A Chemical Biology Approach to Reveal Sirt6-targeted Histone H3 Sites in Nucleosomes

Abstract

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.

SIRT1 and SIRT6 Signaling Pathways in Cardiovascular Disease Protection

“…characterized as a highly specific H3 deacetylase that targets Lys-9 (H3K9), Lys-56 (H3K56), and Lys-18 (H3K18) (Fig. 2) (86,121,122,140,171), as confirmed by Wang et al through a novel approach to generate nucleosomes specifically acetylated on a single specific site (191). It has been hypothesized that, under oxidative and nitrosative stress, SIRT6 activity can be regulated via reactive nitrogen species-mediated posttranslational modifications (71).…”

SIRT6: Novel Mechanisms and Links to Aging and Disease

“…Recently, using a novel approach to generate nucleosomes that are specifically acetylated on a single defined site, Wang and colleagues definitively confirmed the high efficiency and selectivity of SIRT6 in deacetylating its previously described substrates H3K9ac and H3K18ac. In addition, they showed that SIRT6 can also deacetylate nucleosomal H3K27ac, a chromatin mark associated with transcriptional enhancer elements, suggesting potential functions of SIRT6 in enhancer-regulated gene expression programs [14]. Interestingly, the in vitro deacetylation of histone peptides by SIRT6 can be activated up to 35-fold fold by free fatty acids (FFAs; e.g., myristic acid), suggesting that in vivo , efficient deacetylation of known or novel SIRT6 substrates could depend on endogenous FFA activators that fluctuate with metabolic conditions [15].…”

“…They showed that SIRT6 removes long-chain octenoyl-groups on H3K9, H3K18, and H3K27 (and with weaker activity, on H3K4 and H3K23), but was unable to de-octenoylate these residues when acylated free H3 histones or acylated H3-H4 tetramers were used as substrates. Thus, both the deacetylase and long-chain defatty-acylase activities of SIRT6 appear to be much more active on histones in the physiologic context of nucleosomal structure [14]. …”

“…The role of SIRT6 in site-specific histone deacetylation has now been linked to regulation of many processes that are deregulated in aging, cancer, and metabolic syndromes, including DNA repair, heterochromatin silencing, genomic instability, glucose and lipid homeostasis, circadian regulation, and stem cell function. Early concerns about the weak histone deacetylase activity of SIRT6 observed in vitro have been resolved by detection of efficient deacetylase activity using improved assays that better mimic physiologic conditions at chromatin, and by development of approaches with optimized detection of substrate-specific deacetylation [10, 12, 14, 23]. Such assays also provide tools for the identification of novel SIRT6 substrates in the future.…”

Genetically Encoding a Lipidated Amino Acid for Extension of Protein Half‐Life in vivo

“…[8] Thea bility to generate site-specifically lipidated proteins in vivo will greatly facilitate the investigation of the complicated network of membranes and lipidated proteins involved in basic cellular function and human diseases.U aas bearing simple alkyl, alkyl thiol ether, or halo-and alkene-terminated alkyl side chains have been genetically incorporated into proteins, [9] yet their binding with HSA has not been investigated and they may not be ideal candidates for HSA binding due to their insufficient length or terminal derivatization. [8] Thea bility to generate site-specifically lipidated proteins in vivo will greatly facilitate the investigation of the complicated network of membranes and lipidated proteins involved in basic cellular function and human diseases.U aas bearing simple alkyl, alkyl thiol ether, or halo-and alkene-terminated alkyl side chains have been genetically incorporated into proteins, [9] yet their binding with HSA has not been investigated and they may not be ideal candidates for HSA binding due to their insufficient length or terminal derivatization.…”