RESUMO
In addition to its function as an inhibitor of histone acetyltransferases, Nir (Noc2l) binds to p53 and TAp63 to regulate their activity. Here, we show that epidermis-specific ablation of Nir impairs epidermal stratification and barrier function, resulting in perinatal lethality. Nir-deficient epidermis lacks appendages and remains single layered during embryogenesis. Cell proliferation is inhibited, whereas apoptosis and p53 acetylation are increased, indicating that Nir is controlling cell proliferation by limiting p53 acetylation. Transcriptome analysis revealed that Nir regulates the expression of essential factors in epidermis development, such as keratins, integrins and laminins. Furthermore, Nir binds to and controls the expression of p63 and limits H3K18ac at the p63 promoter. Corroborating the stratification defects, asymmetric cell divisions were virtually absent in Nir-deficient mice, suggesting that Nir is required for correct mitotic spindle orientation. In summary, our data define Nir as a key regulator of skin development.
Assuntos
Epiderme/metabolismo , Histona Acetiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Animais , Apoptose/genética , Divisão Celular Assimétrica/genética , Técnicas de Cultura de Células , Divisão Celular , Proliferação de Células/genética , Imunoprecipitação da Cromatina , Epiderme/crescimento & desenvolvimento , Imunofluorescência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
The remarkable capacity of the adult olfactory epithelium (OE) to regenerate fully both neurosensory and nonneuronal cell types after severe epithelial injury depends on life-long persistence of two stem cell populations: the horizontal basal cells (HBCs), which are quiescent and held in reserve, and mitotically active globose basal cells. It has recently been demonstrated that down-regulation of the ΔN form of the transcription factor p63 is both necessary and sufficient to release HBCs from dormancy. However, the mechanisms by which p63 is down-regulated after acute OE injury remain unknown. To identify the cellular source of potential signaling mechanisms, we assessed HBC activation after neuron-only and sustentacular cell death. We found that ablation of sustentacular cells is sufficient for HBC activation to multipotency. By expression analysis, next-generation sequencing, and immunohistochemical examination, down-regulation of Notch pathway signaling is coincident with HBC activation. Therefore, using HBC-specific conditional knockout of Notch receptors and overexpression of N1ICD, we show that Notch signaling maintains p63 levels and HBC dormancy, in contrast to its suppression of p63 expression in other tissues. Additionally, Notch1, but not Notch2, is required to maintain HBC dormancy after selective neuronal degeneration. Taken together, our data indicate that the activation of HBCs observed after tissue injury or sustentacular cell ablation is caused by the reduction/elimination of Notch signaling on HBCs; elimination of Jagged1 expressed by sustentacular cells may be the ligand responsible.
Assuntos
Células-Tronco Neurais/citologia , Mucosa Olfatória/citologia , Fosfoproteínas/metabolismo , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Transativadores/metabolismo , Animais , Morte Celular , Biologia Computacional , Genótipo , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Neurônios Receptores Olfatórios/citologia , Neurônios Receptores Olfatórios/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , TranscriptomaRESUMO
BACKGROUND: The p63 gene is integral to the development of many body parts including limb, palate, teeth, and urogenital tract. Loss of p63 expression may alter developmental rate, which is crucial to normal morphogenesis. To validate a novel, unbiased embryo phenotyping software tool, we tested whether delayed development contributes to the pathological phenotype of a p63 mouse mutant (p63-/- ). We quantified dysmorphology in p63-/- embryos and tested for universal growth delay relative to wild-type (WT) embryos. Fixed embryos (n = 6; p63-/- ) aged day (E) 15.5 were micro-CT scanned and quantitatively analyzed using a digital WT atlas that defined volumetric differences between p63-/- and WT embryos. RESULTS: p63-/- embryos showed a growth delay of approximately 22 hr (0.9 days). Among the E15.5 mutants, overall size was closest to WT E14.6 mice but shape was closest to WT E14.0. The atlas clearly identified in p63-/- embryos malformations of epithelial derivatives including limbs, tail, urogenital structures, brain, face, and tooth. CONCLUSIONS: The software atlas technique described the p63-/- phenotype as a combination of developmental delay (i.e., heterochrony) and malformation (i.e., pathological shape; failed organogenesis). This study identifies for the first time global and local roles for p63 in prenatal growth and development. Developmental Dynamics 247:779-787, 2018. © 2018 Wiley Periodicals, Inc.
Assuntos
Embrião de Mamíferos/metabolismo , Morfogênese/fisiologia , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Animais , Embrião de Mamíferos/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos , Camundongos Knockout , Morfogênese/genética , Fosfoproteínas/genética , Transativadores/genéticaRESUMO
Chromatin structural states and their remodelling, including higher-order chromatin folding and three-dimensional (3D) genome organisation, play an important role in the control of gene expression. The role of 3D genome organisation in the control and execution of lineage-specific transcription programmes during the development and differentiation of multipotent stem cells into specialised cell types remains poorly understood. Here, we show that substantial remodelling of the higher-order chromatin structure of the epidermal differentiation complex (EDC), a keratinocyte lineage-specific gene locus on mouse chromosome 3, occurs during epidermal morphogenesis. During epidermal development, the locus relocates away from the nuclear periphery towards the nuclear interior into a compartment enriched in SC35-positive nuclear speckles. Relocation of the EDC locus occurs prior to the full activation of EDC genes involved in controlling terminal keratinocyte differentiation and is a lineage-specific, developmentally regulated event controlled by transcription factor p63, a master regulator of epidermal development. We also show that, in epidermal progenitor cells, p63 directly regulates the expression of the ATP-dependent chromatin remodeller Brg1, which binds to distinct domains within the EDC and is required for relocation of the EDC towards the nuclear interior. Furthermore, Brg1 also regulates gene expression within the EDC locus during epidermal morphogenesis. Thus, p63 and its direct target Brg1 play an essential role in remodelling the higher-order chromatin structure of the EDC and in the specific positioning of this locus within the landscape of the 3D nuclear space, as required for the efficient expression of EDC genes in epidermal progenitor cells during skin development.
Assuntos
Montagem e Desmontagem da Cromatina/genética , DNA Helicases/metabolismo , Células-Tronco Multipotentes/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Cromatina/metabolismo , DNA Helicases/genética , Células Epidérmicas , Epiderme/embriologia , Epiderme/metabolismo , Fator de Transcrição de Proteínas de Ligação GA/genética , Regulação da Expressão Gênica no Desenvolvimento , Queratinócitos/citologia , Queratinócitos/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética , Fosfoproteínas/genética , Ligação Proteica , Dobramento de Proteína , Interferência de RNA , RNA Interferente Pequeno , Ribonucleoproteínas/metabolismo , Fatores de Processamento de Serina-Arginina , Transativadores/genética , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Lung development is determined by the coordinated expression of several key genes. Previously, we and others have shown the importance of the sex determining region Y-box 2 (Sox2) gene in lung development. Transgenic expression of Sox2 during lung development resulted in cystic airways, and here we show that modulating the timing of ectopic Sox2 expression in the branching regions of the developing lung results in variable cystic lesions resembling the spectrum of the human congenital disorder congenital cystic adenomatoid malformation (CCAM). Sox2 dominantly differentiated naive epithelial cells into the proximal lineage irrespective of the presence of Fgf10. Sox2 directly induced the expression of Trp63, the master switch toward the basal cell lineage and induced the expression of Gata6, a factor involved in the emergence of bronchoalveolar stem cells. We showed that SOX2 and TRP63 are coexpressed in the lungs of human patients with type II CCAM. The combination of premature differentiation toward the proximal cell lineage and the induction of proliferation resulted in the cyst-like structures. Thus, we show that Sox2 is directly responsible for the emergence of two lung progenitor cells: basal cells by regulating the master gene Trp63 and bronchoalveolar stem cells by regulating Gata6.
Assuntos
Malformação Adenomatoide Cística Congênita do Pulmão/metabolismo , Células Epiteliais/metabolismo , Pulmão/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Proteínas Supressoras de Tumor/metabolismo , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Linhagem da Célula , Proliferação de Células , Malformação Adenomatoide Cística Congênita do Pulmão/genética , Malformação Adenomatoide Cística Congênita do Pulmão/patologia , Células Epiteliais/patologia , Fator 10 de Crescimento de Fibroblastos/metabolismo , Fator de Transcrição GATA6/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Idade Gestacional , Células HEK293 , Humanos , Pulmão/patologia , Camundongos , Camundongos Transgênicos , Fenótipo , Fosfoproteínas/genética , Fatores de Transcrição SOXB1/genética , Células-Tronco/patologia , Técnicas de Cultura de Tecidos , Transativadores/genética , Fatores de Transcrição/genética , Transfecção , Proteínas Supressoras de Tumor/genética , Regulação para CimaRESUMO
Human Ectrodactyly, Ectodermal dysplasia, Clefting (EEC) syndrome is an autosomal dominant developmental disorder defined by limb deformities, skin defects, and craniofacial clefting. Although associated with heterozygous missense mutations in TP63, the genetic basis underlying the variable expressivity and incomplete penetrance of EEC is unknown. Here, we show that mice heterozygous for an allele encoding the Trp63 p.Arg318His mutation, which corresponds to the human TP63 p.Arg279His mutation found in patients with EEC, have features of human EEC. Using an allelic series, we discovered that whereas clefting and skin defects are caused by loss of Trp63 function, limb anomalies are due to gain- and/or dominant-negative effects of Trp63. Furthermore, we identify TAp63 as a strong modifier of EEC-associated phenotypes with regard to both penetrance and expressivity.
Assuntos
Fenda Labial/etiologia , Fenda Labial/patologia , Fissura Palatina/etiologia , Fissura Palatina/patologia , Modelos Animais de Doenças , Displasia Ectodérmica/etiologia , Displasia Ectodérmica/patologia , Mutação/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Alelos , Animais , Southern Blotting , Heterozigoto , Humanos , Camundongos , FenótipoRESUMO
Basal tumor propagating cells (TPCs) control squamous cell carcinoma (SCC) growth by self-renewing and differentiating into supra-basal SCC cells, which lack proliferative potential. While transcription factors such as SOX2 and KLF4 can drive these behaviors, their molecular roles and regulatory interactions with each other have remained elusive. Here, we show that PITX1 is specifically expressed in TPCs, where it co-localizes with SOX2 and TRP63 and determines cell fate in mouse and human SCC. Combining gene targeting with chromatin immunoprecipitation sequencing (ChIP-seq) and transcriptomic analyses reveals that PITX1 cooperates with SOX2 and TRP63 to sustain an SCC-specific transcriptional feed-forward circuit that maintains TPC-renewal, while inhibiting KLF4 expression and preventing KLF4-dependent differentiation. Conversely, KLF4 represses PITX1, SOX2, and TRP63 expression to prevent TPC expansion. This bi-stable, multi-input network reveals a molecular framework that explains self-renewal, aberrant differentiation, and SCC growth in mice and humans, providing clues for developing differentiation-inducing therapeutic strategies.
Assuntos
Carcinoma de Células Escamosas/genética , Diferenciação Celular , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Box Pareados/genética , Transcrição Gênica , Animais , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proliferação de Células , Feminino , Humanos , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos Nus , Fatores de Transcrição Box Pareados/metabolismo , Células Tumorais CultivadasRESUMO
Lung and esophageal development and organogenesis involve a complex interplay of signaling pathways and transcriptional factors. Once the lung and esophagus do separate, their epithelial proliferation and differentiation programs share certain common properties that may fuel adaptive responses to injury and subsequent regeneration. Lung and esophageal tissue organogenesis and regeneration provide perspectives on squamous cell cancers and adenocarcinomas in each tissue.
Assuntos
Esôfago/embriologia , Esôfago/fisiologia , Pulmão/embriologia , Pulmão/fisiologia , Regeneração/fisiologia , Carcinogênese , Diferenciação Celular , Redes Reguladoras de Genes , HumanosRESUMO
Altered progesterone responsiveness leads to female infertility and cancer, but underlying mechanisms remain unclear. Mice with uterine-specific ablation of GATA binding protein 2 (Gata2) are infertile, showing failures in embryo implantation, endometrial decidualization, and uninhibited estrogen signaling. Gata2 deficiency results in reduced progesterone receptor (PGR) expression and attenuated progesterone signaling, as evidenced by genome-wide expression profiling and chromatin immunoprecipitation. GATA2 not only occupies at and promotes expression of the Pgr gene but also regulates downstream progesterone responsive genes in conjunction with the PGR. Additionally, Gata2 knockout uteri exhibit abnormal luminal epithelia with ectopic TRP63 expressing squamous cells and a cancer-related molecular profile in a progesterone-independent manner. Lastly, we found a conserved GATA2-PGR regulatory network in both human and mice based on gene signature and path analyses using gene expression profiles of human endometrial tissues. In conclusion, uterine Gata2 regulates a key regulatory network of gene expression for progesterone signaling at the early pregnancy stage.