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PCAF-primed EZH2 acetylation regulates its stability and promotes lung adenocarcinoma progression.

Wan J, Zhan J, Li S, Ma J, Xu W, Liu C, Xue X, Xie Y, Fang W, Chin YE, Zhang H - Nucleic Acids Res. (2015)

Bottom Line: We, herein, report that EZH2 is acetylated by acetyltransferase P300/CBP-associated factor (PCAF) and is deacetylated by deacetylase SIRT1.Mechanistically, K348 acetylation decreases EZH2 phosphorylation at T345 and T487 and increases EZH2 stability without disrupting the formation of polycomb repressive complex 2 (PRC2).Our findings define a new mechanism underlying EZH2 modulation by linking EZH2 acetylation to its phosphorylation that stabilizes EZH2 and promotes lung adenocarcinoma progression.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, and State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, Beijing 100191, China Laboratory of Molecular Cell Biology and Tumor Biology, Department of Anatomy, Histology and Embrology, Peking University Health Science Center, Beijing 100191, China.

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SIRT1 interacts with and deacetylates EZH2 (A and B) EZH2 acetylation is increased in the presence of nicotinamide. H1299 cells were co-transfected with FLAG-EZH2 and FLAG-PCAF and then treated with 3 μM TSA or 5 mM nicotinamide for 12 h. Lysates were co-immunoprecipitated with an anti-FLAG Ab and immunoprecipitates were immunoblotted with the AcK Ab (A). H1299 cells were transfected with FLAG-EZH2 and then treated with 5 mM nicotinamide for 12 h. Lysates were co-immunoprecipitated with an anti-FLAG Ab, then immunoblotted with the AcK Ab (B). (C and D) EZH2 interacts with SIRT1 in vivo. HEK-293T cells were co-transfected with Myc-EZH2, Myc-EZH2 plus FLAG-SIRT1 or Myc-EZH2 plus FLAG-SIRT2. Forty-eight hours post transfection, lysates were co-immunoprecipitated with an anti-FLAG Ab, followed by a western blot with an anti-Myc Ab (C). HEK-293T cells were co-transfected with Myc-EZH2 and FLAG-SIRT1. The lysates were co-immunoprecipitated with an anti-Myc Ab, followed by western blot with an anti-FLAG Ab (D). (E and F) EZH2 is deacetylated by SIRT1. HEK-293T cells were transfected with FLAG-EZH2, FLAG-PCAF and FLAG-SIRT1 as indicated. Lysates were co-immunoprecipitated with an anti-FLAG Ab and detected by the AcK Ab (E). The in vitro deacetylation reaction mixtures were subjected to SDS-PAGE and immunoblotted with the anti-AcK348-EZH2 Ab. The purified GST fusion proteins were detected by Coomassie blue staining (F).
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Figure 4: SIRT1 interacts with and deacetylates EZH2 (A and B) EZH2 acetylation is increased in the presence of nicotinamide. H1299 cells were co-transfected with FLAG-EZH2 and FLAG-PCAF and then treated with 3 μM TSA or 5 mM nicotinamide for 12 h. Lysates were co-immunoprecipitated with an anti-FLAG Ab and immunoprecipitates were immunoblotted with the AcK Ab (A). H1299 cells were transfected with FLAG-EZH2 and then treated with 5 mM nicotinamide for 12 h. Lysates were co-immunoprecipitated with an anti-FLAG Ab, then immunoblotted with the AcK Ab (B). (C and D) EZH2 interacts with SIRT1 in vivo. HEK-293T cells were co-transfected with Myc-EZH2, Myc-EZH2 plus FLAG-SIRT1 or Myc-EZH2 plus FLAG-SIRT2. Forty-eight hours post transfection, lysates were co-immunoprecipitated with an anti-FLAG Ab, followed by a western blot with an anti-Myc Ab (C). HEK-293T cells were co-transfected with Myc-EZH2 and FLAG-SIRT1. The lysates were co-immunoprecipitated with an anti-Myc Ab, followed by western blot with an anti-FLAG Ab (D). (E and F) EZH2 is deacetylated by SIRT1. HEK-293T cells were transfected with FLAG-EZH2, FLAG-PCAF and FLAG-SIRT1 as indicated. Lysates were co-immunoprecipitated with an anti-FLAG Ab and detected by the AcK Ab (E). The in vitro deacetylation reaction mixtures were subjected to SDS-PAGE and immunoblotted with the anti-AcK348-EZH2 Ab. The purified GST fusion proteins were detected by Coomassie blue staining (F).

Mentions: Acetylation is a dynamic process and acetylated proteins, e.g. p53 and FOXO3a can be reversed by specific deacetylases (31,32). To this end, we found that cells treated with the class III SIRT inhibitor nicotinamide greatly enhanced the level of acetylated FLAG-EZH2 with the presence of PCAF, whereas the class I and II HDAC inhibitor TSA displayed no effect on the deacetylation of EZH2 (Figure 4A), indicating that EZH2 is deacetylated mainly by the class III SIRT deacetylases. Importantly, nicotinamide also increased EZH2 acetylation without PCAF overexpression in H1299, HeLa and HEK-293T cells (Figure 4B and Supplementary Figure S1J, K). Next, we selected SIRT1 and SIRT2 from SIRT family to examine if they interact with and deacetylate EZH2. We co-transfected Myc-EZH2 with FLAG-SIRT1 or FLAG-SIRT2, and found that Myc-EZH2 was co-immunoprecipitated by FLAG-SIRT1, not by FLAG-SIRT2 (Figure 4C). Reciprocally, FLAG-SIRT1 was co-immunoprecipitated by Myc-EZH2 (Figure 4D). These findings suggested that EZH2 selectively interacts with SIRT1 not SIRT2, which is in agreement with the previous report (33). We therefore focused on the role of SIRT1 in deacetylation of EZH2. In cells, EZH2 acetylation was completely blocked by SIRT1 and was drastically restored upon addition of nicotinamide (Figure 4E). Note that the self-acetylation of PCAF was also enhanced in the presence of nicotinamide (Figure 4E). Furthermore, in an in vitro deacetylation assay we confirmed that EZH2 is a bona fide substrate of SIRT1 (Figure 4F). Taken together, these results strongly demonstrated that SIRT1 interacts with and deacetylates EZH2 both in vivo and in vitro.


PCAF-primed EZH2 acetylation regulates its stability and promotes lung adenocarcinoma progression.

Wan J, Zhan J, Li S, Ma J, Xu W, Liu C, Xue X, Xie Y, Fang W, Chin YE, Zhang H - Nucleic Acids Res. (2015)

SIRT1 interacts with and deacetylates EZH2 (A and B) EZH2 acetylation is increased in the presence of nicotinamide. H1299 cells were co-transfected with FLAG-EZH2 and FLAG-PCAF and then treated with 3 μM TSA or 5 mM nicotinamide for 12 h. Lysates were co-immunoprecipitated with an anti-FLAG Ab and immunoprecipitates were immunoblotted with the AcK Ab (A). H1299 cells were transfected with FLAG-EZH2 and then treated with 5 mM nicotinamide for 12 h. Lysates were co-immunoprecipitated with an anti-FLAG Ab, then immunoblotted with the AcK Ab (B). (C and D) EZH2 interacts with SIRT1 in vivo. HEK-293T cells were co-transfected with Myc-EZH2, Myc-EZH2 plus FLAG-SIRT1 or Myc-EZH2 plus FLAG-SIRT2. Forty-eight hours post transfection, lysates were co-immunoprecipitated with an anti-FLAG Ab, followed by a western blot with an anti-Myc Ab (C). HEK-293T cells were co-transfected with Myc-EZH2 and FLAG-SIRT1. The lysates were co-immunoprecipitated with an anti-Myc Ab, followed by western blot with an anti-FLAG Ab (D). (E and F) EZH2 is deacetylated by SIRT1. HEK-293T cells were transfected with FLAG-EZH2, FLAG-PCAF and FLAG-SIRT1 as indicated. Lysates were co-immunoprecipitated with an anti-FLAG Ab and detected by the AcK Ab (E). The in vitro deacetylation reaction mixtures were subjected to SDS-PAGE and immunoblotted with the anti-AcK348-EZH2 Ab. The purified GST fusion proteins were detected by Coomassie blue staining (F).
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Figure 4: SIRT1 interacts with and deacetylates EZH2 (A and B) EZH2 acetylation is increased in the presence of nicotinamide. H1299 cells were co-transfected with FLAG-EZH2 and FLAG-PCAF and then treated with 3 μM TSA or 5 mM nicotinamide for 12 h. Lysates were co-immunoprecipitated with an anti-FLAG Ab and immunoprecipitates were immunoblotted with the AcK Ab (A). H1299 cells were transfected with FLAG-EZH2 and then treated with 5 mM nicotinamide for 12 h. Lysates were co-immunoprecipitated with an anti-FLAG Ab, then immunoblotted with the AcK Ab (B). (C and D) EZH2 interacts with SIRT1 in vivo. HEK-293T cells were co-transfected with Myc-EZH2, Myc-EZH2 plus FLAG-SIRT1 or Myc-EZH2 plus FLAG-SIRT2. Forty-eight hours post transfection, lysates were co-immunoprecipitated with an anti-FLAG Ab, followed by a western blot with an anti-Myc Ab (C). HEK-293T cells were co-transfected with Myc-EZH2 and FLAG-SIRT1. The lysates were co-immunoprecipitated with an anti-Myc Ab, followed by western blot with an anti-FLAG Ab (D). (E and F) EZH2 is deacetylated by SIRT1. HEK-293T cells were transfected with FLAG-EZH2, FLAG-PCAF and FLAG-SIRT1 as indicated. Lysates were co-immunoprecipitated with an anti-FLAG Ab and detected by the AcK Ab (E). The in vitro deacetylation reaction mixtures were subjected to SDS-PAGE and immunoblotted with the anti-AcK348-EZH2 Ab. The purified GST fusion proteins were detected by Coomassie blue staining (F).
Mentions: Acetylation is a dynamic process and acetylated proteins, e.g. p53 and FOXO3a can be reversed by specific deacetylases (31,32). To this end, we found that cells treated with the class III SIRT inhibitor nicotinamide greatly enhanced the level of acetylated FLAG-EZH2 with the presence of PCAF, whereas the class I and II HDAC inhibitor TSA displayed no effect on the deacetylation of EZH2 (Figure 4A), indicating that EZH2 is deacetylated mainly by the class III SIRT deacetylases. Importantly, nicotinamide also increased EZH2 acetylation without PCAF overexpression in H1299, HeLa and HEK-293T cells (Figure 4B and Supplementary Figure S1J, K). Next, we selected SIRT1 and SIRT2 from SIRT family to examine if they interact with and deacetylate EZH2. We co-transfected Myc-EZH2 with FLAG-SIRT1 or FLAG-SIRT2, and found that Myc-EZH2 was co-immunoprecipitated by FLAG-SIRT1, not by FLAG-SIRT2 (Figure 4C). Reciprocally, FLAG-SIRT1 was co-immunoprecipitated by Myc-EZH2 (Figure 4D). These findings suggested that EZH2 selectively interacts with SIRT1 not SIRT2, which is in agreement with the previous report (33). We therefore focused on the role of SIRT1 in deacetylation of EZH2. In cells, EZH2 acetylation was completely blocked by SIRT1 and was drastically restored upon addition of nicotinamide (Figure 4E). Note that the self-acetylation of PCAF was also enhanced in the presence of nicotinamide (Figure 4E). Furthermore, in an in vitro deacetylation assay we confirmed that EZH2 is a bona fide substrate of SIRT1 (Figure 4F). Taken together, these results strongly demonstrated that SIRT1 interacts with and deacetylates EZH2 both in vivo and in vitro.

Bottom Line: We, herein, report that EZH2 is acetylated by acetyltransferase P300/CBP-associated factor (PCAF) and is deacetylated by deacetylase SIRT1.Mechanistically, K348 acetylation decreases EZH2 phosphorylation at T345 and T487 and increases EZH2 stability without disrupting the formation of polycomb repressive complex 2 (PRC2).Our findings define a new mechanism underlying EZH2 modulation by linking EZH2 acetylation to its phosphorylation that stabilizes EZH2 and promotes lung adenocarcinoma progression.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, and State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, Beijing 100191, China Laboratory of Molecular Cell Biology and Tumor Biology, Department of Anatomy, Histology and Embrology, Peking University Health Science Center, Beijing 100191, China.

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Related in: MedlinePlus