Human Sir2-related protein SIRT1 associates with the bHLH repressors HES1 and HEY2 and is involved in HES1- and HEY2-mediated transcriptional repression

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Abstract

The Hairy-related bHLH proteins function as transcriptional repressors in most cases and play important roles in diverse aspects of metazoan development. Recently, it was shown that the Drosophila bHLH repressor proteins, Hairy and Deadpan, bind to and function with the NAD+-dependent histone deacetylase, Sir2. Here we demonstrate that the human Sir2 homologue, SIRT1, also physically associates with the human bHLH repressor proteins, hHES1 and hHEY2, both in vitro and in vivo. Moreover, using the reporter assay, we show that both SIRT1-dependent and -independent deacetylase pathways are involved in the transcriptional repressions mediated by these bHLH repressors. These results indicate that the molecular association between bHLH proteins and Sir2-related proteins is conserved among metazoans, from Drosophila to human, and suggest that the Sir2–bHLH interaction also plays important roles in human cells.

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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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