A Chemical Biology Approach to Reveal Sirt6-targeted Histone H3 Sites in Nucleosomes.
A Chemical Biology Approach to Reveal Sirt6-targeted Histone H3 Sites in Nucleosomes.
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DOI：
10.1021/acschembio.6b00243
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发表时间：
2016-07-15
影响因子：
4
通讯作者：
Liu WR
中科院分区：
文献类型：
--
作者：
Wang WW;Zeng Y;Wu B;Deiters A;Liu WR
SIRT6是高度保守的依赖NAD+的组蛋白脱乙酰酶家族的成员，是哺乳动物基因组稳定性、新陈代谢和寿命的关键调节因子。以前的研究表明，SIRT6在H3K9和H3K56上是硬连接的，可以去除组蛋白乙酰化。然而，SIRT6如何识别其核小体底物一直难以捉摸，因为很难获得均一的乙酰核小体，而且SIRT6对肽底物的活性很低。基于SIRT6具有更强的去除赖氨酸长链脂肪酰化的活性，我们开发了一种将组蛋白H3与脂肪酰化赖氨酸N-ε-(7-辛烯基)-赖氨酸(OKK)连接在多个赖氨酸位点上重组合成组蛋白H3的方法，并将这些酰基-H3蛋白组装成具有活性的SIRT6底物。还设计了一种化学生物学方法，可以在核小体中显示Ock，从而允许直接敏化其酰基-核小体底物上的SIRT6活性。通过结合这两种方法，我们发现SIRT6主动地去除H3K9、H3K18和H3K27上的酰化，对H3K4和K3K23活性相对较低，但缓慢地去除H3K14、H3K36、H3K56和H3K79上的酰化。在293T细胞中过表达SIRT6导致H3K18和K3K27乙酰化下调，证实了细胞中这两个新的SIRT6靶向核小体赖氨酸位点。鉴于H3K18乙酰化下调与几种癌症的不良预后相关，以及H3K27乙酰化可拮抗H3K27二甲基化和三甲基化对抑制基因的调控，我们目前的研究提示SIRT6可能成为肿瘤干预和细胞调控途径研究的靶点。
As a member of a highly conserved family of NAD+-dependent histone deacetylases, Sirt6 is a key regulator of mammalian genome stability, metabolism, and life span. Previous studies indicated that Sirt6 is hardwired to remove histone acetylation at H3K9 and H3K56. However, how Sirt6 recognizes its nucleosome substrates has been elusive due to the difficulty of accessing homogenous acetyl-nucleosomes and the low activity of Sirt6 toward peptide substrates. Based on the fact that Sirt6 has an enhanced activity to remove long chain fatty acylation from lysine, we developed an approach to recombinantly synthesize histone H3 with a fatty acylated lysine, Nε-(7-octenoyl)-lysine (OcK), installed at a number of lysine sites and used these acyl-H3 proteins to assemble acyl-nucleosomes as active Sirt6 substrates. A chemical biology approach that visualizes OcK in nucleosomes and therefore allows directly sensitizes Sirt6 activities on its acyl-nucleosome substrates was also formulated. By combining these two approaches, we showed that Sirt6 actively removes acylation from H3K9, H3K18, and H3K27, has relatively low activities toward H3K4 and K3K23, but sluggishly removes acylation at H3K14, H3K36, H3K56, and H3K79. Overexpressing Sirt6 in 293T cells led to downregulated acetylation at H3K18 and K3K27, confirmed these two novel Sirt6-targeted nucleosome lysine sites in cells. Given that downregulation of H3K18 acetylation is correlated with poor prognosis of several cancer types and H3K27 acetylation antagonizes repressive gene regulation by di- and trimethylation at H3K27, our current study implies that Sirt6 may serve as a target for cancer intervention and regulatory pathway investigation in cells.