Supplementary Figure S1. Appearacne of new acetyl groups in acetylated lysines using 2,3-13 C 6 pyruvate as a tracer instead of labeled glucose. (a) Relative levels of both new acetylation and all acetylation (both old and new) in 17 acetylated sites. Two fewer sites were measured (relative to glucose labeling) due to the higher noise levels encountered in pyruvate labeling experiments. (b) Distributive levels of new acetylation for 15 acetylated sites. Data from 3 technical repliates of each were reported with one standard deviation depicted by the error bar. 1
Supplementary Figure S2. Data fitting results of acetylation levels with an exponential model. Both parameters and goodness of fit (R 2 ) are shown for each fit. (a) Distributive levels of new acetylation from sites in Group I were used for the fitting routine. (b) Relative levels of acetylation from sites in Group II were fitted. Two independent fittings were performed for each of two biological replicates by using the mean of three technical replicates. 2
Supplementary Figure S3. Time course data of acetylation levles in response to butyrate treatment for 19 distinct sites. (a, b) Relative levels and fold change of acetylation levels (treatment : control) from 0, 1.5, 3, 6, and 24 h treatments of 5 mm sodium butyrate. Data from 3 technical replicates of each were reported with standard deviation in the error bar. (c) Percentage value of relative acetylation levels measured. (d) Half-maximum values for acetylation levels of Group I sites in response to butyrate were determined by fitting data in (c) with an exponential model. Means from three independent fittings of three technical replicates were presented with the standard deviations shown inside parentheses. 3
Supplementary Figure S4. Acetylation turnover at six specific sites was affected by the modification status of their neighboring site. (a-c) In order to measure the acetylation turnover rate of a particular site in the context where various modifications were present at neighboring site on the same peptide, distributive levels of new acetylation in each site were aggregated from all possible labeled species (refer to Methods for more detail) to calculate the half-lives of 15 combinatorial modification forms from six sites. The six sites examined included H3K9ac, H3K14ac, H3K18ac, H3K23ac, H2AK5ac, and H2AK9ac. Data from two biological replicates with 3 technical replicates of each were reported with SEMs shown as error bars. (d) Venn diagrams were used to visualize the turnover between different modification forms (e.g., turnover of H3K9ac between H3K9ac-K14me1 and H3K9un- K14me1). The stoichiometry of these combinatorial modification forms is shown as the percentage of all forms measured. The overlap regions indicate doubly-modified forms. Arrows with different thicknesses were used to indicate the half-lives (in units of hours) between different modification forms; these half-lives were listed alongside each arrow. 4
Supplementary Figure S5. H3.3 variant specific lysine 36 acetylation. (a) A single nlc-qqq run of digested histone purified from HeLa cells was able to distinguish H3.3 K36ac from K36me3 when its neighboring K27 is unmodified, mono-, or di-methylated by virtue of their different retention times. Both K27 and K36 were in a tryptic peptide encompassing residues 27 to 40. K36ac was not observed in histone H3.1. (b) Two separate runs of nlc-qqq for H3.3 K27un-K36un and H3.3 K27un-K36ac synthetic peptide, respectively. 5
Supplementary Figure S6. Multi-target Mass Spectrometry. (a) Two rounds of derivatization using propionic anhydride before and after trypsin digestion were used to make all lysine residues inaccessible to trypsin regardless of their modification state. (b) Five SRM channels for five analytical targets are shown during a single nlc-qqq run of equal molar mixture of five H3K4 peptides (1 pmol) including unmodified, me1, me2, me3, and ac. Me3 was distinguished from ac by retention time (RT). Fragment ions used in SRM analyses and the retention time of nano-lc-ms runs are indicated with labels for b- and y-type fragment ions. 6
Supplementary Figure S7. Increase of methylation in old histone. Relative levels of unmodified, mono-, di-, trimethylated, and acetylated forms were quantified by combining the peptide forms carrying each modification and dividing this sum by the overall singal from all these modified forms measured for each peptide. Acetylation signal was the sum of both old and new acetylation. K9, K27, and K36 were aggregated from multiple forms created by the different modification status in their neighboring sites. All these data were from old histone (differentiated by metabolic incorporation of +6 Da heavy arginine). Data from two biological replicates with 3 technical replicates of each were reported with SEMs shown as error bars. 7
Supplementary Table Peptide Modified Sequence Precursor Fragment m/z m/z H3 K4un T[+56]K[+56]QTAR 408.732 246.156, 347.204, 475.262 H3 K4me1 T[+56]K[+70]QTAR 415.74 246.156, 347.204, 475.262 H3 K4me2 T[+56]K[+28]QTAR 394.735 175.119, 475.262, 543.314, 614.351 H3 K4me3 T[+56]K[+42]QTAR 401.743 175.119, 456.282, 557.329, 628.366 H3 K4ac T[+56]K[+42]QTAR 401.725 246.156, 347.204, 475.262 H3 K9un-K14ac K[+112.1]STGGK[+42]APR 528.297 241.155, 627.357, 815.437 H3 K9un-K14un K[+112.1]STGGK[+56]APR 535.304 241.155, 742.421, 829.453 H3 K9ac-K14ac K[+98]STGGK[+42]APR 521.288 227.139, 728.405, 815.437 H3 K9ac-K14un K[+98]STGGK[+56]APR 528.296 227.139, 641.373, 829.453 H3 K9me1-K14ac K[+126.1]STGGK[+42]APR 535.305 255.17, 627.357, 815.437 H3 K9me1-K14un K[+126.1]STGGK[+56]APR 542.311 255.17, 641.373, 829.453 H3 K9me2-K14ac K[+84.1]STGGK[+42]APR 514.298 213.16, 685.388, 815.437 H3 K9me2-K14un K[+84.1]STGGK[+56]APR 521.306 213.16, 641.373, 829.453 H3 K9me3-K14ac K[+98.1]STGGK[+42]APR 521.307 529.298, 570.336, 815.437 H3 K9me3-K14un K[+98.1]STGGK[+56]APR 528.314 641.373, 784.456, 829.453 H3 K18un-K23un K[+112.1]QLATK[+56]AAR 577.848 602.362, 673.399, 786.483 H3 K18ac-K23un K[+98]QLATK[+56]AAR 570.84 602.362, 673.399, 786.483 H3 K18un-K23ac K[+112.1]QLATK[+42]AAR 570.841 588.346, 659.384, 772.468 H3 K18ac-K23ac K[+98]QLATK[+42]AAR 563.833 588.346, 659.384, 772.468 H3.1/2 K27un-K36un K[+112.1]SAPATGGVK[+56]K[+56]PHR 829.473 630.365, 1259.722 H3.1/2 K27me1-K36un K[+126.1]SAPATGGVK[+56]K[+56]PHR 836.481 630.365, 1259.722 H3.1/2 K27me2-K36un K[+84.1]SAPATGGVK[+56]K[+56]PHR 543.985 777.473, 754.409 H3.1/2 K27me3-K36un K[+98.1]SAPATGGVK[+56]K[+56]PHR 548.657 768.425, 777.473 H3.1/2-K27ac-K36un K[+98]SAPATGGVK[+56]K[+56]PHR 548.646 630.365, 709.399 H3.1/2 K27un-K36me1 K[+112.1]SAPATGGVK[+70]K[+56]PHR 836.482 637.372, 1273.738 H3.1/2 K27me1-K36me1 K[+126.1]SAPATGGVK[+70]K[+56]PHR 843.488 637.372, 1273.738 H3.1/2 K27me2-K36me1 K[+84.1]SAPATGGVK[+70]K[+56]PHR 548.658 754.409, 791.489 H3.1/2 K27me3-K36me1 K[+98.1]SAPATGGVK[+70]K[+56]PHR 553.33 768.425, 791.489 H3.1/2 K27ac-K36me1 K[+98]SAPATGGVK[+70]K[+56]PHR 553.318 637.372, 716.407 H3.1/2 K27un-K36me2 K[+112.1]SAPATGGVK[+28]K[+56]PHR 543.986 616.367, 695.402 H3.1/2 K27me1-K36me2 K[+126.1]SAPATGGVK[+28]K[+56]PHR 548.659 616.367, 695.402 H3.1/2 K27me2-K36me2 K[+84.1]SAPATGGVK[+28]K[+56]PHR 534.654 616.367, 695.402 H3.1/2 K27me3-K36me2 K[+98.1]SAPATGGVK[+28]K[+56]PHR 539.326 616.367, 768.425 H3.1/2 K27ac-K36me2 K[+98]SAPATGGVK[+28]K[+56]PHR 539.314 616.367, 695.402 H3.1/2 K27un-K36me3 K[+112.1]SAPATGGVK[+42]K[+56]PHR 548.66 623.375, 702.41 H3.1/2 K27me1-K36me3 K[+126.1]SAPATGGVK[+42]K[+56]PHR 553.331 623.375, 702.41 H3.1/2 K27me2-K36me3 K[+84.1]SAPATGGVK[+42]K[+56]PHR 539.327 623.375, 702.41 H3.1/2 K27me3-K36me3 K[+98.1]SAPATGGVK[+42]K[+56]PHR 543.998 623.375, 702.41 H3.3 K27un-K36un K[+112.1]SAPSTGGVK[+56]K[+56]PHR 837.47 638.362, 1275.717 H3.3 K27me1-K36un K[+126.1]SAPSTGGVK[+56]K[+56]PHR 844.478 638.362, 1275.717 H3.3 K27me2-K36un K[+84.1]SAPSTGGVK[+56]K[+56]PHR 549.317 770.404, 777.473 H3.3 K27me3-K36un K[+98.1]SAPSTGGVK[+56]K[+56]PHR 553.989 777.473, 784.42 H3.3 K27ac-K36un K[+98]SAPSTGGVK[+56]K[+56]PHR 553.977 638.362, 717.397 H3.3 K27un-K36me1 K[+112.1]SAPSTGGVK[+70]K[+56]PHR 844.479 645.37, 1289.732 H3.3 K27me1-K36me1 K[+126.1]SAPSTGGVK[+70]K[+56]PHR 851.486 645.37, 1289.732 H3.3 K27me2-K36me1 K[+84.1]SAPSTGGVK[+70]K[+56]PHR 553.99 770.404, 791.489 H3.3 K27me3-K36me1 K[+98.1]SAPSTGGVK[+70]K[+56]PHR 558.661 784.42, 791.489 H3.3 K27ac-K36me1 K[+98]SAPSTGGVK[+70]K[+56]PHR 558.648 645.37, 724.404 H3.3 K27un-K36me2 K[+112.1]SAPSTGGVK[+28]K[+56]PHR 549.318 624.365, 703.399 H3.3 K27me1-K36me2 K[+126.1]SAPSTGGVK[+28]K[+56]PHR 553.991 624.365, 703.399 H3.3 K27me2 K36me2 K[+84.1]SAPSTGGVK[+28]K[+56]PHR 539.986 624.365, 703.399 H3.3 K27me3-K36me2 K[+98.1]SAPSTGGVK[+28]K[+56]PHR 544.658 624.365, 784.42 H3.3 K27ac-K36me2 K[+98]SAPSTGGVK[+28]K[+56]PHR 544.646 624.365, 703.399 H3.3 K27un-K36me3 K[+112.1]SAPSTGGVK[+42]K[+56]PHR 553.992 631.372, 710.407 H3.3 K27un-K36ac K[+112.1]SAPSTGGVK[+42]K[+56]PHR 553.978 631.354, 710.389 H3.3 K27me1-K36me3 K[+126.1]SAPSTGGVK[+42]K[+56]PHR 558.662 631.372, 710.407 H3.3 K27me1-K36ac K[+126.1]SAPSTGGVK[+42]K[+56]PHR 558.649 631.354, 710.389 H3.3 K27me2-K36me3 K[+84.1]SAPSTGGVK[+42]K[+56]PHR 544.659 631.372, 710.407 H3.3 K27me2-K36ac K[+84.1]SAPSTGGVK[+42]K[+56]PHR 544.647 631.354, 710.389 H3.3 K27me3-K36me3 K[+98.1]SAPSTGGVK[+42]K[+56]PHR 549.33 631.372, 710.407 H3 K56un Y[+56]QK[+56]STELLIR 681.885 744.461, 831.493, 1015.615 8
H3 K56ac Y[+56]QK[+42]STELLIR 674.877 744.461, 831.493, 1001.599 H3 K79un E[+56]IAQDFK[+56]TDLR 724.375 288.203, 370.197, 835.467 H3 K79me1 E[+56]IAQDFK[+70]TDLR 731.383 288.203, 299.16, 849.483 H3 K79me2 E[+56]IAQDFK[+28]TDLR 710.378 288.203, 404.24, 1132.552 H3 K79me3 E[+56]IAQDFK[+42]TDLR 717.385 411.248, 821.488, 1146.568 H3 K79ac E[+56]IAQDFK[+42]TDLR 717.367 403.23, 504.278, 288.203 H3 K122un V[+56]TIMPK[+56]DIQLAR 748.929 501.274, 996.584, 1127.624 H3 K122ac V[+56]TIMPK[+42]DIQLAR 741.921 487.299, 600.383, 715.41 H4(4-17) unmodified G[+56]K[+56]GGK[+56]GLGK[+56]GGAK[+56]R 775.954 544.32, 627.37, 785.463 H4 K5ac G[+56]K[+42]GGK[+56]GLGK[+56]GGAK[+56]R 768.946 785.463, 544.32, 627.37 H4 K16ac G[+56]K[+56]GGK[+56]GLGK[+56]GGAK[+42]R 768.947 530.305, 620.362, 771.447 H4 K5+K8 2Ac G[+56]K[+42]GGK[+42]GLGK[+56]GGAK[+56]R 761.938 544.32, 620.362, 785.463 H4 K5+K16 2Ac G[+56]K[+56]GGK[+42]GLGK[+56]GGAK[+42]R 761.939 530.305, 771.447 H4 K12+K16 2Ac G[+56]K[+56]GGK[+56]GLGK[+42]GGAK[+42]R 761.94 613.354, 757.432 H4 K5+K8+K12 3Ac G[+56]K[+42]GGK[+42]GLGK[+42]GGAK[+56]R 754.931 544.32, 613.354, 771.447 H4 K8+K12+K16 3Ac G[+56]K[+56]GGK[+42]GLGK[+42]GGAK[+42]R 754.932 530.305, 606.346. 757.432 H4 K5+K8+K12+K16 4Ac G[+56]K[+42]GGK[+42]GLGK[+42]GGAK[+42]R 747.923 530.305, 606.346, 757.432 H4 K20un K[+112.1]VLR 627.419 241.155, 340.223, 387.271 H4 K20me1 K[+126.1]VLR 321.221 175.119, 255.17, 387.271 H4 K20me2 K[+84.1]VLR 300.216 175.119, 213.16, 387.271 H4 K20me3 K[+98.1]VLR 307.223 175.119, 387.271, 439.328 H4 K20ac K[+98]VLR 613.403 175.119, 227.139, 326.207 H2A K5un-K9un G[+56]K[+56]QGGK[+56]AR 485.277 487.299, 672.379 H2A K5ac-K9un G[+56]K[+42]QGGK[+56]AR 478.27 487.299, 672.379 H2A K5un-K9ac G[+56]K[+56]QGGK[+42]AR 478.271 473.283, 658.363 H2A K5ac-K9ac G[+56]K[+42]QGGK[+42]AR 471.262 473.283, 658.363 H2A K13un-K15un A[+56]K[+56]AK[+56]TR 421.758 312.192, 460.288, 531.325 H2A K13ac-K15un A[+56]K[+42]AK[+56]TR 414.751 298.176, 460.288, 531.325 H2A K13un-K15ac A[+56]K[+56]AK[+42]TR 414.752 312.192, 446.272, 517.309 H2A K13ac-K15ac A[+56]K[+42]AK[+42]TR 407.743 298.176, 446.272, 517.309 H2A K36un K[+112.1]GNYAER 475.241 538.262, 652.305, 709.326 H2A K36ac K[+98]GNYAER 468.233 538.262, 652.305, 709.326 Supplementary Table S1. List of transitions used for targeted and quantitative analysis using SRM to track histone acetylation on 19 individual sites of lysine acetylation on core histones. 9
Supplementary Methods Mass spectrometric method for the multi-target measurement of histone acetylation at specific sites: assay description and validation. The targeted mass spectrometry method uses a SRM technique, where pairs of precursor and fragment ions are used for quantification, has been developed by us previously to measure the methylation kinetics of lysine 27 and 36 localized in the same peptide 14. Here, we adapted our SRM platform to quantify more targets aiming to make a comprehensive measurement for all the known acetylated sites in core histones using the previously established derivatization and digestion protocol (Supplementary Fig. S6a) 33. Although the mass difference between acetylation and trimethylation (only 0.04 Da) is exceeding the mass resolution offered by triple quadrupole mass spectrometry (QqQ), we were able to quantify acetylation by relying on the separation of acetylated peptides from trimethylated peptides using nanocapillary reverse phase liquid chromatography (nlc). As an example, when the mixture of five H3K4 peptides (see Methods for peptide nomenclature used in this paper) present in an equal molar ratio, including unmodified (un), me1, me2, me3, and ac, was subjected to our targeted MS analysis using nlc-qqq, all the peptides were separated from each other except me2 and me3 in our nlc conditions (Supplementary Fig. S6b). Specifically, the H3K4ac peptide eluted about 4 min. later than the H3K4me3 peptide and about 1.5 min. earlier than the H3K4un peptide. For longer peptides, the chromatographic resolution is smaller but the acetylated peptide always eluted later (and baseline resolved from) than the analogous trimethylated peptide. As shown in Supplementary Figure S5a, the elution time of H3K36ac was the same as H3K36un but ~2 min. later than H3K36me3. The separation of acetylation from di-and trimethylation is mainly achieved by the charge difference in the lysine (positive for me2 and me3 vs. neutral for ac, as shown in Supplementary Fig. S6a) as the positive charge weakens the binding affinity with stationary phase of reverse phase chromatography. Overall, we developed a SRM method for 19 sites in histone H3, H4, and H2A from 13 different peptides with 7 of them carrying combinatorial modifications (see 10
Supplementary Table S1 for the full list of peptide and SRMs). The five acetylations in H2B were not measured here because it lacks arginines, which combined with the propionyl-blocking group put on the lysines (see Supplementary Fig. S6a) creates larger peptides harboring multiple sites complicating the nlc-qqq SRM measurement. H3.3 variant-specific K36 acetylation. Amino acid 31 in H3K27-K36 peptide from H3.1 and H3.2 (alanine) is different from that of H3.3 (serine), which enabled us to measure variant specific acetylation for K27 and K36 from H3.3. Surprisingly, direct comparison of K36ac from both H3.1/2 and H3.3 peptides in the same nlc-qqq run only identified K36ac in the H3.3 variant, as shown in Supplementary Figure S5a for the K27un-K36ac form; the single peak in the H3.1/2 K27un-K36ac channel was K36me3 whereas the second peak in H3.3 K27un-K36ac was assigned clearly as K36ac (due to the rationale described above in this supplemental document). Custom-synthesized H3.3 peptide with K36ac was used to confirm that the observed second peak was indeed K36ac (Supplementary Fig. S5b). In addition, H3.3-K36ac was observed when K27 was mono- and di-methylated (Supplementary Fig. S5a). On the contrary, K27ac was found in all three variants of histone H3. Since the H3.3 variant only accounts ~10% of total H3 11, the global level of this variant-specific modification will be 10% of the measurement using H3.3 K27-K36 as the normalization pool. Considerations for limiting analyses to old histones. In addition to being used as the acetyl donor, acetyl-coa is also used by many catabolic and anabolic processes and can be converted to many amino acids via the TCA cycle, which can be used subsequently to synthesize new histone (depicted as dark rectangle in newly synthesized histone in Fig. 1a). Such incorporation is difficult to predict due to the huge heterogeneity created by incomplete labeling of multiple amino acids in multiple sites. In previous radioactive labeling experiment, cyclohexmide was used to inhibit such unwanted incorporation to prevent interference of radioactivity 15-17. By contrast and by virtue of the double- 11
labeling scheme used here, these newly synthesized histones can be easily excluded in the mass spectrometric measurement Turnover in Group II. In this study, we defined turnover of acetylation as old acetylation being replaced by new acetylation. Three possible scenarios can lead to such turnover under our experimental scheme of Figure 1a. First, appearance of new acetylation by adding heavy acetyl through HATs (leading to the increase of overall acetylation in old histone); second, decrease of old acetylation by removing old acetyl through HDACs (leading to the decrease of overall acetylation in old histone); third, combined actions of previous two reactions (leading to the possible stable acetylation in old histone). It is worth emphasizing that modification in old histone is not necessarily in a steady, homeostatic state; as demonstrated by us and others, over-methylated old histone can be buffered by under-methylated new histone to maintain a homeostatic, proper level of global methylation 12,14. Any of the above three actions could be detected by measuring distributive levels of new acetylation (normalized within each acetylation site). For Group II, distributive levels of new acetylation at H4K20 and H3.3-K36 were found to increase over the time course, with H4K20 showing a quick rise peaking at 3 h, followed by subsequent decrease and H3.3-K36 showing a slight decrease until 3 h followed by gradual increase. However, these increases were not the result of new acetylation but simply caused by the decrease of old acetylation with the contribution of noise increasing over the sampling time course. Interestingly, such decrease of overall acetylation was found in multiple sites targeted by methylation, including H3K9, H3.1/2-K27, H3.3-K36, and H4K20 (Fig. 3a). As shown in Supplementary Figure S7, methylation levels in these sites were found increased in old histone, which is consistent with our previous findings for H3K27 and H3K36 using different labeling scheme 14. However the implication of the decrease of acetylation in old histone for these dual-modification sites requires further investigation. The decrease of old acetylation of H4K20 and H3.3 K36 in old histone was quite fast, with 12
half-lives of 1.0 and 1.2 h, respectively. Such decreases should be compensated by the increase of new acetylation in new histone. 13