1. Volume 49, Issue 2, Pages 368-378 (January 2013)
A Map of General and Specialized Chromatin Readers in Mouse Tissues Generated by Label-free Interaction Proteomics 
H. Christian Eberl, Cornelia G. Spruijt, Christian D. Kelstrup, Michiel Vermeulen, Matthias Mann 
Molecular Cell 
Volume 49, Issue 2, Pages (January 2013)
DOI: /j.molcel
Copyright © 2013 Elsevier Inc. Terms and Conditions

2. Figure 1
Label-free Quantification Is as Powerful as SILAC-Based Quantification
(A) Peptide pull-down H3K4me3 versus H3 unmodified SILAC forward and reverse; significant outliers are marked in blue.
(B) Same pull-down in label-free; outliers that show significance in modified t test-based analysis are marked in blue.
(C) Overlap of outliers between SILAC and parallel label-free experiment: blue, outliers that were identified and significant in both; green, outliers that were only identified in one experiment; red, outliers significant in one experiment but not in the other, n.q., not quantified; n.s., not significant.
(D) The workflow for screening chromatin readers from mouse tissue extracts is as follows: nuclear extract pools were prepared from mouse brain, liver, and kidney. Pull-downs were performed with each extract with three different peptides (H3 unmodified, K4me3 and K9me3 modified), resulting in a total of 45 samples. Samples were measured separately, and a label-free quantification algorithm was applied.
(E) Heat map of significant outliers from peptide pull-downs for H3K9me3 and H3K4me3 from brain, kidney, and liver nuclear extracts. Readers with the same pattern are clustered together and are indicated on the right (see also Table S1).
(F) Similar heat map as in (E) for testis. In contrast to (E), whole-cell extracts were used (see also Table S1).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 2
Verification of General and Tissue-Specific Chromatin Readers and Associated Proteins
(A) Peptide pull-down using purified CXXC1 PHD finger 1: specific binding of the SET1 complex subunit CXXC1 to H3K4me3.
(B) Overexpression of GFP-tagged mouse CXXC1 full-length and delta PHD in 293 cells. SET1 coprecipitates with both constructs.
(C) Peptide pull-down with HEK293 nuclear extracts overexpressing CXXC1-GFP WT and delta PHD. CXXC1 WT is enriched on the H3K4me3 peptide compared to the unmodified peptide. The delta PHD mutant only shows background binding. SET1 binding to H3K4me3 is seen in the CXXC1 WT extracts, but not when CXXC1 delta PHD is overexpressed, demonstrating that CXXC1 recruits SET1 to H3K4me3.
(D) HP1 alpha coIP is as follows: ZNF462—which is enriched on H3K9me3—is enriched from brain and kidney but not from liver extracts.
(E) Western blot verification of selected readers.
(F) CHD5-coIP from brain nuclear extracts, followed by label-free quantitative proteomics: CHD5 enriches members of the NuRD complex.
(G) Peptide pull-down using purified CHD5 PHD fingers reveals specific repulsion by H3K4me3.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Interaction Proteomics for Proteins Repelled by H3K4me3
SILAC GFP pull-downs from HeLa nuclear extracts for ZMYND8 (A), ZNF687 (B) and RAI1 (C); proteins are expressed at near-endogenous levels in HeLa cells. Interaction partners can be found on the right lower quadrant and are marked with their names.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Extension of the Proteomic Screen
(A) Label-free interaction screen for readers of H3K4me1 from mouse brain and liver nuclear extracts.
(B) Protein expression profiles of selected chromatin readers. General chromatin readers show nearly equal expression levels over the analyzed tissues, whereas organ-specific chromatin readers show organ-specific expression profiles.
(C) Proteomic expression profiles of chromatin readers identified in this study.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions