1. Volume 61, Issue 2, Pages 247-259 (January 2016)
Modulations of DNA Contacts by Linker Histones and Post-translational Modifications Determine the Mobility and Modifiability of Nucleosomal H3 Tails 
Alexandra Stützer, Stamatios Liokatis, Anja Kiesel, Dirk Schwarzer, Remco Sprangers, Johannes Söding, Philipp Selenko, Wolfgang Fischle 
Molecular Cell 
Volume 61, Issue 2, Pages (January 2016)
DOI: /j.molcel
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2. Molecular Cell 2016 61, 247-259DOI: (10.1016/j.molcel.2015.12.015)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
Nucleosome Incorporation and Linker Histone Binding Impair H3 Modifiability
(A) Schematic representation of the assembly of different nucleosomal complexes used in this study.
(B) Histone acetyltransferase assays of the indicated enzymes using 14C-Acetyl-CoA and free or nucleosomal H3 as substrates. The left image shows fluorograph and Coomassie staining of SDS-PAGE gels of the reactions, and the right image shows densitometric quantification of nucleosomal H3 signal intensity. The data were normalized to the H3 signal on 187 nucleosomes. The averages from at least three independent experiments are shown, and the error bars represent SD.
(C) Histone methyltransferase assays of the indicated enzymes using 3H-SAM and free or nucleosomal H3 as substrates. The reactions were analyzed as described in (B).
(D) Histone kinase assays of the indicated enzymes using ATP and free or nucleosomal H3 as substrates. The left image shows reactions that were analyzed by western blotting. The asterisks mark reactions using reduced enzyme concentrations. The right image shows quantification of the reactions as described in (B).
See also Figures S1 and S2.
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4. Figure 2
Nucleosome Incorporation and Linker Histone Binding Reduce H3 Tail Dynamics
(A) Superposition of 1H/15N NMR spectra (selected region) of the H3 tail in context of free peptide (black), free nucleosomes (187, red), and nucleosomes containing H1 (187+H1.4, blue).
(B and C) Sequence mapping of chemical shift differences calculated between H3 residues 3–35 of free H3 peptide and H3 within 187 nucleosomes (B) or H3 within 187 and 187+H1.4 nucleosomes (C).
(D) Rotational correlation time (τc) of residues of the H3 tail measured in context of free H3 peptide or H3 within 187 and 187+H1.4 nucleosomes as determined by NMR relaxation measurements. The averages and variation (error bars) from two independent experiments are shown.
See also Figure S3.
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5. Figure 3 Transient DNA Contacts Govern Nucleosomal H3 Tail Dynamics
(A) The top image shows NMR chemical shifts of the Thr22, Ser10, Ala7, Ala15, and Ala29 residues of H3-GB1 at increasing concentrations of DNA. The DNA:protein molar ratios are indicated by color code. The bottom image shows NMR chemical shifts of the same H3 residues in context of free H3 peptide or H3 within 187 and 187+H1.4 nucleosomes. The scheme illustrates H3-GB1 fusion protein.
(B) τc of H3 tail residues in H3-GB1 in absence or presence of saturating amounts of DNA. The averages and variation (error bars) from two independent experiments are shown.
(C–E) Gcn5 histone acetyltransferase assays on the indicated nucleosomal substrates. The fluorograph and Coomassie staining of SDS-PAGE gels of the reactions are shown. The schemes illustrate different nucleosomal complexes reconstituted on 187 bp of 601 DNA with (C) core histones and the GD (aa 31–115) of H1.4, with (D) core histones and the C-terminal domain of H1.4 (CTD, aa 110–219) fused to H2A, and (E) on 147 bp of 601 DNA with core histones (E, left) or replacing H2A by the H2A-CTD fusion protein of H1.4 (E, right).
See also Figure S3.
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6. Figure 4 Acetylation and Phosphorylation Modulate H3 Tail Dynamics
(A and B) τc of H3 tail residues upon acetylation by Gcn5 (K14ac), phosphorylation by Aurora B (S10ph S28ph), or acetylation by Gcn5 and phosphorylation by Aurora B (S10ph K14ac) in the context of 187 nucleosomes (A) or free H3 peptide (B). The averages and variation (error bars) from two independent experiments are shown.
(C) NMR chemical shifts of H3 Thr22 of H3-GB1 without modification (left) and after reaction with Aurora B (S10ph S28ph, middle) or Gcn5 (K14ac, right) at increasing concentrations of DNA. The DNA:protein molar ratios are indicated by color code.
(D) τc of H3 tail residues in H3-GB1 in absence or presence of saturating amounts of DNA and upon acetylation by Gcn5 (K14ac) or phosphorylation by Aurora B (S10ph S28ph). The averages and variation (error bars) from two independent experiments are shown.
See also Figure S4.
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7. Figure 5
Acetylation and Phosphorylation Promote Secondary H3 Modifications
(A–C) Time-resolved NMR profiling of consecutive enzymatic reactions. (A) Gcn5 activity on H3 Lys14 in context of 187 nucleosomes unmodified or after pre-phosphorylation with Aurora B (S10ph). The Gcn5 activity on H3 Lys14 in context of H3-GB1 unmodified or after pre-phosphorylation with Aurora B (S10ph) in absence (B) or presence (C) of saturating amounts of DNA is shown. The representative measurement with error bars calculated on the difference between the peak intensity of the corresponding residue and the intensity of the noise (A) or averages and variation from two independent experiments (B and C) are shown.
(D) Histone acetyltransferase assays of Gcn5 using 14C-Acetyl-CoA and nucleosomal substrates without (−) or after (+) pre-phosphorylation by Aurora B. The 187+H5 corresponds to a nucleosome reconstituted on 187 bp of 601 DNA containing native chicken linker histone H5. The fluorograph and Coomassie staining of SDS-PAGE gels of the reactions are shown. The input of the reactions was analyzed by western blotting.
See also Figure S5.
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8. Figure 6
Enhancement of Secondary H3 Modification Reactions Is a General Phenomenon
(A) Histone methyltransferase assays of Set2 and Set7/9 using 3H-SAM and nucleosomal substrates without (−) or after (+) pre-phosphorylation by Aurora B (S10ph S28ph). The fluorograph and Coomassie staining of SDS-PAGE gels of the reactions are shown.
(B) Histone kinase assays of MSK2 using ATP and nucleosomal substrates without (−) or after (+) pre-acetylation by Gcn5 (K14ac). The reactions were analyzed by western blotting.
(C) Histone methyltransferase assays of Set2 using 3H-SAM and nucleosomal substrates without (−) or after (+) pre-phosphorylation by Haspin (T3ph). The fluorograph and Coomassie staining of SDS-PAGE gels of the reactions are shown.
(D and E) Histone acetyltransferase assays of Gcn5 using 14C-Acetyl-CoA (D) or histone methyltransferase assays of Set2 using 3H-SAM with oligonucleosomes (±H1.4) (E) as substrates at the indicated salt concentrations. The reactions were performed without (−) or after (+) pre-phosphorylation by Aurora B (S10ph S28ph). The fluorograph and Coomassie staining of SDS-PAGE gels of the reactions are shown.
See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
H3 Tail Modifications and Linker Histone Occupancy Segregate on a Genome-wide Level
(A) Correlation coefficients between mouse linker histones H1.2 or H1.3 (mouse H1c and H1d, respectively), eight different H3 modifications, and one H4 modification in mESCs. The superscript denotes the origin of the data set: A, Cao et al. (2013); B, Mikkelsen et al. (2007); C, Creyghton et al. (2010); D∗, Karmodiya et al. (2012). The Pearson correlations were computed between log2 ratios of IP over background signals for the entire genome. The data set marked by an asterisk could not be corrected for lab-related batch effects.
(B) Genome browser coverage tracks of library-normalized reads per 500 bp of H1.2, H1.3, and different core histone PTMs along the indicated mouse chromosomes. For histone modifications, which were available in more than one data set, one representative experiment was selected for display.
(C) Schemes illustrate H3 tail-DNA interactions and H3 tail dynamics in context of nucleosomes with and without H1 and after phosphorylation (ph, yellow) or acetylation (ac, green). The incorporation of the CTD of H1 organizes linker DNA into stem-like structures. However, the exact changes in dynamic properties of nucleosomal DNA induced by linker histone binding are not known (schematized by solid and dashed lines in the different nucleosomal complexes).
See also Figure S7.
Molecular Cell  , DOI: ( /j.molcel )
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