1
Site-specific human histone H3
methylation stability: fast K4me3
turnover: Supplementary figure
legends and Supplementary text
Steve M.M. Sweet1a,1,2, Yupeng Zhenga,1, Jeremiah D. Tiptona,3, Paul M. Thomasa, Neil L. Kellehera,4.
a
Departments of Chemistry and Molecular Biosciences, Chemistry of Life Processes Institute,
Northwestern University, Evanston, IL 60208, USA.
1
S.M.M.S and Y.Z. contributed equally to this work. 2Present address: Genome Damage and Stability
Centre, School of Life Sciences, University of Sussex, Brighton, BN1 9RQ, UK. 3Present address: AB
Sciex, Tampa, FL, USA. 4To whom correspondence should be addressed: nkelleher@northwestern.edu
Supp. Figure 1. Mass spectrometric raw data showing efficient incorporation of heavy methionine
and heavy methyl. A representative H3.1 replicate 0 h time-point is shown for the methioninecontaining control peptide VTIMPKDIQLAR (92% ± 0.3 incorporation of heavy methionine) and the
K79me1 peptide (89% ± 0.5 incorporation of heavy methyl).
Supp. Figure 2. K4me1, me2, me3 kinetics of old histones. Each labelled species at histone H3.1 (A)
K4 (B) K9 (C) K27 (D) K36 (E) K79 are normalized to the total old histone signal for each peptide. Data
from two biological replicates with 3 technical replicates of each are reported, with SEMs shown as
error bars.
Supp. Figure 3. Decay of old histone H3.1 fully-old trimethyl species (3:3). Data is normalized to
total old histone signal for each peptide and presented in log scale. Data from two biological
replicates with 3 technical replicates of each are reported, with SEMs shown as error bars.
Supp. Figure 4. Fast methylation turnover of H3K4 was also observed in multiple myeloma cells.
Data from TKO cells were plotted in the same format as shown for HeLa cells, comparing (A) to Supp.
Figure 2 and (B) to the third panel of Supp. Figure 1. Data is from three technical replicates of bulk
histone H3 (error bars indicate ± standard deviation).
Supp. Figure 5. Site-specific overall methyl decay in old histone. (A) Two hypothetical scenarios of
what happens to 10 nucleosomes after the media switch. When there is no demethylation, the
relative levels of me(1:1) and me(2:2) decrease simply because some of them are further methylated
to higher methylation states, i.e., me(2:1) and me(3:2), respectively (upper panel). By contrast,
tracking all the old methyl in old histone avoids such complication (comparing upper panel with lower
panel). (B) Scatter plot of overall old methyl levels vs. time for five major sites in H3.1. Data were
from two biological replicates and three technical replicates of each (error bars indicate ± SEMs).
2
Supp. Figure 6 . Sequential methylation and demethylation model for the dynamics of 10 SILAC
intermediates from three methylation states. Methylation (green arrow) or demethylation (red
arrow) reactions determined all of the interconversion between 10 SILAC intermediates. First order
kinetics used to model these interconversions were described by ten ordinary differential equations
(ODE) containing 6 rate constant (kxy: x for the number of methyl groups in reactant; y for the
number of methyl groups in product) and concentration of 10 species (indicated by square bracket).
The relative levels of each SILAC intermediate can come from single species (as in H3.1 K4 and H3.1
K79) or the aggregated levels from multiple species (as in H3.1 K9, combining K14 unmodified with
K14 acetylated; H3.1 K27 and H3.1 K36, combining all 10 SILAC forms of its neighbouring site). These
levels were used as surrogates of concentration in the kinetic modelling. The solution of methylation
and demethylation rate constants are solved by nonlinear least-squares fitting and ODE45
differential equation solver in Matlab.
Supp. Figure 7. Effect of iron chelators on methylation levels. (A) HeLa Methylation levels in bulk
histone H3 treated with two iron chelators, mimosine and desferoxamine (DFO), with or without the
addition of ferrous sulfate (Fe). CoCl2 treatment is used to control for the effect of an induced
hypoxia response. (B) Fold change compared to untreated. Data from 3 technical replicates of each
were reported with standard deviation shown as error bars.
Supp. Figure 8. FACS analysis of HeLa S3 cells with different treatments. DNA contents were
measured by propidium iodide staining to monitor the cell cycle.
Supp. Figure 9. Supp. Figure 9. Effect of iron chelator on methylation levels: data for K27me1 and
K36me3 peptides. Same dataset as Figure 4. A strong increase is seen for K27me1K36me3. The two
main precursor peptides for this species, K27unmodK36me3 and K27me1K36me2, both show an
initial slight increase, with a subsequent dip, presumably due to conversion to K27me1K36me3).
There is only a tiny increase in K27me2K36me3, consistent with antagonism of further K27
methylation by K36me3. HeLa H3.1 methylation levels of: asynchronous population; doublethymidine G/S1 phase arrest; 0.5 mM mimosine treated for 24; 48 h; and 72 h. Data from 3 technical
replicates of each are reported with standard deviation shown as error bars.
Supp. Figure 10. Methylation levels observed after mimosine treatment compared to levels
predicted for zero demethylation. Data in the top panel is re-plotted from Supp. Figure 7 for ease of
comparison. Methylation levels were predicted using the rate constants determined in Figure 3 using
the initial levels measured from asynchronous H3.1.
Supp. Table 1. SRM transitions.
Supp. Table 2. Co-eluting isobaric species and correction employed.
Supplementary Text
Over 85% of heavy-labelled histones from the asynchronous cell population are from the previous
cell cycle. FACS data shows that 20-25% of our asynchronous HeLa cells are undergoing S-phase at
any time and 10-15% are in G2/M (Supp. Fig, 8). Therefore, 60-65% of heavy-labelled histones are
3
from the previous cell cycle (G1 cells). Of the 20% of cells in S-phase at the transition to light media,
between 100% and 50% of their histones are from the previous S-phase (start of S-phase and end of
S-phase, respectively), so only 25%, i.e. 5% of the total, are from the current S-phase. Half of the
G2/M histones are from the previous cell cycle, so 7.5% of the total are from the latest S-phase.
Therefore 87.5% of heavy-labelled histones are expected to be from the previous cell cycle.