1. Volume 24, Issue 10, Pages 1810-1820 (October 2016)
Structural Insights into the Association of Hif1 with Histones H2A-H2B Dimer and H3-H4 Tetramer 
Mengying Zhang, Hejun Liu, Yongxiang Gao, Zhongliang Zhu, Zijun Chen, Peiyi Zheng, Lu Xue, Jixi Li, Maikun Teng, Liwen Niu 
Structure 
Volume 24, Issue 10, Pages (October 2016)
DOI: /j.str
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2. Structure 2016 24, 1810-1820DOI: (10.1016/j.str.2016.08.001)
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3. Figure 1 Hif1 Binding to the H2A-H2B Dimer and the H3-H4 Tetramer
GST pull-down experiments were performed under gradient ionic strength conditions. Gradients and samples are indicated above each lane. Yeast histones were reconstituted and purified as detailed in the text.
(A) Hif1 pull-down with the H3-H4 tetramer.
(B) Hif1 pull-down with the H2A-H2B dimer.
(C) Thermodynamics of Hif1 binding to histones. The left panel illustrates Hif1 titrating into the H2A-H2B dimer. The right panel depicts Hif1 titrating into the H3-H4 tetramer. The heat (kcal) evolved per mole in each injection is plotted below the raw data with red solid curves representing the best fit in each panel. Affinities for each binding are indicated in data plot windows. Detailed thermodynamic parameters are listed in Table S2. SPR and reverse ITC assays were also applied for confirmation; see Figures S1A and S1B and Table S1.
(D) Hif1-histone complexes as determined by analytical size exclusion chromatography (aSEC). The red dots superimposed on the blue line indicate protein standards of precisely known molecular weights (MWs). Green circles indicate target proteins or protein complexes subjected to analysis. The MWs measured by aSEC are summarized in the bottom left table. The elution profiles of protein standard, Hif1, histones, and Hif1-histone complexes are plotted in Figures S1C and S1D.
Structure  , DOI: ( /j.str )
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4. Figure 2
Structure Analysis and Mutagenesis of Hif1 in Complex with H2A-H2B
Each protein is shown in cartoon mode and indicated by its individual color (A–D). Residues interacting at the interface between Hif1 and the H2A-H2B dimer are shown as stick representations. Magenta dashed lines indicate hydrogen bonds or salt bridges. Black dotted lines indicate grouped mutation sites namely a, b, c, d. And alanine mutants in (F) and (G) are named accordingly above each lane. WT indicates wild-type Hif1.
(A) Overview of Hif1 in complex with the histone H2A-H2B dimer. H2A-H2B is shown in electrostatics potential surface mode with red indicating negatively charged surfaces and blue indicating positively charged surfaces. Hif1 is shown as a cylindrical α helix model. Hif1 involved in TPR extended binding to H2A-H2B is shown in deep green on the left, and Hif1 involved in nucleosomal DNA competitive binding is shown in cyan on the right. Patch B is indicated where it surrounds the TPR cylinders. Missing regions of the acid patch in the crystal structure are presumably free loops as indicated by the yellow beaded line. For crystal packing and physiological binding interfaces, see Figure S2 and Table S3.
(B) TPR extended binding between Hif1 and H2A-H2B.
(C) Nucleosomal DNA competitive binding between Hif1 and H2A-H2B.
(D) Nucleosomal DNA binding region on H2A-H2B. H2A-H2B from the nucleosome (PDB: 1ID3) is shown in the same perspective as in (C) for comparison with its nucleosomal DNA binding sites.
(E) Sequence alignment of histone H2A and H2B from yeast and human. The sequences were downloaded from the UniProt database with entries indicated in Figure S3. Secondary structures and sequence numbers of yeast H2A1 and H2B1 are indicated above the sequence alignment panel. Residues involved in interactions with Hif1 are highlighted by yellow blocks. The blue frames indicate highly conserved residues. Full-length alignment with more species is shown in Figure S3.
(F) Assay of multi-site mutants binding with the H2A-H2B dimer. For single-site mutants, see Figure S4A. For their binding thermodynamics, see Figure S5.
(G) Comparison of multi-site mutants on binding to the H2A-H2B dimer and the H3-H4 tetramer.
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5. Figure 3
Alternate Binding between the H2A-H2B Dimer and the H3-H4 Tetramer
GST pull-down was employed for analysis of the binding between Hif1 mutants or sNASP and histones. Samples are indicated above each lane.
(A) Spatial relationship of the interfaces between Hif1-histone and histone octamer. Hif1 in complex with H2A-H2B is superimposed on the nucleosomal octamer (PDB: 1ID3). For Hif1 involved in nucleosomal DNA competitive binding, the N terminus of patch A and the docking domain of H2A are indicated. Fragments involved in binding with H3-H4 are mapped onto both Hif1 molecules with claret highlights, and potential binding regions for the H3-H4 tetramer are indicated as dotted lines. CBR indicates the cooperative binding region of patch A N-terminal and patch B C-terminal.
(B) Schematic illustration of acid patches. Truncation boundaries of both patch A and B are labeled to clarify various truncations used in (C and D).
(C) Binding effects of various acid patch truncations. Hif1Δ(85-198), truncation with deletion of the whole acid patch; Hif1ΔM(80-158), truncation with deletion of acid patch A; Hif1ΔM(95-158), truncation with deletion of the C-terminal counterpart (95–158 aa, keeping the H3-H4 binding region) of acid patch A; Hif1ΔM( ), truncation with deletion of the C-terminal counterpart (135–158 aa) of acid patch A. Hif1ΔM( ), truncation similar to Hif1ΔM( ) but with excess deletion of N-terminal helixes η1 and η2 of patch B. Each truncation was subjected to pull-down with both the 2A-H2B dimer and the H3-H4 tetramer as indicated above the gel. For the acid patch fragment binding assay, see Figure S4B.
(D) Acid patch fragments binding to the H3-H4 tetramer under high ionic strength conditions. Various fragments of acid patches as indicated in the top panel were tested for their binding to H3-H4 tetramer under high ionic strength conditions (1 M KCl).
(E) Competitive binding to yeast Hif1 or human sNASP between the H2A-H2B dimer and the H3-H4 tetramer. Aliquots of GST-Hif1 (top panel) or GST-sNASP (bottom panel) were incubated first with H2A-H2B dimer on ice. The mixtures were then incubated with increasing amounts of H3-H4 tetramer as indicated in the top bar.
(F) Chaperone specificity of human sNASP. GST pull-down experiments were performed under gradient ionic strength conditions as indicated above each lane for binding to the H3-H4 tetramer (top panel) and the H2A-H2B dimer (bottom panel).
(G) Hif1 pull-down with mononucleosomes ex vivo. Various ionic strength conditions were applied to this pull-down assay as indicated above each lane. Asterisk indicates overdigested mononucleosomes. DNA length of the two batches of mononucleosomes was checked with 3% agarose gel (see Figure S4C).
(H) Hif1 mutants binding to overdigested mononucleosomes. The pull-down assay was performed in the ionic strength condition of 300 mM KCl.
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6. Figure 4
Conservation of Hif1 Binding Patterns and Possible Hif1-Histone Complexes
Structures of specific H2A-H2B (including H2A.Z-H2B) histone-chaperone complexes were superposed onto the Hif1-H2A-H2B complex structure (A–C). Chaperones in these complexes are shown in magenta.
(A) Human Anp32e (top panel) and yeast Chz1 (bottom panel) binding patterns.
(B) Swr1-Z domain binding pattern.
(C) Spt16 C-domain binding pattern.
(D) Proposed schematics of Hif1 involved in nucleosome (dis)assembly. Structures of each component are shown as the surface model with individual colors as indicated by the accompanying wording. Gray solid lines surrounding texts indicate DNA binding to histone components, and gray dotted lines indicate DNA competition with Hif1 for binding to histones. Asterisk (∗) indicates the symmetrical binding to H3-H4 by the CBR site of the newcomer Hif1-H2A-H2B-Hif1 complex.
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