RESUMEN
Active cis-regulatory elements (cREs) in eukaryotes are characterized by nucleosomal depletion and, accordingly, higher accessibility. This property has turned out to be immensely useful for identifying cREs genome-wide and tracking their dynamics across different cellular states and is the basis of numerous methods taking advantage of the preferential enzymatic cleavage/labeling of accessible DNA. ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) has emerged as the most versatile and widely adaptable method and has been widely adopted as the standard tool for mapping open chromatin regions. Here, we discuss the current optimal practices and important considerations for carrying out ATAC-seq experiments, primarily in the context of mammalian systems.
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina , Secuenciación de Nucleótidos de Alto Rendimiento , Animales , Análisis de Secuencia de ADN , Cromatina , Secuencias Reguladoras de Ácidos Nucleicos , Mamíferos/genéticaRESUMEN
The ability to analyze the transcriptomic and epigenomic states of individual single cells has in recent years transformed our ability to measure and understand biological processes. Recent advancements have focused on increasing sensitivity and throughput to provide richer and deeper biological insights at the cellular level. The next frontier is the development of multiomic methods capable of analyzing multiple features from the same cell, such as the simultaneous measurement of the transcriptome and the chromatin accessibility of candidate regulatory elements. In this chapter, we discuss and describe SHARE-seq (Simultaneous high-throughput ATAC, and RNA expression with sequencing) for carrying out simultaneous chromatin accessibility and transcriptome measurements in single cells, together with the experimental and analytical considerations for achieving optimal results.
Asunto(s)
Cromatina , Transcriptoma , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Secuencias Reguladoras de Ácidos Nucleicos , Análisis de la Célula Individual/métodosRESUMEN
A hallmark feature of active cis-regulatory elements (CREs) in eukaryotes is their nucleosomal depletion and, accordingly, higher accessibility to enzymatic treatment. This property has been the basis of a number of sequencing-based assays for genome-wide identification and tracking the activity of CREs across different biological conditions, such as DNAse-seq, ATAC-seq , NOMeseq, and others. However, the fragmentation of DNA inherent to many of these assays and the limited read length of short-read sequencing platforms have so far not allowed the simultaneous measurement of the chromatin accessibility state of CREs located distally from each other. The combination of labeling accessible DNA with DNA modifications and nanopore sequencing has made it possible to develop such assays. Here, we provide a detailed protocol for carrying out the SMAC-seq assay (Single-Molecule long-read Accessible Chromatin mapping sequencing), in its m6A-SMAC-seq and m6A-CpG-GpC-SMAC-seq variants, together with methods for data processing and analysis, and discuss key experimental and analytical considerations for working with SMAC-seq datasets.
Asunto(s)
Cromatina , Secuenciación de Nucleótidos de Alto Rendimiento , Cromatina/genética , Secuenciación de Inmunoprecipitación de Cromatina , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Nucleosomas , Análisis de Secuencia de ADN/métodosRESUMEN
Aging is characterized by an increased vulnerability to infection and the development of inflammatory diseases, such as atherosclerosis, frailty, cancer and neurodegeneration. Here, we find that aging is associated with the loss of diurnally rhythmic innate immune responses, including monocyte trafficking from bone marrow to blood, response to lipopolysaccharide and phagocytosis. This decline in homeostatic immune responses was associated with a striking disappearance of circadian gene transcription in aged compared to young tissue macrophages. Chromatin accessibility was significantly greater in young macrophages than in aged macrophages; however, this difference did not explain the loss of rhythmic gene transcription in aged macrophages. Rather, diurnal expression of Kruppel-like factor 4 (Klf4), a transcription factor (TF) well established in regulating cell differentiation and reprogramming, was selectively diminished in aged macrophages. Ablation of Klf4 expression abolished diurnal rhythms in phagocytic activity, recapitulating the effect of aging on macrophage phagocytosis. Examination of individuals harboring genetic variants of KLF4 revealed an association with age-dependent susceptibility to death caused by bacterial infection. Our results indicate that loss of rhythmic Klf4 expression in aged macrophages is associated with disruption of circadian innate immune homeostasis, a mechanism that may underlie age-associated loss of protective immune responses.
Asunto(s)
Relojes Circadianos/genética , Macrófagos/fisiología , Envejecimiento , Animales , Aterosclerosis/genética , Diferenciación Celular/genética , Regulación de la Expresión Génica/genética , Inmunidad Innata/genética , Inflamación/genética , Factor 4 Similar a Kruppel/genética , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/fisiología , Fagocitosis/genéticaRESUMEN
Mammalian SWI/SNF (BAF) chromatin remodelers play dosage-sensitive roles in many human malignancies and neurologic disorders. The gene encoding the BAF subunit actin-like 6a (ACTL6A) is amplified early in the development of many squamous cell carcinomas (SCCs), but its oncogenic role remains unclear. Here we demonstrate that ACTL6A overexpression leads to its stoichiometric assembly into BAF complexes and drives their interaction and engagement with specific regulatory regions in the genome. In normal epithelial cells, ACTL6A was substoichiometric to other BAF subunits. However, increased ACTL6A levels by ectopic expression or in SCC cells led to near saturation of ACTL6A within BAF complexes. Increased ACTL6A occupancy enhanced polycomb opposition genome-wide to activate SCC genes and facilitated the co-dependent loading of BAF and TEAD-YAP complexes on chromatin. Both mechanisms appeared to be critical and function as a molecular AND gate for SCC initiation and maintenance, thereby explaining the specificity of the role of ACTL6A amplification in SCCs.
Asunto(s)
Actinas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Actinas/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Epigénesis Genética , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Proteínas del Grupo Polycomb/genética , Unión Proteica , Factores de Transcripción de Dominio TEA/genética , Factores de Transcripción de Dominio TEA/metabolismo , Proteínas Señalizadoras YAP/genética , Proteínas Señalizadoras YAP/metabolismoRESUMEN
The germinal center (GC) response is critical for both effective adaptive immunity and establishing peripheral tolerance by limiting autoreactive B cells. Dysfunction in these processes can lead to defective immune responses to infection or contribute to autoimmune disease. To understand the gene regulatory principles underlying the GC response, we generated a single-cell transcriptomic and epigenomic atlas of the human tonsil, a widely studied and representative lymphoid tissue. We characterize diverse immune cell subsets and build a trajectory of dynamic gene expression and transcription factor activity during B cell activation, GC formation, and plasma cell differentiation. We subsequently leverage cell typespecific transcriptomic and epigenomic maps to interpret potential regulatory impact of genetic variants implicated in autoimmunity, revealing that many exhibit their greatest regulatory potential in GC-associated cellular populations. These included gene loci linked with known roles in GC biology (IL21, IL21R, IL4R, and BCL6) and transcription factors regulating B cell differentiation (POU2AF1 and HHEX). Together, these analyses provide a powerful new cell typeresolved resource for the interpretation of cellular and genetic causes underpinning autoimmune disease.
Asunto(s)
Autoinmunidad/inmunología , Centro Germinal/inmunología , Proteínas de Homeodominio/inmunología , Interleucinas/inmunología , Análisis de la Célula Individual , Transactivadores/inmunología , Factores de Transcripción/inmunología , Diferenciación Celular/inmunología , Epigenómica , Proteínas de Homeodominio/genética , Humanos , Interleucinas/genética , Tonsila Palatina/inmunología , Análisis de Secuencia de ARN , Transactivadores/genética , Factores de Transcripción/genética , TranscriptomaRESUMEN
ATAC-seq is a versatile, adaptable, and widely adopted technique for mapping open chromatin regions. However, some biological systems, such as primary neurons, present unique challenges to its application. Conventional ATAC-seq would require the dissociation of the primary neurons after plating but dissociating them leads to rapid cell death and major changes in cell state, affecting ATAC-seq results. We have developed this modified ATAC-seq protocol to address this challenge for primary neurons, providing a high-quality and high-resolution accessible chromatin profile. For complete details on the use and execution of this protocol, please refer to Maor-Nof et al. (2021).
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina , Secuenciación de Nucleótidos de Alto Rendimiento , Animales , Cromatina/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Ratones , Neuronas , Análisis de Secuencia de ADN/métodosRESUMEN
The ATAC-seq assay has emerged as the most useful, versatile, and widely adaptable method for profiling accessible chromatin regions and tracking the activity of cis-regulatory elements (cREs) in eukaryotes. Thanks to its great utility, it is now being applied to map active chromatin in the context of a very wide diversity of biological systems and questions. In the course of these studies, considerable experience working with ATAC-seq data has accumulated and a standard set of computational tasks that need to be carried for most ATAC-seq analyses has emerged. Here, we review and provide examples of common such analytical procedures (including data processing, quality control, peak calling, identifying differentially accessible open chromatin regions, and variable transcription factor (TF) motif accessibility) and discuss recommended optimal practices.
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina/métodos , Cromatina/genética , Eucariontes/genética , Animales , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Secuencias Reguladoras de Ácidos Nucleicos/genética , Análisis de Secuencia de ADN/métodos , Factores de Transcripción/genéticaRESUMEN
The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a GGGGCC repeat expansion in the C9orf72 gene. We developed a platform to interrogate the chromatin accessibility landscape and transcriptional program within neurons during degeneration. We provide evidence that neurons expressing the dipeptide repeat protein poly(proline-arginine), translated from the C9orf72 repeat expansion, activate a highly specific transcriptional program, exemplified by a single transcription factor, p53. Ablating p53 in mice completely rescued neurons from degeneration and markedly increased survival in a C9orf72 mouse model. p53 reduction also rescued axonal degeneration caused by poly(glycine-arginine), increased survival of C9orf72 ALS/FTD-patient-induced pluripotent stem cell (iPSC)-derived motor neurons, and mitigated neurodegeneration in a C9orf72 fly model. We show that p53 activates a downstream transcriptional program, including Puma, which drives neurodegeneration. These data demonstrate a neurodegenerative mechanism dynamically regulated through transcription-factor-binding events and provide a framework to apply chromatin accessibility and transcription program profiles to neurodegeneration.
Asunto(s)
Proteína C9orf72/metabolismo , Expansión de las Repeticiones de ADN/genética , Degeneración Nerviosa/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Axones/metabolismo , Proteína C9orf72/genética , Muerte Celular , Células Cultivadas , Corteza Cerebral/patología , Cromatina/metabolismo , Daño del ADN , Modelos Animales de Enfermedad , Drosophila , Ratones Endogámicos C57BL , Degeneración Nerviosa/patología , Estabilidad Proteica , Transcripción Genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Synaptic activity in neurons leads to the rapid activation of genes involved in mammalian behavior. ATP-dependent chromatin remodelers such as the BAF complex contribute to these responses and are generally thought to activate transcription. However, the mechanisms keeping such "early activation" genes silent have been a mystery. In the course of investigating Mendelian recessive autism, we identified six families with segregating loss-of-function mutations in the neuronal BAF (nBAF) subunit ACTL6B (originally named BAF53b). Accordingly, ACTL6B was the most significantly mutated gene in the Simons Recessive Autism Cohort. At least 14 subunits of the nBAF complex are mutated in autism, collectively making it a major contributor to autism spectrum disorder (ASD). Patient mutations destabilized ACTL6B protein in neurons and rerouted dendrites to the wrong glomerulus in the fly olfactory system. Humans and mice lacking ACTL6B showed corpus callosum hypoplasia, indicating a conserved role for ACTL6B in facilitating neural connectivity. Actl6b knockout mice on two genetic backgrounds exhibited ASD-related behaviors, including social and memory impairments, repetitive behaviors, and hyperactivity. Surprisingly, mutation of Actl6b relieved repression of early response genes including AP1 transcription factors (Fos, Fosl2, Fosb, and Junb), increased chromatin accessibility at AP1 binding sites, and transcriptional changes in late response genes associated with early response transcription factor activity. ACTL6B loss is thus an important cause of recessive ASD, with impaired neuron-specific chromatin repression indicated as a potential mechanism.
Asunto(s)
Trastorno del Espectro Autista/genética , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Hipocampo/patología , Actinas/genética , Adenosina Trifosfato/genética , Animales , Trastorno del Espectro Autista/patología , Conducta Animal/fisiología , Cromatina/genética , Ensamble y Desensamble de Cromatina/genética , Emparejamiento Cromosómico/genética , Emparejamiento Cromosómico/fisiología , Cuerpo Calloso/metabolismo , Cuerpo Calloso/patología , Dendritas/genética , Dendritas/fisiología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Hipocampo/metabolismo , Humanos , Ratones , Ratones Noqueados , Mutación/genética , Neuronas/metabolismo , Neuronas/patología , Factores de Transcripción/genéticaRESUMEN
Mapping open chromatin regions has emerged as a widely used tool for identifying active regulatory elements in eukaryotes. However, existing approaches, limited by reliance on DNA fragmentation and short-read sequencing, cannot provide information about large-scale chromatin states or reveal coordination between the states of distal regulatory elements. We have developed a method for profiling the accessibility of individual chromatin fibers, a single-molecule long-read accessible chromatin mapping sequencing assay (SMAC-seq), enabling the simultaneous, high-resolution, single-molecule assessment of chromatin states at multikilobase length scales. Our strategy is based on combining the preferential methylation of open chromatin regions by DNA methyltransferases with low sequence specificity, in this case EcoGII, an N6-methyladenosine (m6A) methyltransferase, and the ability of nanopore sequencing to directly read DNA modifications. We demonstrate that aggregate SMAC-seq signals match bulk-level accessibility measurements, observe single-molecule nucleosome and transcription factor protection footprints, and quantify the correlation between chromatin states of distal genomic elements.
Asunto(s)
Cromatina/química , Fragmentación del ADN , Saccharomyces cerevisiae/química , Adenosina/análogos & derivados , Adenosina/química , Línea Celular , Inmunoprecipitación de Cromatina , Islas de CpG , Metilación de ADN , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Metilación , Metiltransferasas/genética , Nucleosomas/química , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
Patchy infiltration of tumors by cytotoxic T cells (CTLs) predicts poorer prognosis for cancer patients. The factors limiting intratumoral CTL dissemination, though, are poorly understood. To study CTL dissemination in tumors, we histologically examined human melanoma samples and used mice to image B16-OVA tumors infiltrated by OT-I CTLs using intravital two-photon microscopy. In patients, most CTLs concentrated around peripheral blood vessels, especially in poorly infiltrated tumors. In mice, OT-I CTLs had to cluster around tumor cells to efficiently kill them in a contact-and perforin-dependent manner and cytotoxicity was strictly antigen-specific. OT-I CTLs as well as non-specific CTLs concentrated around peripheral vessels, and cleared the tumor cells around them. This was also the case when CTLs were injected directly into the tumors. CTLs crawled rapidly only in areas within 50 µm of flowing blood vessels and transient occlusion of vessels immediately, though reversibly, stopped their migration. In vitro, oxygen depletion and blockade of oxidative phosphorylation also reduced CTL motility. Taken together, these results suggest that hypoxia limits CTL migration away from blood vessels, providing immune-privileged niches for tumor cells to survive. Normalizing intratumoral vasculature may thus synergize with tumor immunotherapy.
Asunto(s)
Vasos Sanguíneos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Neoplasias Cutáneas/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Antígenos de Neoplasias/inmunología , Movimiento Celular , Citotoxicidad Inmunológica , Humanos , Melanoma/irrigación sanguínea , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales , Neovascularización Patológica , Fosforilación Oxidativa , Perforina/metabolismo , Neoplasias Cutáneas/irrigación sanguíneaRESUMEN
Physical access to DNA is a highly dynamic property of chromatin that plays an essential role in establishing and maintaining cellular identity. The organization of accessible chromatin across the genome reflects a network of permissible physical interactions through which enhancers, promoters, insulators and chromatin-binding factors cooperatively regulate gene expression. This landscape of accessibility changes dynamically in response to both external stimuli and developmental cues, and emerging evidence suggests that homeostatic maintenance of accessibility is itself dynamically regulated through a competitive interplay between chromatin-binding factors and nucleosomes. In this Review, we examine how the accessible genome is measured and explore the role of transcription factors in initiating accessibility remodelling; our goal is to illustrate how chromatin accessibility defines regulatory elements within the genome and how these epigenetic features are dynamically established to control gene expression.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Epigenómica , Genoma Humano/fisiología , Nucleosomas/metabolismo , Animales , Elementos de Facilitación Genéticos/fisiología , Humanos , Nucleosomas/genética , Regiones Promotoras Genéticas/fisiologíaRESUMEN
DNA methylation is a key regulator of embryonic stem cell (ESC) biology, dynamically changing between naïve, primed, and differentiated states. The p53 tumor suppressor is a pivotal guardian of genomic stability, but its contributions to epigenetic regulation and stem cell biology are less explored. We report that, in naïve mouse ESCs (mESCs), p53 restricts the expression of the de novo DNA methyltransferases Dnmt3a and Dnmt3b while up-regulating Tet1 and Tet2, which promote DNA demethylation. The DNA methylation imbalance in p53-deficient (p53-/-) mESCs is the result of augmented overall DNA methylation as well as increased methylation landscape heterogeneity. In differentiating p53-/- mESCs, elevated methylation persists, albeit more mildly. Importantly, concomitant with DNA methylation heterogeneity, p53-/- mESCs display increased cellular heterogeneity both in the "naïve" state and upon induced differentiation. This impact of p53 loss on 5-methylcytosine (5mC) heterogeneity was also evident in human ESCs and mouse embryos in vivo. Hence, p53 helps maintain DNA methylation homeostasis and clonal homogeneity, a function that may contribute to its tumor suppressor activity.
Asunto(s)
Metilación de ADN/genética , Regulación de la Expresión Génica/genética , Heterogeneidad Genética , Homeostasis/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Diferenciación Celular/genética , Células Clonales , ADN (Citosina-5-)-Metiltransferasas/genética , Células Madre Embrionarias , Eliminación de Gen , Humanos , Ratones , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Stable maintenance of gene regulatory programs is essential for normal function in multicellular organisms. Epigenetic mechanisms, and DNA methylation in particular, are hypothesized to facilitate such maintenance by creating cellular memory that can be written during embryonic development and then guide cell-type-specific gene expression. Here we develop new methods for quantitative inference of DNA methylation turnover rates, and show that human embryonic stem cells preserve their epigenetic state by balancing antagonistic processes that add and remove methylation marks rather than by copying epigenetic information from mother to daughter cells. In contrast, somatic cells transmit considerable epigenetic information to progenies. Paradoxically, the persistence of the somatic epigenome makes it more vulnerable to noise, since random epimutations can accumulate to massively perturb the epigenomic ground state. The rate of epigenetic perturbation depends on the genomic context, and, in particular, DNA methylation loss is coupled to late DNA replication dynamics. Epigenetic perturbation is not observed in the pluripotent state, because the rapid turnover-based equilibrium continuously reinforces the canonical state. This dynamic epigenetic equilibrium also explains how the epigenome can be reprogrammed quickly and to near perfection after induced pluripotency.
Asunto(s)
Metilación de ADN , Epigénesis Genética , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Alelos , Línea Celular , Línea Celular Tumoral , Células Clonales/citología , Células Clonales/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Fibroblastos/citología , Genoma Humano/genética , Humanos , Células Madre Pluripotentes Inducidas/citologíaRESUMEN
Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3ß signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Animales , Blastocisto/citología , Reprogramación Celular , Quimera/embriología , Cromatina/metabolismo , Metilación de ADN , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Epigénesis Genética , Femenino , Estratos Germinativos/citología , Histonas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Masculino , Ratones , Mórula/citología , Organogénesis , Regiones Promotoras Genéticas/genética , Medicina Regenerativa , Reproducibilidad de los Resultados , Transducción de Señal , Inactivación del Cromosoma XRESUMEN
Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.