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. 2019 Feb 13;141(6):2462-2473.
doi: 10.1021/jacs.8b12083. Epub 2019 Feb 4.

A Click Chemistry Approach Reveals the Chromatin-Dependent Histone H3K36 Deacylase Nature of SIRT7

Affiliations

A Click Chemistry Approach Reveals the Chromatin-Dependent Histone H3K36 Deacylase Nature of SIRT7

Wesley Wei Wang et al. J Am Chem Soc. .

Abstract

Using an engineered pyrrolysyl-tRNA synthetase mutant together with tRNACUAPyl, we have genetically encoded Nε-(7-azidoheptanoyl)-l-lysine (AzHeK) by amber codon in Escherichia coli for recombinant expression of a number of AzHeK-containing histone H3 proteins. We assembled in vitro acyl-nucleosomes from these recombinant acyl-H3 histones. All these acyl-nucleosomes contained an azide functionality that allowed quick click labeling with a strained alkyne dye for in-gel fluorescence analysis. Using these acyl-nucleosomes as substrates and click labeling as a detection method, we systematically investigated chromatin deacylation activities of SIRT7, a class III NAD+-dependent histone deacylase with roles in aging and cancer biology. Besides confirming the previously reported histone H3K18 deacylation activity, our results revealed that SIRT7 has an astonishingly high activity to catalyze deacylation of H3K36 and is also catalytically active to deacylate H3K37. We further demonstrated that this H3K36 deacylation activity is nucleosome dependent and can be significantly enhanced when appending the acyl-nucleosome substrate with a short double-stranded DNA that mimics the bridging DNA between nucleosomes in native chromatin. By overexpressing SIRT7 in human cells, we verified that SIRT7 natively removes acetylation from histone H3K36. Moreover, SIRT7-deficient cells exhibited H3K36 hyperacetylation in whole cell extracts, at rDNA sequences in nucleoli, and at select SIRT7 target loci, demonstrating the physiologic importance of SIRT7 in determining endogenous H3K36 acetylation levels. H3K36 acetylation has been detected at active gene promoters, but little is understood about its regulation and functions. Our findings establish H3K36 as a physiologic substrate of SIRT7 and implicate this modification in potential SIRT7 pathways in heterochromatin silencing and genomic stability.

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Conflict of interest statement

Notes

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
A click chemistry-based approach to profile SIRT7-targeted chromatin lysine deacylation sites. AzHeK that structurally resembles DeK is site-specifically incorporated into a histone that is further assembled in vitro with other histones and 601 DNA into an acyl-nucleosome. This acyl-nucleosome can be labeled fluorescently with a strained alkyne dye and subsequently visualized in a 1× TBE native-PAGE gel. When SIRT7 is catalytically active to remove fatty acylation from the incorporated AzHeK to recover lysine, incubating the assembled acyl-nucleosome with SIRT7 will remove the fatty acylation and therefore afford a deacylated nucleosome that cannot be labeled fluorescently and then visualized in a gel.
Figure 2.
Figure 2.
The genetic incorporation of AzHeK. (A) A diagram to illustrate the fluorescent labeling of a genetically incorporated AzHeK in a protein with MB488-DBCO, a strained alkyne dye from Click Chemistry Tools LLC. (B) The selective incorporation of AzHeK into ubiquitin at its K48 position to afford UbK48az. We transformed BL21(DE3) cells with plasmid pEVOL-OcKRS coding OcKRS and tRNACUAPyl and plasmid pETDuet-UbK48Am coding a ubiquitin gene with an amber mutation at the K48 coding position and then grew the transformed cells in 2YT media with or without 1 mM AzHeK. (C) The selective labeling of UbK48az with MB488-DBCO. Reaction conditions: we incubated UbK48az with 100 μM DBCO-MB488 for 1 h before doing the in-gel fluorescence analysis. (D) The MALDI-TOF MS spectrum of UbK48az. The calculated molecular weight is 9540.7 Da.
Figure 3.
Figure 3.
SIRT7 actively removes acylation from H3 K18 and H3 K36 in the nucleosome context. (A) SDS-PAGE analysis of eight purified AzHeK-containing H3 proteins. (B) Acyl-nucleosomes that we assembled from eight acyl-H3 proteins, H2A, H2B, H4, and 601 DNA (147 bp). Nu-H3K4az denotes an acyl-nucleosome assembled from H3K4az. All other acyl-nucleosomes are named in the same way. Mononucleosomes are typically shown around the 500 bp DNA position in an EtBr-stained 5% 1× TBE native-PAGE gel. (C) SIRT7 catalyzed deacylation activities on eight acyl-nucleosome substrates. Reaction conditions: we incubated 1 μM acyl-nucleosome with 0.5 μM SIRT7, 0.5 mM BME and 1 mM NAD+ at 37 °C for 2 h before we quenched the reaction by the addition of 20 mM nicotinamide. We then labeled the resulted acyl-nucleosome substrate with 100 μM MB488-DBCO for 1 h and analyzed it fluorescently in an 8% 1× TBE native-PAGE gel. The top panel shows the MB488-DBCO-based fluorescent imaging and the bottom panel shows the EtBr-stained DNA from the same gel. (D) Quantified deacylation levels at eight H3 lysine sites. We repeated experiments show in C thrice and calculated the deacylation level at each site by subtracting average MB488-DBCO-based fluorescence intensity of SIRT7-treated samples from that of untreated controls.
Figure 4.
Figure 4.
Effects of DNA and salt on SIRT7-catalyzed deacylation. (A) Free DNA inhibits SIRT7-catalyzed nucleosome deacylation. Reaction conditions: we incubated 1 μM nu-H3K36az with 0.1 μM SIRT7, a varied concentration of 147 bp DNA, 1 mM NAD+ and 0.5 mM BME at 37 °C for 3 h before we labeled the solution with 100 μM MB488-DBCO and analyzed the acyl-nucleosome substrate fluorescently in a 1× TBE native-PAGE gel. The top panel is MB488-DBCO-based imaging that indicates relative acylation levels and the bottom panel is EtBr-based imaging that confirms the nucleosome integrity. (B) Salt inhibits SIRT7-catalyzed nucleosome deacylation. Conditions were as same as in A except we replaced free DNA with NaCl. (C) Free DNA has a binary role on SIRT7-catalyzed deacylation of the H3K36az-H4 tetramer substrate. Reaction conditions: we incubated 2 μM H3K36az-H4 tetramer with 1 μM SIRT7, a varied concentration of 147 bp DNA, 0.5 mM BME, and 1 mM NAD+ at 37 °C for 2 h before we labeled the solution with 100 μM MB488-DBCO and analyzed the tetramer substrate fluorescently in a SDS-PAGE gel.
Figure 5.
Figure 5.
A linker DNA on an acyl-nucleosome substrate improves SIRT7-catalyzed deacylation. (A) A diagram to illustrate how a linker DNA affiliates the binding of SIRT7 to an acyl-nucleosome substrate. (B) The in vitro assembly of nu-H3K18az and nu-H3K36az with different lengths of linker DNAs. The gel showed the EtBr-stained nucleosomes. (C) The catalytic removal of acylation from nu-H3K36az substrates that contained different lengths of linker DNAs. Reaction conditions: we incubated a 1 μM H3K36az nucleosome with 0.1 μM SIRT7, 1 mM NAD+ and 0.5 mM BME at 37 °C for various times before we quenched the reaction by adding 20 mM nicotinamide, labeled the solution with 100 μM MB488-DBCO for 1 h, and then analyzed the nucleosome substrate fluorescently by 8% 1× TBE native-PAGE gel. (D) The catalytic removal of acylation from nu-H3K18az substrates that contained different lengths of linker DNAs. Reaction conditions were as same as for nu-H3K36az substrates except higher amount of SIRT7 (0.3 μM) was used. (E-F) Quantified deacylation percentage vs time for two acyl-nucleosome substrates. Reactions shown in C and D were repeated thrice. Deacylation was calculated and averaged by subtracting MB488-DBCO-based fluorescence intensity at different time points from that at 0 min.
Figure 6.
Figure 6.
SIRT7 catalyzes deacetylation at H3 K18 and H3 K36 in cells. (A) SIRT7 activities on a number of in vitro assembled acetyl-nucleosomes. Reaction conditions: we incubated an acetyl-nucleosome (1 μM) with or without 0.3 μM SIRT7 for 2 h before we analyzed the reaction by SDS-PAGE and then probed the acetylation level and H3 by anti-H3K36ac antibody and an anti-H3 antibody, respectively. We tested both a nu-H3K36ac nucleosome without a linker DNA and the one with a 20 bp linker DNA. All other acetyl-nucleosomes contained a 20 bp linker DNA. (B) Acetylation levels detected by Western blotting at K9, K14, K18, K23, K27, and K36 of histone H3 in SIRT7-transfected cells with respect to those in control cells that expressed just EGFP or an inactive SIRT7 mutant. Signals were probed by site-specific Kac antibodies.
Figure 7.
Figure 7.
SIRT7 is a physiologic nucleolar and nucleoplasmic H3K36 deacetylase. (A) Immunoblot showing increased H3K36ac levels in whole cell lysates from SIRT7 knock-down (KD) U2OS cells compared to control cells. (B) ChIP-PCR showing increased H3K36ac levels at promoters of SIRT7 target genes RPS20 and MRPS18b, but not COPS2, in SIRT7 KD versus control U2OS cells. Two independent H3K36ac antibodies (Ab 1 and 2) were used. Data show the average of three technical replicates +/− SEM, and are representative of 2–4 biological replicates. Statistical significance was calculated performing Student’s t-test. (C) Immunoblot of nucleolar fractionation showing increased levels of H3K36ac in SIRT7 KD cells versus control cells. β-Tubulin, p84, and FBL or UBF are shown as marker controls for cytoplasmic, nucleoplasmic, and nucleolar fractions, respectively. (D) Immunoblot of nucleolar fractionation of SIRT7 knockout versus control U2OS cells. (E) ChIP-PCR showing increased levels of H3K36ac at 18S rDNA sequences in SIRT7 KD cells. Data show the average of three biological replicates +/− SEM. Statistical significance was calculated performing Student’s t-test. (F) ChIP-seq enrichment analysis of H3K36ac at repetitive sequences in SIRT7 KD versus control U2OS cells. Bar plot of log10(p-value) shows statistically significant enrichment of H3K36ac in SIRT7-KD cells at rDNA (18S, 28S) and telomeric (TTAGGG) repeats, but not at repetitive sequences from 20 specific centromeres (CT), 6 specific pericentromeres (PCT), or 4 centromeric and 4 pericentric consensus sequences. P-values were calculated performing Chi-squared test.
Figure 8.
Figure 8.
Lysine acetylation at H3 K36 improves DNA unwrapping from the nucleosome core. (A) A diagram to illustrate how H3K36ac increases DNA unwrapping from the nucleosome core and subsequent Pst1 access to an embedded Pst1 restriction site. (B) The in vitro assembly of three nucleosomes, nu’, nu’-H3K18ac, nu’-H3K36ac, and nu’-H3K56ac from corresponding histones and a mutant 601 DNA that had a Pst1 restriction site and a linker DNA. The gel shows EtBr-stained nucleosomes. (C) Pst1 digestion progress of four nucleosomes versus time. Reaction conditions: we incubated a 0.49 μM nucleosome with 2 U/uL Pst1 at 37 °C for various times before the reaction was stopped and the solution was analyzed by 5% 1× TBE native-PAGE. The gel was stained by EtBr. The top band in a lane shows the nucleosome with the linker DNA and the bottom band represents the one with the linker DNA removed. (D) The calculated ratio of digested nucleosome to undigested nucleosome vs time. We calculated and averaged the ratios based on integrated fluorescence intensities of original and digested nucleosome bands in gels.
Figure 9.
Figure 9.
SIRT7 deacetylates H3K37ac in vitro. (A) In vitro deacetylation of nu-H3K37ac by SIRT7. Reaction conditions: nu-H3K37ac (1 μM) was incubated with or without 0.3 μM SIRT7 for 2 h, and then analyzed by SDS-PAGE and Western blot. (B-D) Time-based deacetylation of nu-H3K37ac, nu-H3K36ac, and nu-H3K18ac by SIRT7. Reaction conditions: 1 μM acetyl-nucleosomes were incubated with 0.1 μM SIRT7, 1 mM NAD+ and 0.5 mM BME at 37 °C for various times followed by reaction quenching in 20 mM nicotinamide, and acetylation levels were analyzed by Western blot. Reactions were repeated thrice and average acetylation levels at different times are presented in the bottom panel diagram. (E) Immunoblot of nucleoplasmic and nucleolar fractions from control and SIRT7 knockout cell lines. Data showed global increase in H3K36ac but not H3K18ac or H3K37ac levels. p84 and FBL are shown as marker controls for nucleoplasmic and nucleolar fractions, respectively.

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