Sequential Binding Research Articles

A sequential binding mechanism for 5′ splice site recognition and modulation for the human U1 snRNP

Splice site recognition is essential for defining the transcriptome. Drugs like risdiplam and branaplam change how human U1 snRNP recognizes particular 5′ splice sites (5′SS) and promote U1 snRNP binding and splicing at these locations. Despite the therapeutic potential of 5′SS modulators, the complexity of their interactions and snRNP substrates have precluded defining a mechanism for 5′SS modulation. We have determined a sequential binding mechanism for modulation of −1A bulged 5′SS by branaplam using a combination of ensemble kinetic measurements and colocalization single molecule spectroscopy (CoSMoS). Our mechanism establishes that U1-C protein binds reversibly to U1 snRNP, and branaplam binds to the U1 snRNP/U1-C complex only after it has engaged with a −1A bulged 5′SS. Obligate orders of binding and unbinding explain how reversible branaplam interactions cause formation of long-lived U1 snRNP/5′SS complexes. Branaplam targets a ribonucleoprotein, not only an RNA duplex, and its action depends on fundamental properties of 5′SS recognition.

Structural proteomics defines a sequential priming mechanism for the progesterone receptor.

The progesterone receptor (PR) is a steroid-responsive nuclear receptor with two isoforms: PR-A and PR-B. Disruption of PR-A:PR-B signaling is associated with breast cancer through interactions with oncogenic co-regulatory proteins (CoRs). However, molecular details of isoform-specific PR-CoR interactions remain poorly understood. Using structural mass spectrometry, we investigate the sequential binding mechanism of purified full-length PR and intact CoRs, steroid receptor coactivator 3 (SRC3) and p300, as complexes on target DNA. Our findings reveal selective CoR NR-box binding by PR and unique interaction surfaces between PR and CoRs during complex assembly, providing a structural basis for CoR sequential binding on PR. Antagonist-bound PR showed persistent CoR interactions, challenging the classical model of nuclear receptor activation and repression. Collectively, we offer a peptide-level perspective on the organization of the PR transcriptional complex and infer the mechanisms behind the interactions of these proteins, both in active and inactive conformations.

First-principles study of vacancy-impurity (C, N, O) interactions in vanadium

Abstract Interstitial impurities of C, N, and O typically exist in V and its alloys, which have significant effects on the structure and properties of the materials due to their strong interaction with the vacancies. In this study, the structure and energy properties of vacancy-impurity complexes in V are comprehensively investigated by first principles calculations. The effects of impurity concentration and temperature on the concentration distribution of the complexes were studied using statistical methods. The results showed that the Vac n X 2 complexes exhibit a high sequential binding energy. There is an evident mutual attraction between the vacancy and the O atom, and it has a higher affinity than the C or N atom. The simple defect complexes of Vac 1 X 1–2 have always been the most abundant type of complex defect in V. The trapping effect of vacancy on C, N, and O atom is very significant at low temperatures rather than at high temperatures. This study would broaden the understanding of vacancy-impurity interactions in vanadium and provide new insights into the influence of C, N, and O on vacancy defect concentrations.

Effect of cyclodextrin modification on complexation thermodynamics with hexadecyltrimethylammonium cation with emphasis on subsequent surfactant micellization

Surfactant-cyclodextrin based complexes, are ideal guest-hosts for fundamental studies since both surfactant hydrophobic region with head group, and cyclodextrin cavity can be systematically varied. This allows, better understanding of the contributions that different chemical entities do to the stabilization of the complex and subsequent surfactant micellization. We studied systematically the interaction of hexadecyltrimethylammonium bromide (▪) with native α-, β- and γ-CDs, 2-hydroxypropylated and methylated α- and β-CDs, using isothermal titration calorimetry (ITC), measuring below critical micellization concentration (CMC) at least at three temperatures in the range (288.15 to 318.15) K. Data were treated simultaneously allowing the estimation of thermodynamically consistent temperature dependences of the equilibrium constant, the enthalpy and heat capacity for the inclusion complex formation. The data for cation/CD interaction were analysed mostly by the sequential binding model with 1:1 and 1:2 (cation:CD) stoichiometries, while some cation-CD combinations were examined using only 1:1, or more complicated 1:1, 2:1 and 2:2 stoichiometries. Thermodynamic quantities for complexation are discussed in terms of structural features, their temperature dependence with previous literature being examined. The shift of the CMC in presence of CDs was calculated founding large discrepancies with data in the literature. An extended mass-action chemical equilibrium model is proposed including free surfactant, free CD, complexes of the above mention stoichiometries, also including micelle, surfactant-cyclodextrin complexes of higher order stoichiometry, similar to that found for sodium dodecylsulfate-CDs systems. The new model provides a qualitative match with our own calorimetric titration curves measured above CMC for all studied ▪/CD combinations, and matches quantitatively with literature CMC values.

Structural basis of TMPRSS2 zymogen activation and recognition by the HKU1 seasonal coronavirus

The human seasonal coronavirus HKU1-CoV, which causes common colds worldwide, relies on the sequential binding to surface glycans and transmembrane serine protease 2 (TMPRSS2) for entry into target cells. TMPRSS2 is synthesized as a zymogen that undergoes autolytic activation to process its substrates. Several respiratory viruses, in particular coronaviruses, use TMPRSS2 for proteolytic priming of their surface spike protein to drive membrane fusion upon receptor binding. We describe the crystal structure of the HKU1-CoV receptor binding domain in complex with TMPRSS2, showing that it recognizes residues lining the catalytic groove. Combined mutagenesis of interface residues and comparison across species highlight positions 417 and 469 as determinants of HKU1-CoV host tropism. The structure of a receptor-blocking nanobody in complex with zymogen or activated TMPRSS2 further provides the structural basis of TMPRSS2 activating conformational change, which alters loops recognized by HKU1-CoV and dramatically increases binding affinity.

Analytical Aspects of ANSA-BSA Association: A Thermodynamic and Conformational Approach.

Many studies have demonstrated the manner in which ANS interacts with bovine serum albumin (BSA), although they are limited by the extremely low solubility of dye. The present study demonstrates the binding of ANSA dye with BSA, and since this dye can easily replace ANS, it not only simplifies research but also improves sensor accuracy for serum albumin. A combination of calorimetry and spectroscopy has been employed to establish the thermodynamic signatures associated with the interaction of ANSA with the protein and the consequent conformational changes in the latter. The results of differential scanning calorimetry reveal that when the concentration of ANSA in solution is increased, the thermal stability of the protein increases substantially. The fluorescence data demonstrated a decrease in the binding affinity of ANSA with the protein when pH increased but was unable to identify a change in the mode of interaction of the ligand. ITC has demonstrated that the mode of interaction between ANSA and the protein varies from a single set of binding sites at pH 5 and 7.4 to a sequential binding site at pH 10, emphasizing the potential relevance of protein conformational changes. TCSPC experiments suggested a dynamic type in the presence of ANSA. Molecular docking studies suggest that ANSA molecules are able to find ionic centers in the hydrophobic pockets of BSA. The findings further imply that given its ease of use in experiments, ANSA may be a useful probe for tracking the presence of serum albumin and partially folded protein states.

Heliorhodopsin-mediated light-modulation of ABC transporter

Heliorhodopsins (HeRs) have been hypothesized to have widespread functions. Recently, the functions for few HeRs have been revealed; however, the hypothetical functions remain largely unknown. Herein, we investigate light-modulation of heterodimeric multidrug resistance ATP-binding cassette transporters (OmrDE) mediated by Omithinimicrobium cerasi HeR. In this study, we classifiy genes flanking the HeR-encoding genes and identify highly conservative residues for protein–protein interactions. Our results reveal that the interaction between OcHeR and OmrDE shows positive cooperatively sequential binding through thermodynamic parameters. Moreover, light-induced OcHeR upregulates OmrDE drug transportation. Hence, the binding may be crucial to drug resistance in O. cerasi as it survives in a drug-containing habitat. Overall, we unveil a function of HeR as regulatory rhodopsin for multidrug resistance. Our findings suggest potential applications in optogenetic technology.

Exploring the interdependence of calcium and chloride activation of O2 evolution in photosystem II.

Calcium and chloride are activators of oxygen evolution in photosystem II (PSII), the light-absorbing water oxidase of higher plants, algae, and cyanobacteria. Calcium is an essential part of the catalytic Mn4CaO5 cluster that carries out water oxidation and chloride has two nearby binding sites, one of which is associated with a major water channel. The co-activation of oxygen evolution by the two ions is examined in higher plant PSII lacking the extrinsic PsbP and PsbQ subunits using a bisubstrate enzyme kinetics approach. Analysis of three different preparations at pH 6.3 indicates that the Michaelis constant, KM, for each ion is less than the dissociation constant, KS, and that the affinity of PSII for Ca2+ is about ten-fold greater than for Cl-, in agreement with previous studies. Results are consistent with a sequential binding model in which either ion can bind first and each promotes the activation by the second ion. At pH 5.5, similar results are found, except with a higher affinity for Cl- and lower affinity for Ca2+. Observation of the slow-decaying Tyr Z radical, YZ•, at 77 K and the coupled S2YZ• radical at 10 K, which are both associated with Ca2+ depletion, shows that Cl- is necessary for their observation. Given the order of electron and proton transfer events, this indicates that chloride is required to reach the S3 state preceding Ca2+ loss and possibly for stabilization of YZ• after it forms. Interdependence through hydrogen bonding is considered in the context of the water environment that intervenes between Cl- at the Cl-1 site and the Ca2+/Tyr Z region.

Identification of the potassium-binding site in serotonin transporter

Clearance of serotonin (5-hydroxytryptamine, 5-HT) from the synaptic cleft after neuronal signaling is mediated by serotonin transporter (SERT), which couples this process to the movement of a Na+ ion down its chemical gradient. After release of 5-HT and Na+ into the cytoplasm, the transporter faces a rate-limiting challenge of resetting its conformation to be primed again for 5-HT and Na+ binding. Early studies of vesicles containing native SERT revealed that K+ gradients can provide an additional driving force, via K+ antiport. Moreover, under appropriate conditions, a H+ ion can replace K+. Intracellular K+ accelerates the resetting step. Structural studies of SERT have identified two binding sites for Na+ ions, but the K+ site remains enigmatic. Here, we show that K+ antiport can drive substrate accumulation into vesicles containing SERT extracted from a heterologous expression system, allowing us to study the residues responsible for K+ binding. To identify candidate binding residues, we examine many cation binding configurations using molecular dynamics simulations, predicting that K+ binds to the so-called Na2 site. Site-directed mutagenesis of residues in this site can eliminate the ability of both K+ and H+ to drive 5-HT accumulation into vesicles and, in patch clamp recordings, prevent the acceleration of turnover rates and the formation of a channel-like state by K+ or H+. In conclusion, the Na2 site plays a pivotal role in orchestrating the sequential binding of Na+ and then K+ (or H+) ions to facilitate 5-HT uptake in SERT.

The apo-acyl coenzyme A binding protein of Leishmania major forms a unique ‘AXXA’ motif mediated dimer

Acyl-Coenzyme A binding domain containing proteins (ACBDs) are ubiquitous in nearly all eukaryotes. They can exist as a free protein, or a domain of a large, multidomain, multifunctional protein. Besides modularity, ACBDs also display multiplicity. The same organism may have multiple ACBDs, differing in sequence and organization. By virtue of this diversity, ACBDs perform functions ranging from transport, synthesis, trafficking, signal transduction, transcription, and gene regulation. In plants and some microorganisms, these ACBDs are designated ACBPs (acyl-CoA binding proteins). The simplest ACBD/ACBP is a small, ∼10 kDa, soluble protein, comprising the acyl-CoA binding (ACB) domain. Most of these small ACBDs exist as monomers, while a few show a tendency to oligomerize. In sync with those studies, we report the crystal structure of two ACBDs from Leishmania major, named ACBP103, and ACBP96 based on the number of residues present. Interestingly, ACBP103 crystallized as a monomer and a dimer under different crystallization conditions. Careful examination of the dimer disclosed an exposed ‘AXXA’ motif in the helix I of the two ACBP103 monomers, aligned in a head-to-tail arrangement in the dimer. Glutaraldehyde cross-linking studies confirm that apo-ACBP103 can self-associate in solution. Isothermal titration calorimetry studies further show that ACBP103 can bind ligands ranging from C8 – to C20-CoA, and the data could be best fit to a ‘two sets of sites’/sequential binding site model. Taken together, our studies show that Leishmania major ACBP103 can self-associate in the apo-form through a unique dimerization motif, an interaction that may play an important role in its function.

ARID1A orchestrates SWI/SNF-mediated sequential binding of transcription factors with ARID1A loss driving pre-memory B cell fate and lymphomagenesis

ARID1A, a subunit of the canonical BAF nucleosome remodeling complex, is commonly mutated in lymphomas. We show that ARID1A orchestrates B cell fate during the germinal center (GC) response, facilitating cooperative and sequential binding of PU.1 and NF-kB at crucial genes for cytokine and CD40 signaling. The absence of ARID1A tilts GC cell fate toward immature IgM+CD80-PD-L2- memory B cells, known for their potential to re-enter new GCs. When combined with BCL2 oncogene, ARID1A haploinsufficiency hastens the progression of aggressive follicular lymphomas (FLs) in mice. Patients with FL with ARID1A-inactivating mutations preferentially display an immature memory B cell-like state with increased transformation risk to aggressive disease. These observations offer mechanistic understanding into the emergence of both indolent and aggressive ARID1A-mutant lymphomas through the formation of immature memory-like clonal precursors. Lastly, we demonstrate that ARID1A mutation induces synthetic lethality to SMARCA2/4 inhibition, paving the way for potential precision therapy for high-risk patients.

Binding order of substrate and cofactor in sulfonamide monooxygenase during sulfa drug degradation: in silico studies

For decades, sulfonamide antibiotics have been used across industries such as agriculture and animal husbandry. However, the use and inadvertent misuse of these antibiotics have resulted in the advent of sulfonamide-drug-resistant strains due to antibiotic pollution. Enzymatic bioremediation of antibiotics remains a potential emerging solution to combat antibiotic pollution. Here, we propose an enzymatic model for the degradation of sulfonamides by Microbacterium sp. We have employed a multi-pronged computational strategy involving – protein structure modelling, ligand docking and molecular dynamics simulations to decipher a plausible binding order for the enzymatic degradation of sulfonamides by the bacterial sulfonamide monooxygenase, SulX. Our results enable us to predict that this degradation is achieved through the sequential binding of the antibiotic sulfonamide followed by the reduced flavin cofactor FMNH2, thereby laying the computational foundation for further advancements in enzyme-mediated degradation of the antibiotic. We also provide a list of experiments which may be performed to verify and follow-up on our in-silico studies. Communicated by Ramaswamy H. Sarma

DNA looping mediates cooperative transcription activation.

Transcription factors respond to multilevel stimuli and co-occupy promoter regions of target genes to activate RNA polymerase (RNAP) in a cooperative manner. To decipher the molecular mechanism, here we report two cryo-electron microscopy structures of Anabaena transcription activation complexes (TACs): NtcA-TAC composed of RNAP holoenzyme, promoter and a global activator NtcA, and NtcA-NtcB-TAC comprising an extra context-specific regulator, NtcB. Structural analysis showed that NtcA binding makes the promoter DNA bend by ∼50°, which facilitates RNAP to contact NtcB at the distal upstream NtcB box. The sequential binding of NtcA and NtcB induces looping back of promoter DNA towards RNAP, enabling the assembly of a fully activated TAC bound with two activators. Together with biochemical assays, we propose a 'DNA looping' mechanism of cooperative transcription activation in bacteria.

Microscale Thermophoresis Analysis of Chromatin Interactions.

Architectural DNA-binding proteins are key to the organization and compaction of genomic DNA inside cells. The activity of architectural proteins is often subject to further modulation and regulation through the interaction with a diverse array of other protein factors. Detailed knowledge on the binding modes involved is crucial for our understanding of how these protein-protein and protein-DNA interactions shape the functional landscape of chromatin in all kingdoms of life: bacteria, archaea, and eukarya.Microscale thermophoresis (MST) is a biophysical technique for the study of biomolecular interactions. It has seen increasing application in recent years thanks to its solution-based nature, rapid application, modest sample demand, and the sensitivity of the thermophoresis effect to binding events.Here, we describe the use of MST in the study of chromatin interactions. The emphasis lies on the wide range of ways in which these experiments are set up and the diverse types of information they reveal. These aspects are illustrated with four very different systems: the sequence-dependent DNA compaction by architectural protein HMfB, the sequential binding of core histone complexes to histone chaperone APLF, the impact of the nucleosomal context on the recognition of histone modifications, and the binding of a viral peptide to the nucleosome. Special emphasis is given to the key steps in the design, execution, and analysis of MST experiments in the context of the provided examples.

Gauging Dynamics-driven Allostery Using a New Computational Tool: A CAP Case Study

In this study, we utilize Protein Residue Networks (PRNs), constructed using Local Spatial Pattern (LSP) alignment, to explore the dynamic behavior of Catabolite Activator Protein (CAP) upon the sequential binding of cAMP. We employed the Degree Centrality of these PRNs to investigate protein dynamics on a sub-nanosecond time scale, hypothesizing that it would reflect changes in CAP's entropy related to its thermal motions. We show that the binding of the first cAMP led to an increase in stability in the Cyclic-Nucleotide Binding Domain A (CNBD-A) and destabilization in CNBD-B, agreeing with previous reports explaining the negative cooperativity of cAMP binding in terms of an entropy-driven allostery. LSP-based PRNs also allow for the study of Betweenness Centrality, another graph-theoretical characteristic of PRNs, providing insights into global residue connectivity within CAP. Using this approach, we were able to correctly identify amino acids that were shown to be critical in mediating allosteric interactions in CAP. The agreement between our studies and previous experimental reports validates our method, particularly with respect to the reliability of Degree Centrality as a proxy for entropy related to protein thermal dynamics. Because LSP-based PRNs can be easily extended to include dynamics of small organic molecules, polynucleotides, or other allosteric proteins, the methods presented here mark a significant advancement in the field, positioning them as vital tools for a fast, cost-effective, and accurate analysis of entropy-driven allostery and identification of allosteric hotspots.

Structural Basis of the Bivalency of the TRPV1 Agonist DkTx.

Bivalency is a prevalent natural mechanism to enhance receptor avidity. Various two-domain disulfide-rich peptides exhibiting bivalent action have been identified from animal venoms. A unique characteristic of these peptides is that they induce a pharmacological response different from that provoked by any of the constituent domains. The enhanced potency and avidity of such peptides is therefore a consequence of their domain fusion by a peptide linker. The role of the linker itself, beyond conjugation, remains unclear. Here, we investigate how the linker affects the bivalency of the capsaicin receptor (TRPV1) agonist DkTx. We recombinantly produced isotope labelled DkTx using a protein splicing approach, to solve the high-resolution solution structure of DkTx, revealing residual linker order stabilised by linker-domain interactions leading to biased domain orientations. The significance of this was studied using a combination of mutagenesis, spin relaxation studies and electrophysiology measurements. Our results reveal that disrupting the pre-organisation of the domains of DkTx is accompanied by reductions in potency and onset of avidity. Our findings support a model of pre-configured two-domain binding, in favour of the previously suggested sequential binding model. This highlights the significance of ordered elements in linker design and the natural evolution of these in bivalent toxins.

Structural Basis of the Bivalency of the TRPV1 Agonist DkTx

AbstractBivalency is a prevalent natural mechanism to enhance receptor avidity. Various two‐domain disulfide‐rich peptides exhibiting bivalent action have been identified from animal venoms. A unique characteristic of these peptides is that they induce a pharmacological response different from that provoked by any of the constituent domains. The enhanced potency and avidity of such peptides is therefore a consequence of their domain fusion by a peptide linker. The role of the linker itself, beyond conjugation, remains unclear. Here, we investigate how the linker affects the bivalency of the capsaicin receptor (TRPV1) agonist DkTx. We recombinantly produced isotope labelled DkTx using a protein splicing approach, to solve the high‐resolution solution structure of DkTx, revealing residual linker order stabilised by linker‐domain interactions leading to biased domain orientations. The significance of this was studied using a combination of mutagenesis, spin relaxation studies and electrophysiology measurements. Our results reveal that disrupting the pre‐organisation of the domains of DkTx is accompanied by reductions in potency and onset of avidity. Our findings support a model of pre‐configured two‐domain binding, in favour of the previously suggested sequential binding model. This highlights the significance of ordered elements in linker design and the natural evolution of these in bivalent toxins.

Abstract C114: Dose escalation results from a first-in-human, phase I/II study of GB263T, a novel EGFR/cMET/cMET trispecific antibody, in patients with advanced EGFR-mutated (EGFRm) non-small cell lung cancer (NSCLC)

Abstract Background: GB263T, a novel trispecific antibody directed against EGFR and cMET, adopts the design of two humanized VHH antibodies that recognize two different cMET epitopes, in which a conformational change induced by the first VHH binding exposes the second epitope for the second VHH to bind. Sequential binding of the two VHH antibodies to cMET increases tumor specificity, which results in widening of the safety window. In vitro studies show that GB263T induces better antigen-antibody molecular endocytosis, more completely blocks the signal transduction pathway, and has better ADCC activity compared to a benchmark. In addition, GB263T has demonstrated promising pre-clinical activity in models with various EGFR/cMET alterations, including those with high medical need. Here, we report updated dose escalation results of the first-in-human phase I/II study of GB263T in patients (pts) with advanced EGFRm NSCLC (NCT05332574). Methods: This multicenter (China and Australia), phase I/II study was conducted using an accelerated titration method followed by a traditional 3+3 design for dose escalation. Pts with EGFRm NSCLC with prior EGFR TKI and platinum-based chemotherapy were enrolled. GB263T was given at 140-1680 mg (5 dose cohorts) IV weekly for the first two 28-day cycles and biweekly thereafter. Objectives were to evaluate the safety and tolerability, determine the maximum tolerated dose (MTD) and the recommended Phase 2 dose (RP2D), assess the pharmacokinetics, immunogenicity, and preliminary efficacy of GB263T. Results: As of July 5, 2023, 13 pts were treated: 140mg (n=1), 420mg (n=1), 840mg (n=3), 1260mg (n=3), 1680mg (n=5). The enrolment of the 1680mg cohort is ongoing. All pts had received previous third-generation EGFR-TKI and platinum-based chemotherapy. Median number of prior lines of systemic therapy was 3 (range 1-7). One patient at 1680mg experienced DLT (grade 3 oral mucositis, which has resolved after symptomatic treatment). The most common treatment-related adverse events (TRAEs) were rash (61.5%), infusion related reaction (38.5%), fatigue (30.8%) and myalgia (23.1%), and all are mild (grade 1/2). Only one patient developed ≥grade 3 TRAE (grade 3 oral mucositis). There were no treatment-related discontinuations. Among 10 response-evaluable pts, two achieved partial response (PR) and four achieved stable disease (SD) with tumor shrinkage observed in 3/4 SD pts. The disease control rate (DCR) is 60%. The objective response rate (ORR) at the therapeutic dose range (1260-1680mg) is 40% (2/5). Two PR pts and 2/4 SD pts remained on treatment at data cutoff. Conclusions: Results to date demonstrate a good safety profile with promising efficacy at the therapeutic dose range (1260-1680mg). Continued dose escalation and clinical investigation of GB263T is ongoing.Clinical trial information: NCT05332574. Research Sponsor: Genor Biopharma Co. Ltd. Citation Format: Jin-Ji Yang, Nick Pavlakis, Fan Xie, Tong Li, Yi-Long Wu. Dose escalation results from a first-in-human, phase I/II study of GB263T, a novel EGFR/cMET/cMET trispecific antibody, in patients with advanced EGFR-mutated (EGFRm) non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C114.

Dimensional interaction is driven by feature integration: A binding theory of garner interference.

For nearly half a century now, Garner interference has been serving as the gold standard measure of dimensional interaction and selective attention. But the mechanisms that generate Garner interference are still not well understood. The current study proposes a novel theory that ascribes the interference (and dimensional interaction in general) to episodic feature integration processes at the micro (trial-to-trial) level. The novel account builds on earlier well-established notions of "feature integration" and "object files," and is augmented by formal derivations. The sequential binding account predicts that the magnitude of Garner interference is related to the strength of feature integration along consecutive trials. Three experiments were set to test this novel binding theory. Experiment 1 and Experiment 2 tested performance with integral dimensions (chroma and value, and width and height of rectangles); whereas Experiment 3 examined performance with a pair of separable dimensions (circle's size and angle of a diameter). In addition, the time lag ensuing between consecutive trials was manipulated. The results strongly supported the predictions of the sequential binding account: (a) with integral dimensions, substantial amounts of Garner interference were correlated with large partial repetition costs (e.g., consensual markers of feature integration), but this pattern was not observed with separable dimensions, and (b) the magnitude of both Garner interference and partial repetition costs diminished as a function of the ensuing time lag between consecutive trials, pointing to a common time-dependent memory mechanism. These results adduce strong support in the predictions of the feature binding theory of Garner interference, giving currency to the idea that dimensional interaction is driven by feature integration. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

SWI/SNF Chromatin Remodeling Complex Orchestrates Sequential Binding of Key Transcription Factors in B Cells and Restricts Aggressive Lymphoma

Mutations in the SWI/SNF chromatin remodeling complex BAF are a recurring feature in many cancers, with a high prevalence in follicular lymphoma (FL; >19%) and diffuse large B-cell lymphoma (DLBCL; >34%). Despite this, the mechanism that links mutations in ARID1A, most frequently mutated BAF subunit, to the development of lymphoma is still not understood. To this aim, we crossed Arid1a-floxed mice with Cγ1-Cre mice to yield offspring with a conditional (cKO) Arid1a deletion in germinal center (GC) B cells, FL and DLBCL cell-of-origin. We immunized WT/WT ( Arid1a+/+; Cγ1Cre/+) and cKO/WT ( Arid1a+/-; Cγ1Cre/+) mice with sheep red blood cells and analyzed 10 days later at the peak of the GC reaction. Upon Arid1a deletion, we found a decrease in the total number of GC B cells relative to total B cells (WT/WT vs. cKO/WT; p<0.001). However, we observed skewing of GC polarity manifesting as increased proportions of proliferating centroblasts (CB) vs. centrocytes (CC) (WT/WT vs. cKO/WT; p=0.004). These observations imply a substantial dysfunction at the CC stage, which could account for the overall reduced number of GC B-cells. To investigate this, we performed a detailed molecular characterization of these cells. We performed the ATAC-seq assay on sorted CB and CC from our mouse model and observed an extensive loss of chromatin accessibility. Changes in chromatin accessibility are thought to be dependent on the recruitment of the BAF complex by specific transcription factors. We discovered strong enrichment for DNA motifs of PU.1 and NF-kB factors in closing chromatin (p-value <0.001), indicating their dependence on the BAF complex. Furthermore, we noted that this chromatin closing occurred in proximity to canonical GC exit programs, including genes induced by the CD40, NF-kB signaling, IRF4, STAT3, IL2, IL4, IL6 and Notch pathways (hypergeometric p-value <0.001), indicating GC-exit perturbation. Expanding upon our findings, we leveraged a combined single-cell RNA and ATAC assay (Multiome) to scrutinize subtle shifts within GC populations. Determining the chromatin accessibility within the pseudo-time trajectory of GC transitions, we found that PU.1 and NF-kB factors are chronologically dependent on ARID1A with PU.1 accessibility initially compromised, followed by a subsequent decrease at binding sites for NF-kB factors. This sequence reveals ARID1A's unrecognized function as a regulator of temporal dynamics of these key transcription factors. Strikingly, our Multiome data revealed expansion of the pre-memory B cells accompanied by a decrease in the proportion of pre-plasma cells upon Arid1a deletion (p-value <0.001), suggesting that Arid1a loss favors GC exit towards the memory cell fate. To validate our findings, we performed immunophenotyping and observed an increase in memory B cells in cKO/WT mice (p=0.019), and a decrease in long-lived plasma cells (p=0.012) upon NP-KLH immunization. Furthermore, we observed that the absence of ARID1A tilts GC cell-fate towards immature IgM+CD80-PDL2- memory B cells (p=0.0016), known for their potential to re-enter new GCs. Linking this critical role of ARID1A in chromatin regulation to lymphomagenesis, we observed a reduction in overall survival for mice carrying VavP-Bcl2; Arid1a+/- allele (compared to VavP-Bcl2; Arid1a+/+; p=0.0087). Remarkably, we further show that FL patients with ARID1A-inactivating mutations display an immature memory B-cell-like state with increased transformation risk to aggressive disease (p=0.0391). These observations offer mechanistic understanding into the emergence of both indolent and aggressive lymphomas in ARID1A-mutant patients through formation of immature memory-like clonal precursors.