RESUMO
Vertebrate postembryonic development is regulated by thyroid hormone (T3). Of particular interest is anuran metamorphosis, which offers several unique advantages for studying the role of T3 and its two nuclear receptor genes, TRα and TRß, during postembryonic development. We have recently generated TR double knockout (TRDKO) Xenopus tropicalis animals and reported that TR is essential for the completion of metamorphosis. Furthermore, TRDKO tadpoles are stalled at the climax of metamorphosis before eventual death. Here we show that TRDKO intestine lacked larval epithelial cell death and adult stem cell formation/proliferation during natural metamorphosis. Interestingly, TRDKO tadpole intestine had premature formation of adult-like epithelial folds and muscle development. In addition, T3 treatment of premetamorphic TRDKO tadpoles failed to induce any metamorphic changes in the intestine. Furthermore, RNA-seq analysis revealed that TRDKO altered the expression of many genes in biological pathways such as Wnt signaling and the cell cycle that likely underlay the inhibition of larval epithelial cell death and adult stem cell development caused by removing both TR genes. Our data suggest that liganded TR is required for larval epithelial cell degeneration and adult stem cell formation, whereas unliganded TR prevents precocious adult tissue morphogenesis such as smooth-muscle development and epithelial folding.
Assuntos
Células-Tronco Adultas/metabolismo , Proteínas de Anfíbios/genética , Células Epiteliais/metabolismo , Intestinos/citologia , Larva/genética , Receptores dos Hormônios Tireóideos/genética , Hormônios Tireóideos/genética , Xenopus/genética , Células-Tronco Adultas/citologia , Células-Tronco Adultas/efeitos dos fármacos , Proteínas de Anfíbios/classificação , Proteínas de Anfíbios/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/genética , Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Ontologia Genética , Redes Reguladoras de Genes , Intestinos/efeitos dos fármacos , Intestinos/crescimento & desenvolvimento , Larva/citologia , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Redes e Vias Metabólicas/genética , Metamorfose Biológica , Anotação de Sequência Molecular , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Receptores dos Hormônios Tireóideos/deficiência , Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/farmacologia , Via de Sinalização Wnt/genética , Xenopus/crescimento & desenvolvimento , Xenopus/metabolismoRESUMO
The maintenance of the intestinal epithelium is ensured by the controlled proliferation of intestinal stem cells (ISCs) and differentiation of their progeny into various cell types, including enterocytes (ECs) that both mediate nutrient absorption and provide a barrier against pathogens. The signals that regulate transition of proliferative ISCs into differentiated ECs are not fully understood. IRBIT is an evolutionarily conserved protein that regulates ribonucleotide reductase (RNR), an enzyme critical for the generation of DNA precursors. Here, we show that IRBIT expression in ISC progeny within the Drosophila midgut epithelium cells regulates their differentiation via suppression of RNR activity. Disruption of this IRBIT-RNR regulatory circuit causes a premature loss of intestinal tissue integrity. Furthermore, age-related dysplasia can be reversed by suppression of RNR activity in ISC progeny. Collectively, our findings demonstrate a role of the IRBIT-RNR pathway in gut homeostasis.
RESUMO
BACKGROUND: Structural rearrangements of the genome resulting in genic imbalance due to copy number change are often deleterious at the organismal level, but are common in immortalized cell lines and tumors, where they may be an advantage to cells. In order to explore the biological consequences of copy number changes in the Drosophila genome, we resequenced the genomes of 19 tissue-culture cell lines and generated RNA-Seq profiles. RESULTS: Our work revealed dramatic duplications and deletions in all cell lines. We found three lines of evidence indicating that copy number changes were due to selection during tissue culture. First, we found that copy numbers correlated to maintain stoichiometric balance in protein complexes and biochemical pathways, consistent with the gene balance hypothesis. Second, while most copy number changes were cell line-specific, we identified some copy number changes shared by many of the independent cell lines. These included dramatic recurrence of increased copy number of the PDGF/VEGF receptor, which is also over-expressed in many cancer cells, and of bantam, an anti-apoptosis miRNA. Third, even when copy number changes seemed distinct between lines, there was strong evidence that they supported a common phenotypic outcome. For example, we found that proto-oncogenes were over-represented in one cell line (S2-DRSC), whereas tumor suppressor genes were under-represented in another (Kc167). CONCLUSION: Our study illustrates how genome structure changes may contribute to selection of cell lines in vitro. This has implications for other cell-level natural selection progressions, including tumorigenesis.
Assuntos
Linhagem Celular , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Evolução Molecular , Dosagem de Genes , Animais , Sobrevivência Celular , DNA/análise , Proteínas de Drosophila/genética , Feminino , Aptidão Genética , Variação Genética , Masculino , MicroRNAs/genética , Receptores Proteína Tirosina Quinases/genética , Seleção Genética , Análise de Sequência de DNA , Cromossomos Sexuais/genética , Técnicas de Cultura de TecidosRESUMO
E2F and RB proteins regulate the expression of genes involved in cell cycle progression, apoptosis, differentiation, and development. Recent studies indicate that they function as part of an evolutionarily conserved multiprotein complex termed dREAM/DREAM/LINC. Here we characterize the role of the Drosophila complex, dREAM, in the regulation of differentiation-specific E2F target genes in actively proliferating cells. These genes are regulated differently from cell cycle-related E2F targets, they do not depend on E2F activation, and E2F/RB repression is maintained throughout the cell cycle. In proliferating cells, their repression is dependent on dREAM. We find that dREAM plays a dual role in their regulation. First, it is required for the stability of the repressive dE2F2/RBF complexes at their promoters during S phase. Second, we find that dREAM is indispensable for both transcriptional repression mechanisms employed at these genes.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Fatores de Transcrição E2F/genética , Proteína do Retinoblastoma/genética , Animais , Ciclo Celular , Diferenciação Celular/genética , Regulação da Expressão Gênica , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismoRESUMO
The RB and E2F proteins play important roles in the regulation of cell division, cell death, and development by controlling the expression of genes involved in these processes. The mechanisms of repression by the retinoblastoma protein (pRB) have been extensively studied at cell cycle-regulated promoters. However, little is known about developmentally regulated E2F/RB genes. Here, we have taken advantage of the simplicity of the E2F/RB pathway in flies to inspect the regulation of differentiation-specific target genes. These genes are repressed by dE2F2/RBF and a recently identified RB-containing complex, dREAM/MMB, in a cell type- and cell cycle-independent manner. Our studies indicate that the mechanism of repression differs from that of cell cycle-regulated genes. We find that two different activities are involved in their regulation and that in proliferating cells, both are required to maintain repression. First, dE2F2/RBF and dREAM/MMB employ histone deacetylase (HDAC) activities at promoter regions. Remarkably, we have also uncovered an unconventional mechanism of repression by the Polycomb group (PcG) protein Enhancer of zeste [E(Z)], which is involved in silencing of these genes through the dimethylation of histone H3 Lys27 at nucleosomes located downstream of the transcription start sites (TSS).