in HSC, the Polycomb marked DNA methylation Canyons (DMC) form multi-Mb size long-range interactions. DMC interactions in general decrease in primary AMLs. AMLs with IDH1 or TET2 mutations shows the biggest reduction in DMC interactions. Hypermethylation in the DMCs is observed in the AML samples with IDH1/2 or TET2 mutations, suggesting DNA methylation level in DMCs controls DMC 3D interactions directly. In leukemia cell lines, the DMC interactions almost disappear, with further hypermethylation in DMCs. Compared with normal HSC, we found in AML, the AML-specific H3K27ac marked regions form leukemia specific loops and transcription stripes in both cell lines and primary samples. Particularly in MLL-r primary leukemias, we found broad H3K27ac covered, hyperacetylated domains (10kb to 200kb). 22 such hyperacetylated domains were identified and associated with leukemogenic genes such as SATB1, ZEB2 and HOXA. All these domains formed distinct 3D micro TAD in the MLL-r primary leukemia in comparison with the HSPC, and CTCFs are not located at the border of these domains. Taken together, suggest active leukemia specific transcription created new 3D genomic interactions which is independent of cohesion-CTCF mediated loop extrusion. Interestingly, in HOXA cluster, we found a geneless DMC 1.3MB upstream of HOXA switched from Polycomb binding site to active enhancer site in the leukemia cells. By applying CRISPR/Cas9 editing, we found this canyon is essential for survival of HOXA high expressing leukemia cell lines like OCI-AML3 and MV4:11. In summary, we found the 3D chromatin organization in human leukemia significantly alters in two opposite way 1. The significant loss of Polycomb marked DMC interactions caused by the DNA hypermethylation and 2. The leukemic specific hyperacetylated domains form its own distinct micro TAD and stripes in the 3D chromatin organization. Disclosures No relevant conflicts of interest to declare.

Experimental Research on the Hysteretic Behaviour of Pressurized Artificial Muscles Made from Elastomers with Aramid Fibre Insertions

Inherent hysteresis behaviour of pressurized artificial muscles is complicated to understand and handle, calling for experimental research that allows the modelling of this phenomenon. The paper presents the results of the experimental study of the hysteretic behaviour of a small-size pneumatic muscle. The specific hysteresis loops were revealed by isotonic and isometric tests. Starting from hypothesis according to that the tube used for the pneumatic muscle is made entirely of aramid fibres enveloped by an elastomer material that merely ensures their airtightness, the paper presents the hysteresis curves that describe the radial and axial dimensional modifications as well as the variation of the developed forces for different feed pressures. The obtained third-degree polynomial equations underlie the configuration of high-performance positioning systems.

Cooperative effects on the compaction of DNA fragments by the nucleoid protein H-NS and the crowding agent PEG probed by Magnetic Tweezers

BackgroundDNA bridging promoted by the H-NS protein, combined with the compaction induced by cellular crowding, plays a major role in the structuring of the E. coli genome. However, only few studies consider the effects of the physical interplay of these two factors in a controlled environment. MethodsWe apply a single molecule technique (Magnetic Tweezers) to study the nanomechanics of compaction and folding kinetics of a 6 kb DNA fragment, induced by H-NS bridging and/or PEG crowding. ResultsIn the presence of H-NS alone, the DNA shows a step-wise collapse driven by the formation of multiple bridges, and little variations in the H-NS concentration-dependent unfolding force. Conversely, the DNA collapse force observed with PEG was highly dependent on the volume fraction of the crowding agent. The two limit cases were interpreted considering the models of loop formation in a pulled chain and pulling of an equilibrium globule respectively. ConclusionsWe observed an evident cooperative effect between H-NS activity and the depletion of forces induced by PEG. General SignificanceOur data suggest a double role for H-NS in enhancing compaction while forming specific loops, which could be crucial in vivo for defining specific mesoscale domains in chromosomal regions in response to environmental changes.

Highly conserved extracellular residues mediate interactions between pore-forming and regulatory subunits of the yeast Ca2+ channel related to the animal VGCC/NALCN family

Yeasts and fungi generate Ca2+ signals in response to environmental stresses through Ca2+ channels essentially composed of Cch1 and Mid1. Cch1 is homologous to the pore-forming α1 subunit of animal voltage-gated Ca2+ channels (VGCCs) and sodium leak channels nonselective (NALCNs), whereas Mid1 is a membrane-associated protein similar to the regulatory α2/δ subunit of VGCCs and the regulatory subunit of NALCNs. Although the physiological roles of Cch1/Mid1 channels are known, their molecular regulation remains elusive, including subunit interactions regulating channel functionality. Herein, we identify amino acid residues involved in interactions between the pore-forming Cch1 subunit and the essential regulatory Mid1 subunit of Saccharomyces cerevisiae. In vitro mutagenesis followed by functional assays and co-immunoprecipitation experiments reveal that three residues present in a specific extracellular loop in the repeat III region of Cch1 are required for interaction with Mid1, and that one essential Mid1 residue is required for interaction with Cch1. Importantly, these residues are necessary for Ca2+ channel activity and are highly conserved in fungal and animal counterparts. We discuss that this unique subunit interaction-based regulatory mechanism for Cch1 differs from that of VGCCs/NALCNs.

Future perspective: high-throughput construction of new ultrasensitive cytokine and virion liquid chips for high-throughput screening (HTS) of anti-inflammatory drugs or clinical diagnosis and treatment of inflammatory diseases.

Pathogen–host cell interactions play an important role in many human infectious and inflammatory diseases. Several pathogens, including Escherichia coli (E. coli), Mycobacterium tuberculosis (M. tb), and even the recent 2019 novel coronavirus (2019-nCoV), can cause serious breathing and brain disorders, tissue injury and inflammation, leading to high rates of mortality and resulting in great loss to human physical and mental health as well as the global economy. These infectious diseases exploit the microbial and host factors to induce serious inflammatory and immunological symptoms. Thus the development of anti-inflammatory drugs targeting bacterial/viral infection is an urgent need. In previous studies, YojI-IFNAR2, YojI-IL10RA, YojI-NRP1,YojI-SIGLEC7, and YojI-MC4R membrane–protein interactions were found to mediate E. coli invasion of the blood–brain barrier (BBB), which activated the downstream anti-inflammatory proteins NACHT, LRR and PYD domains-containing protein 2(NLRP2), using a proteomic chip conjugated with cell immunofluorescence labeling. However, the studies of pathogen (bacteria/virus)–host cell interactions mediated by membrane protein interactions did not extend their principles to broad biomedical applications such as 2019-nCoV infectious disease therapy. The first part of this feature article presents in-depth analysis of the cross-talk of cellular anti-inflammatory transduction signaling among interferon membrane protein receptor II (IFNAR2), interleukin-10 receptor subunit alpha (IL-10RA), NLRP2 and [Ca2+]-dependent phospholipase A2 (PLA2G5), based on experimental results and important published studies, which lays a theoretical foundation for the high-throughput construction of the cytokine and virion solution chip. The paper then moves on to the construction of the novel GPCR recombinant herpes virion chip and virion nano-oscillators for profiling membrane protein functions, which drove the idea of constructing the new recombinant virion and cytokine liquid chips for HTS of leading drugs. Due to the different structural properties of GPCR, IFNAR2, ACE2 and Spike of 2019-nCoV, their ligands will either bind the extracellular domain of IFNAR2/ACE2/Spike or the specific loops of the GPCR on the envelope of the recombinant herpes virions to induce dynamic charge distribution changes that lead to the variable electron transition for detection. Taken together, the combined overview of two of the most innovative and exciting developments in the immunoinflammatory field provides new insight into high-throughput construction of ultrasensitive cytokine and virion liquid chips for HTS of anti-inflammatory drugs or clinical diagnosis and treatment of inflammatory diseases including infectious diseases, acute or chronic inflammation (acute gouty arthritis or rheumatoid arthritis), cardiovascular disease, atheromatosis, diabetes, obesity, tissue injury and tumors. It has significant value in the prevention and treatment of these serious and painful diseases. Graphical abstract

Insight into Structural Aspects of Histidine 284 of Daphnia magna Arginine Kinase.

Arginine kinase (AK), a bioenergy-related enzyme, is distributed widely in invertebrates. The role of highly conserved histidines in AKs is still unascertained. In this study, the highly conserved histidine 284 (H284) in AK of Daphnia magna (DmAK) was replaced with alanine to elucidate the role of H284. We examined the alteration of catalytic activity and structural changes of H284A in DmAK. The catalytic activity of H284A was reduced dramatically compared to that in wild type (WT). Thus the crystal structure of H284A displayed several structural changes, including the alteration of D324, a hydrogen-bonding network around H284, and the disruption of π-stacking between the imidazole group of the H284 residue and the adenine ring of ATP. These findings suggest that such alterations might affect a conformational change of the specific loop consisting of G310-V322 at the antiparallel β-sheet region. Thus, we speculated that the H284 residue might play an important role in the conformational change of the specific loop when ATP binds to the substrate-binding site of DmAK

Post-transcriptional modulation of the SigF regulon in Mycobacterium smegmatis by the PhoH2 toxin-antitoxin.

PhoH2 proteins are highly conserved across bacteria and archaea yet their biological function is poorly characterised. We examined the growth profiles of Mycobacterium smegmatis strains mc2155 and mc2155 ΔphoH2 and observed the same growth profile and growth rate in a variety of conditions. In light of the comparable growth, we used RNAseq to provide a snapshot of the differences between the transcriptomes of M. smegmatis mc2155 and M. smegmatis mc2155 ΔphoH2 during normal growth. At 48 hours, elevated expression of the sigF regulon was observed in ΔphoH2 relative to wild type. In biochemical assays, PhoH2 showed activity toward sigF mRNA insinuating a role of PhoH2 in modulating the pool of sigF mRNA in the cell during normal growth, adding further complexity to the repertoire of reported mechanisms of post-translational regulation. Multiple copies of the preferred target site of PhoH2 were identified in loops of the sigF mRNA structure, leading us to propose a mechanism for the activity of PhoH2 that is initiated after assembly on specific single-stranded loops of RNA. We hypothesise that PhoH2 is a toxin-antitoxin that contributes to the regulation of SigF at a post-transcriptional level through targeted activity on sigF mRNA. This work presents the first evidence for post-transcriptional regulation of SigF along with the biological function of PhoH2 from M. smegmatis. This has implications for the highly conserved PhoH2 toxin-antitoxin module across the mycobacteria including the important human pathogen M. tuberculosis.

The level of expression of miR-196a in patients with chronic viral hepatitis C with the first genotype of HCV according to previous experience of antiviral therapy.

The authors present the study of the level of expression of miR-196a in 74 patients with chronic viral hepatitis C with the 1st genotype of HCV, based on previous experience in patients with antiviral therapy regimens containing interferon. The patients were divided into two groups, depending on the previous experience of antiviral therapy with circuits containing interferon – 21 patients who failed after antiviral therapy regimens containing interferon (group 1) and the comparison group (group 2) – 53 naive patients. To study the level of expression of miR-196a (miR-196a), a two-stage study according to the manufacturer's protocol was used. First, total RNA was isolated from the plasma by the method of phenol-chloroform extraction. Futher reverse transcription was performed using a kit for reverse transcription of miR TaqMan® (Applied Biosystems, USA), specific loop primers to achieve mature miRNA, snRNA U6 (as an endogenous control gene), and 10 ng of total RNA. Real-time quantitative PCR was performed using TaqMan® miRNA analysis. In order to optimize the prediction of response to antiviral therapy and the use of optimal treatment regimens for problem patients with treatment failure regimens containing interferon, analysis using ROC curves was used. The average level of expression of miR-196a in patients with chronic hepatitis C was evaluated. In the 1st group of patients it was 0.011 (IQR: 0.002; 0.310) and in the comparison group – 0.346 (IQR: 0.054; 1.239) at p=0.012 by U criterion. The conducted ROC analysis showed that the studied miR-196a could differentiate patients with chronic viral hepatitis C with HCV genotype 1, depending on previous experience of antiviral therapy, namely, patients with treatment failure regimens containing interferons and naive. AUC=0.688 (95% CI 0.570-0.791; p=0.017), J=0.40, Se=57.14%, Sp=83.02%. It gives additional opportunities for correction of therapeutic tactics. Therefore, the level of expression of miR-196a (<-1,75) may be an additional biomarker in the pathogenesis of HCV-infection, which can then be used in the monitoring and treatment of patients. Low expression of miR-196a may be the basis for prescribing more effective direct-acting antiviral therapy to patients and will allow personalizing therapeutic tactics in patients with chronic viral hepatitis C.

Optimizing OpenCL-Based CNN Design on FPGA with Comprehensive Design Space Exploration and Collaborative Performance Modeling

Recent success in applying convolutional neural networks (CNNs) to object detection and classification has sparked great interest in accelerating CNNs using hardware-like field-programmable gate arrays (FPGAs). However, finding an efficient FPGA design for a given CNN model and FPGA board is not trivial since a strong background in hardware design and detailed knowledge of the target board are required. In this work, we try to solve this problem by design space exploration with a collaborative framework. Our framework consists of three main parts: FPGA design generation, coarse-grained modeling, and fine-grained modeling. In the FPGA design generation, we propose a novel data structure, LoopTree, to capture the details of the FPGA design for CNN applications without writing down the source code. Different LoopTrees, which indicate different FPGA designs, are automatically generated in this process. A coarse-grained model will evaluate LoopTrees at the operation level, e.g., add, mult, and so on, so that the most efficient LoopTrees can be selected. A fine-grained model, which is based on the source code, will then refine the selected design in a cycle-accurate manner. A set of comprehensive OpenCL-based designs have been implemented on board to verify our framework. An average estimation error of 8.87% and 4.8% has been observed for our coarse-grained model and fine-grained model, respectively. This is much lower than the prevalent operation-statistics-based estimation, which is obtained according to a predefined formula for specific loop schedules.

Predictor-Based Control of LTI Remote Systems With Estimated Time-Varying Delays

This letter provides a practical control solution to remote control systems with unknown time-varying delays. An external signal which can be considered as a specific communication loop is added to estimate the unknown round-trip delay in finite time. Then the system is stabilized with the practical delay estimation technique and a predictor-based controller. The main results of this letter also provide an alternative way to design predictor-based controller for systems with known input and output time-varying delays. The theoretical results of this letter are illustrated by simulation results.

Molecular genetics of maternally-controlled cell divisions.

Forward genetic screens remain at the forefront of biology as an unbiased approach for discovering and elucidating gene function at the organismal and molecular level. Past mutagenesis screens targeting maternal-effect genes identified a broad spectrum of phenotypes ranging from defects in oocyte development to embryonic patterning. However, earlier vertebrate screens did not reach saturation, anticipated classes of phenotypes were not uncovered, and technological limitations made it difficult to pinpoint the causal gene. In this study, we performed a chemically-induced maternal-effect mutagenesis screen in zebrafish and identified eight distinct mutants specifically affecting the cleavage stage of development and one cleavage stage mutant that is also male sterile. The cleavage-stage phenotypes fell into three separate classes: developmental arrest proximal to the mid blastula transition (MBT), irregular cleavage, and cytokinesis mutants. We mapped each mutation to narrow genetic intervals and determined the molecular basis for two of the developmental arrest mutants, and a mutation causing male sterility and a maternal-effect mutant phenotype. One developmental arrest mutant gene encodes a maternal specific Stem Loop Binding Protein, which is required to maintain maternal histone levels. The other developmental arrest mutant encodes a maternal-specific subunit of the Minichromosome Maintenance Protein Complex, which is essential for maintaining normal chromosome integrity in the early blastomeres. Finally, we identify a hypomorphic allele of Polo-like kinase-1 (Plk-1), which results in a male sterile and maternal-effect phenotype. Collectively, these mutants expand our molecular-genetic understanding of the maternal regulation of early embryonic development in vertebrates.

The Role of the Flexible Loop in Substrate Recognition and Catalysis in Malate Dehydrogenase

All enzymes do three things: they bind a specific substrate, catalyze a reaction, and release the product. In addition to providing insight into structure‐function relationships, understanding the molecular basis of steps involved in substrate binding can provide insight into 1.) how to design specific ligands to inhibit, or 2.) how to manipulate substrate specificity to be used for biotech purposes. Malate dehydrogenase (MDH), present in all organisms, plays critical metabolic roles in the cell and catalyzes the interconversion of malate and oxaloacetate. Within the active site of MDH, there is a flexible loop (P119‐N137) that contains two of three arginines whose positive charge interact with the negatively charged 4‐carbon dicarboxylic acid substrate to coordinate its orientation for catalysis. In LDH, (which evolved from MDH) there is a similar flexible loop region which binds pyruvate (a 3‐carbon mono carboxylic acid), Previous work with Ecoli MDH, creation of an R124Q mutation in the flexible loop, led to a change in substrate specificity and also an increased affinity for pyruvate, supporting the suggestion that the flexible loop of MDH is important in discrimination between substrates. Citrate, a 6 carbon tricarboxylic acid has been shown to bind to the active site of MDH in a half‐sites manner, and is thought to regulate activity in some MDHs. We hypothesize that other residues in the loop region are involved in fine tuning both binding and catalysis. To explore this hypothesis we have used watermelon glyoxysomal MDH (WMgMDH) as a model and constructed a variety of mutations throughout the loop to explore how the loop is involved in both discriminating between ligands (including substrate, citrate, and pyruvate) and contributing to catalysis. To explore potential interactions that residues in the loop makes, HINT computational analysis was used to predict the interactions individual residues make with the rest of the protein or substrates. A series of site‐directed mutants were created: A120G, G121A, P123S, F134S, D132A, L133S, F134S, and N137A. using Quikchange mutagenesis. Mutants were expressed and purified using Ni‐NTA affinity chromatography and characterized. Michaelis Menten Kinetic studies were used to determine overall catalytic efficiency, substrate binding and specificity, and cofactor binding, and compared to wild‐type. D132A, L133S, F134S, and N137A had a significant decrease in specific activity. A120G, G121A, L133S, and N137A showed anomalous NADH saturation. G121A, P123S, M128A, D132A, L133S, F134S, and N137A showed significant changes in oxaloacetate binding. An interesting trend for the four mutants that were farther away from R124 and R130 (D132A, L133S, F134S, and N137A) was that those that had the highest affinity for NADH had the lowest affinity for OAA and vice versa. These results suggest that the “specificity loop” of Malate Dehydrogenase plays additional roles in cofactor binding and catalysis than previously thought, and could provide new insights that could be exploited for biotech purposes as well as furthering our knowledge of structure‐function relationships in this important metabolic enzyme.Support or Funding InformationThis work was supported by NSF Grants 1726932 and 0448905

DNA sequence, physics, and promoter function: Analysis of high-throughput data On T7 promoter variants activity.

RNA polymerase/promoter recognition represents a basic problem of molecular biology. Decades-long efforts were made in the area, and yet certain challenges persist. The usage of certain most suitable model subjects is pivotal for the research. System of T7 bacteriophage RNA-polymerase/T7 native promoter represents an exceptional example for the purpose. Moreover, it has been studied the most and successfully applied to aims of biotechnology and bioengineering. Both structural simplicity and high specificity of this molecular duo are the reason for this. Despite highly similar sequences of distinct T7 native promoters, the T7 RNA-polymerase enzyme is capable of binding respective promoter in a highly specific and adjustable manner. One explanation here is that the process relies primarily on DNA physical properties rather than nucleotide sequence. Here, we address the issue by analyzing massive data recently published by Komura and colleagues. This initial study employed Next Generation Sequencing (NGS) in order to quantify activity of promoter variants including ones with multiple substitutions. As a result of our work substantial bias in simultaneous occurrence of single-nucleotide sequence alterations was found: the highest rate of co-occurrence was evidenced within specificity loop of binding region while the lowest- in initiation region of promoter. If both location and a kind of nucleotides involved in replacement (both initial and resulting) are taken into consideration, one can easily note that N to A substitutions are most preferred ones across the whole 19 b.p.-long sequence. At the same time, N to C are tolerated only at crucial position in recognition loop of binding region, and N to G are uniformly least tolerable. Later in this work the complete set of variants was split into groups with mutations (1) exclusively in binding region; (2) exclusively in melting region; (3) in both regions. Among these three groups second comprises extremely few variants (at triple-digit rate lesser than in two other groups, 46 versus over one and six thousand). Yet these are all promoter with substantial to high activity. This group two appeared heterogenous by primary sequence; indeed, upon further subdivision into above versus below average activity subgroups first one was found to comprise promoters with negligible conservation at 2 position of melting region; the second was hardly conserved in this region at all. This draws our attention to perfect consensus sequence of class III T7 promoter with 2 nucleotide randomized (all four are present by one to several copies in the previously published source dataset), the picture becomes even more pronounced. We therefore suggest that mutations at the position therefore do not cause significant changes in terms of promoter activity. At the same time, such modifications dramatically change DNA physical properties which were calculated in our study (namely electrostatic potential and propensity to bend). One possible suggestion here is that 2 nucleotide might function as a generic switch; if so, substitution 2A to 2T has important regulatory consequences. The fact that that 2 b.p. is the most evidently different nucleotide between class II versus class III promoters of T7 genome and that it also distinguishes the class III promoter in T7 genome versus promoters of its relative but reproductively isolated bacteriophage T3. In other words, it appears feasible that mutation at 2 nucleotide does not impede promoter activity yet alter its physical properties thus affecting differential RNA polymerase/promoter interaction.

Proximity-dependent biotin labelling reveals CP190 as an EcR/Usp molecular partner

Proximity-dependent biotin labelling revealed undescribed participants of the ecdysone response in Drosophila. Two labelling enzymes (BioID2 and APEX2) were fused to EcR or Usp to biotin label the surrounding proteins. The EcR/Usp heterodimer was found to collaborate with nuclear pore subunits, chromatin remodelers, and architectural proteins. Many proteins identified through proximity-dependent labelling with EcR/Usp were described previously as functional components of an ecdysone response, corroborating the potency of this labelling method. A link to ecdysone response was confirmed for some newly discovered regulators by immunoprecipitation of prepupal nuclear extract with anti-EcR antibodies and functional experiments in Drosophila S2 cells. A more in-depth study was conducted to clarify the association of EcR/Usp with one of the detected proteins, CP190, a well-described cofactor of Drosophila insulators. CP190 was found to co-immunoprecipitate with the EcR subunit of EcR/Usp in a 20E-independent manner. ChIP-Seq experiments revealed only partial overlapping between CP190 and EcR bound sites in the Drosophila genome and complete absence of CP190 binding at 20E-dependent enhancers. Analysis of Hi-C data demonstrated an existence of remote interactions between 20E-dependent enhancers and CP190 sites which suggests formation of a protein complex between EcR/Usp and CP190 through the space. Our results support the previous concept that CP190 has a role in stabilization of specific chromatin loops for proper activation of transcription of genes regulated by 20E hormone.

Nuclear pore complex-mediated gene expression in Arabidopsis thaliana.

Nuclear pore complexes (NPCs) are large multi-protein complexes that control bidirectional trafficking of macromolecules between the nucleus and cytoplasm. This trafficking is highly regulated and participates in a considerably broader range of cellular activities, including defense responses against pathogens in plants. Recently, NPC is emerging as a platform to physically associate the underlying chromatin with the nuclear periphery, thus regulating chromatin structure and gene expression. For instance, NPC components have been shown to promote the formation of specific genomics loops, which is linked to transcriptional memory for rapid reactivation of genes. With newly developed techniques and tools, our insight in this area has been substantially advanced. This review summarizes recent works on the molecular function of NPC machinery as hubs for transcriptional regulation and compares systems between plant and non-plant organisms.