摘要
Objective： Id2 is a natural inhibitor of the basic helix-loop-helix（bHLH） transcription factors. Although it is well known that active ld2 prevents differentiation and promotes cell cycle progression and tumorigenesis, the molecular events that regulate Id2 activity remain to be investigated. Methods： Yeast two-hybrid, mammalian two-hybrid, GST-pulldown and immunoprecipitation （ColP） assays were used to screen and identify novel Id2 interactors. Luciferase assays were used to detect E47-mediated transcription activity. Colony formation and BrdU incorporation assays were used to determine cellular proliferation abilities. Northorn blot, western blot and quantitative PCR methods were used to measure gene expression levels. Electrophoretic mobility shift assays （EMSAs） were performed to investigate protein/DNA binding. Results： The LIM-only protein FHL2 （four-and-a-half-LIM-only protein 2） was identified to be a novel Id2 interactor. The HLH domain within Id2 is not required for its interaction with FHL2. FHL2 antagonizes the inhibitory effect of Id2 on the basic helix-loop-helix protein E47-mediated transcription. FHL2 prevents the formation of Id2-E47 heterdimer, thus releasing E47 to its target DNA and restoring its transcriptional activity. FHL2 expression was remarkably up-regulated during retinoic acid-induced differentiation of neuroblastoma cells, during which the expression of Id2 is opposite to that. Ectopic FHL2 expression in neuroblastoma cells markedly reduces the transcriptional and cell-cycle promoting functions of Id2. Conclusion： These results indicate that FHL2 is an important repressor of the oncogenic activity of Id2 in neuroblastoma cells.
Objective： Id2 is a natural inhibitor of the basic helix-loop-helix（bHLH） transcription factors. Although it is well known that active ld2 prevents differentiation and promotes cell cycle progression and tumorigenesis, the molecular events that regulate Id2 activity remain to be investigated. Methods： Yeast two-hybrid, mammalian two-hybrid, GST-pulldown and immunoprecipitation （ColP） assays were used to screen and identify novel Id2 interactors. Luciferase assays were used to detect E47-mediated transcription activity. Colony formation and BrdU incorporation assays were used to determine cellular proliferation abilities. Northorn blot, western blot and quantitative PCR methods were used to measure gene expression levels. Electrophoretic mobility shift assays （EMSAs） were performed to investigate protein/DNA binding. Results： The LIM-only protein FHL2 （four-and-a-half-LIM-only protein 2） was identified to be a novel Id2 interactor. The HLH domain within Id2 is not required for its interaction with FHL2. FHL2 antagonizes the inhibitory effect of Id2 on the basic helix-loop-helix protein E47-mediated transcription. FHL2 prevents the formation of Id2-E47 heterdimer, thus releasing E47 to its target DNA and restoring its transcriptional activity. FHL2 expression was remarkably up-regulated during retinoic acid-induced differentiation of neuroblastoma cells, during which the expression of Id2 is opposite to that. Ectopic FHL2 expression in neuroblastoma cells markedly reduces the transcriptional and cell-cycle promoting functions of Id2. Conclusion： These results indicate that FHL2 is an important repressor of the oncogenic activity of Id2 in neuroblastoma cells.
基金
supported by the National Natural Science Foundation of China (No.30870507)
partially supported by a grant from the Ministry of Science and Technology of China (No.2005CB522603)
