RESUMEN
Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory specificity is DNA-guided TF cooperativity. Although in vitro studies suggest that it may be common, examples of such cooperativity remain scarce in cellular contexts. Here, we demonstrate how "Coordinator," a long DNA motif composed of common motifs bound by many basic helix-loop-helix (bHLH) and homeodomain (HD) TFs, uniquely defines the regulatory regions of embryonic face and limb mesenchyme. Coordinator guides cooperative and selective binding between the bHLH family mesenchymal regulator TWIST1 and a collective of HD factors associated with regional identities in the face and limb. TWIST1 is required for HD binding and open chromatin at Coordinator sites, whereas HD factors stabilize TWIST1 occupancy at Coordinator and titrate it away from HD-independent sites. This cooperativity results in the shared regulation of genes involved in cell-type and positional identities and ultimately shapes facial morphology and evolution.
Asunto(s)
Proteínas de Unión al ADN , Desarrollo Embrionario , Factores de Transcripción , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sitios de Unión , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Mesodermo/metabolismo , Factores de Transcripción/metabolismo , Humanos , Animales , Ratones , Extremidades/crecimiento & desarrolloRESUMEN
A ubiquitous feature of eukaryotic transcriptional regulation is cooperative self-assembly between transcription factors (TFs) and DNA cis-regulatory motifs. It is thought that this strategy enables specific regulatory connections to be formed in gene networks between otherwise weakly interacting, low-specificity molecular components. Here, using synthetic gene circuits constructed in yeast, we find that high regulatory specificity can emerge from cooperative, multivalent interactions among artificial zinc-finger-based TFs. We show that circuits "wired" using the strategy of cooperative TF assembly are effectively insulated from aberrant misregulation of the host cell genome. As we demonstrate in experiments and mathematical models, this mechanism is sufficient to rescue circuit-driven fitness defects, resulting in genetic and functional stability of circuits in long-term continuous culture. Our naturally inspired approach offers a simple, generalizable means for building high-fidelity, evolutionarily robust gene circuits that can be scaled to a wide range of host organisms and applications.
Asunto(s)
Redes Reguladoras de Genes , Factores de Transcripción , Factores de Transcripción/genética , Saccharomyces cerevisiae/genética , GenomaRESUMEN
The mechanisms underlying ribonucleoprotein (RNP) granule assembly, including the basis for establishing and maintaining RNP granules with distinct composition, are unknown. One prominent type of RNP granule is the stress granule (SG), a dynamic and reversible cytoplasmic assembly formed in eukaryotic cells in response to stress. Here, we show that SGs assemble through liquid-liquid phase separation (LLPS) arising from interactions distributed unevenly across a core protein-RNA interaction network. The central node of this network is G3BP1, which functions as a molecular switch that triggers RNA-dependent LLPS in response to a rise in intracellular free RNA concentrations. Moreover, we show that interplay between three distinct intrinsically disordered regions (IDRs) in G3BP1 regulates its intrinsic propensity for LLPS, and this is fine-tuned by phosphorylation within the IDRs. Further regulation of SG assembly arises through positive or negative cooperativity by extrinsic G3BP1-binding factors that strengthen or weaken, respectively, the core SG network.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Ribonucleoproteínas/metabolismo , Línea Celular Tumoral , Citoplasma/metabolismo , Estructuras Citoplasmáticas/metabolismo , Células HEK293 , Humanos , Fosforilación , ARN/metabolismoRESUMEN
Ribosome assembly is an efficient but complex and heterogeneous process during which ribosomal proteins assemble on the nascent rRNA during transcription. Understanding how the interplay between nascent RNA folding and protein binding determines the fate of transcripts remains a major challenge. Here, using single-molecule fluorescence microscopy, we follow assembly of the entire 3' domain of the bacterial small ribosomal subunit in real time. We find that co-transcriptional rRNA folding is complicated by the formation of long-range RNA interactions and that r-proteins self-chaperone the rRNA folding process prior to stable incorporation into a ribonucleoprotein (RNP) complex. Assembly is initiated by transient rather than stable protein binding, and the protein-RNA binding dynamics gradually decrease during assembly. This work questions the paradigm of strictly sequential and cooperative ribosome assembly and suggests that transient binding of RNA binding proteins to cellular RNAs could provide a general mechanism to shape nascent RNA folding during RNP assembly.
Asunto(s)
Pliegue del ARN , ARN Ribosómico/metabolismo , Proteínas de Unión al ARN/metabolismo , Modelos Biológicos , Conformación de Ácido Nucleico , Unión Proteica , Estabilidad del ARN , ARN Ribosómico/química , Transcripción GenéticaRESUMEN
Phase-separated multi-molecular assemblies provide a general regulatory mechanism to compartmentalize biochemical reactions within cells. We propose that a phase separation model explains established and recently described features of transcriptional control. These features include the formation of super-enhancers, the sensitivity of super-enhancers to perturbation, the transcriptional bursting patterns of enhancers, and the ability of an enhancer to produce simultaneous activation at multiple genes. This model provides a conceptual framework to further explore principles of gene control in mammals.
Asunto(s)
Regulación de la Expresión Génica , Modelos Biológicos , Transcripción Genética , Animales , Elementos de Facilitación Genéticos , Células Eucariotas/metabolismo , Humanos , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
Single-molecule imaging inside living cells has revealed that transcription factors (TFs) bind to DNA transiently, but a long-standing question is how this transient binding is related to transcription activation. Here, we devised a microscopy method to simultaneously measure transient TF binding at a single locus and the effect of these binding events on transcription. We show that DNA binding of the yeast TF Gal4 activates transcription of a target gene within a few seconds, with at least â¼20% efficiency and with a high initiation rate of â¼1 RNA/s. Gal4 DNA dissociation decreases transcription rapidly. Moreover, at a gene with multiple binding sites, individual Gal4 molecules only rarely stay bound throughout the entire burst but instead frequently exchange during a burst to increase the transcriptional burst duration. Our results suggest a mechanism for enhancer regulation in more complex eukaryotes, where TF cooperativity and exchange enable robust and responsive transcription regulation.
Asunto(s)
Regulación de la Expresión Génica , Factores de Transcripción , Factores de Transcripción/metabolismo , Unión Proteica , Sitios de Unión , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Activación Transcripcional , ADN/metabolismoRESUMEN
Despite the unique ability of pioneer factors (PFs) to target nucleosomal sites in closed chromatin, they only bind a small fraction of their genomic motifs. The underlying mechanism of this selectivity is not well understood. Here, we design a high-throughput assay called chromatin immunoprecipitation with integrated synthetic oligonucleotides (ChIP-ISO) to systematically dissect sequence features affecting the binding specificity of a classic PF, FOXA1, in human A549 cells. Combining ChIP-ISO with in vitro and neural network analyses, we find that (1) FOXA1 binding is strongly affected by co-binding transcription factors (TFs) AP-1 and CEBPB; (2) FOXA1 and AP-1 show binding cooperativity in vitro; (3) FOXA1's binding is determined more by local sequences than chromatin context, including eu-/heterochromatin; and (4) AP-1 is partially responsible for differential binding of FOXA1 in different cell types. Our study presents a framework for elucidating genetic rules underlying PF binding specificity and reveals a mechanism for context-specific regulation of its binding.
Asunto(s)
Factor Nuclear 3-alfa del Hepatocito , Unión Proteica , Factor de Transcripción AP-1 , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Factor Nuclear 3-alfa del Hepatocito/genética , Humanos , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción AP-1/genética , Sitios de Unión , Células A549 , Cromatina/metabolismo , Cromatina/genética , Inmunoprecipitación de Cromatina , Oligonucleótidos/metabolismo , Oligonucleótidos/genéticaRESUMEN
Pioneer transcription factors are essential for cell fate changes by targeting closed chromatin. OCT4 is a crucial pioneer factor that can induce cell reprogramming. However, the structural basis of how pioneer factors recognize the in vivo nucleosomal DNA targets is unknown. Here, we determine the high-resolution structures of the nucleosome containing human LIN28B DNA and its complexes with the OCT4 DNA binding region. Three OCT4s bind the pre-positioned nucleosome by recognizing non-canonical DNA sequences. Two use their POUS domains while the other uses the POUS-loop-POUHD region; POUHD serves as a wedge to unwrap â¼25 base pair DNA. Our analysis of previous genomic data and determination of the ESRRB-nucleosome-OCT4 structure confirmed the generality of these structural features. Moreover, biochemical studies suggest that multiple OCT4s cooperatively open the H1-condensed nucleosome array containing the LIN28B nucleosome. Thus, our study suggests a mechanism of how OCT4 can target the nucleosome and open closed chromatin.
Asunto(s)
Cromatina , Nucleosomas , Factor 3 de Transcripción de Unión a Octámeros , Proteínas de Unión al ARN , Humanos , Secuencia de Bases , Reprogramación Celular , Cromatina/genética , ADN/metabolismo , Nucleosomas/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismoRESUMEN
The androgen receptor (AR) is a nuclear receptor that governs gene expression programs required for prostate development and male phenotype maintenance. Advanced prostate cancers display AR hyperactivation and transcriptome expansion, in part, through AR amplification and interaction with oncoprotein cofactors. Despite its biological importance, how AR domains and cofactors cooperate to bind DNA has remained elusive. Using single-particle cryo-electron microscopy, we isolated three conformations of AR bound to DNA, showing that AR forms a non-obligate dimer, with the buried dimer interface utilized by ancestral steroid receptors repurposed to facilitate cooperative DNA binding. We identify novel allosteric surfaces which are compromised in androgen insensitivity syndrome and reinforced by AR's oncoprotein cofactor, ERG, and by DNA-binding motifs. Finally, we present evidence that this plastic dimer interface may have been adopted for transactivation at the expense of DNA binding. Our work highlights how fine-tuning AR's cooperative interactions translate to consequences in development and disease.
Asunto(s)
Neoplasias de la Próstata , Receptores Androgénicos , Microscopía por Crioelectrón , ADN/metabolismo , Dimerización , Humanos , Masculino , Neoplasias de la Próstata/genética , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Activación TranscripcionalRESUMEN
Enhancers harbor binding motifs that recruit transcription factors (TFs) for gene activation. While cooperative binding of TFs at enhancers is known to be critical for transcriptional activation of a handful of developmental enhancers, the extent of TF cooperativity genome-wide is unknown. Here, we couple high-resolution nuclease footprinting with single-molecule methylation profiling to characterize TF cooperativity at active enhancers in the Drosophila genome. Enrichment of short micrococcal nuclease (MNase)-protected DNA segments indicates that the majority of enhancers harbor two or more TF-binding sites, and we uncover protected fragments that correspond to co-bound sites in thousands of enhancers. From the analysis of co-binding, we find that cooperativity dominates TF binding in vivo at the majority of active enhancers. Cooperativity is highest between sites spaced 50 bp apart, indicating that cooperativity occurs without apparent protein-protein interactions. Our findings suggest nucleosomes promoting cooperativity because co-binding may effectively clear nucleosomes and promote enhancer function.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Unión Proteica/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Sitios de Unión/genética , Línea Celular , Drosophila/genética , Drosophila/metabolismo , Genoma/genética , Nucleasa Microcócica/genética , Nucleosomas/genética , Nucleosomas/metabolismo , Mapas de Interacción de Proteínas/genética , Activación Transcripcional/genéticaRESUMEN
Gene activation requires the cooperative activity of multiple transcription factors at cis-regulatory elements (CREs). Yet, most transcription factors have short residence time, questioning the requirement of their physical co-occupancy on DNA to achieve cooperativity. Here, we present a DNA footprinting method that detects individual molecular interactions of transcription factors and nucleosomes with DNA in vivo. We apply this strategy to quantify the simultaneous binding of multiple transcription factors on single DNA molecules at mouse CREs. Analysis of the binary occupancy patterns at thousands of motif combinations reveals that high DNA co-occupancy occurs for most types of transcription factors, in the absence of direct physical interaction, at sites of competition with nucleosomes. Perturbation of pairwise interactions demonstrates the function of molecular co-occupancy in binding cooperativity. Our results reveal the interactions regulating CREs at molecular resolution and identify DNA co-occupancy as a widespread cooperativity mechanism used by transcription factors to remodel chromatin.
Asunto(s)
Huella de ADN/métodos , ADN/genética , Nucleosomas/química , Elementos Reguladores de la Transcripción , Factores de Transcripción/genética , Animales , Sitios de Unión , ADN/química , ADN/metabolismo , Masculino , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Nucleosomas/metabolismo , Unión Proteica , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The coactivator p300/CREB-binding protein (CBP) regulates genes by facilitating the assembly of transcriptional machinery and by acetylating histones and other factors. However, it remains mostly unclear how both functions of p300 are dynamically coordinated during gene control. Here, we showed that p300 can orchestrate two functions through the formation of dynamic clusters with certain transcription factors (TFs), which is mediated by the interactions between a TF's transactivation domain (TAD) and the intrinsically disordered regions of p300. Co-condensation can enable spatially defined, all-or-none activation of p300's catalytic activity, priming the recruitment of coactivators, including Brd4. We showed that co-condensation can modulate transcriptional initiation rate and burst duration of target genes, underlying nonlinear gene regulatory functions. Such modulation is consistent with how p300 might shape gene bursting kinetics globally. Altogether, these results suggest an intriguing gene regulation mechanism, in which TF and p300 co-condensation contributes to transcriptional bursting regulation and cooperative gene control.
Asunto(s)
Proteína p300 Asociada a E1A/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Activación Transcripcional/genética , Acetilación , Animales , Células CHO , Proteína de Unión a CREB/metabolismo , Línea Celular , Cricetulus , Regulación de la Expresión Génica/genética , Células HEK293 , Histonas/metabolismo , Humanos , Cinética , Ratones , Transactivadores/metabolismoRESUMEN
Circadian clocks evolved in diverse organisms as an adaptation to the daily swings in ambient light and temperature that derive from Earth's rotation. These timing systems, based on intracellular molecular oscillations, synchronize organisms' behavior and physiology with the 24-h environmental rhythm. The cyanobacterial clock serves as a special model for understanding circadian rhythms because it can be fully reconstituted in vitro. This review summarizes recent advances that leverage new biochemical, biophysical, and mathematical approaches to shed light on the molecular mechanisms of cyanobacterial Kai proteins that support the clock, and their homologues in other bacteria. Many questions remain in circadian biology, and the tools developed for the Kai system will bring us closer to the answers.
Asunto(s)
Relojes Circadianos , Cianobacterias , Proteínas Bacterianas/metabolismo , Ritmo Circadiano , Cianobacterias/metabolismo , Péptidos y Proteínas de Señalización del Ritmo Circadiano/genéticaRESUMEN
Enhancers are DNA elements that are bound by transcription factors (TFs), which recruit coactivators and the transcriptional machinery to genes. Phase-separated condensates of TFs and coactivators have been implicated in assembling the transcription machinery at particular enhancers, yet the role of DNA sequence in this process has not been explored. We show that DNA sequences encoding TF binding site number, density, and affinity above sharply defined thresholds drive condensation of TFs and coactivators. A combination of specific structured (TF-DNA) and weak multivalent (TF-coactivator) interactions allows for condensates to form at particular genomic loci determined by the DNA sequence and the complement of expressed TFs. DNA features found to drive condensation promote enhancer activity and transcription in cells. Our study provides a framework to understand how the genome can scaffold transcriptional condensates at specific loci and how the universal phenomenon of phase separation might regulate this process.
Asunto(s)
Cromatina/genética , Elementos de Facilitación Genéticos , Factores de Transcripción/genética , Transcripción Genética , Animales , Secuencia de Bases/genética , Sitios de Unión/genética , ADN/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Genómica , Ratones , Células Madre Embrionarias de RatonesRESUMEN
Cooperative interactions between amino acids are critical for protein function. A genetic reflection of cooperativity is epistasis, which is when a change in the amino acid at one position changes the sequence requirements at another position. To assess epistasis within an enzyme active site, we utilized CTX-M ß-lactamase as a model system. CTX-M hydrolyzes ß-lactam antibiotics to provide antibiotic resistance, allowing a simple functional selection for rapid sorting of modified enzymes. We created all pairwise mutations across 17 active site positions in the ß-lactamase enzyme and quantitated the function of variants against two ß-lactam antibiotics using next-generation sequencing. Context-dependent sequence requirements were determined by comparing the antibiotic resistance function of double mutations across the CTX-M active site to their predicted function based on the constituent single mutations, revealing both positive epistasis (synergistic interactions) and negative epistasis (antagonistic interactions) between amino acid substitutions. The resulting trends demonstrate that positive epistasis is present throughout the active site, that epistasis between residues is mediated through substrate interactions, and that residues more tolerant to substitutions serve as generic compensators which are responsible for many cases of positive epistasis. Additionally, we show that a key catalytic residue (Glu166) is amenable to compensatory mutations, and we characterize one such double mutant (E166Y/N170G) that acts by an altered catalytic mechanism. These findings shed light on the unique biochemical factors that drive epistasis within an enzyme active site and will inform enzyme engineering efforts by bridging the gap between amino acid sequence and catalytic function.
Asunto(s)
Escherichia coli , beta-Lactamasas , Escherichia coli/genética , Dominio Catalítico/genética , Mutación , Sustitución de Aminoácidos , beta-Lactamasas/químicaRESUMEN
The cooperative action of the subunits in oligomeric receptors enables fine-tuning of receptor activation, as demonstrated for the regulation of voltage-activated HCN pacemaker ion channels by relating cAMP binding to channel activation in ensemble signals. HCN channels generate electric rhythmicity in specialized brain neurons and cardiomyocytes. There is conflicting evidence on whether binding cooperativity does exist independent of channel activation or not, as recently reported for detergent-solubilized receptors positioned in zero-mode waveguides. Here, we show positive cooperativity in ligand binding to closed HCN2 channels in native cell membranes by following the binding of individual fluorescence-labeled cAMP molecules. Kinetic modeling reveals that the affinity of the still empty binding sites rises with increased degree of occupation and that the transition of the channel to a flip state is promoted accordingly. We conclude that ligand binding to the subunits in closed HCN2 channels not pre-activated by voltage is already cooperative. Hence, cooperativity is not causally linked to channel activation by voltage. Our analysis also shows that single-molecule binding measurements at equilibrium can quantify cooperativity in ligand binding to receptors in native membranes.
Asunto(s)
Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Activación del Canal Iónico , Ligandos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Activación del Canal Iónico/fisiología , AMP Cíclico/metabolismo , Fenómenos Biofísicos , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismoRESUMEN
Protein-protein and protein-water hydrogen bonding interactions play essential roles in the way a protein passes through the transition state during folding or unfolding, but the large number of these interactions in molecular dynamics (MD) simulations makes them difficult to analyze. Here, we introduce a state space representation and associated "rarity" measure to identify and quantify transition state passage (transit) events. Applying this representation to a long MD simulation trajectory that captured multiple folding and unfolding events of the GTT WW domain, a small protein often used as a model for the folding process, we identified three transition categories: Highway (faster), Meander (slower), and Ambiguous (intermediate). We developed data sonification and visualization tools to analyze hydrogen bond dynamics before, during, and after these transition events. By means of these tools, we were able to identify characteristic hydrogen bonding patterns associated with "Highway" versus "Meander" versus "Ambiguous" transitions and to design algorithms that can identify these same folding pathways and critical protein-water interactions directly from the data. Highly cooperative hydrogen bonding can either slow down or speed up transit. Furthermore, an analysis of protein-water hydrogen bond dynamics at the surface of WW domain shows an increase in hydrogen bond lifetime from folded to unfolded conformations with Ambiguous transitions as an outlier. In summary, hydrogen bond dynamics provide a direct window into the heterogeneity of transits, which can vary widely in duration (by a factor of 10) due to a complex energy landscape.
Asunto(s)
Enlace de Hidrógeno , Simulación de Dinámica Molecular , Pliegue de Proteína , Proteínas , Proteínas/química , Proteínas/metabolismo , Agua/química , Dominios WW , Conformación Proteica , AlgoritmosRESUMEN
The consistency of energy landscape theory predictions with available experimental data, as well as direct evidence from molecular simulations, have shown that protein folding mechanisms are largely determined by the contacts present in the native structure. As expected, native contacts are generally energetically favorable. However, there are usually at least as many energetically favorable nonnative pairs owing to the greater number of possible nonnative interactions. This apparent frustration must therefore be reduced by the greater cooperativity of native interactions. In this work, we analyze the statistics of contacts in the unbiased all-atom folding trajectories obtained by Shaw and coworkers, focusing on the unfolded state. By computing mutual cooperativities between contacts formed in the unfolded state, we show that native contacts form the most cooperative pairs, while cooperativities among nonnative or between native and nonnative contacts are typically much less favorable or even anticooperative. Furthermore, we show that the largest network of cooperative interactions observed in the unfolded state consists mainly of native contacts, suggesting that this set of mutually reinforcing interactions has evolved to stabilize the native state.
Asunto(s)
Pliegue de Proteína , Proteínas , Proteínas/química , Termodinámica , Conformación Proteica , Modelos Moleculares , Simulación de Dinámica MolecularRESUMEN
Virus infection induces stochastic activation of the interferon-ß gene. Three previously identified Alu-like DNA elements called NRCs (NF-κB reception centers) function by capturing and delivering NF-κB to the IFNB1 enhancer via stochastic interchromosomal interactions. We show that the transcription factor ThPOK binds cooperatively with NF-κB to NRCs and mediates their physical proximity with the IFNB1 gene via its ability to oligomerize when bound to DNA. ThPOK knockdown significantly decreased the frequency of interchromosomal interactions, NF-κB DNA binding to the IFNB1 enhancer, and virus-induced IFNB1 gene activation. We also demonstrate that cooperative DNA binding between ThPOK and NF-κB on the same face of the double DNA helix is required for interchromosomal interactions and distinguishes NRCs from various other Alu elements bearing κB sites. These studies show how DNA binding cooperativity of stereospecifically aligned transcription factors provides the necessary ultrasensitivity for the all-or-none stochastic cell responses to virus infection.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Interferón beta/metabolismo , Factores de Transcripción/metabolismo , Elementos Alu , Cromosomas/genética , Cromosomas/metabolismo , Proteínas de Unión al ADN/genética , Elementos de Facilitación Genéticos , Células HEK293 , Células HeLa , Humanos , Interferón beta/genética , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , Procesos Estocásticos , Factores de Transcripción/genética , Transcripción Genética , Virosis/metabolismoRESUMEN
Proper control of the mitochondrial Ca2+ uniporter's pore (MCU) is required to allow Ca2+-dependent activation of oxidative metabolism and to avoid mitochondrial Ca2+ overload and cell death. The MCU's gatekeeping and cooperative activation is mediated by the Ca2+-sensing MICU1 protein, which has been proposed to form dimeric complexes anchored to the EMRE scaffold of MCU. We unexpectedly find that MICU1 suppresses inhibition of MCU by ruthenium red/Ru360, which bind to MCU's DIME motif, the selectivity filter. This led us to recognize in MICU1's sequence a putative DIME interacting domain (DID), which is required for both gatekeeping and cooperative activation of MCU and for cell survival. Thus, we propose that MICU1 has to interact with the D-ring formed by the DIME domains in MCU to control the uniporter.