RESUMEN
Naïve epiblast cells in the embryo and pluripotent stem cells in vitro undergo developmental progression to a formative state competent for lineage specification. During this transition, transcription factors and chromatin are rewired to encode new functional features. Here, we examine the role of mitogen-activated protein kinase (ERK1/2) signalling in pluripotent state transition. We show that a primary consequence of ERK activation in mouse embryonic stem cells is elimination of Nanog, which precipitates breakdown of the naïve state gene regulatory network. Variability in pERK dynamics results in heterogeneous loss of Nanog and metachronous state transition. Knockdown of Nanog allows exit without ERK activation. However, transition to formative pluripotency does not proceed and cells collapse to an indeterminate identity. This outcome is due to failure to maintain expression of the central pluripotency factor Oct4. Thus, during formative transition ERK signalling both dismantles the naïve state and preserves pluripotency. These results illustrate how a single signalling pathway can both initiate and secure transition between cell states.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros , Células Madre Pluripotentes , Animales , Proteína Homeótica Nanog/metabolismo , Proteína Homeótica Nanog/genética , Ratones , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Diferenciación Celular/genética , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/citología , Regulación del Desarrollo de la Expresión Génica , Estratos Germinativos/metabolismo , Estratos Germinativos/citología , Redes Reguladoras de Genes , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genéticaRESUMEN
The control of eukaryotic gene expression is intimately connected to highly dynamic chromatin structures. Gene regulation relies on activator and repressor transcription factors (TFs) that induce local chromatin opening and closing. However, it is unclear how nucleus-wide chromatin organization responds dynamically to the activity of specific TFs. Here, we examined how two TFs with opposite effects on local chromatin accessibility modulate chromatin dynamics nucleus-wide. We combine high-resolution diffusion mapping and dense flow reconstruction and correlation in living cells to obtain an imaging-based, nanometer-scale analysis of local diffusion processes and long-range coordinated movements of both chromatin and TFs. We show that the expression of either an individual transcriptional activator (CDX2) or repressor (SIX6) with large numbers of binding sites increases chromatin mobility nucleus-wide, yet they induce opposite coherent chromatin motions at the micron scale. Hi-C analysis of higher-order chromatin structures shows that induction of the pioneer factor CDX2 leads both to changes in local chromatin interactions and the distribution of A and B compartments, thus relating the micromovement of chromatin with changes in compartmental structures. Given that inhibition of transcription initiation and elongation by RNA Pol II has a partial impact on the global chromatin dynamics induced by CDX2, we suggest that CDX2 overexpression alters chromatin structure dynamics both dependently and independently of transcription. Our biophysical analysis shows that sequence-specific TFs can influence chromatin structure on multiple architectural levels, arguing that local chromatin changes brought by TFs alter long-range chromatin mobility and its organization.
Asunto(s)
Cromatina , Factores de Transcripción , Cromatina/metabolismo , Cromatina/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Humanos , Factor de Transcripción CDX2/metabolismo , Factor de Transcripción CDX2/genética , Regulación de la Expresión Génica , Núcleo Celular/metabolismo , Sitios de Unión , Ensamble y Desensamble de CromatinaRESUMEN
The level of a given protein is determined by the synthesis and degradation rates of its mRNA and protein. While several studies have quantified the contribution of different gene expression steps in regulating protein levels, these are limited by using equilibrium approximations in out-of-equilibrium biological systems. Here, we introduce gene expression flux analysis to quantitatively dissect the dynamics of the expression level for specific proteins and use it to analyze published transcriptomics and proteomics datasets. Our analysis reveals distinct regulatory modalities shared by sets of genes with clear functional signatures. We also find that protein degradation plays a stronger role than expected in the adaptation of protein levels. These findings suggest that shared regulatory strategies can lead to versatile responses at the protein level and highlight the importance of going beyond equilibrium approximations to dissect the quantitative contribution of different steps of gene expression to protein dynamics.
RESUMEN
Protein turnover (PT) has been formally defined only in equilibrium conditions, which is ill-suited to quantify PT during dynamic processes that occur during embryogenesis or (extra) cellular signaling. In this Hypothesis, we propose a definition of PT in an out-of-equilibrium regime that allows the quantification of PT in virtually any biological context. We propose a simple mathematical and conceptual framework applicable to a broad range of available data, such as RNA sequencing coupled with pulsed-SILAC datasets. We apply our framework to a published dataset and show that stimulation of mouse dendritic cells with LPS leads to a proteome-wide change in PT. This is the first quantification of PT out-of-equilibrium, paving the way for the analysis of biological systems in other contexts.
Asunto(s)
Proteoma , Animales , Ratones , ProteolisisRESUMEN
Transcription factors (TFs) bind to specific DNA motifs to regulate the expression of target genes. To reach their binding sites, TFs diffuse in 3D and perform local motions such as 1D sliding, hopping, or intersegmental transfer. TF-DNA interactions depend on multiple parameters, such as the chromatin environment, TF partitioning into distinct subcellular regions, and cooperativity with other DNA-binding proteins. In this review, how current understanding of the search process has initially been shaped by prokaryotic studies is discussed, as well as what is known about the parameters regulating TF search efficiency in the context of the complex eukaryotic chromatin landscape.
Asunto(s)
ADN/metabolismo , Factores de Transcripción/metabolismo , Animales , Regulación de la Expresión Génica , Genoma , Humanos , Modelos Biológicos , Nucleosomas/metabolismo , Factores de Transcripción/genéticaRESUMEN
The pioneer activity of transcription factors allows for opening of inaccessible regulatory elements and has been extensively studied in the context of cellular differentiation and reprogramming. In contrast, the function of pioneer activity in self-renewing cell divisions and across the cell cycle is poorly understood. Here we assessed the interplay between OCT4 and SOX2 in controlling chromatin accessibility of mouse embryonic stem cells. We found that OCT4 and SOX2 operate in a largely independent manner even at co-occupied sites, and that their cooperative binding is mostly mediated indirectly through regulation of chromatin accessibility. Controlled protein degradation strategies revealed that the uninterrupted presence of OCT4 is required for post-mitotic re-establishment and interphase maintenance of chromatin accessibility, and that highly OCT4-bound enhancers are particularly vulnerable to transient loss of OCT4 expression. Our study sheds light on the constant pioneer activity required to maintain the dynamic pluripotency regulatory landscape in an accessible state.
Asunto(s)
Ciclo Celular/fisiología , Cromatina/metabolismo , Factores de Transcripción/metabolismo , Animales , División Celular/fisiología , Línea Celular , Ácidos Indolacéticos/farmacología , Interfase , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción SOXB1/metabolismoRESUMEN
SOX2 and OCT4 are pioneer transcription factors playing a key role in embryonic stem (ES) cell self-renewal and differentiation. How temporal fluctuations in their expression levels bias lineage commitment is unknown. Here, we generated knock-in reporter fusion ES cell lines allowing to monitor endogenous SOX2 and OCT4 protein fluctuations in living cells and to determine their impact on mesendodermal and neuroectodermal commitment. We found that small differences in SOX2 and OCT4 levels impact cell fate commitment in G1 but not in S phase. Elevated SOX2 levels modestly increased neuroectodermal commitment and decreased mesendodermal commitment upon directed differentiation. In contrast, elevated OCT4 levels strongly biased ES cells towards both neuroectodermal and mesendodermal fates in undirected differentiation. Using ATAC-seq on ES cells gated for different endogenous SOX2 and OCT4 levels, we found that high OCT4 levels increased chromatin accessibility at differentiation-associated enhancers. This suggests that small endogenous fluctuations of pioneer transcription factors can bias cell fate decisions by concentration-dependent priming of differentiation-associated enhancers.
Asunto(s)
Diferenciación Celular/genética , Factor 3 de Transcripción de Unión a Octámeros , Células Madre Pluripotentes/fisiología , Factores de Transcripción SOXB1 , Animales , Línea Celular , Endodermo/citología , Endodermo/metabolismo , Elementos de Facilitación Genéticos/genética , Técnicas de Sustitución del Gen/métodos , Ratones , Placa Neural/citología , Placa Neural/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismoRESUMEN
Phenotypically identical mammalian cells often display considerable variability in transcript levels of individual genes. How transcriptional activity propagates in cell lineages, and how this varies across genes is poorly understood. Here we combine live-cell imaging of short-lived transcriptional reporters in mouse embryonic stem cells with mathematical modelling to quantify the propagation of transcriptional activity over time and across cell generations in phenotypically homogenous cells. In sister cells we find mean transcriptional activity to be strongly correlated and transcriptional dynamics tend to be synchronous; both features control how quickly transcriptional levels in sister cells diverge in a gene-specific manner. Moreover, mean transcriptional activity is transmitted from mother to daughter cells, leading to multi-generational transcriptional memory and causing inter-family heterogeneity in gene expression.
Asunto(s)
Linaje de la Célula/genética , Regulación de la Expresión Génica/genética , Modelos Biológicos , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Línea Celular , Células HEK293 , Humanos , Microscopía Intravital , Ratones , Microscopía Fluorescente , Células Madre Embrionarias de Ratones , Análisis de la Célula Individual , Imagen de Lapso de TiempoRESUMEN
The transduction of extracellular signals through signaling pathways that culminate in a transcriptional response is central to many biological processes. However, quantitative relationships between activities of signaling pathway components and transcriptional output of target genes remain poorly explored. Here we developed a dual bioluminescence imaging strategy allowing simultaneous monitoring of nuclear translocation of the SMAD4 and SMAD2 transcriptional activators upon TGF-ß stimulation, and the transcriptional response of the endogenous connective tissue growth factor (ctgf) gene. Using cell lines allowing to vary exogenous SMAD4/2 expression levels, we performed quantitative measurements of the temporal profiles of SMAD4/2 translocation and ctgf transcription kinetics in hundreds of individual cells at high temporal resolution. We found that while nuclear translocation efficiency had little impact on initial ctgf transcriptional activation, high total cellular SMAD4 but not SMAD2 levels increased the probability of cells to exhibit a sustained ctgf transcriptional response. The approach we present here allows time-resolved single cell quantification of transcription factor dynamics and transcriptional responses and thereby sheds light on the quantitative relationship between SMADs and target gene responses.
Asunto(s)
Regulación de la Expresión Génica , Expresión Génica , Análisis de la Célula Individual , Proteína Smad2/metabolismo , Proteína Smad4/metabolismo , Transporte Biológico , Humanos , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Mammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequence-specific/non-specific DNA binding. How these properties affect their ability to occupy specific genomic sites and modify the epigenetic landscape is unclear. The association of TFs with mitotic chromosomes observed by fluorescence microscopy is largely mediated by non-specific DNA interactions and differs broadly between TFs. Here we combine quantitative measurements of mitotic chromosome binding (MCB) of 501 TFs, TF mobility measurements by fluorescence recovery after photobleaching, single molecule imaging of DNA binding, and mapping of TF binding and chromatin accessibility. TFs associating to mitotic chromosomes are enriched in DNA-rich compartments in interphase and display slower mobility in interphase and mitosis. Remarkably, MCB correlates with relative TF on-rates and genome-wide specific site occupancy, but not with TF residence times. This suggests that non-specific DNA binding properties of TFs regulate their search efficiency and occupancy of specific genomic sites.
Asunto(s)
Cromatina/metabolismo , Cromosomas/metabolismo , Interfase/fisiología , Mitosis/fisiología , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Cromosomas/genética , ADN/genética , ADN/metabolismo , Humanos , Interfase/genética , Mitosis/genética , Unión Proteica , Factores de Transcripción/genéticaRESUMEN
Transcription factors (TFs) regulate gene expression in both prokaryotes and eukaryotes by recognizing and binding to specific DNA promoter sequences. In higher eukaryotes, it remains unclear how the duration of TF binding to DNA relates to downstream transcriptional output. Here, we address this question for the transcriptional activator NF-κB (p65), by live-cell single molecule imaging of TF-DNA binding kinetics and genome-wide quantification of p65-mediated transcription. We used mutants of p65, perturbing either the DNA binding domain (DBD) or the protein-protein transactivation domain (TAD). We found that p65-DNA binding time was predominantly determined by its DBD and directly correlated with its transcriptional output as long as the TAD is intact. Surprisingly, mutation or deletion of the TAD did not modify p65-DNA binding stability, suggesting that the p65 TAD generally contributes neither to the assembly of an "enhanceosome," nor to the active removal of p65 from putative specific binding sites. However, TAD removal did reduce p65-mediated transcriptional activation, indicating that protein-protein interactions act to translate the long-lived p65-DNA binding into productive transcription.
Asunto(s)
FN-kappa B/genética , Factor de Transcripción ReIA/genética , Factores de Transcripción/genética , Activación Transcripcional/genética , Proteínas de Unión al ADN/genética , Expresión Génica/genética , Genoma Humano/genética , Células HeLa , Humanos , Cinética , Proteínas Mutantes/química , Proteínas Mutantes/genética , FN-kappa B/química , Dominios y Motivos de Interacción de Proteínas/genética , Imagen Individual de Molécula , Factor de Transcripción ReIA/química , Factores de Transcripción/químicaRESUMEN
Many mammalian genes are transcribed during short bursts of variable frequencies and sizes that substantially contribute to cell-to-cell variability. However, which molecular mechanisms determine bursting properties remains unclear. To probe putative mechanisms, we combined temporal analysis of transcription along the circadian cycle with multiple genomic reporter integrations, using both short-lived luciferase live microscopy and single-molecule RNA-FISH. Using the Bmal1 circadian promoter as our model, we observed that rhythmic transcription resulted predominantly from variations in burst frequency, while the genomic position changed the burst size. Thus, burst frequency and size independently modulated Bmal1 transcription. We then found that promoter histone-acetylation level covaried with burst frequency, being greatest at peak expression and lowest at trough expression, while remaining unaffected by the genomic location. In addition, specific deletions of ROR-responsive elements led to constitutively elevated histone acetylation and burst frequency. We then investigated the suggested link between histone acetylation and burst frequency by dCas9p300-targeted modulation of histone acetylation, revealing that acetylation levels influence burst frequency more than burst size. The correlation between acetylation levels at the promoter and burst frequency was also observed in endogenous circadian genes and in embryonic stem cell fate genes. Thus, our data suggest that histone acetylation-mediated control of transcription burst frequency is a common mechanism to control mammalian gene expression.
Asunto(s)
Factores de Transcripción ARNTL/biosíntesis , Ritmo Circadiano/fisiología , Regulación de la Expresión Génica/fisiología , Histonas/metabolismo , Modelos Biológicos , Regiones Promotoras Genéticas/fisiología , Transcripción Genética/fisiología , Factores de Transcripción ARNTL/genética , Acetilación , Animales , Ratones , Células 3T3 NIHRESUMEN
The regulation of fundamental processes such as gene expression or cell differentiation involves chromatin states, demarcated by combinatorial histone post-translational modification (PTM) patterns. The subnuclear organization and dynamics of chromatin states is not well understood, as tools for their detection and modulation in live cells are lacking. Here, we report the development of genetically encoded chromatin-sensing multivalent probes, cMAPs, selective for bivalent chromatin, a PTM pattern associated with pluripotency in embryonic stem cells (ESCs). cMAPs were engineered from a set of PTM-binding (reader) proteins and optimized using synthetic nucleosomes carrying defined PTMs. Applied in live ESCs, cMAPs formed discrete subnuclear foci, revealing the organization of bivalent chromatin into local clusters. Moreover, cMAPs enabled direct monitoring of the loss of bivalency upon treatment with small-molecule epigenetic modulators. cMAPs thus provide a versatile platform to monitor chromatin state dynamics in live cells.
Asunto(s)
Cromatina/metabolismo , Células Madre Embrionarias/metabolismo , Histonas/metabolismo , Proteínas Luminiscentes/metabolismo , Ingeniería de Proteínas , Cromatina/genética , Humanos , Proteínas Luminiscentes/genética , Estructura Molecular , Procesamiento Proteico-PostraduccionalRESUMEN
During mitosis, gene transcription stops, and the bulk of DNA-binding proteins are excluded from condensed chromosomes. While most gene-specific transcription factors are largely evicted from mitotic chromosomes, a subset remains bound to specific and non-specific DNA sites. Here, we review the current knowledge on the mechanisms leading to the retention of a subset of transcription factors on mitotic chromosomes and discuss the implications in gene expression regulation and their potential as an epigenetic mechanism controlling stem cell self-renewal and differentiation.
Asunto(s)
Cromosomas Humanos/metabolismo , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética/fisiología , Mitosis/fisiología , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Animales , Humanos , Células Madre/citologíaRESUMEN
The ability of some transcription factors to remain bound to specific genes on condensed mitotic chromosomes has been suggested to play a role in their rapid transcriptional reactivation upon mitotic exit. We have recently shown that SOX2 and OCT4 remain associated to mitotic chromosomes, and that depletion of SOX2 at the mitosis-G1 (M-G1) transition impairs its ability to maintain pluripotency and drive neuroectodermal commitment. Here we report on the role of SOX2 at the M-G1 transition in regulating transcriptional activity of embryonic stem cells. Using single cell time-lapse analysis of reporter constructs for STAT3 and SOX2/OCT4 activity, we show that SOX2/OCT4 do not lead to more rapid transcriptional reactivation in G1 than STAT3, a transcription factor that is excluded from mitotic chromosomes. We also report that only few endogenous target genes show decreased pre-mRNA levels after mitotic exit or in other cell cycle phases in the absence of SOX2 at the M-G1 transition. This suggests that bookmarked SOX2 target genes are not differently regulated than non-bookmarked target genes, and we discuss an alternative hypothesis on how mitotic bookmarking by SOX2 and other sequence-specific transcription factors could be involved in transcriptional regulation.
Asunto(s)
Regulación de la Expresión Génica , Mitosis/genética , Factores de Transcripción SOXB1/metabolismo , Transcripción Genética , Animales , Exones/genética , Intrones/genética , Luciferasas/metabolismo , Ratones , Células 3T3 NIH , Nocodazol/farmacología , Plásmidos/metabolismo , Precursores del ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Mitotic bookmarking transcription factors remain bound to chromosomes during mitosis and were proposed to regulate phenotypic maintenance of stem and progenitor cells at the mitosis-to-G1 (M-G1) transition. However, mitotic bookmarking remains largely unexplored in most stem cell types, and its functional relevance for cell fate decisions remains unclear. Here we screened for mitotic chromosome binding within the pluripotency network of embryonic stem (ES) cells and show that SOX2 and OCT4 remain bound to mitotic chromatin through their respective DNA-binding domains. Dynamic characterization using photobleaching-based methods and single-molecule imaging revealed quantitatively similar specific DNA interactions, but different nonspecific DNA interactions, of SOX2 and OCT4 with mitotic chromatin. Using ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) to assess the genome-wide distribution of SOX2 on mitotic chromatin, we demonstrate the bookmarking activity of SOX2 on a small set of genes. Finally, we investigated the function of SOX2 mitotic bookmarking in cell fate decisions and show that its absence at the M-G1 transition impairs pluripotency maintenance and abrogates its ability to induce neuroectodermal differentiation but does not affect reprogramming efficiency toward induced pluripotent stem cells. Our study demonstrates the mitotic bookmarking property of SOX2 and reveals its functional importance in pluripotency maintenance and ES cell differentiation.
Asunto(s)
Diferenciación Celular/genética , Mitosis/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Animales , Reprogramación Celular/genética , Cromatina/metabolismo , Células Madre Embrionarias , Fase G1 , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Células 3T3 NIH , Placa Neural/citología , Placa Neural/fisiología , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Unión ProteicaRESUMEN
Superresolution microscopy based on single-molecule centroid determination has been widely applied to cellular imaging in recent years. However, quantitative imaging of the mammalian nucleus has been challenging due to the lack of 3D optical sectioning methods for normal-sized cells, as well as the inability to accurately count the absolute copy numbers of biomolecules in highly dense structures. Here we report a reflected light-sheet superresolution microscopy method capable of imaging inside the mammalian nucleus with superior signal-to-background ratio as well as molecular counting with single-copy accuracy. Using reflected light-sheet superresolution microscopy, we probed the spatial organization of transcription by RNA polymerase II (RNAP II) molecules and quantified their global extent of clustering inside the mammalian nucleus. Spatiotemporal clustering analysis that leverages on the blinking photophysics of specific organic dyes showed that the majority (>70%) of the transcription foci originate from single RNAP II molecules, and no significant clustering between RNAP II molecules was detected within the length scale of the reported diameter of "transcription factories." Colocalization measurements of RNAP II molecules equally labeled by two spectrally distinct dyes confirmed the primarily unclustered distribution, arguing against a prevalent existence of transcription factories in the mammalian nucleus as previously proposed. The methods developed in our study pave the way for quantitative mapping and stoichiometric characterization of key biomolecular species deep inside mammalian cells.
Asunto(s)
Núcleo Celular/metabolismo , Microscopía Fluorescente/métodos , Imagen Molecular/métodos , ARN Polimerasa II/metabolismo , Transcripción Genética/fisiología , Línea Celular Tumoral , Humanos , Plásmidos/genéticaRESUMEN
Mammalian genes are often transcribed discontinuously as short bursts of RNA synthesis followed by longer silent periods. However, how these "on" and "off" transitions, together with the burst sizes, are modulated in single cells to increase gene expression upon stimulation is poorly characterized. By combining single-cell time-lapse luminescence imaging with stochastic modeling of the time traces, we quantified the transcriptional responses of the endogenous connective tissue growth factor gene to different physiological stimuli: serum and TGF-ß1. Both stimuli caused a rapid and acute increase in burst sizes. Whereas TGF-ß1 showed prolonged transcriptional activation mediated by an increase of transcription rate, serum stimulation resulted in a large and temporally tight first transcriptional burst, followed by a refractory period in the range of hours. Our study thus reveals how different physiological stimuli can trigger kinetically distinct transcriptional responses of the same gene.
Asunto(s)
Modelos Biológicos , Transcripción Genética/fisiología , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Ratones , Células 3T3 NIH , Procesos EstocásticosRESUMEN
Imaging single fluorescent proteins in living mammalian cells is challenged by out-of-focus fluorescence excitation. To reduce out-of-focus fluorescence we developed reflected light-sheet microscopy (RLSM), a fluorescence microscopy method allowing selective plane illumination throughout the nuclei of living mammalian cells. A thin light sheet parallel to the imaging plane and close to the sample surface is generated by reflecting an elliptical laser beam incident from the top by 90° with a small mirror. The thin light sheet allows for an increased signal-to-background ratio superior to that in previous illumination schemes and enables imaging of single fluorescent proteins with up to 100-Hz time resolution. We demonstrated the single-molecule sensitivity of RLSM by measuring the DNA-bound fraction of glucocorticoid receptor (GR) and determining the residence times on DNA of various oligomerization states and mutants of GR and estrogen receptor-α (ER), which permitted us to resolve different modes of DNA binding of GR. We demonstrated two-color single-molecule imaging by observing the spatiotemporal colocalization of two different protein pairs. Our single-molecule measurements and statistical analysis revealed dynamic properties of transcription factors.