RESUMEN
Loss-of-function mutations in MECP2 cause Rett syndrome (RTT), a severe neurological disorder that mainly affects girls. Mutations in MECP2 do occur in males occasionally and typically cause severe encephalopathy and premature lethality. Recently, we identified a missense mutation (c.353G>A, p.Gly118Glu [G118E]), which has never been seen before in MECP2, in a young boy who suffered from progressive motor dysfunction and developmental delay. To determine whether this variant caused the clinical symptoms and study its functional consequences, we established two disease models, including human neurons from patient-derived iPSCs and a knock-in mouse line. G118E mutation partially reduces MeCP2 abundance and its DNA binding, and G118E mice manifest RTT-like symptoms seen in the patient, affirming the pathogenicity of this mutation. Using live-cell and single-molecule imaging, we found that G118E mutation alters MeCP2's chromatin interaction properties in live neurons independently of its effect on protein levels. Here we report the generation and characterization of RTT models of a male hypomorphic variant and reveal new insight into the mechanism by which this pathological mutation affects MeCP2's chromatin dynamics. Our ability to quantify protein dynamics in disease models lays the foundation for harnessing high-resolution single-molecule imaging as the next frontier for developing innovative therapies for RTT and other diseases.
Asunto(s)
Cromatina , Síndrome de Rett , Femenino , Humanos , Masculino , Ratones , Animales , Cromatina/metabolismo , Encéfalo/metabolismo , Proteína 2 de Unión a Metil-CpG/genética , Síndrome de Rett/genética , Mutación , Neuronas/metabolismoRESUMEN
Comparative genome analyses reveal that organismal complexity scales not with gene number but with gene regulation. Recent efforts indicate that the human genome likely contains hundreds of thousands of enhancers, with a typical gene embedded in a milieu of tens of enhancers. Proliferation of cis-regulatory DNAs is accompanied by increased complexity and functional diversification of transcriptional machineries recognizing distal enhancers and core promoters and by the high-order spatial organization of genetic elements. We review progress in unraveling one of the outstanding mysteries of modern biology: the dynamic communication of remote enhancers with target promoters in the specification of cellular identity.
Asunto(s)
Diferenciación Celular , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Animales , ARN Polimerasas Dirigidas por ADN/metabolismo , Humanos , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Whereas folding of genomes at the large scale of epigenomic compartments and topologically associating domains (TADs) is now relatively well understood, how chromatin is folded at finer scales remains largely unexplored in mammals. Here, we overcome some limitations of conventional 3C-based methods by using high-resolution Micro-C to probe links between 3D genome organization and transcriptional regulation in mouse stem cells. Combinatorial binding of transcription factors, cofactors, and chromatin modifiers spatially segregates TAD regions into various finer-scale structures with distinct regulatory features including stripes, dots, and domains linking promoters-to-promoters (P-P) or enhancers-to-promoters (E-P) and bundle contacts between Polycomb regions. E-P stripes extending from the edge of domains predominantly link co-expressed loci, often in the absence of CTCF and cohesin occupancy. Acute inhibition of transcription disrupts these gene-related folding features without altering higher-order chromatin structures. Our study uncovers previously obscured finer-scale genome organization, establishing functional links between chromatin folding and gene regulation.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Cromatina/química , Cromatina/metabolismo , Transcripción Genética , Animales , Factor de Unión a CCCTC/genética , Cromatina/genética , ADN Polimerasa II/genética , ADN Polimerasa II/metabolismo , Células Madre Embrionarias/fisiología , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Componentes Genómicos , Ratones , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Mammalian genomes are folded into topologically associating domains (TADs), consisting of chromatin loops anchored by CTCF and cohesin. Some loops are cell-type specific. Here we asked whether CTCF loops are established by a universal or locus-specific mechanism. Investigating the molecular determinants of CTCF clustering, we found that CTCF self-association in vitro is RNase sensitive and that an internal RNA-binding region (RBRi) mediates CTCF clustering and RNA interaction in vivo. Strikingly, deleting the RBRi impairs about half of all chromatin loops in mESCs and causes deregulation of gene expression. Disrupted loop formation correlates with diminished clustering and chromatin binding of RBRi mutant CTCF, which in turn results in a failure to halt cohesin-mediated extrusion. Thus, CTCF loops fall into at least two classes: RBRi-independent and RBRi-dependent loops. We speculate that evidence for RBRi-dependent loops may provide a molecular mechanism for establishing cell-specific CTCF loops, potentially regulated by RNA(s) or other RBRi-interacting partners.
Asunto(s)
Factor de Unión a CCCTC/metabolismo , Cromatina/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Animales , Factor de Unión a CCCTC/química , Factor de Unión a CCCTC/genética , Línea Celular , Cromatina/química , Cromatina/genética , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones Transgénicos , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-ActividadRESUMEN
The transcriptional activators Oct4, Sox2, and Nanog cooperate with a wide array of cofactors to orchestrate an embryonic stem (ES) cell-specific gene expression program that forms the molecular basis of pluripotency. Here, we report using an unbiased in vitro transcription-biochemical complementation assay to discover a multisubunit stem cell coactivator complex (SCC) that is selectively required for the synergistic activation of the Nanog gene by Oct4 and Sox2. Purification, identification, and reconstitution of SCC revealed this coactivator to be the trimeric XPC-nucleotide excision repair complex. SCC interacts directly with Oct4 and Sox2 and is recruited to the Nanog and Oct4 promoters as well as a majority of genomic regions that are occupied by Oct4 and Sox2. Depletion of SCC/XPC compromised both pluripotency in ES cells and somatic cell reprogramming of fibroblasts to induced pluripotent stem (iPS) cells. This study identifies a transcriptional coactivator with diversified functions in maintaining ES cell pluripotency and safeguarding genome integrity.
Asunto(s)
Células Madre Embrionarias/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción SOXB1/metabolismo , Animales , Línea Celular , Reprogramación Celular , Reparación del ADN , Células Madre Embrionarias/citología , Inestabilidad Genómica , Células HeLa , Proteínas de Homeodominio/genética , Humanos , Ratones , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismoRESUMEN
Faithful resetting of the epigenetic memory of a somatic cell to a pluripotent state during cellular reprogramming requires DNA methylation to silence somatic gene expression and dynamic DNA demethylation to activate pluripotency gene transcription. The removal of methylated cytosines requires the base excision repair enzyme TDG, but the mechanism by which TDG-dependent DNA demethylation occurs in a rapid and site-specific manner remains unclear. Here we show that the XPC DNA repair complex is a potent accelerator of global and locus-specific DNA demethylation in somatic and pluripotent stem cells. XPC cooperates with TDG genome-wide to stimulate the turnover of essential intermediates by overcoming slow TDG-abasic product dissociation during active DNA demethylation. We further establish that DNA demethylation induced by XPC expression in somatic cells overcomes an early epigenetic barrier in cellular reprogramming and facilitates the generation of more robust induced pluripotent stem cells, characterized by enhanced pluripotency-associated gene expression and self-renewal capacity. Taken together with our previous studies establishing the XPC complex as a transcriptional coactivator, our findings underscore two distinct but complementary mechanisms by which XPC influences gene regulation by coordinating efficient TDG-mediated DNA demethylation along with active transcription during somatic cell reprogramming.
Asunto(s)
Reprogramación Celular/genética , Metilación de ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Madre Pluripotentes/fisiología , Animales , Células Madre Embrionarias , Epigénesis Genética/genética , Fibroblastos/fisiología , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Células HEK293 , Humanos , Ratones , Timina ADN Glicosilasa/genética , Timina ADN Glicosilasa/metabolismoRESUMEN
Transcription factor (TF)-directed enhanceosome assembly constitutes a fundamental regulatory mechanism driving spatiotemporal gene expression programs during animal development. Despite decades of study, we know little about the dynamics or order of events animating TF assembly at cis-regulatory elements in living cells and the long-range molecular "dialog" between enhancers and promoters. Here, combining genetic, genomic, and imaging approaches, we characterize a complex long-range enhancer cluster governing Krüppel-like factor 4 (Klf4) expression in naïve pluripotency. Genome editing by CRISPR/Cas9 revealed that OCT4 and SOX2 safeguard an accessible chromatin neighborhood to assist the binding of other TFs/cofactors to the enhancer. Single-molecule live-cell imaging uncovered that two naïve pluripotency TFs, STAT3 and ESRRB, interrogate chromatin in a highly dynamic manner, in which SOX2 promotes ESRRB target search and chromatin-binding dynamics through a direct protein-tethering mechanism. Together, our results support a highly dynamic yet intrinsically ordered enhanceosome assembly to maintain the finely balanced transcription program underlying naïve pluripotency.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica/genética , Factores de Transcripción de Tipo Kruppel/genética , Células Madre Pluripotentes/fisiología , Animales , Sitios de Unión , Cromatina/metabolismo , Células Madre Embrionarias , Factor 4 Similar a Kruppel , Ratones , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Unión Proteica , Receptores de Estrógenos/metabolismo , Factores de Transcripción SOXB1/metabolismo , Factor de Transcripción STAT3/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The enormous size of mammalian genomes means that for a DNA-binding protein the number of nonspecific, off-target sites vastly exceeds the number of specific, cognate sites. How mammalian DNA-binding proteins overcome this challenge to efficiently locate their target sites is not known. Here, through live-cell single-molecule tracking, we show that CCCTC-binding factor, CTCF, is repeatedly trapped in small zones that likely correspond to CTCF clusters, in a manner that is largely dependent on an internal RNA-binding region (RBRi). We develop a new theoretical model called anisotropic diffusion through transient trapping in zones to explain CTCF dynamics. Functionally, transient RBRi-mediated trapping increases the efficiency of CTCF target search by ~2.5-fold. Overall, our results suggest a 'guided' mechanism where CTCF clusters concentrate diffusing CTCF proteins near cognate binding sites, thus increasing the local ON-rate. We suggest that local guiding may allow DNA-binding proteins to more efficiently locate their target sites.
Asunto(s)
Factor de Unión a CCCTC/metabolismo , Imagen Individual de Molécula/métodos , Animales , Sitios de Unión/fisiología , Factor de Unión a CCCTC/fisiología , Línea Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Femenino , Humanos , Masculino , Ratones , Unión Proteica/fisiología , Proteínas Represoras/metabolismoRESUMEN
Transcription is apparently risky business. Its intrinsic mutagenic potential must be kept in check by networks of DNA repair factors that monitor the transcription process to repair DNA lesions that could otherwise compromise transcriptional fidelity and genome integrity. Intriguingly, recent studies point to an even more direct function of DNA repair complexes as coactivators of transcription and the unexpected role of "scheduled" DNA damage/repair at gene promoters. Paradoxically, spontaneous DNA double-strand breaks also induce ectopic transcription that is essential for repair. Thus, transcription, DNA damage, and repair may be more physically and functionally intertwined than previously appreciated.
Asunto(s)
Reparación del ADN/genética , ADN/genética , Proteínas/genética , Transcripción Genética , Animales , Roturas del ADN de Doble Cadena , Daño del ADN/genética , Inestabilidad Genómica , Humanos , Regiones Promotoras GenéticasRESUMEN
The general transcription factor P-TEFb, a master regulator of RNA polymerase (Pol) II elongation, phosphorylates the C-terminal domain (CTD) of Pol II and negative elongation factors to release Pol II from promoter-proximal pausing. We show here that P-TEFb surprisingly inhibits the myoblast differentiation into myotubes, and that P-TEFb and its two positive complexes are eliminated in this process. In contrast, DYRK1A, another CTD kinase known to control transcription of a subset of genes important for development and tissue homeostasis, is found to activate transcription of key myogenic genes. We show that active DYRK1A exists in a complex with the WD40-repeat protein DCAF7 that stabilizes and tethers DYRK1A to Pol II, so that DYRK1A-DCAF7 can co-migrate with and phosphorylate Pol II along the myogenic gene loci. Thus, DCAF7 modulates the kinase signaling output of DYRK1A on Pol II to stimulate myogenic transcription after active P-TEFb function is shut off.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Desarrollo de Músculos/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Transcripción Genética , Animales , Diferenciación Celular/genética , Ciclina T/genética , Quinasa 9 Dependiente de la Ciclina/genética , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Ratones , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Proteínas Nucleares/genética , ARN Polimerasa II/química , ARN Polimerasa II/genética , Canales de Translocación SEC/genética , Factores de Transcripción/genética , Quinasas DyrKRESUMEN
The embryonic stem cell (ESC) state is transcriptionally controlled by OCT4, SOX2, and NANOG with cofactors, chromatin regulators, noncoding RNAs, and other effectors of signaling pathways. Uncovering components of these regulatory circuits and their interplay provides the knowledge base to deploy ESCs and induced pluripotent stem cells. We recently identified the DNA-repair complex xeroderma pigmentosum C (XPC)-RAD23B-CETN2 as a stem cell coactivator (SCC) required for OCT4/SOX2 transcriptional activation. Here we investigate the role of SCC genome-wide in murine ESCs by mapping regions bound by RAD23B and analyzing transcriptional profiles of SCC-depleted ESCs. We establish OCT4 and SOX2 as the primary transcription factors recruiting SCC to regulatory regions of pluripotency genes and identify the XPC subunit as essential for interaction with the two proteins. The present study reveals new mechanistic and functional aspects of SCC transcriptional activity, and thus underscores the diversified functions of this regulatory complex.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica , Animales , Sitios de Unión , Diferenciación Celular , Linaje de la Célula , Reparación del ADN , Genoma , Células HEK293 , Humanos , Inmunoglobulina G/química , Lentivirus/metabolismo , Ratones , Ratones Noqueados , Células Madre Pluripotentes/citología , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The human NuA4/TIP60 co-activator complex, a fusion of the yeast SWR1 and NuA4 complexes, both incorporates the histone variant H2A.Z into nucleosomes and acetylates histones H4/H2A/H2A.Z to regulate gene expression and maintain genome stability. Our cryo-electron microscopy studies show that, within the NuA4/TIP60 complex, the EP400 subunit serves as a scaffold holding the different functional modules in specific positions, creating a unique arrangement of the ARP module. EP400 interacts with the TRRAP subunit using a footprint that overlaps with that of the SAGA acetyltransferase complex, preventing the formation of a hybrid complex. Loss of the TRRAP subunit leads to mislocalization of NuA4/TIP60, resulting in the redistribution of H2A.Z and its acetylation across the genome, emphasizing the dual functionality of NuA4/TIP60 as a single macromolecular assembly.
RESUMEN
The vast majority of biologic-based therapeutics operate within serum, on the cell surface, or within endocytic vesicles, in large part because proteins and nucleic acids fail to efficiently cross cell or endosomal membranes. The impact of biologic-based therapeutics would expand exponentially if proteins and nucleic acids could reliably evade endosomal degradation, escape endosomal vesicles, and remain functional. Using the cell-permeant mini-protein ZF5.3, here we report the efficient nuclear delivery of functional Methyl-CpG-binding-protein 2 (MeCP2), a transcriptional regulator whose mutation causes Rett syndrome (RTT). We report that ZF-tMeCP2, a conjugate of ZF5.3 and MeCP2(Δaa13-71, 313-484), binds DNA in a methylation-dependent manner in vitro, and reaches the nucleus of model cell lines intact to achieve an average concentration of 700 nM. When delivered to live cells, ZF-tMeCP2 engages the NCoR/SMRT corepressor complex, selectively represses transcription from methylated promoters, and colocalizes with heterochromatin in mouse primary cortical neurons. We also report that efficient nuclear delivery of ZF-tMeCP2 relies on an endosomal escape portal provided by HOPS-dependent endosomal fusion. The Tat conjugate of MeCP2 (Tat-tMeCP2), evaluated for comparison, is degraded within the nucleus, is not selective for methylated promoters, and trafficks in a HOPS-independent manner. These results support the feasibility of a HOPS-dependent portal for delivering functional macromolecules to the cell interior using the cell-penetrant mini-protein ZF5.3. Such a strategy could broaden the impact of multiple families of biologic-based therapeutics.
RESUMEN
Integration of retroviral vectors in the human genome follows nonrandom patterns that favor insertional deregulation of gene expression and increase the risk of their use in clinical gene therapy. The molecular basis of retroviral target site selection is still poorly understood. We used deep sequencing technology to build genomewide, high-definition maps of > 60 000 integration sites of Moloney murine leukemia virus (MLV)- and HIV-based retroviral vectors in the genome of human CD34(+) multipotent hematopoietic progenitor cells (HPCs) and used gene expression profiling, chromatin immunoprecipitation, and bioinformatics to associate integration to genetic and epigenetic features of the HPC genome. Clusters of recurrent MLV integrations identify regulatory elements (alternative promoters, enhancers, evolutionarily conserved noncoding regions) within or around protein-coding genes and microRNAs with crucial functions in HPC growth and differentiation, bearing epigenetic marks of active or poised transcription (H3K4me1, H3K4me2, H3K4me3, H3K9Ac, Pol II) and specialized chromatin configurations (H2A.Z). Overall, we mapped 3500 high-frequency integration clusters, which represent a new resource for the identification of transcriptionally active regulatory elements. High-definition MLV integration maps provide a rational basis for predicting genotoxic risks in gene therapy and a new tool for genomewide identification of promoters and regulatory elements controlling hematopoietic stem and progenitor cell functions.
Asunto(s)
Genoma Humano , Células Madre Hematopoyéticas/fisiología , Elementos Reguladores de la Transcripción/genética , Retroviridae/genética , Integración Viral/genética , Biomarcadores/metabolismo , Células Cultivadas , Cromatina/genética , Inmunoprecipitación de Cromatina , Epigenómica , Sangre Fetal/citología , Perfilación de la Expresión Génica , VIH/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Virus de la Leucemia Murina de Moloney/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genéticaRESUMEN
Integration of retroviral vectors in the human genome follows non random patterns that favor insertional deregulation of gene expression and may cause risks of insertional mutagenesis when used in clinical gene therapy. Understanding how viral vectors integrate into the human genome is a key issue in predicting these risks. We provide a new statistical method to compare retroviral integration patterns. We identified the positions where vectors derived from the Human Immunodeficiency Virus (HIV) and the Moloney Murine Leukemia Virus (MLV) show different integration behaviors in human hematopoietic progenitor cells. Non-parametric density estimation was used to identify candidate comparative hotspots, which were then tested and ranked. We found 100 significative comparative hotspots, distributed throughout the chromosomes. HIV hotspots were wider and contained more genes than MLV ones. A Gene Ontology analysis of HIV targets showed enrichment of genes involved in antigen processing and presentation, reflecting the high HIV integration frequency observed at the MHC locus on chromosome 6. Four histone modifications/variants had a different mean density in comparative hotspots (H2AZ, H3K4me1, H3K4me3, H3K9me1), while gene expression within the comparative hotspots did not differ from background. These findings suggest the existence of epigenetic or nuclear three-dimensional topology contexts guiding retroviral integration to specific chromosome areas.
Asunto(s)
Vectores Genéticos/genética , Genoma Humano , VIH/genética , Modelos Genéticos , Virus de la Leucemia Murina de Moloney/genética , Integración Viral , Antígenos CD34/genética , Cromosomas Humanos Par 6 , Sitios Genéticos , Antígenos HLA/genética , Células Madre Hematopoyéticas , Histonas/genética , Humanos , Reproducibilidad de los ResultadosRESUMEN
Clinical trials have demonstrated the potential of ex vivo hematopoietic stem cell gene therapy to treat X-linked severe combined immunodeficiency (SCID-X1) using γ-retroviral vectors, leading to immune system functionality in the majority of treated patients without pretransplant conditioning. The success was tempered by insertional oncogenesis in a proportion of the patients. To reduce the genotoxicity risk, a self-inactivating (SIN) lentiviral vector (LV) with improved expression of a codon optimized human interleukin-2 receptor γ gene (IL2RG) cDNA (coγc), regulated by its 1.1 kb promoter region (γcPr), was compared in efficacy to the viral spleen focus forming virus (SF) and the cellular phosphoglycerate kinase (PGK) promoters. Pretransplant conditioning of Il2rg(-/-) mice resulted in long-term reconstitution of T and B lymphocytes, normalized natural antibody titers, humoral immune responses, ConA/IL-2 stimulated spleen cell proliferation, and polyclonal T-cell receptor gene rearrangements with a clear integration preference of the SF vector for proto-oncogenes, contrary to the PGK and γcPr vectors. We conclude that SIN lentiviral gene therapy using coγc driven by the γcPr or PGK promoter corrects the SCID phenotype, potentially with an improved safety profile, and that low-dose conditioning proved essential for immune competence, allowing for a reduced threshold of cell numbers required.
Asunto(s)
Codón , Terapia Genética , Trasplante de Células Madre Hematopoyéticas , Subunidad gamma Común de Receptores de Interleucina/genética , Lentivirus/genética , Inmunodeficiencia Combinada Grave/terapia , Animales , Formación de Anticuerpos , Linfocitos B/inmunología , Ratones , Ratones SCID , Receptores de Antígenos de Linfocitos T/inmunología , Inmunodeficiencia Combinada Grave/inmunología , Linfocitos T/inmunologíaRESUMEN
Vector-associated side effects in clinical gene therapy have provided insights into the molecular mechanisms of hematopoietic regulation in vivo. Surprisingly, many retrovirus insertion sites (RIS) present in engrafted cells have been found to cluster nonrandomly in close association with specific genes. Our data demonstrate that these genes directly influence the in vivo fate of hematopoietic cell clones. Analysis of insertions thus far has been limited to individual clinical studies. Here, we studied >7,000 insertions retrieved from various studies. More than 40% of all insertions found in engrafted gene-modified cells were clustered in the same genomic areas covering only 0.36% of the genome. Gene classification analyses displayed significant overrepresentation of genes associated with hematopoietic functions and relevance for cell growth and survival in vivo. The similarity of insertion distributions indicates that vector insertions in repopulating cells cluster in predictable patterns. Thus, insertion analyses of preclinical in vitro and murine in vivo studies as well as vector insertion repertoires in clinical trials yielded concerted results and mark a small number of interesting genomic loci and genes that warrants further investigation of the biological consequences of vector insertions.
Asunto(s)
Gammaretrovirus/genética , Terapia Genética/efectos adversos , Vectores Genéticos/efectos adversos , Genoma , Integración Viral , Animales , Mapeo Cromosómico , Redes Reguladoras de Genes , Trasplante de Células Madre Hematopoyéticas , Humanos , Ratones , Primates , Trasplantes , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/genética , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/terapiaRESUMEN
3D genome mapping aims at connecting the physics of chromatin folding to the underlying biological events, and applications of various chromosomal conformation capture (3C) assays continue to discover critical roles of genome folding in regulating nuclear functions. To interrogate the full spectrum of chromatin folding ranging from the level of nucleosomes to full chromosomes in mammals, we developed an enhanced 3C-based method called Micro-C. The protocol employs Micrococcal nuclease (MNase) to fragment the genome, which overcomes the resolution limit of restriction enzyme-based methods, enabling the estimation of contact frequencies between proximal nucleosomes. Such improvements successfully resolve the fine-scale level of chromatin folding, including enhancer-promoter or promoter-promoter interactions, genic and nucleosomal folding, and boost the signal-to-noise ratio in detecting loops and substructures underlying TADs. In this chapter, we will thoroughly discuss the details of the Micro-C protocol and critical parameters to consider for generating high-quality Micro-C maps.
Asunto(s)
Genoma , Nucleosomas , Animales , Cromatina/genética , Mapeo Cromosómico/métodos , Mamíferos/genética , Nucleasa Microcócica , Nucleosomas/genéticaRESUMEN
It remains unclear why acute depletion of CTCF (CCCTC-binding factor) and cohesin only marginally affects expression of most genes despite substantially perturbing three-dimensional (3D) genome folding at the level of domains and structural loops. To address this conundrum, we used high-resolution Micro-C and nascent transcript profiling in mouse embryonic stem cells. We find that enhancer-promoter (E-P) interactions are largely insensitive to acute (3-h) depletion of CTCF, cohesin or WAPL. YY1 has been proposed as a structural regulator of E-P loops, but acute YY1 depletion also had minimal effects on E-P loops, transcription and 3D genome folding. Strikingly, live-cell, single-molecule imaging revealed that cohesin depletion reduced transcription factor (TF) binding to chromatin. Thus, although CTCF, cohesin, WAPL or YY1 is not required for the short-term maintenance of most E-P interactions and gene expression, our results suggest that cohesin may facilitate TFs to search for and bind their targets more efficiently.
Asunto(s)
Proteínas , Animales , Ratones , Factor de Unión a CCCTC/genética , CohesinasRESUMEN
Transcription factors (TFs) are classically attributed a modular construction, containing well-structured sequence-specific DNA-binding domains (DBDs) paired with disordered activation domains (ADs) responsible for protein-protein interactions targeting co-factors or the core transcription initiation machinery. However, this simple division of labor model struggles to explain why TFs with identical DNA-binding sequence specificity determined in vitro exhibit distinct binding profiles in vivo. The family of hypoxia-inducible factors (HIFs) offer a stark example: aberrantly expressed in several cancer types, HIF-1α and HIF-2α subunit isoforms recognize the same DNA motif in vitro - the hypoxia response element (HRE) - but only share a subset of their target genes in vivo, while eliciting contrasting effects on cancer development and progression under certain circumstances. To probe the mechanisms mediating isoform-specific gene regulation, we used live-cell single particle tracking (SPT) to investigate HIF nuclear dynamics and how they change upon genetic perturbation or drug treatment. We found that HIF-α subunits and their dimerization partner HIF-1ß exhibit distinct diffusion and binding characteristics that are exquisitely sensitive to concentration and subunit stoichiometry. Using domain-swap variants, mutations, and a HIF-2α specific inhibitor, we found that although the DBD and dimerization domains are important, another main determinant of chromatin binding and diffusion behavior is the AD-containing intrinsically disordered region (IDR). Using Cut&Run and RNA-seq as orthogonal genomic approaches, we also confirmed IDR-dependent binding and activation of a specific subset of HIF target genes. These findings reveal a previously unappreciated role of IDRs in regulating the TF search and binding process that contribute to functional target site selectivity on chromatin.