Biochemical and Biophysical Research Communications
Human Sir2-related protein SIRT1 associates with the bHLH repressors HES1 and HEY2 and is involved in HES1- and HEY2-mediated transcriptional repression
Section snippets
Materials and methods
Cell cultures. All cells were cultured with supplementation of 10% FCS, l-glutamine, penicillin, and streptomycin and grown in 5% CO2 at 37 °C. PBS(−) (phosphate-buffered saline) without magnesium and calcium was used in this work. FuGENE6 (Roche) was used to transfect various cell lines.
Isolation of SIRT1 gene. Human SIRT1 gene was obtained by screening the cDNA library prepared from poly(A)+ RNA of PA-1 cells using the ZAP-cDNA Synthesis Kit and the lambda ZAP II Predigested EcoRI/CIAP-Treated
Physical interactions of human SIRT1 with hHES1 and hHEY2 in vivo
To investigate whether SIRT1 interacts physically with hHES1 in vivo, we performed co-immunoprecipitation experiments (Figs. 1A and B). 293T cells were transiently transfected with FLAG-tagged SIRT1 and/or HA-tagged hHES1. The cell lysates were immunoprecipitated with anti-HA or anti-FLAG antibodies and the precipitated fractions were analyzed by Western blotting using anti-FLAG or anti-HA antibodies, respectively. Human SIRT1-FLAG was co-immunoprecipitated with anti-HA antibodies only when
Discussion
It has been recently reported that Drosophila Sir2 (dSir2) physically associates in vitro with Drosophila Hairy and Deadpan (Dpn), but not with E(spl) proteins [30]. Genetic experiments in the same study clearly showed that these interactions have functional roles in vivo. Interestingly, dSir2-interacting Hairy and Dpn are classified as members of the Hairy subfamily and non-interacting E(spl) is classified as a member of another subfamily E(spl). Therefore, it appears that the ability of
Acknowledgements
We are grateful to Dr. Ryoichiro Kageyama (Kyoto University) for pβA-luc. The excellent secretarial work of F. Nishizaki, K. Saito, K. Yokoyama, A. Orii, M. Ohmaki, M. Sakamoto, Y. Tada, and A. Katayama and the technical assistance by M. Tamura are also acknowledged. This work was supported by a COE Grant and a Grant-in-Aid for Cancer Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, a Health and Labour Sciences Research Grants from the Ministry of Health,
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