RESUMEN
The transcription factor C/EBPα is essential for myeloid differentiation and is frequently dysregulated in acute myeloid leukemia. Although studied extensively, the precise regulation of its gene by upstream factors has remained largely elusive. Here, we investigated its transcriptional activation during myeloid differentiation. We identified an evolutionarily conserved octameric sequence, CCCAGCAG, â¼100 bases upstream of the CEBPA transcription start site, and demonstrated through mutational analysis that this sequence is crucial for C/EBPα expression. This sequence is present in the genes encoding C/EBPα in humans, rodents, chickens, and frogs and is also present in the promoters of other C/EBP family members. We identified that ZNF143, the human homolog of the Xenopus transcriptional activator STAF, specifically binds to this 8-bp sequence to activate C/EBPα expression in myeloid cells through a mechanism that is distinct from that observed in liver cells and adipocytes. Altogether, our data suggest that ZNF143 plays an important role in the expression of C/EBPα in myeloid cells.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/genética , Células Mieloides/citología , Regiones Promotoras Genéticas , Transactivadores/metabolismo , Activación Transcripcional , Secuencia de Bases , Línea Celular , Secuencia Conservada , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis , Humanos , Células Mieloides/metabolismo , Unión ProteicaRESUMEN
Mutations in mouse and human Nfe2, Fli1 and Runx1 cause thrombocytopenia. We applied genome-wide chromatin dynamics and ChIP-seq to determine these transcription factors' (TFs) activities in terminal megakaryocyte (MK) maturation. Enhancers with H3K4me2-marked nucleosome pairs were most enriched for NF-E2, FLI and RUNX sequence motifs, suggesting that this TF triad controls much of the late MK program. ChIP-seq revealed NF-E2 occupancy near previously implicated target genes, whose expression is compromised in Nfe2-null cells, and many other genes that become active late in MK differentiation. FLI and RUNX were also the motifs most enriched near NF-E2 binding sites and ChIP-seq implicated FLI1 and RUNX1 in activation of late MK, including NF-E2-dependent, genes. Histones showed limited activation in regions of single TF binding, while enhancers that bind NF-E2 and either RUNX1, FLI1 or both TFs gave the highest signals for TF occupancy and H3K4me2; these enhancers associated best with genes activated late in MK maturation. Thus, three essential TFs co-occupy late-acting cis-elements and show evidence for additive activity at genes responsible for platelet assembly and release. These findings provide a rich dataset of TF and chromatin dynamics in primary MK and explain why individual TF losses cause thrombopocytopenia.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Megacariocitos/metabolismo , Subunidad p45 del Factor de Transcripción NF-E2/genética , Proteína Proto-Oncogénica c-fli-1/genética , Activación Transcripcional/genética , Animales , Cromatina/genética , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica/genética , Histonas/genética , Humanos , Ratones , Regiones Promotoras Genéticas , Unión Proteica/genéticaRESUMEN
Tissues may adopt diverse strategies to establish specific transcriptional programs in daughter lineages. In intestinal crypts, enhancers for genes expressed in both major cell types appear broadly permissive in stem and specified progenitor cells. In blood, another self-renewing tissue, it is unclear when chromatin becomes permissive for transcription of genes expressed in distinct terminal lineages. Using chromatin immunoprecipitation (ChIP) combined with deep sequencing (ChIP-seq) to profile activating histone marks, we studied enhancer dynamics in primary mouse blood stem, progenitor, and specified cells. Stem and multipotent progenitor cells show scant H3K4me2 marking at enhancers bound by specific transcription factors in their committed progeny. Rather, enhancers are modulated dynamically and serially, with substantial loss and gain of H3K4me2, at each cellular transition. Quantitative analysis of these dynamics accurately modeled hematopoiesis according to Waddington's notion of epigenotypes. Delineation of enhancers in terminal blood lineages coincides with cell specification, and enhancers active in single lineages show well-positioned H3K4me2- and H3K27ac-marked nucleosomes and DNaseI hypersensitivity in other cell types, revealing limited lineage fidelity. These findings demonstrate that enhancer chronology in blood cells differs markedly from that in intestinal crypts. Chromatin dynamics in hematopoiesis provide a useful foundation to consider classical observations such as cellular reprogramming and multilineage locus priming.
Asunto(s)
Células Sanguíneas/citología , Células Sanguíneas/metabolismo , Elementos de Facilitación Genéticos/genética , Hematopoyesis/genética , Animales , Diferenciación Celular , Linaje de la Célula , Epigénesis Genética , Histonas/metabolismo , Ratones , Células Madre/citología , Células Madre/metabolismoRESUMEN
Cells differentiate when transcription factors bind accessible cis-regulatory elements to establish specific gene expression programs. In differentiating embryonic stem cells, chromatin at lineage-restricted genes becomes sequentially accessible, probably by means of 'pioneer' transcription factor activity, but tissues may use other strategies in vivo. Lateral inhibition is a pervasive process in which one cell forces a different identity on its neighbours, and it is unclear how chromatin in equipotent progenitors undergoing lateral inhibition quickly enables distinct, transiently reversible cell fates. Here we report the chromatin and transcriptional underpinnings of differentiation in mouse small intestine crypts, where notch signalling mediates lateral inhibition to assign progenitor cells into absorptive or secretory lineages. Transcript profiles in isolated LGR5(+) intestinal stem cells and secretory and absorptive progenitors indicated that each cell population was distinct and the progenitors specified. Nevertheless, secretory and absorptive progenitors showed comparable levels of H3K4me2 and H3K27ac histone marks and DNase I hypersensitivity--signifying accessible, permissive chromatin-at most of the same cis-elements. Enhancers acting uniquely in progenitors were well demarcated in LGR5(+) intestinal stem cells, revealing early priming of chromatin for divergent transcriptional programs, and retained active marks well after lineages were specified. On this chromatin background, ATOH1, a secretory-specific transcription factor, controls lateral inhibition through delta-like notch ligand genes and also drives the expression of numerous secretory lineage genes. Depletion of ATOH1 from specified secretory cells converted them into functional enterocytes, indicating prolonged responsiveness of marked enhancers to the presence or absence of a key transcription factor. Thus, lateral inhibition and intestinal crypt lineage plasticity involve interaction of a lineage-restricted transcription factor with broadly permissive chromatin established in multipotent stem cells.
Asunto(s)
Diferenciación Celular/genética , Cromatina/genética , Cromatina/metabolismo , Regulación de la Expresión Génica , Intestino Delgado/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Linaje de la Célula/genética , Desoxirribonucleasa I/metabolismo , Elementos de Facilitación Genéticos/genética , Enterocitos/citología , Enterocitos/metabolismo , Femenino , Histonas/metabolismo , Intestino Delgado/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores Notch/metabolismo , Células Madre/citología , Células Madre/metabolismo , Transcripción GenéticaRESUMEN
The protein kinase C and casein kinase 2 substrate in neurons (Pacsin) is a subfamily of membrane-binding proteins that participates in vesicle trafficking and cytoskeleton organization. Here, we studied Pacsin 2 in kidney development and repair following injury. In the postnatal developing kidneys, Pacsin 2 was found to be expressed in both ureteric bud- and mesenchyme-derived structures including proximal and distal tubules, Bowman's capsule, and the glomerular tuft. In the adult kidney, its expression was decreased in proximal tubules but increased in glomerular tuft when compared to that in the developing kidneys. Interestingly, Pacsin 2 expression was significantly upregulated during the repair phase after ischemia-reperfusion injury, especially on the apical brush border of proximal tubules that experienced massive damage. Pacsin 2 localized to the primary cilia of renal epithelial cells. Knockdown of Pacsin 2 by shRNA did not affect the cell cycle or cell polarity; however, it increased the length of primary cilia, and resulted in significant tubulogenic defects in three-dimensional cell culture. Thus, we propose that Pacsin 2 contributes to kidney development and repair in a nephron-specific manner.
Asunto(s)
Riñón/embriología , Proteínas/fisiología , Daño por Reperfusión/fisiopatología , Proteínas Adaptadoras Transductoras de Señales , Animales , Ciclo Celular , Polaridad Celular , Proliferación Celular , Proteínas del Citoesqueleto , Células Epiteliales/química , Riñón/química , Túbulos Renales Colectores/citología , Ratones , Proteínas/análisisRESUMEN
Epiboly, the spreading and the thinning of the blastoderm to cover the yolk cell and close the blastopore in fish embryos, is central to the process of gastrulation. Despite its fundamental importance, little is known about the molecular mechanisms that control this coordinated cell movement. By a combination of knockdown studies and rescue experiments in zebrafish (Danio rerio), we show that epiboly relies on the molecular networking of syntenin with syndecan heparan sulphate proteoglycans, which act as co-receptors for adhesion molecules and growth factors. Furthermore, we show that the interaction of syntenin with phosphatidylinositol 4,5-bisphosphate (PIP2) and with the small GTPase ADP-ribosylation factor 6 (Arf6), which regulate the endocytic recycling of syndecan, is necessary for epiboly progression. Analysis of the earliest cellular defects suggests a role for syntenin in the autonomous vegetal expansion of the yolk syncytial layer and the rearrangement of the actin cytoskeleton in extra-embryonic tissues, but not in embryonic cell fate determination. This study identifies the importance of the syntenin-syndecan-PIP2-Arf6 complex for the progression of fish epiboly and establishes its key role in directional cell movements during early development.
Asunto(s)
Gastrulación/fisiología , Sindecanos/metabolismo , Sinteninas/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/metabolismo , Animales , Movimiento Celular/fisiología , Citoesqueleto/genética , Técnicas de Silenciamiento del Gen , Ratones , Datos de Secuencia Molecular , Fosfatidilinositol 4,5-Difosfato/metabolismo , Sinteninas/genética , Pez Cebra/metabolismo , Pez Cebra/fisiología , Proteínas de Pez Cebra/genéticaRESUMEN
Mutations in PKD1, which encodes polycystin-1 (PC1), contribute to >85% of cases of autosomal dominant polycystic kidney disease (ADPKD). The planar cell polarity (PCP) pathway is necessary for the oriented cell division and convergent extension that establishes and maintains the structure of kidney tubules, but the role of this pathway in the pathophysiology of ADPKD is incompletely understood. Here, we show that inactivation of Pkd1 in postnatal developing mouse kidneys leads to a defect in oriented cell division in precystic kidney tubules. We also observed this defect in precystic Pkd1-inactivated mature kidneys subjected to ischemia-reperfusion injury as a "third hit." Cystic kidneys exhibited striking upregulation and activation of Frizzled 3 (Fz3), a regulator of PCP, and its downstream effector, CDC42. Precystic kidneys demonstrated upregulation of CDC42, but the localization of the polarity proteins Par3 and Par6 was similar to control. Fz3 was expressed on the cilia of cystic kidneys but barely detected on the cilia of normal kidneys. In vitro, PC1 and Fz3 antagonized each other to control CDC42 expression and the rate of cell migration in HEK293T cells. Taken together, our data suggest that PC1 controls oriented cell division and that aberrant PCP signaling contributes to cystogenesis.
Asunto(s)
Túbulos Renales/patología , Enfermedades Renales Poliquísticas/patología , Animales , División Celular , Línea Celular , Polaridad Celular , Receptores Frizzled/fisiología , Humanos , Ratones , Receptores Acoplados a Proteínas G/fisiología , Canales Catiónicos TRPP/fisiología , Proteína de Unión al GTP cdc42/fisiologíaAsunto(s)
Receptores Frizzled/metabolismo , Dominios PDZ , Transducción de Señal/fisiología , Proteínas Wnt/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Polaridad Celular , Receptores Frizzled/genética , Proteínas de la Matriz de Golgi , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Modelos Biológicos , Sinteninas/genética , Sinteninas/metabolismo , Proteínas Wnt/genéticaRESUMEN
Wnt signaling pathways are essential for embryonic patterning, and they are disturbed in a wide spectrum of diseases, including cancer. An unresolved question is how the different Wnt pathways are supported and regulated. We previously established that the postsynaptic density 95/disc-large/zona occludens (PDZ) protein syntenin binds to syndecans, Wnt coreceptors, and known stimulators of protein kinase C (PKC)alpha and CDC42 activity. Here, we show that syntenin also interacts with the C-terminal PDZ binding motif of several Frizzled Wnt receptors, without compromising the recruitment of Dishevelled, a key downstream Wnt-signaling component. Syntenin is coexpressed with cognate Frizzled during early development in Xenopus. Overexpression and down-regulation of syntenin disrupt convergent extension movements, supporting a role for syntenin in noncanonical Wnt signaling. Syntenin stimulates c-jun phosphorylation and modulates Frizzled 7 signaling, in particular the PKCalpha/CDC42 noncanonical Wnt signaling cascade. The syntenin-Frizzled 7 binding mode indicates syntenin can accommodate Frizzled 7-syndecan complexes. We propose that syntenin is a novel component of the Wnt signal transduction cascade and that it might function as a direct intracellular link between Frizzled and syndecans.