1. Volume 50, Issue 5, Pages 723-735 (June 2013)
A Method for Systematic Mapping of Protein Lysine Methylation Identifies Functions for HP1β in DNA Damage Response 
Huadong Liu, Marek Galka, Eiichiro Mori, Xuguang Liu, Yu-fen Lin, Ran Wei, Paula Pittock, Courtney Voss, Gurpreet Dhami, Xing Li, Masaaki Miyaji, Gilles Lajoie, Benjamin Chen, Shawn Shun-Cheng Li 
Molecular Cell 
Volume 50, Issue 5, Pages (June 2013)
DOI: /j.molcel
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2. Figure 1 A Strategy to Identify Protein Lys Methylation
Nuclear proteins are affinity purified by an immobilized Kme-binding domain (KMBD), and the associated proteins are identified by MS/MS. In complementary experiments, the specificity profile of the KMBD, determined by peptide arrays, is used to predict, via the computer program SMALI, Kme sites from the pool of proteins identified by AP-MS. Kme sites/peptides scored above a cutoff value are confirmed by LC-MRM-MS. See also Figure S7.
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3. Figure 2 An HP1β Interactome Identified by AP-MS/MS Analysis
Proteins in the interactome are grouped according to functions as annotated in the UniProt database (2012). Color codes are used as follows: green, RNA splicing; red, DNA damage repair; blue, ribosomal proteins; gray, miscellaneous; yellow, RNA splicing and DNA damage repair. Purple circles denote proteins that contain Kme sites as determined by subsequent LC-MRM-MS analysis (see also Table 1). Interacting proteins are connected via lines based on the STRING database (Szklarczyk et al., 2011). Proteins that coimmunoprecipitated with HP1β (Figure 4B) are identified in cyan. This figure was generated with NAViGaTOR (Brown et al., 2009). See also Figures S1 and S7, Table S1, and Document S3.
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4. Figure 3 Prediction and Confirmation of Lys Methylation Sites
(A) Binding profile of the HP1β CD to a permutation peptide array based on the H3K9me3 peptide RKQTAR[Kme3]STGG.
(B) Correlation between the experimental binding signal as reported by Liu et al. (2010) and the SMALI score for a group of Lys methylated histone peptides.
(C) Determination of SMALI cutoff for Kme site prediction. At the cutoff of 7, 80% of peptides contained an Ala or Gly at the position −2.
(D) Distribution of Lys-containing peptides in the HP1β interactome relative to the SMALI scores. Peptides with a score over 7.0 were predicted to contain a methyllysine. The remaining peptides (>98%) were filtered out.
(E) A diagram showing the MRM transitions detected for a K1150me3-containing precursor peptide from DNA-PKcs.
(F) Extracted ion chromatographs (XICs) showing the MRM transitions detected for the K1150me3 peptide.
See also Figures S2–S4 and S7 and Table S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Validation of the HP1β Interactome
(A) A pie chart summary of the results from MRM-MS analysis of the HP1β immunoprecipitates showing that 28/29 of the methylated proteins and 49/80 of the nonmethylated proteins were detected.
(B) Validation of representative interactions in the HP1-β interactome by coimmunoprecipitation from HEK293T cells.
(C) Coimmunoprecipitation of endogenous DNA-PKcs with HP1β from U2OS cells.
(D) DNA damage promoted the DNA-PKcs-HP1β interaction.
(E) CoIP of HP1β with Ku80 from WT CHO or the DNA-PKcs-deficient V3 cells.
(F) (His)6-HP1β, but not the W42A mutant, pulled down DNA-PKcs from the U2OS cell lysate.
(G) The HP1β CD bound to methylated, but not the unmethylated peptides from DNA-PKcs. The H3K9 and H3K23 histone peptides were included as controls.
(H) Binding curves of the HP1β CD to the K1150 peptide in different methylation states as measured by fluorescence polarization (FP) in solution. The H3K9me2 peptide was included in the analysis as a positive control.
See also Figure S7 and Tables S3 and S4.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
The Lys Methylation Sites in DNA-PKcs and HP1β Play Important Roles in DDR
(A) A western blot showing the protein levels of DNA-PKcs and the K-to-R mutants in V3 cells stably transfected with the corresponding expression constructs.
(B) Sensitivity of the CHO-V3 cells or cells stably expressing the WT DNA-PKcs or a K-to-R mutant to ionizing radiation. Data represent mean ± SEM (n = 3).
(C) CHO-V3 cells expressing wild-type DNA-PKcs or K-to-R mutants were subjected to 1 Gy γ ray irradiation and γH2AX immunostaining. DSB repair kinetics by γH2AX foci formation was analyzed. Each point represents the mean ± SEM of γH2AX foci from more than 100 cells.
(D) HP1β depletion affected DSB repair. The γH2AX level was compared by western bloting for HP1β-depleted or control U2OS cells with or without etoposide treatment. UNC0638, a G9a/GLP inhibitor.
(E) U2OS cells were irradiated with 20 Gy γ rays and harvested at the indicated time points for neutral comet assay. More than 50 cells were randomly selected and assayed for tail moment. Columns and error bars represent mean ± SEM (**p < 0.01, n = 3).
See also Figures S5 and S7.
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7. Figure 6 HP1β Plays a Role in the Localization of DNA-PKcs to DSBs
(A) HP1β is involved in the recruitment of the activated DNA-PKcs to DSB foci (marked by γH2AX). U2OS cells transfected with an HP1β-specific siRNA or the scrambled control, treated or not with etoposide or UNC0638, were immunostained for γH2AX and pT2609-DNA-PKcs and imaged.
(B) Quantification of data in (A). The confocal images were quantified by the Pearson correlation coefficient with the JACoP plugin in the ImageJ software. Data represent mean ± SEM (*p < 0.005, n = 5).
(C and D) Depletion of HP1β did not affect the localization of 53BP1 to the γH2AX foci significantly. Data in (D) represent mean ± SEM (n = 5).
See also Figures S6 and S7.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 Lysine Methylation Is a Dynamic PTM
(A) The dynamic change in the DNA-PKcs- K1150me3 induced by DNA damage. Shown are XICs of the MRM transitions corresponding to the deuterated (red trace) and the natural abundance K1150me3 peptide (purple trace).
(B) Dynamic changes in methylation observed for 16 Lys residues in different proteins of the HP1β interactome during DDR. Shown are ratios of methylation between etoposide-treated and the control samples. A negative number denotes a reduction whereas a positive number indicates an increase in the methylation level. Proteins/sites are color coded according to function: red, DNA damage response; green, RNA splicing; gray, miscellaneous.
See also Figure S7.
Molecular Cell  , DOI: ( /j.molcel )
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