Project description:Atoh8 is a transcription factor of the basic-helix-loop-helix (bHLH) family that is expressed in multiple tissues during embryonic development but whose specific functions remain unknown. The gene encoding Atoh8 is induced by various lineage- determining bHLH transcription factors in cell culture, suggesting a possible common role of this gene in multiple bHLH-driven differentiation programs. In the pancreas, the pro-endocrine bHLH factors Neurogenin3 (Neurog3) and NeuroD1 activate Atoh8 expression. Moreover, Atoh8 is expressed in the embryonic pancreas confirming its participation in the pancreatic transcriptional cascade. This work aims at gaining insight into the molecular function of Atoh8 during the endocrine differentiation program initiated by Neurog3 in the pancreas. To this aim, we have generated a recombinant adenovirus encoding an Atoh8-specific shRNA (Ad-shAtoh8) and used it to down-regulate expression of the Atoh8 gene in Neurog3-expressing pancreatic ductal cells (mPAC), a cellular model of endocrine cell differentiation. Thus, we have compared global changes in gene expression profiles between cells treated with Ad-Neurog3+shControl and cells treated with Ad-Neurog3+shAtoh8 using Affymetrix microarrays. Our results show that Atoh8 silencing significantly affects the expression of 293 genes in Neurog3-expressing mPAC cells. Gene Ontology analysis has revealed cell cycle as the biological function most significantly represented among the modified genes. These results uncover a potential function of Math6 as a regulator of cell cycle progression and provide novel insights into the link between Neurog3 and the regulation of the cell cycle. mPAC cells (mouse pancreatic duct cells) overexpressing Neurogenin3 were divided in 2 groups according to the shRNA encoded by the recombinant adenoviruses used: 1) Scramble shRNA (control for the infection effect), 2) Atonal Homolog 8-specific shRNA. Experiment was performed three independent times (3 independent biological replicates).
Project description:Atoh8 is a transcription factor of the basic-helix-loop-helix (bHLH) family that is expressed in multiple tissues during embryonic development but whose specific functions remain unknown. The gene encoding Atoh8 is induced by various lineage- determining bHLH transcription factors in cell culture, suggesting a possible common role of this gene in multiple bHLH-driven differentiation programs. In the pancreas, the pro-endocrine bHLH factors Neurogenin3 (Neurog3) and NeuroD1 activate Atoh8 expression. Moreover, Atoh8 is expressed in the embryonic pancreas confirming its participation in the pancreatic transcriptional cascade. This work aims at gaining insight into the molecular function of Atoh8 during the endocrine differentiation program initiated by Neurog3 in the pancreas. To this aim, we have generated a recombinant adenovirus encoding an Atoh8-specific shRNA (Ad-shAtoh8) and used it to down-regulate expression of the Atoh8 gene in Neurog3-expressing pancreatic ductal cells (mPAC), a cellular model of endocrine cell differentiation. Thus, we have compared global changes in gene expression profiles between cells treated with Ad-Neurog3+shControl and cells treated with Ad-Neurog3+shAtoh8 using Affymetrix microarrays. Our results show that Atoh8 silencing significantly affects the expression of 293 genes in Neurog3-expressing mPAC cells. Gene Ontology analysis has revealed cell cycle as the biological function most significantly represented among the modified genes. These results uncover a potential function of Math6 as a regulator of cell cycle progression and provide novel insights into the link between Neurog3 and the regulation of the cell cycle.
Project description:The basic helix-loop-helix (bHLH) transcription factors of the Drosophila’s atonal-related superfamily Neurogenin3 (Neurog3) and NeuroD1 promote endocrine differentiation in the gastrointestinal tract. Atonal Homolog 8 (Atoh8/Math6) is a newly identified member of the atonal-related family whose expression is induced by Neurog3 and NeuroD1 in cell culture, indicating a possible role for this gene in the endocrine differentiation program downstream of these two pro-endocrine factors. Intriguingly, available experimental evidence based on a reduced number of genes suggests that Atoh8 may negatively regulate Neurog3-targeting events. In this study, we have analyzed global changes in gene expression profiles upon exogenous expression of Atoh8 alone or in combination with Neurog3 in mouse pancreatic duct (mPAC) cells. These cells activate neuroendocrine-specific gene expression in response to Neurog3 and NeuroD1 and thus serve as an optimal model to evaluate the proendocrine activity of Atoh8. We have compared transcriptional profiles between mPAC cells treated with a recombinant adenovirus expressing Atoh8 (Ad-Atoh8) or a control adenovirus encoding B-galactosidase (Ad-Bgal), and between cells treated with Ad-Neurog3+Ad-Bgal or cells treated with Ad-Neurog3+Ad-Atoh8. The results obtained show that Atoh8 exhibits a very modest transcriptional activity in these cells thus confirming that Atoh8 does not function as a proendocrine gene. Furthermore, our data also confirm the ability of Atoh8 to block Neurog3-dependent transcriptional activation events. However, since repression is only seen for a small subset of Neurog3 gene targets, we discard a general role of Atoh8 as a negative regulator of Neurog3 pro-endocrine activity.
Project description:Here, we report in vivo reprogramming of pancreatic ductal cells through intra-ductal delivery of an adenoviral vector expressing the transcription factors Pdx1, Neurog3 and Mafa (adPNM). To better understand the extent of reprogramming in induced pancreatic ductal cells, comparative single cell RNA sequencing was performed on insulin+ pancreatic ductal cells and pancreatic β-cells. Compared with pancreatic β cells, we show that insulin+ pancreatic ductal cells had established a β-cell gene expression program, albeit with immature features.
Project description:Transcription profiling by array of Neurogenin-3-overexpressing mouse pancreatic ductal cells with shRNA knockdown of Atoh8 against non-knockdown controls to study the role of Atoh8 during the endocrine differentiation program initiated by Neurog3 in the pancreas
Project description:ATOH8 inhibits cell proliferation through transcriptional regulating cell cycle related genes, and ATOH8 and SMAD3 form a transcriptional complex to induce an anti-cell cycle signature and prevent cell cycle progression
Project description:Long noncoding RNA (lncRNA) play important roles in the pathogenesis of cancer. LncRNA SBF2-AS1is unregulated in lung cancer tissues, while its biological function and molecular mechanism are largely unknown. RNA sequencing results suggest cell cycle-related genes are altered after SBF2-AS1 knockdown. In vivo and in vitro experiments confirm SBF2-AS1 could promote tumorigenesis of lung cancer. Further experiments prove SBF2-AS1 could bind with miR-338-3p and miR-362-3p to regulate various cell cycle-related genes, including E2F1. These results indicate the existence of ceRNA network driven by SBF2-AS1 through sponging miRNAs.
Project description:Neurog3 and NeuroD1 share the ability to induce endocrine differentiation when expressed ectopically in vivo (Apelqvist et al., 1999; Schwitzgebel et al., 2000; Grapin-Botton et al., 2001) and in vitro (Heremans et al., 2002; Gasa et al., 2004), suggesting that they target a common set of genes. To test this hypothesis, we directly compared in the same cells the effects of Neurog3 and NeuroD1 on global gene expression patterns. Normalized M and A values [limmaGUI output] for the neurogenin3 (Ngn3) and neuroD1 (Nd1) experiments (individual or combined) have been linked below as Supplementary files. Note that M=log2(test/ref), eg, log2(Nd1/Bgal); A=[log2(test*ref)]/2, eg, [log2(Nd1*Bgal)]/2. Additional measures of reliability/probability are also included within the supplementary files. Mouse pancreatic ductal cells (mPAC cells) were infected with equal titers of recombinant adenoviruses encoding human Neurogenin3 (AdCMV-NEUROG3) or mouse NeuroD1 (AdCMV-NeuroD1), and 48 hrs later their transcriptomes were compared with that of control cells infected with an adenovirus encoding betagalactosidase (beta-gal; AdCMV-Bgal) using cDNA microarrays. Differences in the activation patterns of the two transcription factors were then determined by comparing the fold-change (FC) values of the pair-wise hybridizations Neurog3/Bgal and NeuroD1/beta-gal.