RESUMEN
Kidney formation and nephrogenesis are controlled by precise spatiotemporal gene expression programs, which are coordinately regulated by cell-cycle, cell type-specific transcription factors and epigenetic/chromatin regulators. However, the roles of epigenetic/chromatin regulators in kidney development and disease remain poorly understood. In this study, we investigated the impact of deleting the chromatin remodeling factor Smarca4 (Brg1), a human Wilms tumor-associated gene, in Wnt4-expressing cells. Smarca4 deficiency led to severe tubular defects and a shortened medulla. Through unbiased single-cell RNA sequencing analyses, we identified multiple types of Wnt4 Cre-labeled interstitial cells, along with nephron-related cells. Smarca4 deficiency increased interstitial cells but markedly reduced tubular cells, resulting in cells with mixed identity and elevated expression of cell-cycle regulators and genes associated with extracellular matrix and epithelial-to-mesenchymal transition/fibrosis. We found that Smarca4 loss induced a significant upregulation of the oncogene Pttg1 and hyperproliferation of Wnt4 Cre-labeled cells. These changes in the cellular state could hinder the cellular transition into characteristic tubular structures, eventually leading to fibrosis. In conclusion, our findings shed light on novel cell types and genes associated with Wnt4 Cre-labeled cells and highlight the critical role of Smarca4 in regulating tubular cell differentiation and the expression of the cancer-causing gene Pttg1 in the kidney. These findings may provide valuable insights into potential therapeutic strategies for renal cell carcinoma resulting from SMARCA4 deficiency.
RESUMEN
Members of the Eya family, which are a class of transcription factors with phosphatase activity, are widely expressed in cranial sensory organs during development. However, it is unclear whether these genes are expressed in the taste system during development and whether they play any role in specifying taste cell fate. In this study, we report that Eya1 is not expressed during embryonic tongue development but that Eya1-expressing progenitors in somites or pharyngeal endoderm give rise to tongue musculature or taste organs, respectively. In the Eya1-deficient tongues, these progenitors do not proliferate properly, resulting in a smaller tongue at birth, impaired growth of taste papillae, and disrupted expression of Six1 in the papillary epithelium. On the other hand, Eya2 is specifically expressed in endoderm-derived circumvallate and foliate papillae located on the posterior tongue during development. In adult tongues, Eya1 is predominantly expressed in IP3R3-positive taste cells in the taste buds of the circumvallate and foliate papillae, while Eya2 is persistently expressed in these papillae at higher levels in some epithelial progenitors and at lower levels in some taste cells. We found that conditional knockout of Eya1 in the third week or Eya2 knockout reduced Pou2f3+, Six1+ and IP3R3+ taste cells. Our data define for the first time the expression patterns of Eya1 and Eya2 during the development and maintenance of the mouse taste system and suggest that Eya1 and Eya2 may act together to promote lineage commitment of taste cell subtypes.
RESUMEN
Eya1 is critical for establishing and maintaining nephron progenitor cells (NPCs). It belongs to a family of proteins called phosphatase-transcriptional activators but without intrinsic DNA-binding activity. However, the spectrum of the Eya1-centered networks is underexplored. Here, we combined transcriptomic, genomic and proteomic approaches to characterize gene regulation by Eya1 in the NPCs. We identified Eya1 target genes, associated cis-regulatory elements and partner proteins. Eya1 preferentially occupies promoter sequences and interacts with general transcription factors (TFs), RNA polymerases, different types of TFs, chromatin-remodeling factors with ATPase or helicase activity, and DNA replication/repair proteins. Intriguingly, we identified REST-binding motifs in 76% of Eya1-occupied sites without H3K27ac-deposition, which were present in many Eya1 target genes upregulated in Eya1-deficient NPCs. Eya1 copurified REST-interacting chromatin-remodeling factors, histone deacetylase/lysine demethylase, and corepressors. Coimmunoprecipitation validated physical interaction between Eya1 and Rest/Hdac1/Cdyl/Hltf in the kidneys. Collectively, our results suggest that through interactions with chromatin-remodeling factors and specialized DNA-binding proteins, Eya1 may modify chromatin structure to facilitate the assembly of regulatory complexes that regulate transcription positively or negatively. These findings provide a mechanistic basis for how Eya1 exerts its activity by forming unique multiprotein complexes in various biological processes to maintain the cellular state of NPCs.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Nefronas/citología , Proteínas Nucleares/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Adenosina Trifosfatasas/genética , Animales , Cromatina/genética , Proteínas Co-Represoras , Proteínas de Unión al ADN/genética , Histona Desacetilasas/metabolismo , Ratones , Complejos Multiproteicos/genética , Nefronas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Proteómica , Factores Generales de Transcripción/genéticaRESUMEN
The vertebrate Six1 and Six2 arose by gene duplication from the Drosophila sine oculis and have since diverged in their developmental expression patterns. Both genes are expressed in nephron progenitors of human fetal kidneys, and mutations in SIX1 or SIX2 cause branchio-oto-renal syndrome or renal hypodysplasia respectively. Since â¼80% of SIX1 target sites are shared by SIX2, it is speculated that SIX1 and SIX2 may be functionally interchangeable by targeting common downstream genes. In contrast, in mouse kidneys, Six1 expression in the metanephric mesenchyme lineage overlaps with Six2 only transiently, while Six2 expression is maintained in the nephron progenitors throughout development. This non-overlapping expression between Six1 and Six2 in mouse nephron progenitors promoted us to examine if Six1 can replace Six2. Surprisingly, forced expression of Six1 failed to rescue Six2-deficient kidney phenotype. We found that Six1 mediated Eya1 nuclear translocation and inhibited premature epithelialization of the progenitors but failed to rescue the proliferation defects and cell death caused by Six2-knockout. Genome-wide binding analyses showed that Six1 selectively occupied a small subset of Six2 target sites, but many Six2-bound loci crucial to the renewal and differentiation of nephron progenitors lacked Six1 occupancy. Altogether, these data indicate that Six1 cannot substitute Six2 to drive nephrogenesis in mouse kidneys, thus demonstrating that the difference in physiological roles of Six1 and Six2 in kidney development stems from both transcriptional regulations of the genes and divergent biochemical properties of the proteins.
RESUMEN
BACKGROUND: Eya1 is a critical regulator of nephron progenitor cell specification and interacts with Six2 to promote NPC self-renewal. Haploinsufficiency of these genes causes kidney hypoplasia. However, how the Eya1-centered network operates remains unknown. METHODS: We engineered a 2×HA-3×Flag-Eya1 knock-in mouse line and performed coimmunoprecipitation with anti-HA or -Flag to precipitate the multitagged-Eya1 and its associated proteins. Loss-of-function, transcriptome profiling, and genome-wide binding analyses for Eya1's interacting chromatin-remodeling ATPase Brg1 were carried out. We assayed the activity of the cis-regulatory elements co-occupied by Brg1/Six2 in vivo. RESULTS: Eya1 and Six2 interact with the Brg1-based SWI/SNF complex during kidney development. Knockout of Brg1 results in failure of metanephric mesenchyme formation and depletion of nephron progenitors, which has been linked to loss of Eya1 expression. Transcriptional profiling shows conspicuous downregulation of important regulators for nephrogenesis in Brg1-deficient cells, including Lin28, Pbx1, and Dchs1-Fat4 signaling, but upregulation of podocyte lineage, oncogenic, and cell death-inducing genes, many of which Brg1 targets. Genome-wide binding analysis identifies Brg1 occupancy to a distal enhancer of Eya1 that drives nephron progenitor-specific expression. We demonstrate that Brg1 enrichment to two distal intronic enhancers of Pbx1 and a proximal promoter region of Mycn requires Six2 activity and that these Brg1/Six2-bound enhancers govern nephron progenitor-specific expression in response to Six2 activity. CONCLUSIONS: Our results reveal an essential role for Brg1, its downstream pathways, and its interaction with Eya1-Six2 in mediating the fine balance among the self-renewal, differentiation, and survival of nephron progenitors.
Asunto(s)
Ensamble y Desensamble de Cromatina , ADN Helicasas/fisiología , Elementos de Facilitación Genéticos , Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Nefronas/citología , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiología , Proteínas Tirosina Fosfatasas/metabolismo , Células Madre/citología , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , Diferenciación Celular , Autorrenovación de las Células , Inmunoprecipitación de Cromatina , Técnicas de Sustitución del Gen , Células HEK293 , Proteínas de Homeodominio/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Riñón/embriología , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Complejos Multiproteicos , Proteínas Nucleares/genética , Mapeo de Interacción de Proteínas , Proteínas Tirosina Fosfatasas/genética , Células Madre/metabolismo , Factores de Transcripción/genética , TranscriptomaRESUMEN
BACKGROUND: This study aimed to investigate the clinicopathological significance of sine oculis homeobox homolog 1 (SIX1) and eyes absent 1 (EYA1) in patients with chronic hepatitis B (CHB) and other liver diseases. METHODS: SIX1 and EYA1 levels were detected in human serum and liver tissues by enzyme linked immunosorbent assay (ELISA) and immunofluorescent staining method, respectively. RESULTS: The serum SIX1 and EYA1 levels in 313 CHB patients were 7.24±0.11 and 25.21±0.51 ng/mL, respectively, and these values were significantly higher than those in 33 healthy controls (2.84±0.15 and 13.11±1.01 ng/mL, respectively; P<0.05). Serum SIX1 and EYA1 levels were also markedly increased in patients with numerous other liver diseases, including liver fibrosis, hepatocellular carcinoma, fatty liver disease, alcoholic liver disease, fulminant hepatic failure, autoimmune liver disease, and hepatitis C, compared to the healthy controls (P<0.05). Dynamic observation of these proteins over time in 35 selected CHB patients revealed that SIX1 and EYA1 serum levels increased over an interval. Immunofluorescent staining revealed that both SIX1 and EYA1 were only expressed in hepatic stellate cells (HSCs), and their increased expression was evident in CHB liver tissue. CONCLUSIONS: SIX1 and EYA1 are novel biomarkers of liver damage in patients of CHB and other liver diseases, with potential clinical utility.
RESUMEN
Specification of Sox2+ proneurosensory progenitors within otic ectoderm is a prerequisite for the production of sensory cells and neurons for hearing. However, the underlying molecular mechanisms driving this lineage specification remain unknown. Here, we show that the Brg1-based SWI/SNF chromatin-remodeling complex interacts with the neurosensory-specific transcriptional regulators Eya1/Six1 to induce Sox2 expression and promote proneurosensory-lineage specification. Ablation of the ATPase-subunit Brg1 or both Eya1/Six1 results in loss of Sox2 expression and lack of neurosensory identity, leading to abnormal apoptosis within the otic ectoderm. Brg1 binds to two of three distal 3' Sox2 enhancers occupied by Six1, and Brg1-binding to these regions depends on Eya1-Six1 activity. We demonstrate that the activity of these Sox2 enhancers in otic neurosensory cells specifically depends on binding to Six1. Furthermore, genome-wide and transcriptome profiling indicate that Brg1 may suppress apoptotic factor Map3k5 to inhibit apoptosis. Together, our findings reveal an essential role for Brg1, its downstream pathways, and their interactions with Six1/Eya1 in promoting proneurosensory fate induction in the otic ectoderm and subsequent neuronal lineage commitment and survival of otic cells.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/genética , Cromatina/metabolismo , Ectodermo/embriología , Ectodermo/metabolismo , Diferenciación Celular/genética , Linaje de la Célula/genética , Biología Computacional/métodos , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Neuronas/citología , Neuronas/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismoRESUMEN
BACKGROUND: Eya2 expression during mouse development has been studied by in situ hybridization and it has been shown to be involved skeletal muscle development and limb formation. Here, we generated Eya2 knockout (Eya2- ) and a lacZ knockin reporter (Eya2lacZ ) mice and performed a detailed expression analysis for Eya2lacZ at different developmental stages to trace Eya2lacZ -positive cells in Eya2-null mice. We describe that Eya2 is not only expressed in cranial sensory and dorsal root ganglia, retina and olfactory epithelium, and somites as previously reported, but also Eya2 is specifically detected in other organs during mouse development. RESULTS: We found that Eya2 is expressed in ocular and trochlear motor neurons. In the inner ear, Eya2lacZ is specifically expressed in differentiating hair cells in both vestibular and cochlear sensory epithelia of the inner ear and Eya2-/- or Eya2lacZ/lacZ mice displayed mild hearing loss. Furthermore, we detected Eya2 expression during both salivary gland and thymus development and Eya2-null mice had a smaller thymus. CONCLUSIONS: As Eya2 is coexpressed with other members of the Eya family genes, these results together highlight that Eya2 as a potential regulator may act synergistically with other Eya genes to regulate the differentiation of the inner ear sensory hair cells and the formation of the salivary gland and thymus.
Asunto(s)
Oído Interno/metabolismo , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Pérdida Auditiva/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Animales , Diferenciación Celular/fisiología , Oído Interno/embriología , Pérdida Auditiva/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas Nucleares/genética , Proteínas Tirosina Fosfatasas/genéticaRESUMEN
Age-related hearing loss (ARHL) is the most prevalent sensory deficit. ARHL reduces the quality of life of the growing population, setting seniors up for the enhanced mental decline. The size of the needy population, the structural deficit, and a likely research strategy for effective treatment of chronic neurosensory hearing in the elderly are needed. Although there has been profound advancement in auditory regenerative research, there remain multiple challenges to restore hearing loss. Thus, additional investigations are required, using novel tools. We propose how the (1) flat epithelium, remaining after the organ of Corti has deteriorated, can be converted to the repaired-sensory epithelium, using Sox2. This will include (2) developing an artificial gene regulatory network transmitted by (3) large viral vectors to the flat epithelium to stimulate remnants of the organ of Corti to restore hair cells. We hope to unite with our proposal toward the common goal, eventually restoring a functional human hearing organ by transforming the flat epithelial cells left after the organ of Corti loss.
Asunto(s)
Envejecimiento/patología , Cóclea/patología , Presbiacusia/patología , Calidad de Vida , Factores de Transcripción SOXB1/metabolismo , Anciano , Células Ciliadas Auditivas/patología , Pérdida Auditiva , HumanosRESUMEN
The transcription factor Six1 is essential for induction of sensory cell fate and formation of auditory sensory epithelium, but how it activates gene expression programs to generate distinct cell-types remains unknown. Here, we perform genome-wide characterization of Six1 binding at different stages of auditory sensory epithelium development and find that Six1-binding to cis-regulatory elements changes dramatically at cell-state transitions. Intriguingly, Six1 pre-occupies enhancers of cell-type-specific regulators and effectors before their expression. We demonstrate in-vivo cell-type-specific activity of Six1-bound novel enhancers of Pbx1, Fgf8, Dusp6, Vangl2, the hair-cell master regulator Atoh1 and a cascade of Atoh1's downstream factors, including Pou4f3 and Gfi1. A subset of Six1-bound sites carry consensus-sequences for its downstream factors, including Atoh1, Gfi1, Pou4f3, Gata3 and Pbx1, all of which physically interact with Six1. Motif analysis identifies RFX/X-box as one of the most significantly enriched motifs in Six1-bound sites, and we demonstrate that Six1-RFX proteins cooperatively regulate gene expression through binding to SIX:RFX-motifs. Six1 targets a wide range of hair-bundle regulators and late Six1 deletion disrupts hair-bundle polarity. This study provides a mechanistic understanding of how Six1 cooperates with distinct cofactors in feedforward loops to control lineage-specific gene expression programs during progressive differentiation of the auditory sensory epithelium.
Asunto(s)
Diferenciación Celular/genética , Linaje de la Célula/genética , Epitelio/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Ciliadas Auditivas/citología , Células Ciliadas Auditivas/metabolismo , Proteínas de Homeodominio/metabolismo , Secuencia de Bases , Polaridad Celular , Secuencia de Consenso , ADN/metabolismo , Fosfatasa 6 de Especificidad Dual/genética , Fosfatasa 6 de Especificidad Dual/metabolismo , Elementos de Facilitación Genéticos/genética , Factor 8 de Crecimiento de Fibroblastos/genética , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Sitios Genéticos , Genoma , Células Ciliadas Auditivas/ultraestructura , Humanos , Complejos Multiproteicos/metabolismo , Motivos de Nucleótidos/genética , Unión Proteica , Transducción de Señal/genéticaRESUMEN
During neural development, stem and precursor cells can divide either symmetrically or asymmetrically. The transition between symmetric and asymmetric cell divisions is a major determinant of precursor cell expansion and neural differentiation, but the underlying mechanisms that regulate this transition are not well understood. Here, we identify the Sonic hedgehog (Shh) pathway as a critical determinant regulating the mode of division of cerebellar granule cell precursors (GCPs). Using partial gain and loss of function mutations within the Shh pathway, we show that pathway activation determines spindle orientation of GCPs, and that mitotic spindle orientation correlates with the mode of division. Mechanistically, we show that the phosphatase Eya1 is essential for implementing Shh-dependent GCP spindle orientation. We identify atypical protein kinase C (aPKC) as a direct target of Eya1 activity and show that Eya1 dephosphorylates a critical threonine (T410) in the activation loop. Thus, Eya1 inactivates aPKC, resulting in reduced phosphorylation of Numb and other components that regulate the mode of division. This Eya1-dependent cascade is critical in linking spindle orientation, cell cycle exit and terminal differentiation. Together these findings demonstrate that a Shh-Eya1 regulatory axis selectively promotes symmetric cell divisions during cerebellar development by coordinating spindle orientation and cell fate determinants.
Asunto(s)
División Celular/fisiología , Cerebelo/metabolismo , Proteínas Hedgehog/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis/fisiología , Proteínas Nucleares/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Animales , Cerebelo/embriología , Cerebelo/crecimiento & desarrollo , Ratones , Ratones Mutantes , Células-Madre Neurales/citología , Transducción de Señal/fisiologíaRESUMEN
Craniofacial morphogenesis requires proper development of pharyngeal arches and epibranchial placodes. We show that the epibranchial placodes, in addition to giving rise to cranial sensory neurons, generate a novel lineage-related non-neuronal cell population for mouse pharyngeal arch development. Eya1 is essential for the development of epibranchial placodes and proximal pharyngeal arches. We identify an Eya1-Notch regulatory axis that specifies both the neuronal and non-neuronal commitment of the epibranchial placode, where Notch acts downstream of Eya1 and promotes the non-neuronal cell fate. Notch is regulated by the threonine phosphatase activity of Eya1. Eya1 dephosphorylates p-threonine-2122 of the Notch1 intracellular domain (Notch1 ICD), which increases the stability of Notch1 ICD and maintains Notch signaling activity in the non-neuronal epibranchial placodal cells. Our data unveil a more complex differentiation program in epibranchial placodes and an important role for the Eya1-Notch axis in craniofacial morphogenesis.
Asunto(s)
Región Branquial/embriología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Morfogénesis , Proteínas Nucleares/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal , Animales , Diferenciación Celular , RatonesRESUMEN
The organ of Corti in the cochlea is a two-cell layered epithelium: one cell layer of mechanosensory hair cells that align into one row of inner and three rows of outer hair cells interdigitated with one cell layer of underlying supporting cells along the entire length of the cochlear spiral. These two types of epithelial cells are derived from common precursors in the four- to five-cell layered primordium and acquire functionally important shapes during terminal differentiation through the thinning process and convergent extension. Here, we have examined the role of Six1 in the establishment of the auditory sensory epithelium. Our data show that prior to terminal differentiation of the precursor cells, deletion of Six1 leads to formation of only a few hair cells and defective patterning of the sensory epithelium. Previous studies have suggested that downregulation of Sox2 expression in differentiating hair cells must occur after Atoh1 mRNA activation in order to allow Atoh1 protein accumulation due to antagonistic effects between Atoh1 and Sox2. Our analysis indicates that downregulation of Sox2 in the differentiating hair cells depends on Six1 activity. Furthermore, we found that Six1 is required for the maintenance of Fgf8 expression and dynamic distribution of N-cadherin and E-cadherin in the organ of Corti during differentiation. Together, our analyses uncover essential roles of Six1 in hair cell differentiation and formation of the organ of Corti in the mammalian cochlea.
Asunto(s)
Diferenciación Celular/genética , Células Ciliadas Auditivas/metabolismo , Proteínas de Homeodominio/genética , Órgano Espiral/crecimiento & desarrollo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Cadherinas/genética , Cóclea/crecimiento & desarrollo , Cóclea/metabolismo , Epitelio/crecimiento & desarrollo , Epitelio/metabolismo , Factor 8 de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/biosíntesis , Ratones , Morfogénesis/genética , Órgano Espiral/metabolismo , Factores de Transcripción SOXB1/genéticaRESUMEN
The adult mammalian cochlear sensory epithelium houses two major types of cells, mechanosensory hair cells and underlying supporting cells, and lacks regenerative capacity. Recent evidence indicates that a subset of supporting cells can spontaneously regenerate hair cells after ablation only within the first week postparturition. Here in vivo clonal analysis of mouse inner ear cells during development demonstrates clonal relationship between hair and supporting cells in sensory organs. We report the identification in mouse of a previously unknown population of multipotent stem/progenitor cells that are capable of not only contributing to the hair and supporting cells but also to other cell types, including glia, in cochlea undergoing development, maturation and repair in response to damage. These multipotent progenitors originate from Eya1-expressing otic progenitors. Our findings also provide evidence for detectable regenerative potential in the postnatal cochlea beyond 1 week of age.
Asunto(s)
Células Ciliadas Auditivas/citología , Audición/fisiología , Células Laberínticas de Soporte/citología , Células Madre Multipotentes/citología , Neuroglía/citología , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular , Embrión de Mamíferos , Femenino , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Ciliadas Auditivas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Laberínticas de Soporte/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Células Madre Multipotentes/metabolismo , Miosina VIIa , Miosinas/genética , Miosinas/metabolismo , Neuroglía/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteína Fluorescente RojaRESUMEN
EYA1 is known to be overexpressed in human breast cancer, in which the Myc protein is also accumulated in association with decreased phospho-T58 (pT58) levels. We have recently reported that EYA1 functions as a unique protein phosphatase to dephosphorylate Myc at pT58 to regulate Myc levels. However, it remains unclear whether EYA1-mediated Myc dephosphorylation on T58 is a critical function in regulating Myc protein stability in breast cancer. Furthermore, EYA1's substrate specificity has remained elusive. In this study, we have investigated these questions, and here, we report that depletion of EYA1 using short hairpin RNA (shRNA) in breast cancer cells destabilizes the Myc protein and increases pT58 levels, leading to an increase in the doubling time and impairment of cell cycle progression. In correlation with EYA1-mediated stabilization of cMyc and reduced levels of pT58, EYA1 greatly reduced cMyc-FBW7 binding and cMyc ubiquitination, thus providing novel insight into how EYA1 acts to regulate the FBW7-mediated Myc degradation machinery. We found that the conserved C-terminal haloacid dehalogenase domain of EYA1, which has been reported to have only tyrosine phosphatase activity, has dual phosphatase activities, and both the N- and C-terminal domains interact with substrates to increase the catalytic activity of EYA1. Enzymatic assay and nuclear magnetic resonance (NMR) analysis demonstrated that EYA1 has a striking conformation preference for phospho-T58 of Myc. Together, our results not only provide novel structural evidence about the conformation specificity of EYA1 in dephosphorylating phosphothreonine in Myc but also reveal an important mechanism contributing to Myc deregulation in human breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ciclo Celular , Línea Celular Tumoral , Proteína 7 que Contiene Repeticiones F-Box-WD , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Modelos Moleculares , Fosforilación , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-myc/química , Treonina/metabolismoRESUMEN
Mouse N-ethyl-N-nitrosourea (ENU) mutagenesis has generated many useful animal models for human diseases. Here we describe the identification of a novel ENU-induced mouse mutant strain Turner (Tur) that displays circling and headtossing behavior and progressive hearing loss. Tur/Tur homozygous animals lack Preyer and righting reflexes and display severe headtossing and reaching response defect. We mapped the Tur mutation to a critical region of 11 cM on chromosome 9 that includes myosin VI. Direct sequence analysis revealed a c.820A>T substitution in exon 8 of the Myo6 gene that changes amino acid Asn200 to Ile (p.N200I) in the motor domain. Analysis of inner ear hair cells by immunohistochemistry, scanning electron microscopy and histology revealed degeneration of hair cells in the inner ear and structural malformation of the stereocilia in the cochlea of Turner homozygous mutant mice. Our data indicate that this novel mouse strain provides a useful model for future studies on the function of myosin VI in mammalian auditory and non-auditory systems and in human syndromes.
Asunto(s)
Sordera/genética , Sordera/fisiopatología , Mutación/genética , Cadenas Pesadas de Miosina/genética , Vestíbulo del Laberinto/fisiopatología , Animales , Umbral Auditivo , Secuencia de Bases , Conducta Animal , Cromosomas de los Mamíferos/genética , Epitelio/patología , Epitelio/ultraestructura , Etilnitrosourea , Potenciales Evocados Auditivos del Tronco Encefálico , Genes Dominantes , Células Ciliadas Auditivas/patología , Células Ciliadas Auditivas/ultraestructura , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Penetrancia , Vestíbulo del Laberinto/ultraestructuraRESUMEN
BACKGROUND: Specification of the metanephric mesenchyme is a central step of kidney development as this mesenchyme promotes nephric duct induction to form a ureteric bud near its caudal end. Before ureteric bud formation, the caudal nephric duct swells to form a pseudostratified epithelial domain that later emerges as the tip of the bud. However, the signals that promote the formation of the transient epithelial domain remain unclear. Here, we investigated the early roles of the mesenchymal factor Six family and its cofactor Eya on the initial induction of nephric duct development. RESULTS: The nephrogenic progenitor population is initially present but significantly reduced in mice lacking both Six1 and Six4 and undertakes an abnormal cell death pathway to be completely eliminated by â¼E10.5-E11.0, similar to that observed in Eya1(-/-) embryos. Consequently, the nephric duct fails to be induced to undergo normal proliferation to pseudostratify and form the ureteric bud in Six1(-/-) ;Six4(-/-) or Eya1(-/-) embryos. CONCLUSIONS: Our data support a model where Eya-Six may form a complex to regulate nephron progenitor cell development before metanephric specification and are critical mesenchymal factors for inducing nephric duct development.
Asunto(s)
Proliferación Celular/fisiología , Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Nefronas/embriología , Proteínas Nucleares/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Células Madre/metabolismo , Transactivadores/metabolismo , Uréter/embriología , Animales , Proteínas de Homeodominio/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Noqueados , Modelos Biológicos , Nefronas/citología , Proteínas Nucleares/genética , Proteínas Tirosina Fosfatasas/genética , Células Madre/citología , Transactivadores/genética , Uréter/citologíaRESUMEN
Sonic hedgehog (Shh) signaling is critical in development and oncogenesis, but the mechanisms regulating this pathway remain unclear. Although protein phosphorylation clearly affects Shh signaling, little is known about phosphatases governing the pathway. Here, we conducted a small hairpin RNA (shRNA) screen of the phosphatome and identified Eya1 as a positive regulator of Shh signaling. We find that the catalytically active phosphatase Eya1 cooperates with the DNA-binding protein Six1 to promote gene induction in response to Shh and that Eya1/Six1 together regulate Gli transcriptional activators. We show that Eya1, which is mutated in a human deafness disorder, branchio-oto-renal syndrome, is critical for Shh-dependent hindbrain growth and development. Moreover, Eya1 drives the growth of medulloblastoma, a Shh-dependent hindbrain tumor. Together, these results identify Eya1 and Six1 as key components of the Shh transcriptional network in normal development and in oncogenesis.
Asunto(s)
Carcinogénesis/patología , Proteínas Hedgehog/metabolismo , Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Factores de Transcripción de Tipo Kruppel/fisiología , Meduloblastoma/patología , Proteínas Nucleares/fisiología , Proteínas Tirosina Fosfatasas/fisiología , Receptores de Superficie Celular/genética , Rombencéfalo/citología , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Carcinogénesis/metabolismo , Células Cultivadas , Inmunoprecipitación de Cromatina , Perfilación de la Expresión Génica , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Hedgehog/genética , Proteínas de Homeodominio/genética , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Meduloblastoma/genética , Meduloblastoma/metabolismo , Ratones , Ratones Noqueados , Mutación/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores Patched , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Superficie Celular/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rombencéfalo/metabolismo , Transducción de Señal , Proteína con Dedos de Zinc GLI1RESUMEN
Self-renewal and proliferation of nephron progenitor cells and the decision to initiate nephrogenesis are crucial events directing kidney development. Despite recent advancements in defining lineage and regulators for the progenitors, fundamental questions about mechanisms driving expansion of the progenitors remain unanswered. Here we show that Eya1 interacts with Six2 and Myc to control self-renewing cell activity. Cell fate tracing reveals a developmental restriction of the Eya1(+) population within the intermediate mesoderm to nephron-forming cell fates and a common origin shared between caudal mesonephric and metanephric nephrons. Conditional inactivation of Eya1 leads to loss of Six2 expression and premature epithelialization of the progenitors. Six2 mediates translocation of Eya1 to the nucleus, where Eya1 uses its threonine phosphatase activity to control Myc phosphorylation/dephosphorylation and function in the progenitor cells. Our results reveal a functional link between Eya1, Six2, and Myc in driving the expansion and maintenance of the multipotent progenitors during nephrogenesis.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Nefronas/citología , Nefronas/metabolismo , Proteínas Nucleares/metabolismo , Organogénesis/fisiología , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Células Madre/citología , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular/fisiología , Mesodermo/metabolismo , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Madre/metabolismoRESUMEN
Proper formation of ureteral smooth muscle cells (SMCs) during embryogenesis is essential for ureter peristalsis that propels urine from the kidney to the bladder in mammals. Currently the molecular factors that regulate differentiation of ureteral mesenchymal cells into SMCs are incompletely understood. A recent study has reported that Smad4 deficiency reduces the number of ureteral SMCs. However, its precise role in the ureteral smooth muscle development remains largely unknown. Here, we used Tbx18:Cre knock-in mouse line to delete Smad4 to examine its requirement in the development of ureteral mesenchyme and SMC differentiation. We found that mice with specific deletion of Smad4 in Tbx18-expressing ureteral mesenchyme exhibited hydroureter and hydronephrosis at embryonic day (E) 16.5, and the mutant mesenchymal cells failed to differentiate into SMCs with increased apoptosis and decreased proliferation. Molecular markers for SMCs including alpha smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SM-MHC) were absent in the mutant ureters. Moreover, disruption of Smad4 significantly reduced the expression of genes, including Sox9, Tbx18 and Myocardin associated with SMC differentiation. These findings suggest that Smad4 is essential for initiating the SMC differentiation program during ureter development.