SIRT1 Regulates The Expression Of EZH2 And Affects The Transcriptional Regulation Of The Target Gene SATB1 By PcG

Polycomb group genes (PcGs) encode a family of proteins that are usually considered as transcriptional repressors that are required for a series of processes during embryonic development. Therefore, this class of proteins has been extensively studied in recent years. It is now known that Polycomb group proteins play critical roles in forming the anterior-posterior Hox gene expression pattern along the body axis, and promoting neurogenesis and haematopoiesis. It has also been confirmed that PcG proteins participate in maintaining the pluripotency of human embryonic stem cells. A recent large scale chip analysis carried out in mammalian systems, mouse and human embryonic cells, identified more than 1000 potential PcG silenced genes, including upstream transcription factors and other regulatory factors in cellular signaling transduction pathways. Previous studies proved that PcG proteins form different complexes and maintain silencing state of target genes through modifying specific histone tails which results in changes of higher order chromatin structures. Polycomb Repressive Complexes (PRC) 2 is such a complex bearing histone methyltransferase activity specific for H3K27. PRC2 is composed of EZH2, Suzl2 and EED, among which EZH2 is the only catalytically active component.Mammalian SIRT1 (Sirtuin 1) belongs to Class III histone deacetylase family, which requires NAD+ for its catalytic activity. Under the conditions of energy restriction or in response to stress, SIRT1 has been shown to maintain genomic stability, modulate cellular responses to stimuli, increase energy production and metabolism, promote DNA repair and extend life time of the cells. SIRT1 can exert its functions through interactions with many important transcription factors or co-factors, including MyoD, p300, PGC-1?, PPARy, NF-?B, Ku70, p53 and FOXO. SIRT1 was also reported to be recruited to its own gene promoter by binding to HIC1 and negatively regulates the transcription. In addition, Sir2 or SIRT1 has been found to interact with EZH2 in Drosophila or human cancer cells and such interaction may change substrate preference of the complexes. Therefore, in this study we ask if SIRT1 affects PcG functions in other aspects. We employed RNA interference technology to knockdown SIRT1 in HeLa cells and then detected the expression level of EZH2. Since published chip data indicate that SATB1 is a potential PcG target gene, we determined this in our study. Then we examined the SIRT1 deficient Hela cells for its expression of the well documentated EZH2 target HoxA9 along with SATB1, which is confirmed in this study as a direct target of EZH2. Furthermore, we detected the specific histone modification status of SATB1 and HoxA9 promoters, and the recruitment of histone modifying enzymes as well. The results show that EZH2 protein level increases in SIRT1 knockdown cells and the transcription of SATB1 and HoxA9 was decreased, which is consistent with the increase of H3K27me3 modification and EZH2 recruitment at their promoter regions. Meanwhile, the H4K16Ac status at the promoters shows no apparent changes and no SIRT1 recruitment to these promoters was found. To further explore the mechanisms, we detected the transcription level of EZH2 but found no significant changes after SIRT1 down-regulation. However, the depletion of SIRT1 results in mild increase of EZH2 stability, which may at least partially explain the increased protein level.To summarize, we found that depletion of SIRT1 in HeLa cells increases EZH2 protein level and enhances EZH2 mediated suppression on HoxA9 and SATB1 genes, while such effects are independent of the H4K16Ac deacetylase activity by SIRT1.