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1.
Cell ; 185(2): 328-344.e26, 2022 01 20.
Artículo en Inglés | MEDLINE | ID: mdl-35063074

RESUMEN

Locomotion is a complex behavior required for animal survival. Vertebrate locomotion depends on spinal interneurons termed the central pattern generator (CPG), which generates activity responsible for the alternation of flexor and extensor muscles and the left and right side of the body. It is unknown whether multiple or a single neuronal type is responsible for the control of mammalian locomotion. Here, we show that ventral spinocerebellar tract neurons (VSCTs) drive generation and maintenance of locomotor behavior in neonatal and adult mice. Using mouse genetics, physiological, anatomical, and behavioral assays, we demonstrate that VSCTs exhibit rhythmogenic properties and neuronal circuit connectivity consistent with their essential role in the locomotor CPG. Importantly, optogenetic activation and chemogenetic silencing reveals that VSCTs are necessary and sufficient for locomotion. These findings identify VSCTs as critical components for mammalian locomotion and provide a paradigm shift in our understanding of neural control of complex behaviors.


Asunto(s)
Locomoción/fisiología , Mamíferos/fisiología , Neuronas Motoras/citología , Tractos Espinocerebelares/citología , Animales , Axones/fisiología , Fenómenos Electrofisiológicos , Uniones Comunicantes/metabolismo , Silenciador del Gen , Ácido Glutámico/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/metabolismo , Interneuronas/fisiología , Vértebras Lumbares/metabolismo , Ratones , Propiocepción , Natación , Sinapsis/fisiología , Factores de Transcripción/metabolismo
2.
Cell ; 184(4): 899-911.e13, 2021 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-33545089

RESUMEN

Changes in appendage structure underlie key transitions in vertebrate evolution. Addition of skeletal elements along the proximal-distal axis facilitated critical transformations, including the fin-to-limb transition that permitted generation of diverse modes of locomotion. Here, we identify zebrafish mutants that form supernumerary long bones in their pectoral fins. These new bones integrate into musculature, form joints, and articulate with neighboring elements. This phenotype is caused by activating mutations in previously unrecognized regulators of appendage patterning, vav2 and waslb, that function in a common pathway. This pathway is required for appendage development across vertebrates, and loss of Wasl in mice causes defects similar to those seen in murine Hox mutants. Concordantly, formation of supernumerary bones requires Hox11 function, and mutations in the vav2/wasl pathway drive enhanced expression of hoxa11b, indicating developmental homology with the forearm. Our findings reveal a latent, limb-like pattern ability in fins that is activated by simple genetic perturbation.


Asunto(s)
Huesos/embriología , Extremidades/embriología , Pez Cebra/embriología , Actinas/metabolismo , Aletas de Animales/embriología , Animales , Secuencia de Bases , Tipificación del Cuerpo , Sistemas CRISPR-Cas/genética , Linaje de la Célula , Epistasis Genética , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Genes Reporteros , Células HeLa , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Mutación/genética , Fenotipo , Filogenia , Transducción de Señal/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
3.
Cell ; 184(7): 1724-1739.e16, 2021 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-33667348

RESUMEN

Divergence of gene function is a hallmark of evolution, but assessing functional divergence over deep time is not trivial. The few alleles available for cross-species studies often fail to expose the entire functional spectrum of genes, potentially obscuring deeply conserved pleiotropic roles. Here, we explore the functional divergence of WUSCHEL HOMEOBOX9 (WOX9), suggested to have species-specific roles in embryo and inflorescence development. Using a cis-regulatory editing drive system, we generate a comprehensive allelic series in tomato, which revealed hidden pleiotropic roles for WOX9. Analysis of accessible chromatin and conserved cis-regulatory sequences identifies the regions responsible for this pleiotropic activity, the functions of which are conserved in groundcherry, a tomato relative. Mimicking these alleles in Arabidopsis, distantly related to tomato and groundcherry, reveals new inflorescence phenotypes, exposing a deeply conserved pleiotropy. We suggest that targeted cis-regulatory mutations can uncover conserved gene functions and reduce undesirable effects in crop improvement.


Asunto(s)
Genes de Plantas , Pleiotropía Genética/genética , Proteínas de Homeodominio/genética , Proteínas de Plantas/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Alelos , Arabidopsis/genética , Sistemas CRISPR-Cas/genética , Cromatina/metabolismo , Regulación de la Expresión Génica de las Plantas , Inflorescencia/genética , Solanum lycopersicum/genética , Mutagénesis , Desarrollo de la Planta/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras Genéticas , Solanaceae/genética , Solanaceae/crecimiento & desarrollo
4.
Cell ; 184(21): 5465-5481.e16, 2021 10 14.
Artículo en Inglés | MEDLINE | ID: mdl-34582787

RESUMEN

In vivo cell fate conversions have emerged as potential regeneration-based therapeutics for injury and disease. Recent studies reported that ectopic expression or knockdown of certain factors can convert resident astrocytes into functional neurons with high efficiency, region specificity, and precise connectivity. However, using stringent lineage tracing in the mouse brain, we show that the presumed astrocyte-converted neurons are actually endogenous neurons. AAV-mediated co-expression of NEUROD1 and a reporter specifically and efficiently induces reporter-labeled neurons. However, these neurons cannot be traced retrospectively to quiescent or reactive astrocytes using lineage-mapping strategies. Instead, through a retrograde labeling approach, our results reveal that endogenous neurons are the source for these viral-reporter-labeled neurons. Similarly, despite efficient knockdown of PTBP1 in vivo, genetically traced resident astrocytes were not converted into neurons. Together, our results highlight the requirement of lineage-tracing strategies, which should be broadly applied to studies of cell fate conversions in vivo.


Asunto(s)
Astrocitos/citología , Diferenciación Celular , Linaje de la Célula , Neuronas/citología , Animales , Astrocitos/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Encéfalo/patología , Lesiones Encefálicas/patología , Línea Celular Tumoral , Reprogramación Celular , Dependovirus/metabolismo , Regulación hacia Abajo , Regulación de la Expresión Génica , Genes Reporteros , Proteína Ácida Fibrilar de la Glía/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Integrasas/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/metabolismo , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo
5.
Cell ; 183(1): 211-227.e20, 2020 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-32937106

RESUMEN

The striosome compartment within the dorsal striatum has been implicated in reinforcement learning and regulation of motivation, but how striosomal neurons contribute to these functions remains elusive. Here, we show that a genetically identified striosomal population, which expresses the Teashirt family zinc finger 1 (Tshz1) and belongs to the direct pathway, drives negative reinforcement and is essential for aversive learning in mice. Contrasting a "conventional" striosomal direct pathway, the Tshz1 neurons cause aversion, movement suppression, and negative reinforcement once activated, and they receive a distinct set of synaptic inputs. These neurons are predominantly excited by punishment rather than reward and represent the anticipation of punishment or the motivation for avoidance. Furthermore, inhibiting these neurons impairs punishment-based learning without affecting reward learning or movement. These results establish a major role of striosomal neurons in behaviors reinforced by punishment and moreover uncover functions of the direct pathway unaccounted for in classic models.


Asunto(s)
Reacción de Prevención/fisiología , Cuerpo Estriado/fisiología , Proteínas de Homeodominio/genética , Proteínas Represoras/genética , Animales , Ganglios Basales , Femenino , Proteínas de Homeodominio/metabolismo , Aprendizaje/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Motivación , Neuronas/fisiología , Castigo , Refuerzo en Psicología , Proteínas Represoras/metabolismo
6.
Cell ; 181(5): 1062-1079.e30, 2020 05 28.
Artículo en Inglés | MEDLINE | ID: mdl-32386547

RESUMEN

Expansions of amino acid repeats occur in >20 inherited human disorders, and many occur in intrinsically disordered regions (IDRs) of transcription factors (TFs). Such diseases are associated with protein aggregation, but the contribution of aggregates to pathology has been controversial. Here, we report that alanine repeat expansions in the HOXD13 TF, which cause hereditary synpolydactyly in humans, alter its phase separation capacity and its capacity to co-condense with transcriptional co-activators. HOXD13 repeat expansions perturb the composition of HOXD13-containing condensates in vitro and in vivo and alter the transcriptional program in a cell-specific manner in a mouse model of synpolydactyly. Disease-associated repeat expansions in other TFs (HOXA13, RUNX2, and TBP) were similarly found to alter their phase separation. These results suggest that unblending of transcriptional condensates may underlie human pathologies. We present a molecular classification of TF IDRs, which provides a framework to dissect TF function in diseases associated with transcriptional dysregulation.


Asunto(s)
Expansión de las Repeticiones de ADN/genética , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Alanina/genética , Animales , Secuencia de Bases/genética , Expansión de las Repeticiones de ADN/fisiología , Modelos Animales de Enfermedad , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Ratones , Mutación/genética , Linaje , Sindactilia/genética , Factores de Transcripción/metabolismo
7.
Cell ; 183(7): 2020-2035.e16, 2020 12 23.
Artículo en Inglés | MEDLINE | ID: mdl-33326746

RESUMEN

Thousands of proteins localize to the nucleus; however, it remains unclear which contain transcriptional effectors. Here, we develop HT-recruit, a pooled assay where protein libraries are recruited to a reporter, and their transcriptional effects are measured by sequencing. Using this approach, we measure gene silencing and activation for thousands of domains. We find a relationship between repressor function and evolutionary age for the KRAB domains, discover that Homeodomain repressor strength is collinear with Hox genetic organization, and identify activities for several domains of unknown function. Deep mutational scanning of the CRISPRi KRAB maps the co-repressor binding surface and identifies substitutions that improve stability/silencing. By tiling 238 proteins, we find repressors as short as ten amino acids. Finally, we report new activator domains, including a divergent KRAB. These results provide a resource of 600 human proteins containing effectors and demonstrate a scalable strategy for assigning functions to protein domains.


Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Sistemas CRISPR-Cas/genética , Femenino , Silenciador del Gen , Genes Reporteros , Células HEK293 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células K562 , Lentivirus/fisiología , Anotación de Secuencia Molecular , Mutación/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Dominios Proteicos , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Reproducibilidad de los Resultados , Transcripción Genética , Dedos de Zinc
8.
Nat Immunol ; 23(8): 1256-1272, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35902638

RESUMEN

The recombination-activating genes (RAG) 1 and 2 are indispensable for diversifying the primary B cell receptor repertoire and pruning self-reactive clones via receptor editing in the bone marrow; however, the impact of RAG1/RAG2 on peripheral tolerance is unknown. Partial RAG deficiency (pRD) manifesting with late-onset immune dysregulation represents an 'experiment of nature' to explore this conundrum. By studying B cell development and subset-specific repertoires in pRD, we demonstrate that reduced RAG activity impinges on peripheral tolerance through the generation of a restricted primary B cell repertoire, persistent antigenic stimulation and an inflammatory milieu with elevated B cell-activating factor. This unique environment gradually provokes profound B cell dysregulation with widespread activation, remarkable extrafollicular maturation and persistence, expansion and somatic diversification of self-reactive clones. Through the model of pRD, we reveal a RAG-dependent 'domino effect' that impacts stringency of tolerance and B cell fate in the periphery.


Asunto(s)
Linfocitos B , Proteínas de Unión al ADN , Proteínas de Homeodominio , Proteínas Nucleares , Diferenciación Celular , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Homeodominio/genética , Humanos , Tolerancia Inmunológica , Recuento de Linfocitos , Proteínas Nucleares/deficiencia
9.
Cell ; 177(3): 654-668.e15, 2019 04 18.
Artículo en Inglés | MEDLINE | ID: mdl-30929900

RESUMEN

New neurons arise from quiescent adult neural progenitors throughout life in specific regions of the mammalian brain. Little is known about the embryonic origin and establishment of adult neural progenitors. Here, we show that Hopx+ precursors in the mouse dentate neuroepithelium at embryonic day 11.5 give rise to proliferative Hopx+ neural progenitors in the primitive dentate region, and they, in turn, generate granule neurons, but not other neurons, throughout development and then transition into Hopx+ quiescent radial glial-like neural progenitors during an early postnatal period. RNA-seq and ATAC-seq analyses of Hopx+ embryonic, early postnatal, and adult dentate neural progenitors further reveal common molecular and epigenetic signatures and developmental dynamics. Together, our findings support a "continuous" model wherein a common neural progenitor population exclusively contributes to dentate neurogenesis throughout development and adulthood. Adult dentate neurogenesis may therefore represent a lifelong extension of development that maintains heightened plasticity in the mammalian hippocampus.


Asunto(s)
Células Madre Embrionarias/metabolismo , Neurogénesis , Animales , Diferenciación Celular , Giro Dentado/metabolismo , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/citología , Femenino , Regulación del Desarrollo de la Expresión Génica , Hipocampo/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo
10.
Cell ; 178(6): 1437-1451.e14, 2019 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-31491387

RESUMEN

CCCTC-binding factor (CTCF) and cohesin are key players in three-dimensional chromatin organization. The topologically associating domains (TADs) demarcated by CTCF are remarkably well conserved between species, although genome-wide CTCF binding has diverged substantially following transposon-mediated motif expansions. Therefore, the CTCF consensus motif poorly predicts TADs, and additional factors must modulate CTCF binding and subsequent TAD formation. Here, we demonstrate that the ChAHP complex (CHD4, ADNP, HP1) competes with CTCF for a common set of binding motifs. In Adnp knockout cells, novel insulated regions are formed at sites normally bound by ChAHP, whereas proximal canonical boundaries are weakened. These data reveal that CTCF-mediated loop formation is modulated by a distinct zinc-finger protein complex. Strikingly, ChAHP-bound loci are mainly situated within less diverged SINE B2 transposable elements. This implicates ChAHP in maintenance of evolutionarily conserved spatial chromatin organization by buffering novel CTCF binding sites that emerged through SINE expansions.


Asunto(s)
Factor de Unión a CCCTC/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN Helicasas/metabolismo , Células Madre Embrionarias/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Retroelementos , Animales , Sitios de Unión , Línea Celular , Homólogo de la Proteína Chromobox 5 , Células Madre Embrionarias/citología , Ratones , Unión Proteica , Dominios Proteicos
11.
Cell ; 176(5): 1158-1173.e16, 2019 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-30712869

RESUMEN

Homeostatic regulation of the intestinal enteroendocrine lineage hierarchy is a poorly understood process. We resolved transcriptional changes during enteroendocrine differentiation in real time at single-cell level using a novel knockin allele of Neurog3, the master regulator gene briefly expressed at the onset of enteroendocrine specification. A bi-fluorescent reporter, Neurog3Chrono, measures time from the onset of enteroendocrine differentiation and enables precise positioning of single-cell transcriptomes along an absolute time axis. This approach yielded a definitive description of the enteroendocrine hierarchy and its sub-lineages, uncovered differential kinetics between sub-lineages, and revealed time-dependent hormonal plasticity in enterochromaffin and L cells. The time-resolved map of transcriptional changes predicted multiple novel molecular regulators. Nine of these were validated by conditional knockout in mice or CRISPR modification in intestinal organoids. Six novel candidate regulators (Sox4, Rfx6, Tox3, Myt1, Runx1t1, and Zcchc12) yielded specific enteroendocrine phenotypes. Our time-resolved single-cell transcriptional map presents a rich resource to unravel enteroendocrine differentiation.


Asunto(s)
Linaje de la Célula/genética , Células Enteroendocrinas/metabolismo , Perfilación de la Expresión Génica/métodos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/genética , Linaje de la Célula/fisiología , Células Enteroendocrinas/fisiología , Colorantes Fluorescentes , Proteínas de Homeodominio/genética , Mucosa Intestinal/citología , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Imagen Óptica/métodos , Organoides , Fenotipo , Análisis de la Célula Individual/métodos , Células Madre , Factores de Transcripción/genética , Transcriptoma/genética
12.
Cell ; 179(5): 1144-1159.e15, 2019 11 14.
Artículo en Inglés | MEDLINE | ID: mdl-31708126

RESUMEN

The colonic epithelium can undergo multiple rounds of damage and repair, often in response to excessive inflammation. The responsive stem cell that mediates this process is unclear, in part because of a lack of in vitro models that recapitulate key epithelial changes that occur in vivo during damage and repair. Here, we identify a Hopx+ colitis-associated regenerative stem cell (CARSC) population that functionally contributes to mucosal repair in mouse models of colitis. Hopx+ CARSCs, enriched for fetal-like markers, transiently arose from hypertrophic crypts known to facilitate regeneration. Importantly, we established a long-term, self-organizing two-dimensional (2D) epithelial monolayer system to model the regenerative properties and responses of Hopx+ CARSCs. This system can reenact the "homeostasis-injury-regeneration" cycles of epithelial alterations that occur in vivo. Using this system, we found that hypoxia and endoplasmic reticulum stress, insults commonly present in inflammatory bowel diseases, mediated the cyclic switch of cellular status in this process.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Colon/patología , Células Madre/patología , Células 3T3 , Animales , Colitis/patología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Proteínas de Homeodominio/metabolismo , Ratones , Modelos Biológicos , Oxígeno/farmacología , Regeneración/efectos de los fármacos , Células Madre/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos
13.
Nat Immunol ; 22(10): 1268-1279, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34556885

RESUMEN

Metabolic inflammation is closely linked to obesity, and is implicated in the pathogenesis of metabolic diseases. FTO harbors the strongest genetic association with polygenic obesity, and IRX3 mediates the effects of FTO on body weight. However, in what cells and how IRX3 carries out this control are poorly understood. Here we report that macrophage IRX3 promotes metabolic inflammation to accelerate the development of obesity and type 2 diabetes. Mice with myeloid-specific deletion of Irx3 were protected against diet-induced obesity and metabolic diseases via increasing adaptive thermogenesis. Mechanistically, macrophage IRX3 promoted proinflammatory cytokine transcription and thus repressed adipocyte adrenergic signaling, thereby inhibiting lipolysis and thermogenesis. JNK1/2 phosphorylated IRX3, leading to its dimerization and nuclear translocation for transcription. Further, lipopolysaccharide stimulation stabilized IRX3 by inhibiting its ubiquitination, which amplified the transcriptional capacity of IRX3. Together, our findings identify a new player, macrophage IRX3, in the control of body weight and metabolic inflammation, implicating IRX3 as a therapeutic target.


Asunto(s)
Proteínas de Homeodominio/metabolismo , Inflamación/metabolismo , Macrófagos/metabolismo , Obesidad/metabolismo , Factores de Transcripción/metabolismo , Adipocitos/metabolismo , Adulto , Animales , Peso Corporal/fisiología , Línea Celular , Diabetes Mellitus Tipo 2/metabolismo , Dieta/métodos , Células HEK293 , Humanos , Masculino , Enfermedades Metabólicas/metabolismo , Ratones , Células RAW 264.7 , Células THP-1 , Termogénesis/fisiología , Transcripción Genética/fisiología , Adulto Joven
14.
Cell ; 172(3): 491-499.e15, 2018 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-29358049

RESUMEN

Non-coding "ultraconserved" regions containing hundreds of consecutive bases of perfect sequence conservation across mammalian genomes can function as distant-acting enhancers. However, initial deletion studies in mice revealed that loss of such extraordinarily constrained sequences had no immediate impact on viability. Here, we show that ultraconserved enhancers are required for normal development. Focusing on some of the longest ultraconserved sites genome wide, located near the essential neuronal transcription factor Arx, we used genome editing to create an expanded series of knockout mice lacking individual or combinations of ultraconserved enhancers. Mice with single or pairwise deletions of ultraconserved enhancers were viable and fertile but in nearly all cases showed neurological or growth abnormalities, including substantial alterations of neuron populations and structural brain defects. Our results demonstrate the functional importance of ultraconserved enhancers and indicate that remarkably strong sequence conservation likely results from fitness deficits that appear subtle in a laboratory setting.


Asunto(s)
Secuencia Conservada , Desarrollo Embrionario/genética , Elementos de Facilitación Genéticos , Animales , Encéfalo/anomalías , Encéfalo/embriología , Encéfalo/metabolismo , Femenino , Eliminación de Gen , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Masculino , Ratones , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
15.
Cell ; 172(4): 667-682.e15, 2018 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-29425489

RESUMEN

Walking is the predominant locomotor behavior expressed by land-dwelling vertebrates, but it is unknown when the neural circuits that are essential for limb control first appeared. Certain fish species display walking-like behaviors, raising the possibility that the underlying circuitry originated in primitive marine vertebrates. We show that the neural substrates of bipedalism are present in the little skate Leucoraja erinacea, whose common ancestor with tetrapods existed ∼420 million years ago. Leucoraja exhibits core features of tetrapod locomotor gaits, including left-right alternation and reciprocal extension-flexion of the pelvic fins. Leucoraja also deploys a remarkably conserved Hox transcription factor-dependent program that is essential for selective innervation of fin/limb muscle. This network encodes peripheral connectivity modules that are distinct from those used in axial muscle-based swimming and has apparently been diminished in most modern fish. These findings indicate that the circuits that are essential for walking evolved through adaptation of a genetic regulatory network shared by all vertebrates with paired appendages. VIDEO ABSTRACT.


Asunto(s)
Proteínas Aviares , Pollos/fisiología , Evolución Molecular , Proteínas de Peces , Proteínas de Homeodominio , Red Nerviosa/fisiología , Rajidae/fisiología , Factores de Transcripción , Caminata/fisiología , Pez Cebra/fisiología , Aletas de Animales/fisiología , Animales , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Embrión de Pollo , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Músculo Esquelético/fisiología , Natación/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
16.
Cell ; 174(2): 391-405.e19, 2018 07 12.
Artículo en Inglés | MEDLINE | ID: mdl-29937225

RESUMEN

Transposable elements represent nearly half of mammalian genomes and are generally described as parasites, or "junk DNA." The LINE1 retrotransposon is the most abundant class and is thought to be deleterious for cells, yet it is paradoxically highly expressed during early development. Here, we report that LINE1 plays essential roles in mouse embryonic stem cells (ESCs) and pre-implantation embryos. In ESCs, LINE1 acts as a nuclear RNA scaffold that recruits Nucleolin and Kap1/Trim28 to repress Dux, the master activator of a transcriptional program specific to the 2-cell embryo. In parallel, LINE1 RNA mediates binding of Nucleolin and Kap1 to rDNA, promoting rRNA synthesis and ESC self-renewal. In embryos, LINE1 RNA is required for Dux silencing, synthesis of rRNA, and exit from the 2-cell stage. The results reveal an essential partnership between LINE1 RNA, Nucleolin, Kap1, and peri-nucleolar chromatin in the regulation of transcription, developmental potency, and ESC self-renewal.


Asunto(s)
Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Diferenciación Celular , Línea Celular , Autorrenovación de las Células , Inmunoprecipitación de Cromatina , Retrovirus Endógenos/genética , Femenino , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Hibridación Fluorescente in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Oligorribonucleótidos Antisentido/metabolismo , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Interferencia de ARN , ARN Ribosómico/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Proteína 28 que Contiene Motivos Tripartito/antagonistas & inhibidores , Proteína 28 que Contiene Motivos Tripartito/genética , Proteína 28 que Contiene Motivos Tripartito/metabolismo , Regulación hacia Arriba , Nucleolina
17.
Cell ; 173(4): 1003-1013.e15, 2018 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-29681457

RESUMEN

The majority of newly diagnosed prostate cancers are slow growing, with a long natural life history. Yet a subset can metastasize with lethal consequences. We reconstructed the phylogenies of 293 localized prostate tumors linked to clinical outcome data. Multiple subclones were detected in 59% of patients, and specific subclonal architectures associate with adverse clinicopathological features. Early tumor development is characterized by point mutations and deletions followed by later subclonal amplifications and changes in trinucleotide mutational signatures. Specific genes are selectively mutated prior to or following subclonal diversification, including MTOR, NKX3-1, and RB1. Patients with low-risk monoclonal tumors rarely relapse after primary therapy (7%), while those with high-risk polyclonal tumors frequently do (61%). The presence of multiple subclones in an index biopsy may be necessary, but not sufficient, for relapse of localized prostate cancer, suggesting that evolution-aware biomarkers should be studied in prospective studies of low-risk tumors suitable for active surveillance.


Asunto(s)
Neoplasias de la Próstata/patología , Biomarcadores de Tumor/sangre , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Clasificación del Tumor , Recurrencia Local de Neoplasia , Polimorfismo de Nucleótido Simple , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Neoplasias de la Próstata/clasificación , Neoplasias de la Próstata/genética , Proteínas de Unión a Retinoblastoma/genética , Proteínas de Unión a Retinoblastoma/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo
18.
Cell ; 174(3): 576-589.e18, 2018 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-30033361

RESUMEN

Genome-wide association studies (GWAS) have identified rs11672691 at 19q13 associated with aggressive prostate cancer (PCa). Here, we independently confirmed the finding in a cohort of 2,738 PCa patients and discovered the biological mechanism underlying this association. We found an association of the aggressive PCa-associated allele G of rs11672691 with elevated transcript levels of two biologically plausible candidate genes, PCAT19 and CEACAM21, implicated in PCa cell growth and tumor progression. Mechanistically, rs11672691 resides in an enhancer element and alters the binding site of HOXA2, a novel oncogenic transcription factor with prognostic potential in PCa. Remarkably, CRISPR/Cas9-mediated single-nucleotide editing showed the direct effect of rs11672691 on PCAT19 and CEACAM21 expression and PCa cellular aggressive phenotype. Clinical data demonstrated synergistic effects of rs11672691 genotype and PCAT19/CEACAM21 gene expression on PCa prognosis. These results provide a plausible mechanism for rs11672691 associated with aggressive PCa and thus lay the ground work for translating this finding to the clinic.


Asunto(s)
Neoplasias de la Próstata/genética , ARN Largo no Codificante/genética , ARN no Traducido/genética , Adulto , Alelos , Línea Celular Tumoral , Cromosomas Humanos Par 19/genética , Estudios de Cohortes , Regulación Neoplásica de la Expresión Génica/genética , Frecuencia de los Genes/genética , Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo , Genotipo , Proteínas de Homeodominio , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genética , Pronóstico
19.
Cell ; 174(3): 564-575.e18, 2018 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-30033362

RESUMEN

The prostate cancer (PCa) risk-associated SNP rs11672691 is positively associated with aggressive disease at diagnosis. We showed that rs11672691 maps to the promoter of a short isoform of long noncoding RNA PCAT19 (PCAT19-short), which is in the third intron of the long isoform (PCAT19-long). The risk variant is associated with decreased and increased levels of PCAT19-short and PCAT19-long, respectively. Mechanistically, the risk SNP region is bifunctional with both promoter and enhancer activity. The risk variants of rs11672691 and its LD SNP rs887391 decrease binding of transcription factors NKX3.1 and YY1 to the promoter of PCAT19-short, resulting in weaker promoter but stronger enhancer activity that subsequently activates PCAT19-long. PCAT19-long interacts with HNRNPAB to activate a subset of cell-cycle genes associated with PCa progression, thereby promoting PCa tumor growth and metastasis. Taken together, these findings reveal a risk SNP-mediated promoter-enhancer switching mechanism underlying both initiation and progression of aggressive PCa.


Asunto(s)
Neoplasias de la Próstata/genética , ARN Largo no Codificante/genética , Alelos , Línea Celular Tumoral , Elementos de Facilitación Genéticos/genética , Regulación Neoplásica de la Expresión Génica/genética , Frecuencia de los Genes/genética , Predisposición Genética a la Enfermedad/genética , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Isoformas de ARN/genética , Factores de Riesgo , Factores de Transcripción/metabolismo , Factor de Transcripción YY1/metabolismo
20.
Cell ; 174(1): 102-116.e14, 2018 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-29804837

RESUMEN

RAG endonuclease initiates antibody heavy chain variable region exon assembly from V, D, and J segments within a chromosomal V(D)J recombination center (RC) by cleaving between paired gene segments and flanking recombination signal sequences (RSSs). The IGCR1 control region promotes DJH intermediate formation by isolating Ds, JHs, and RCs from upstream VHs in a chromatin loop anchored by CTCF-binding elements (CBEs). How VHs access the DJHRC for VH to DJH rearrangement was unknown. We report that CBEs immediately downstream of frequently rearranged VH-RSSs increase recombination potential of their associated VH far beyond that provided by RSSs alone. This CBE activity becomes particularly striking upon IGCR1 inactivation, which allows RAG, likely via loop extrusion, to linearly scan chromatin far upstream. VH-associated CBEs stabilize interactions of D-proximal VHs first encountered by the DJHRC during linear RAG scanning and thereby promote dominant rearrangement of these VHs by an unanticipated chromatin accessibility-enhancing CBE function.


Asunto(s)
Factor de Unión a CCCTC/metabolismo , Cromatina/metabolismo , Proteínas de Homeodominio/metabolismo , Recombinación V(D)J , Animales , Línea Celular , ADN Intergénico/genética , ADN Intergénico/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Mutagénesis , Señales de Clasificación de Proteína , ARN Guía de Kinetoplastida/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo
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