Cis Interactions Research Articles

Cohesin-dependent chromosome loop extrusion is limited by transcription and stalled replication forks.

Genome function depends on regulated chromosome folding, and loop extrusion by the protein complex cohesin is essential for this multilayered organization. The chromosomal positioning of cohesin is controlled by transcription, and the complex also localizes to stalled replication forks. However, the role of transcription and replication in chromosome looping remains unclear. Here, we show that reduction of chromosome-bound RNA polymerase weakens normal cohesin loop extrusion boundaries, allowing cohesin to form new long-range chromosome cis interactions. Stress response genes induced by transcription inhibition are also shown to act as new loop extrusion boundaries. Furthermore, cohesin loop extrusion during early S phase is jointly controlled by transcription and replication units. Together, the results reveal that replication and transcription machineries are chromosome-folding regulators that block the progression of loop-extruding cohesin, opening for new perspectives on cohesin’s roles in genome function and stability.

Thy-1-Integrin Interactions in cis and Trans Mediate Distinctive Signaling.

Thy-1 is a cell surface glycosylphosphatidylinositol (GPI)-anchored glycoprotein that bears a broad mosaic of biological roles across various cell types. Thy-1 displays strong physiological and pathological implications in development, cancer, immunity, and tissue fibrosis. Quite uniquely, Thy-1 is capable of mediating integrin-related signaling through direct trans- and cis-interaction with integrins. Both interaction types have shown distinctive roles, even when interacting with the same type of integrin, where binding in trans or in cis often yields divergent signaling events. In this review, we will revisit recent progress and discoveries of Thy-1–integrin interactions in trans and in cis, highlight their pathophysiological consequences and explore other potential binding partners of Thy-1 within the integrin regulation/signaling paradigm.

Lymphocyte activation gene-3-associated protein networks are associated with HDL-cholesterol and mortality in the Trans-omics for Precision Medicine program

Deficiency of the immune checkpoint lymphocyte activation gene-3 (LAG3) protein is significantly associated with both elevated HDL-cholesterol (HDL-C) and myocardial infarction risk. We determined the association of genetic variants within ±500 kb of LAG3 with plasma LAG3 and defined LAG3-associated plasma proteins with HDL-C and clinical outcomes. Whole genome sequencing and plasma proteomics were obtained from the Multi-Ethnic Study of Atherosclerosis (MESA) and the Framingham Heart Study (FHS) cohorts as part of the Trans-Omics for Precision Medicine program. In situ Hi-C chromatin capture was performed in EBV-transformed cell lines isolated from four MESA participants. Genetic association analyses were performed in MESA using multivariate regression models, with validation in FHS. A LAG3-associated protein network was tested for association with HDL-C, coronary heart disease, and all-cause mortality. We identify an association between the LAG3 rs3782735 variant and plasma LAG3 protein. Proteomics analysis reveals 183 proteins significantly associated with LAG3 with four proteins associated with HDL-C. Four proteins discovered for association with all-cause mortality in FHS shows nominal associations in MESA. Chromatin capture analysis reveals significant cis interactions between LAG3 and C1S, LRIG3, TNFRSF1A, and trans interactions between LAG3 and B2M. A LAG3-associated protein network has significant associations with HDL-C and mortality.

Combining Coarse-Grained Simulations and Single Molecule Analysis Reveals a Three-State Folding Model of the Guanidine-II Riboswitch.

Riboswitch RNAs regulate gene expression by conformational changes induced by environmental conditions and specific ligand binding. The guanidine-II riboswitch is proposed to bind the small molecule guanidinium and to subsequently form a kissing loop interaction between the P1 and P2 hairpins. While an interaction was shown for isolated hairpins in crystallization and electron paramagnetic resonance experiments, an intrastrand kissing loop formation has not been demonstrated. Here, we report the first evidence of this interaction in cis in a ligand and Mg2+ dependent manner. Using single-molecule FRET spectroscopy and detailed structural information from coarse-grained simulations, we observe and characterize three interconvertible states representing an open and kissing loop conformation as well as a novel Mg2+ dependent state for the guanidine-II riboswitch from E. coli. The results further substantiate the proposed switching mechanism and provide detailed insight into the regulation mechanism for the guanidine-II riboswitch class. Combining single molecule experiments and coarse-grained simulations therefore provides a promising perspective in resolving the conformational changes induced by environmental conditions and to yield molecular insights into RNA regulation.

Lfng and Dll3 cooperate to modulate protein interactions in cis and coordinate oscillatory Notch pathway activation in the segmentation clock

In mammalian development, oscillatory activation of Notch signaling is required for segmentation clock function during somitogenesis. Notch activity oscillations are synchronized between neighboring cells in the presomitic mesoderm (PSM) and have a period that matches the rate of somite formation. Normal clock function requires cyclic expression of the Lunatic fringe (LFNG) glycosyltransferase, as well as expression of the inhibitory Notch ligand Delta-like 3 (DLL3). How these factors coordinate Notch activation in the clock is not well understood. Recent evidence suggests that LFNG can act in a signal-sending cell to influence Notch activity in the clock, raising the possibility that in this context, glycosylation of Notch pathway proteins by LFNG may affect ligand activity. Here we dissect the genetic interactions of Lfng and Dll3 specifically in the segmentation clock and observe distinctions in the skeletal and clock phenotypes of mutant embryos showing that paradoxically, loss of Dll3 is associated with strong reductions in Notch activity in the caudal PSM. The patterns of Notch activity in the PSM suggest that the loss of Dll3 is epistatic to the loss of Lfng in the segmentation clock, and we present direct evidence for the modification of several DLL1 and DLL3 EGF-repeats by LFNG. We further demonstrate that DLL3 expression in cells co-expressing DLL1 and NOTCH1 can potentiate a cell's signal-sending activity and that this effect is modulated by LFNG, suggesting a mechanism for coordinated regulation of oscillatory Notch activation in the clock by glycosylation and cis-inhibition.

Rapid cloning of antigen-specific T-cell receptors by leveraging the cis activation of T cells.

It is commonly understood that T cells are activated via trans interactions between antigen-specific T-cell receptors (TCRs) and antigenic peptides presented on major histocompatibility complex (MHC) molecules on antigen-presenting cells. By analysing a large number of T cells at the single-cell level on a microwell array, we show that T-cell activation can occur via cis interactions (where TCRs on the T cell interact with the antigenic peptides presented on MHC class-I molecules on the same cell), and that such cis activation can be used to detect antigen-specific T cells and clone their TCR within 4 d. We used the detection-and-cloning system to clone a tumour-antigen-specific TCR from peripheral blood mononuclear cells of healthy donors. TCR cloning by leveraging the cis activation of T cells may facilitate the development of TCR-engineered T cells for cancer therapy.

Oncogenic gene expression and epigenetic remodeling of cis-regulatory elements in ASXL1-mutant chronic myelomonocytic leukemia

Myeloid neoplasms are clonal hematopoietic stem cell disorders driven by the sequential acquisition of recurrent genetic lesions. Truncating mutations in the chromatin remodeler ASXL1 (ASXL1MT) are associated with a high-risk disease phenotype with increased proliferation, epigenetic therapeutic resistance, and poor survival outcomes. We performed a multi-omics interrogation to define gene expression and chromatin remodeling associated with ASXL1MT in chronic myelomonocytic leukemia (CMML). ASXL1MT are associated with a loss of repressive histone methylation and increase in permissive histone methylation and acetylation in promoter regions. ASXL1MT are further associated with de novo accessibility of distal enhancers binding ETS transcription factors, targeting important leukemogenic driver genes. Chromatin remodeling of promoters and enhancers is strongly associated with gene expression and heterogenous among overexpressed genes. These results provide a comprehensive map of the transcriptome and chromatin landscape of ASXL1MT CMML, forming an important framework for the development of novel therapeutic strategies targeting oncogenic cis interactions.

How clustered protocadherin binding specificity is tuned for neuronal self-/nonself-recognition.

The stochastic expression of fewer than 60 clustered protocadherin (cPcdh) isoforms provides diverse identities to individual vertebrate neurons and a molecular basis for self-/nonself-discrimination. cPcdhs form chains mediated by alternating cis and trans interactions between apposed membranes, which has been suggested to signal self-recognition. Such a mechanism requires that cPcdh cis dimers form promiscuously to generate diverse recognition units, and that trans interactions have precise specificity so that isoform mismatches terminate chain growth. However, the extent to which cPcdh interactions fulfill these requirements has not been definitively demonstrated. Here, we report biophysical experiments showing that cPcdh cis interactions are promiscuous, but with preferences favoring formation of heterologous cis dimers. Trans homophilic interactions are remarkably precise, with no evidence for heterophilic interactions between different isoforms. A new C-type cPcdh crystal structure and mutagenesis data help to explain these observations. Overall, the interaction characteristics we report for cPcdhs help explain their function in neuronal self-/nonself-discrimination.

Long non-coding RNAs in Sus scrofa ileum under starvation stress.

ObjectiveIn this study, we aimed to identify long non-coding RNAs (lncRNAs) that play important roles in starvation stress, analyze their functions, and discover potential molecular targets to alleviate starvation stress to provide a theoretical reference for subsequent in-depth research.MethodsWe generated a piglet starvation stress animal model. Nine Yorkshire weaned piglets were randomly divided into a long-term starvation stress group (starved for 72 h), short-term starvation stress group (starved for 48 h), and the control group. LncRNA libraries were constructed using high-throughput sequencing of piglet ileums.ResultsWe obtained 11,792 lncRNAs, among which, 2,500 lncRNAs were novel. In total, 509 differentially expressed (DE)lncRNAs were identified in this study. Target genes of DElncRNAs were predicted via cis and trans interactions, and functional and pathway analyses were performed. Gene ontology functions and Kyoto encyclopedia of genes and genomes analysis revealed that lncRNA-targeted genes mainly participated in metabolic pathways, cellular processes, immune system processes, digestive systems, and transport activities. To reveal the mechanism underlying starvation stress, the interaction network between lncRNAs and their targets was constructed based on 26 DElncRNAs and 72 DEmRNAs. We performed an interaction network analysis of 121 DElncRNA–DEmRNA pairs with a Pearson correlation coefficient greater than 0.99.ConclusionWe found that MSTRG.19894.13, MSTRG.16726.3, and MSTRG.12176.1 might play important roles in starvation stress. This study not only generated a library of enriched lncRNAs in piglets, but its outcomes also provide a strong foundation to screen key lncRNAs involved in starvation stress and a reference for subsequent in-depth research.

Collective mechanical responses of cadherin-based adhesive junctions as predicted by simulations

Cadherin-based adherens junctions and desmosomes help stabilize cell-cell contacts with additional function in mechano-signaling, while clustered protocadherin junctions are responsible for directing neuronal circuits assembly. Structural models for adherens junctions formed by epithelial cadherin (CDH1) proteins indicate that their long, curved ectodomains arrange to form a periodic, two-dimensional lattice stabilized by tip-to-tip trans interactions (across junction) and lateral cis contacts. Less is known about the exact architecture of desmosomes, but desmoglein (DSG) and desmocollin (DSC) cadherin proteins are also thought to form ordered junctions. In contrast, clustered protocadherin (PCDH)-based cell-cell contacts in neuronal tissues are thought to be responsible for self-recognition and avoidance, and structural models for clustered PCDH junctions show a linear arrangement in which their long and straight ectodomains form antiparallel overlapped trans complexes. Here, we report all-atom molecular dynamics simulations testing the mechanics of minimalistic adhesive junctions formed by CDH1, DSG2 coupled to DSC1, and PCDHγB4, with systems encompassing up to 3.7 million atoms. Simulations generally predict a favored shearing pathway for the adherens junction model and a two-phased elastic response to tensile forces for the adhesive adherens junction and the desmosome models. Complexes within these junctions first unbend at low tensile force and then become stiff to unbind without unfolding. However, cis interactions in both the CDH1 and DSG2-DSC1 systems dictate varied mechanical responses of individual dimers within the junctions. Conversely, the clustered protocadherin PCDHγB4 junction lacks a distinct two-phased elastic response. Instead, applied tensile force strains trans interactions directly, as there is little unbending of monomers within the junction. Transient intermediates, influenced by new cis interactions, are observed after the main rupture event. We suggest that these collective, complex mechanical responses mediated by cis contacts facilitate distinct functions in robust cell-cell adhesion for classical cadherins and in self-avoidance signaling for clustered PCDHs.

Complete modeling of PrPc in the cis interaction employing novel transpeptidation

The pathogenesis of prion diseases is initiated when the cellular prion protein (PrPC) is misfolded into the scrapie prion protein (PrPSc). PrPC is composed of two major domains, the N-terminal intrinsically disordered tail and the C-terminal globular domain. These domains interact with each other via a copper(II)-driven cis interaction. The proposed model of this tertiary structure is a Cu2+ ion coordinated to three histidines in the N-terminal tail and one histidine in the C-terminal domain. To test this hypothesis, the N-terminal tail of PrPC is segmentally 15N-isoptically labeled via transpeptidation by 7M sortase A enzyme.

Bioinformatic Analyses of the Ferroptosis-Related lncRNAs Signature for Ovarian Cancer.

Both ferroptosis and lncRNAs are significant for ovarian cancer (OC). Whereas, the study of ferroptosis-related lncRNAs (FRLs) still few in ovarian cancer. We first constructed an FRL-signature for patients with OC in the study. A total of 548 FRLs were identified for univariate Cox regression analysis, and 21 FRLs with significant prognosis were identified. The prognostic characteristics of nine FRLs was constructed and validated, showing opposite prognosis in two subgroups based on risk scores. The multivariate Cox regression analysis and nomogram further verified the prognostic value of the risk model. By calculating ferroptosis score through ssGSEA, we found that patients with higher risk scores exhibited higher ferroptosis scores, and high ferroptosis score was a risk factor. There were 40 microenvironment cells with significant differences in the two groups, and the difference of Stromal score between the two groups was statistically significant. Six immune checkpoint genes were expressed at different levels in the two groups. In addition, five m6A regulators (FMR1, HNRNPC, METTL16, METTL3, and METTL5) were higher expressed in the low-risk group. GSEA revealed that the risk model was associated with tumor-related pathways and immune-associated pathway. We compared the sensitivity of chemotherapy drugs between the two risk groups. We also explored the co-expression, ceRNA relation, cis and trans interaction of ferroptosis-related genes and lncRNAs, providing a new idea for the regulatory mechanisms of FRLs. Moreover, the nine FRLs were selected for detecting their expression levels in OC cells and tissues.

Eph/Ephrin-Based Protein Complexes: The Importance of cis Interactions in Guiding Cellular Processes.

Although intracellular signal transduction is generally represented as a linear process that transmits stimuli from the exterior of a cell to the interior via a transmembrane receptor, interactions with additional membrane-associated proteins are often critical to its success. These molecules play a pivotal role in mediating signaling via the formation of complexes in cis (within the same membrane) with primary effectors, particularly in the context of tumorigenesis. Such secondary effectors may act to promote successful signaling by mediating receptor-ligand binding, recruitment of molecular partners for the formation of multiprotein complexes, or differential signaling outcomes. One signaling family whose contact-mediated activity is frequently modulated by lateral interactions at the cell surface is Eph/ephrin (EphA and EphB receptor tyrosine kinases and their ligands ephrin-As and ephrin-Bs). Through heterotypic interactions in cis, these molecules can promote a diverse range of cellular activities, including some that are mutually exclusive (cell proliferation and cell differentiation, or adhesion and migration). Due to their broad expression in most tissues and their promiscuous binding within and across classes, the cellular response to Eph:ephrin interaction is highly variable between cell types and is dependent on the cellular context in which binding occurs. In this review, we will discuss interactions between molecules in cis at the cell membrane, with emphasis on their role in modulating Eph/ephrin signaling.

Determination of protein-protein interactions at the single-molecule level using optical tweezers.

Biomolecular interactions are at the base of all physical processes within living organisms; the study of these interactions has led to the development of a plethora of different methods. Among these, single-molecule (in singulo) experiments have become relevant in recent years because these studies can give insight into mechanisms and interactions that are hidden for ensemble-based (in multiplo) methods. The focus of this review is on optical tweezer (OT) experiments, which can be used to apply and measure mechanical forces in molecular systems. OTs are based on optical trapping, where a laser is used to exert a force on a dielectric bead; and optically trap the bead at a controllable position in all three dimensions. Different experimental approaches have been developed to study protein–protein interactions using OTs, such as: (1) refolding and unfolding in trans interaction where one protein is tethered between the beads and the other protein is in the solution; (2) constant force in cis interaction where each protein is bound to a bead, and the tension is suddenly increased. The interaction may break after some time, giving information about the lifetime of the binding at that tension. And (3) force ramp in cis interaction where each protein is attached to a bead and a ramp force is applied until the interaction breaks. With these experiments, parameters such as kinetic constants (koff, kon), affinity values (KD), energy to the transition state ΔG≠, distance to the transition state Δx≠ can be obtained. These parameters characterize the energy landscape of the interaction. Some parameters such as distance to the transition state can only be obtained from force spectroscopy experiments such as those described here.

Thy-1-Integrin Interactions in cis and Trans Mediate Distinctive Signaling

Thy-1-Integrin Interactions in cis and Trans Mediate Distinctive Signaling

Development of lacto-series ganglioside fluorescent probe using late-stage sialylation and behavior analysis with single-molecule imaging.

Gangliosides are a family of sialic-acid-containing glycosphingolipids that form dynamic domains (lipid rafts) with proteins in cell plasma membranes (PMs), and are involved in various biological processes. The dynamic behavior of gangliosides can be elucidated by analyzing fluorescently-labeled molecules with a powerful technique known as single-molecule imaging. We previously developed fluorescent probes for ganglioside subfamilies such as the ganglio- and globo-series, and investigated their behavior in cell PMs. This study targeted a lacto-series ganglioside, sialyl-lactotetraosylceramide, whose behavior in PMs has not yet been investigated. We applied a recently reported method for the direct sialylation of oligosaccharyl lipid acceptors to synthesize the fluorescent ganglioside probes. The glycolipid acceptor exhibited high solubility in organic solvents owing to the installation of a large quantity of p-tert-butylbenzoyl protecting groups, which ensured direct α-sialylation at relatively low temperatures. Biophysical evaluation of the synthesized probe determined that it behaved as a raft molecule in cell PMs. Furthermore, single-molecule imaging revealed cis interactions between the lacto-series ganglioside and a major raft molecule (GPI-anchored protein CD59). Moreover, the fluorescent non-sialylated (asialyl) lactotetraosylceramide behaved similarly to its sialyl counterpart.

Molecular self-avoidance in synaptic neurexin complexes.

Synapses are thought to be organized by interactions of presynaptic neurexins with postsynaptic ligands, particularly with neuroligins and cerebellins. However, when a neuron forms adjacent pre- and postsynaptic specializations, as in dendrodendritic or axo-axonic synapses, nonfunctional cis neurexin/ligand interactions would be energetically favored. Here, we reveal an organizational principle for preventing synaptic cis interactions (“self-avoidance”). Using dendrodendritic synapses between mitral and granule cells in the olfactory bulb as a paradigm, we show that, owing to its higher binding affinity, cerebellin-1 blocks the cis interaction of neurexins with neuroligins, thereby enabling trans neurexin/neuroligin interaction. In mitral cells, ablating either cerebellin-1 or neuroligins severely impaired granule cell➔mitral cell synapses, as did overexpression of wild-type neurexins but not of mutant neurexins unable to bind to neuroligins. Our data uncover a molecular interaction network that organizes the self-avoidance of nonfunctional neurexin/ligand cis interactions, thus allowing assembly of physiological trans interactions.

New locus underlying auriculocondylar syndrome (ARCND): 430 kb duplication involving TWIST1 regulatory elements

BackgroundAuriculocondylar syndrome (ARCND) is a rare genetic disease that affects structures derived from the first and second pharyngeal arches, mainly resulting in micrognathia and auricular malformations. To date, pathogenic variants...

Global profiling of RNA-chromatin interactions reveals co-regulatory gene expression networks in Arabidopsis.

It is increasingly evident that various RNAs can bind chromatin to regulate gene expression and genome organization. Here we adapted a sequencing-based technique to profile RNA-chromatin interactions at a genome-wide scale in Arabidopsis seedlings. We identified more than 10,000 RNA-chromatin interactions mediated by protein-coding RNAs and non-coding RNAs. Cis and intra-chromosomal interactions are mainly mediated by protein-coding RNAs, whereas inter-chromosomal interactions are primarily mediated by non-coding RNAs. Many RNA-chromatin interactions tend to positively correlate with DNA-DNA interactions, suggesting their mutual influence and reinforcement. We further show that some RNA-chromatin interactions undergo alterations in response to biotic and abiotic stresses and that altered RNA-chromatin interactions form co-regulatory networks. Our study provides a global view on RNA-chromatin interactions in Arabidopsis and a rich resource for future investigations of regulatory roles of RNAs in gene expression and genome organization.

Comprehensive analyses of glycolysis-related lncRNAs for ovarian cancer patients

BackgroundNot only glycolysis but also lncRNAs play a significant role in the growth, proliferation, invasion and metastasis of of ovarian cancer (OC). However, researches about glycolysis -related lncRNAs (GRLs) remain unclear in OC. Herein, we first constructed a GRL-based risk model for patients with OC.MethodsThe processed RNA sequencing (RNA-seq) profiles with clinicopathological data were downloaded from TCGA and glycolysis-related genes (GRGs) were obtained from MSigDB. Pearson correlation coefficient between glycolysis-related genes (GRGs) and annotated lncRNAs (|r| > 0.4 and p < 0.05) were calculated to identify GRLs. After screening prognostic GRLs, a risk model based on five GRLs was constructed using Univariate and Cox regression. The identified risk model was validated by two validation sets. Further, the differences in clinicopathology, biological function, hypoxia score, immune microenvironment, immune checkpoint, immune checkpoint blockade, chemotherapy drug sensitivity, N6-methyladenosine (m6A) regulators, and ferroptosis-related genes between risk groups were explored by abundant algorithms. Finally, we established networks based on co-expression, ceRNA, cis and trans interaction.ResultsA total of 535 GRLs were gained and 35 GRLs with significant prognostic value were identified. The prognostic signature containing five GRLs was constructed and validated and can predict prognosis. The nomogram proved the accuracy of the model for predicting prognosis. After computing hypoxia score of each sample by ssGSEA, we found patients with higher risk scores exhibited higher hypoxia score and high hypoxia score was a risk factor. It was revealed that a total of 21 microenvironment cells (such as Central memory CD4 T cell, Neutrophil, Regulatory T cell and so on) and Stromal score had significant differences between the two groups. Four immune checkpoint genes (CD274, LAG3, VTCN1, and CD47) showed disparate expression levels in the two groups. Besides, 16 m6A regulators and 126 ferroptosis-related genes were expressed higher in the low-risk group. GSEA revealed that the risk groups were associated with tumor-related pathways. The two risk groups were confirmed to be sensitive to several chemotherapeutic agents and patients in the low-risk group were more sensitive to ICB therapy. The networks based on co-expression, ceRNA, cis and trans interaction provided insights into the regulatory mechanisms of GRLs.ConclusionsOur identified and validated risk model based on five GRLs is an independent prognostic factor for OC patients. Through comprehensive analyses, findings of our study uncovered potential biomarker and therapeutic target for the risk model based on the GRLs.