AC-motif: a DNA motif containing adenine and cytosine repeat plays a role in gene regulation.

I-motif or C4 is a four-stranded DNA structure with a protonated cytosine:cytosine base pair (C+:C) found in cytosine-rich sequences. We have found that oligodeoxynucleotides containing adenine and cytosine repeats form a stable secondary structure at a physiological pH with magnesium ion, which is similar to i-motif structure, and have named this structure 'adenine:cytosine-motif (AC-motif)'. AC-motif contains C+:C base pairs intercalated with putative A+:C base pairs between protonated adenine and cytosine. By investigation of the AC-motif present in the CDKL3 promoter (AC-motifCDKL3), one of AC-motifs found in the genome, we confirmed that AC-motifCDKL3 has a key role in regulating CDKL3 gene expression in response to magnesium. This is further supported by confirming that genome-edited mutant cell lines, lacking the AC-motif formation, lost this regulation effect. Our results verify that adenine-cytosine repeats commonly present in the genome can form a stable non-canonical secondary structure with a non-Watson-Crick base pair and have regulatory roles in cells, which expand non-canonical DNA repertoires.

NMR Structure and Biophysical Characterization of Thermophilic Single-Stranded DNA Binding Protein from Sulfolobus Solfataricus.

Proteins from Sulfolobus solfataricus (S. solfataricus), an extremophile, are active even at high temperatures. The single-stranded DNA (ssDNA) binding protein of S. solfataricus (SsoSSB) is overexpressed to protect ssDNA during DNA metabolism. Although SsoSSB has the potential to be applied in various areas, its structural and ssDNA binding properties at high temperatures have not been studied. We present the solution structure, backbone dynamics, and ssDNA binding properties of SsoSSB at 50 °C. The overall structure is consistent with the structures previously studied at room temperature. However, the loop between the first two ß sheets, which is flexible and is expected to undergo conformational change upon ssDNA binding, shows a difference from the ssDNA bound structure. The ssDNA binding ability was maintained at high temperature, but different interactions were observed depending on the temperature. Backbone dynamics at high temperature showed that the rigidity of the structured region was well maintained. The investigation of an N-terminal deletion mutant revealed that it is important for maintaining thermostability, structure, and ssDNA binding ability. The structural and dynamic properties of SsoSSB observed at high temperature can provide information on the behavior of proteins in thermophiles at the molecular level and guide the development of new experimental techniques.

Determinants of replication protein A subunit interactions revealed using a phosphomimetic peptide.

Replication protein A (RPA) is a eukaryotic ssDNA-binding protein and contains three subunits: RPA70, RPA32, and RPA14. Phosphorylation of the N-terminal region of the RPA32 subunit plays an essential role in DNA metabolism in processes such as replication and damage response. Phosphorylated RPA32 (pRPA32) binds to RPA70 and possibly regulates the transient RPA70-Bloom syndrome helicase (BLM) interaction to inhibit DNA resection. However, the structural details and determinants of the phosphorylated RPA32-RPA70 interaction are still unknown. In this study, we provide molecular details of the interaction between RPA70 and a mimic of phosphorylated RPA32 (pmRPA32) using fluorescence polarization and NMR analysis. We show that the N-terminal domain of RPA70 (RPA70N) specifically participates in pmRPA32 binding, whereas the unphosphorylated RPA32 does not bind to RPA70N. Our NMR data revealed that RPA70N binds pmRPA32 using a basic cleft region. We also show that at least 6 negatively charged residues of pmRPA32 are required for RPA70N binding. By introducing alanine mutations into hydrophobic positions of pmRPA32, we found potential points of contact between RPA70N and the N-terminal half of pmRPA32. We used this information to guide docking simulations that suggest the orientation of pmRPA32 in complex with RPA70N. Our study demonstrates detailed features of the domain-domain interaction between RPA70 and RPA32 upon phosphorylation. This result provides insight into how phosphorylation tunes transient bindings between RPA and its partners in DNA resection.

Molecular Diagnostic System Using Engineered Fusion Protein-Conjugated Magnetic Nanoparticles.

To effectively control the spread of new infectious diseases, there is a need for highly sensitive diagnostic methods to detect viral nucleic acids rapidly. This study outlines a universal and simple detection strategy that uses magnetic nanoparticles (MNPs) and a novel MagR-MazE fusion protein for molecular diagnostics to facilitate sensitive detection. This study has engineered a novel MNP conjugate that can be generated easily, without using many chemical reagents. The technique is a nucleic acid detection method, using MagR-MazE fusion protein-conjugated MNPs, where the results can be visualized with the naked eye, regardless of the oligonucleotide sequences of the target in the lateral flow assay. This method could sensitively detect polymerase chain reaction (PCR) products of 16S ribosomal RNA (rRNA) and the 2019-nCoV-N-positive control gene in 5 min. It shows a low limit of detection (LoD) of 0.013 ng/µL for dsDNA. It is simpler and more rapid, sensitive, and versatile than other techniques, making it suitable for point-of-care testing. The proposed detection system and MNP conjugation strategy using a fusion protein can be widely applied to various fields requiring rapid on-site diagnosis.

Loop-mediated fluorescent probes for selective discrimination of parallel and antiparallel G-Quadruplexes.

Herein we report simple pyridinium (1-3) and quinolinium (4) salts for the selective recognition of G-quadruplexes (G4s). Among them, the probe 1, interestingly, selectively discriminated parallel (c-KIT-1, c-KIT-2, c-MYC) G4s from anti-parallel/hybrid (22AG, HRAS-1, BOM-17, TBA) G4s at pH 7.2, through a switch on response in the far-red window. Significant changes in the absorption (broad 575 nm â†’ sharp 505 nm) and emission of probe 1 at 620 nm, attributed to selective interaction with parallel G4s, resulted in complete disaggregation-induced monomer emission. Symmetrical push/pull molecular confinements across the styryl units in probe 1 enhanced the intramolecular charge transfer (ICT) by restricting the free rotation of CC units in the presence of sterically less hindered and highly accessible G4 surface/bottom tetrads in the parallel G4s, which is relatively lower extent in antiparallel/hybrid G4s. We confirm that the disaggregation of probe 1 was very effective in the presence of parallel G4-forming ODNs, due to the presence of highly available free surface area, resulting in additional π-stacking interactions. The selective sensing capabilities of probe 1 were analyzed using UV-Vis spectroscopy, fluorescence spectroscopy, molecular dynamics (MD)-based simulation studies, and 1H NMR spectroscopy. This study should afford insights for the future design of selective compounds targeting parallel G4s.

Structural basis for the inhibition of PDK2 by novel ATP- and lipoyl-binding site targeting compounds.

Pyruvate dehydrogenase kinase (PDK) controls the activity of pyruvate decarboxylase complex (PDC) by phosphorylating key serine residues on the E1 subunit, which leads to a decreased oxidative phosphorylation in mitochondria. Inhibition of PDK activity by natural/synthetic compounds has been shown to reverse the Warburg effect, a characteristic metabolism in cancer cells. PDK-PDC axis also has been associated with diabetes and heart disease. Therefore, regulation of PDK activity has been considered as a promising strategy to treat related diseases. Here we present the X-ray crystal structure of PDK2 complexed with a recently identified PDK4 inhibitor, compound 8c, which has been predicted to bind at the lipoyl-binding site and interrupt intermolecular interactions with the E2-E3bp subunits of PDC. The co-crystal structure confirmed the specific binding location of compound 8c and revealed the remote conformational change in the ATP-binding pocket. In addition, two novel 4,5-diarylisoxazole derivatives, GM10030 and GM67520, were synthesized and used for structural studies, which target the ATP-binding site of PDK2. These compounds bind to PDK2 with a sub-100nM affinity as determined by isothermal titration calorimetry experiments. Notably, the crystal structure of the PDK2-GM10030 complex displays unprecedented asymmetric conformation of human PDK2 dimer, especially in the ATP-lids and C-terminal tails.

Dynamics Studies of DNA with Non-canonical Structure Using NMR Spectroscopy.

The non-canonical structures of nucleic acids are essential for their diverse functions during various biological processes. These non-canonical structures can undergo conformational exchange among multiple structural states. Data on their dynamics can illustrate conformational transitions that play important roles in folding, stability, and biological function. Here, we discuss several examples of the non-canonical structures of DNA focusing on their dynamic characterization by NMR spectroscopy: (1) G-quadruplex structures and their complexes with target proteins; (2) i-motif structures and their complexes with proteins; (3) triplex structures; (4) left-handed Z-DNAs and their complexes with various Z-DNA binding proteins. This review provides insight into how the dynamic features of non-canonical DNA structures contribute to essential biological processes.

Development of DNA Aptamers against the Nucleocapsid Protein of Severe Fever with Thrombocytopenia Syndrome Virus for Diagnostic Application: Catalytic Signal Amplification using Replication Protein A-Conjugated Liposomes.

Most prevalent infectious diseases worldwide are caused by mediators such as insects and characterized by high mortality and morbidity, thereby creating a global public health concern. Therefore, a sensitive, selective detection platform for diagnosing diseases in the early stages of infection is needed to prevent disease spread and to protect public health. Here, we developed novel DNA aptamers specific to the nucleocapsid protein (NP) of the severe fever with thrombocytopenia syndrome (SFTS) virus and synthesized ssDNA-binding protein-conjugated liposomes encapsulated with horseradish peroxidase (HRP) for application in a simple and universal platform. This platform achieved highly sensitive detection of the NP by measuring the colorimetric signal following lysis of the HRP encapsulated liposomes, mediated by a mixture of 3,3',5,5'-tetramethylbenzidine and H2O2 solution. The limit of detection was 0.009 ng·mL-1, and NP was successfully detected in diluted human serum with a high recovery rate. Moreover, this method was specific and did not exhibit cross-reactivity among NPs of other virus types. These results demonstrated the efficacy of the proposed method as a highly sensitive, specific, and universal diagnostic tool for potential application in monitoring of the early stages of infectious diseases.

Development of Replication Protein A-Conjugated Gold Nanoparticles for Highly Sensitive Detection of Disease Biomarkers.

Paper-based lateral flow immunoassays (LFIAs) using conventional sandwich-type immunoassays are one of the most commonly used point-of-care (PoC) tests. However, the application of gold nanoparticles (AuNPs) in LFIAs does not meet sensitivity requirements for the detection of infectious diseases or biomarkers present at low concentrations in body fluids because of the limited number of AuNPs that can bind to the target. To overcome this problem, we first developed a single-stranded DNA binding protein (RPA70A, DNA binding domain A of human Replication Protein A 70 kDa) conjugated to AuNPs for a sandwich assay using a capture antibody immobilized in the LFIA and an aptamer as a detection probe, thus, enabling signal intensity enhancement by attaching several AuNPs per aptamer. We applied this method to detect the influenza nucleoprotein (NP) and cardiac troponin I (cTnI). We visually detected spiked targets at a low femtomolar range, with limits of detection for NP in human nasal fluid and for cTnI in serum of 0.26 and 0.23 pg·mL-1, respectively. This technique showed significantly higher sensitivity than conventional methods that are widely used in LFIAs involving antibody-conjugated AuNPs. These results suggest that the proposed method can be universally applied to the detection of substances requiring high sensitivity and can be used in the field of PoC testing for early disease diagnosis.

A Simple and Label-Free Detection of As3+ using 3-nitro-L-tyrosine as an As3+-chelating Ligand.

A simple and rapid As3+ detection method using 3-nitro-L-tyrosine (N-Tyr) is reported. We discovered the specific property of N-Tyr, which specifically chelates As3+. The reaction between As3+ and N-Tyr induces a prompt color change to vivid yellow, concomitantly increasing the absorbance at 430 nm. The selectivity for As3+ is confirmed by competitive binding experiments with various metal ions (Hg2+, Pb2+, Cd2+, Cr3+, Mg2+, Ni2+, Cu2+, Fe2+, Ca2+, Zn2+, and Mn2+). Also, the N-Tyr binding site, binding affinity, and As3+/N-Tyr reaction stoichiometry are investigated. The specific reaction is utilized to design a sensor that enables the quantitative detection of As3+ in the 0.1-100 µM range with good linearity (R2 = 0.995). Furthermore, the method's applicability for the analysis of real samples, e.g., tap and river water, is successfully confirmed, with good recoveries (94.32-109.15%) using As3+-spiked real water samples. We believe that our discovering and its application for As3+ analysis can be effectively utilized in environmental analyses such as those conducted in water management facilities, with simplicity, rapidity, and ease.

Solution structure of the Z-DNA binding domain of PKR-like protein kinase from Carassius auratus and quantitative analyses of the intermediate complex during B-Z transition.

Z-DNA binding proteins (ZBPs) play important roles in RNA editing, innate immune response and viral infection. Structural and biophysical studies show that ZBPs initially form an intermediate complex with B-DNA for B-Z conversion. However, a comprehensive understanding of the mechanism of Z-DNA binding and B-Z transition is still lacking, due to the absence of structural information on the intermediate complex. Here, we report the solution structure of the Zα domain of the ZBP-containing protein kinase from Carassius auratus(caZαPKZ). We quantitatively determined the binding affinity of caZαPKZ for both B-DNA and Z-DNA and characterized its B-Z transition activity, which is modulated by varying the salt concentration. Our results suggest that the intermediate complex formed by caZαPKZ and B-DNA can be used as molecular ruler, to measure the degree to which DNA transitions to the Z isoform.

NMR elucidation of reduced B-Z transition activity of PKZ protein kinase at high NaCl concentration.

A Z-DNA binding protein (ZBP)-containing protein kinase (PKZ) in fish species has an important role in the innate immune response. Previous structural studies of the Zα domain of the PKZ from Carassius auratus (caZαPKZ) showed that the protein initially binds to B-DNA and induces B-Z transition of double stranded DNA in a salt concentration-dependent manner. However, the significantly reduced B-Z transition activity of caZαPKZ at high salt concentration was not fully understood. In this study, we present the binding affinity of the protein for B-DNA and Z-DNA and characterize its extremely low B-Z transition activity at 250 mM NaCl. Our results emphasize that the B-DNA-bound form of caZαPKZ can be used as molecular ruler to measure the degree of B-Z transition.

Comparison of backbone dynamics of the type III antifreeze protein and antifreeze-like domain of human sialic acid synthase.

Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 3(10)-helices, and two ß-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins.

Solution structure of the RecQ C-terminal domain of human Bloom syndrome protein.

RecQ C-terminal (RQC) domain is known as the main DNA binding module of RecQ helicases such as Bloom syndrome protein (BLM) and Werner syndrome protein (WRN) that recognizes various DNA structures. Even though BLM is able to resolve various DNA structures similarly to WRN, BLM has different binding preferences for DNA substrates from WRN. In this study, we determined the solution structure of the RQC domain of human BLM. The structure shares the common winged-helix motif with other RQC domains. However, half of the N-terminal has unstructured regions (α1-α2 loop and α3 region), and the aromatic side chain on the top of the ß-hairpin, which is important for DNA duplex strand separation in other RQC domains, is substituted with a negatively charged residue (D1165) followed by the polar residue (Q1166). The structurally distinctive features of the RQC domain of human BLM suggest that the DNA binding modes of the BLM RQC domain may be different from those of other RQC domains.

Protective roles of highly conserved motif 1 in tardigrade cytosolic-abundant heat soluble protein in extreme environments.

Tardigrades are remarkable microscopic animals that survive harsh conditions such as desiccation and extreme temperatures. Tardigrade-specific intrinsically disordered proteins (TDPs) play an essential role in the survival of tardigrades in extreme environments. Cytosolic-abundant heat soluble (CAHS) protein, a key TDP, is known to increase desiccation tolerance and to protect the activity of several enzymes under dehydrated conditions. However, the function and properties of each CAHS domain have not yet been elucidated in detail. Here, we aimed to elucidate the protective role of highly conserved motif 1 of CAHS in extreme environmental conditions. To examine CAHS domains, three protein constructs, CAHS Full (1-229), CAHS ∆Core (1-120_184-229), and CAHS Core (121-183), were engineered. The highly conserved CAHS motif 1 (124-142) in the CAHS Core formed an amphiphilic α helix, reducing the aggregate formation and protecting lactate dehydrogenase activity during dehydration-rehydration and freeze-thaw treatments, indicating that CAHS motif 1 in the CAHS Core was essential for maintaining protein solubility and stability. Aggregation assays and confocal microscopy revealed that the intrinsically disordered N- and C-terminal domains were more prone to aggregation under our experimental conditions. By explicating the functions of each domain in CAHS, our study proposes the possibility of using engineered proteins or peptides derived from CAHS as a potential candidate for biological applications in extreme environmental stress responses.

Kinetic mechanism of human histidine triad nucleotide binding protein 1.

Human histidine triad nucleotide binding protein 1 (hHint1) is a member of a ubiquitous and ancient branch of the histidine triad protein superfamily. hHint1 is a homodimeric protein that catalyzes the hydrolysis of model substrates, phosphoramidate and acyl adenylate, with a high efficiency. Recently, catalytically inactive hHint1 has been identified as the cause of inherited peripheral neuropathy [Zimon, M., et al. (2012) Nat. Genet. 44, 1080-1083]. We have conducted the first detailed kinetic mechanistic studies of hHint1 and have found that the reaction mechanism is consistent with a double-displacement mechanism, in which the active site nucleophile His112 is first adenylylated by the substrate, followed by hydrolysis of the AMP-enzyme intermediate. A transient burst phase followed by a linear phase from the stopped-flow fluorescence assay indicated that enzyme adenylylation was faster than the subsequent intermediate hydrolysis and product release. Solvent viscosity experiments suggested that both chemical transformation and diffusion-sensitive events (product release or protein conformational change) limit the overall turnover. The catalytic trapping experiments and data simulation indicated that the true koff rate of the final product AMP is unlikely to control the overall kcat. Therefore, a protein conformational change associated with product release is likely rate-limiting. In addition, the rate of Hint1 adenylylation was found to be dependent on two residues with pKa values of 6.5 and 8, with the former pKa agreeing well with the nuclear magnetic resonance titration results for the pKa of the active site nucleophile His112. In comparison to the uncatalyzed rates, hHint1 was shown to enhance acyl-AMP and AMP phosphoramidate hydrolysis by 10(6)-10(8)-fold. Taken together, our analysis indicates that hHint1 catalyzes the hydrolysis of phosphoramidate and acyl adenylate with high efficiency, through a mechanism that relies on rapid adenylylation of the active residue, His112, while being partially rate-limited by intermediate hydrolysis and product release associated with a conformational change. Given the high degree of sequence homology of Hint proteins across all kingdoms of life, it is likely that their kinetic and catalytic mechanisms will be similar to those elucidated for hHint1.

Controlling the Chameleonic Behavior and Membrane Permeability of Cyclosporine Derivatives via Backbone and Side Chain Modifications.

Some macrocycles exhibit enhanced membrane permeability through conformational switching in different environmental polarities, a trait known as chameleonic behavior. In this study, we demonstrate specific backbone and side chain modifications that can control chameleonic behavior and passive membrane permeability using a cyclosporin O (CsO) scaffold. To quantify chameleonic behavior, we used a ratio of the population of the closed conformation obtained in polar solvent and nonpolar solvent for each CsO derivative. We found that ß-hydroxylation at position 1 (1 and 3) can encode chameleonicity and improve permeability. However, the conformational stabilization induced by adding an additional transannular H-bond (2 and 5) leads to a much slower rate of membrane permeation. Our CsO scaffold provides a platform for the systematic study of the relationship among conformation, membrane permeability, solubility, and protein binding. This knowledge contributes to the discovery of potent beyond the rule of five (bRo5) macrocycles capable of targeting undruggable targets.

NMR study on the B-Z junction formation of DNA duplexes induced by Z-DNA binding domain of human ADAR1.

Z-DNA is produced in a long genomic DNA by Z-DNA binding proteins, through formation of two B-Z junctions with the extrusion of one base pair from each junction. To answer the question of how Z-DNA binding proteins induce B-Z transitions in CG-rich segments while maintaining the B-conformation of surrounding segments, we investigated the kinetics and thermodynamics of base-pair openings of a 13-bp DNA in complex with the Z-DNA binding protein, Zα(ADAR1). We also studied perturbations in the backbone of Zα(ADAR1) upon binding to DNA. Our study demonstrates the initial contact conformation as an intermediate structure during B-Z junction formation induced by Zα(ADAR1), in which the Zα(ADAR1) protein displays unique backbone conformational changes, but the 13-bp DNA duplex maintains the B-form helix. We also found the unique structural features of the 13-bp DNA duplex in the initial contact conformation: (i) instability of the AT-rich region II and (ii) longer lifetime for the opening state of the CG-rich region I. Our findings suggest a three-step mechanism of B-Z junction formation: (i) Zα(ADAR1) specifically interacts with a CG-rich DNA segment maintaining B-form helix via a unique conformation; (ii) the neighboring AT-rich region becomes very unstable, and the CG-rich DNA segment is easily converted to Z-DNA; and (iii) the AT-rich regions are base-paired again, and the B-Z junction structure is formed.

Biophysical investigation of the dual binding surfaces of human transcription factors FOXO4 and p53.

Cellular senescence is protective against external oncogenic stress, but its accumulation causes aging-related diseases. Forkhead box O4 (FOXO4) and p53 are human transcription factors known to promote senescence by interacting with each other and activating p21 transcription. Inhibition of the interaction is a strategy for inducing apoptosis of senescent cells, but the binding surfaces that mediate the FOXO4-p53 interaction remain elusive. Here, we investigated two binding sites involved in the interaction between FOXO4 and p53 by NMR spectroscopy. NMR chemical shift perturbation analysis showed that the binding between FOXO4's forkhead domain (FHD) and p53's transactivation domain (TAD), and between FOXO4's C-terminal transactivation domain (CR3) and p53's DNA-binding domain (DBD), mediate the FOXO4-p53 interaction. Isothermal titration calorimetry data showed that both interactions have micromolar Kd values, and FOXO4 FHD-p53 TAD interaction has a higher binding affinity. We also showed that the intramolecular CR3-binding surface of FOXO4 FHD interacts with p53 TAD2, and FOXO4 CR3 interacts with the DNA/p53 TAD-binding surface of p53 DBD, suggesting a network of potentially competitive and/or coordinated interactions. Based on these results, we propose that a network of intramolecular and intermolecular interactions contributes to the two transcription factors' proper localisation on the p21 promoter and consequently promotes p21 transcription and cell senescence. This work provides structural information at the molecular level that is key to understanding the interplay of two proteins responsible for cellular senescence.

FOXO4 Transactivation Domain Interaction with Forkhead DNA Binding Domain and Effect on Selective DNA Recognition for Transcription Initiation.

Forkhead box O4 (FOXO4) is a human transcription factor (TF) that participates in cell homeostasis. While the structure and DNA binding properties of the conserved forkhead domain (FHD) have been thoroughly investigated, how the transactivation domain (TAD) regulates the DNA binding properties of the protein remains elusive. Here, we investigated the role of TAD in modulating the DNA binding properties of FOXO4 using solution NMR. We found that TAD and FHD form an intramolecular complex mainly governed by hydrophobic interaction. Remarkably, TAD and DNA share the same surface of FHD for binding. While FHD did not differentiate binding to target and non-target DNA, the FHD-TAD complex showed different behaviors depending on the DNA sequence. In the presence of TAD, free and DNA-bound FHD exhibited a slow exchange with target DNA and a fast exchange with non-target DNA. The interaction of the two domains affected the kinetic function of FHD depending on the type of DNA. Based on these findings, we suggest a transcription initiation model by which TAD modulates FOXO4 recognition of its target promoter DNA sequences. This study describes the function of TAD in FOXO4 and provides a new kinetic perspective on target sequence selection by TFs.