Murphy KJ , Cutter AR , Fang H , Postnikov YV , Bustin M , Hayes JJ .

R01 GM052426 NIGMS NIH HHS , T32 GM068411 NIGMS NIH HHS

             chromatin remodeling
             nucleosome binding

	Figure 1. HMGN1 reduces the H1-dependent stabilization of nucleosome array self-association. Nucleosomes arrays were prepared and the fraction of the sample remaining soluble in the presence of increasing MgCl2 determined as described in the ‘Materials and Methods’ section. (A) MgCl2-dependent self-association of 12-mer arrays in 50 mM NaCl binding buffer is unaffected by HMGN1, at a ratio of 2 HMGN per nucleosome. (B) The stimulation of H1 array oligomerization is partially abrogated by HMGN1, at ratios of 1 and 2 HMGN1s per nucleosome. Note that H1 significantly increases oligomerization of arrays into higher-order chromatin structures at a ratio of 1 H1 per nucleosome in 50 mM NaCl binding buffer (3). Errors reported are ±standard error, N = 3.
	Figure 2. Mobility shift assays showing specific binding of HMGN1 and 2 to nucleosomes (169 bp) and nucleosome core particles (NCPs) (147 bp). (A) HMGN1 binding to 147 bp NCPs. Lanes 1–8 show nucleosomes (2 nM) incubated with 0, 0.1, 0.2, 0.5, 1, 2, 4 and 20 nM HMGN1, respectively. (B) HMGN1 binding to 169 bp nucleosomes. Lane 1, nucleosomes with sodium dodecyl sulphate (SDS) loading buffer, lanes 2–8, nucleosomes (2 nM) with 0, 0.2, 0.5, 1, 2, 4, 8, 15, nM HMGN1, respectively. (C) HMGN2 binding to 2 nM NCPs (147 bp) is competed by LANA peptide. Lanes 1 and 2 contain 147 bp nucleosomes in the presence and absence of SDS, respectively. Lanes 3–8 and 9–14 contain 0.5, 1, 2, 4, 8 and 15 nM HMGN2, respectively. LANA peptide is present at a concentration of 0.1 μM in samples run in lanes 9–14. Note small shift in nucleosome position due to LANA binding. All analyses were on native 5% acrylamide-0.5× TBE gels. Note that LANA binding directly to the nucleosome causes a small shift in the nucleosome band.
	Figure 3. HMGN1 and H1 bind simultaneously to a nucleosome. (A) HMGN1 supershifts H1-bound nucleosomes. Nucleosomes (∼2 nM) were incubated with either 0, 1 or 5 nM H1 (lanes 1–3), or 2.5, 5 or 10 nM HMGN1 (lanes 4–6). Lanes 7–9 show nucleosomes first incubated with 5 nM H1, then 2.5, 5 or 10 nM HMGN1. (B) H1 supershifts HMGN1-bound nucleosomes. Experiment was carried out as in (A) except that HMGN1 was incubated with nucleosomes prior to addition of H1 in lanes 7–9. Shown are autoradiographs of native 0.7% agarose nucleoprotein gels.
	Figure 4. HMGN1 does not displace H1 from nucleosomes. (A) H1 G101C-APB binds to nucleosomes as indicated by mobility shift on native gels (top) and formation of a covalently crosslinked species (bottom). Nucleosomes (5 nM) assembled with radiolabeled 217 bp DNA templates were incubated with increasing amounts of H1 G101C-APB then half of each sample loaded directly onto the native gel (top) and half irradiated with UV light to induce crosslinking, and loaded onto an SDS-agarose gel (bottom). Lane 1, nucleosome alone, lanes 2–6 contain ∼0.5, 1, 1.5, 3 and 6 nM H1 G101C-APB, respectively. (B) HMGN1 does not displace H1–DNA interactions within the nucleosome. Nucleosomes (5 nM) were incubated with H1 G101C-APB (3 nM), then challenged with increasing HMGN1 or unmodified H1, irradiated and crosslinked H1–DNA products separated on SDS-agarose gels. Lanes 1 and 2, nucleosome alone, lanes 3–9, nucleosomes incubated with H1 G101C-APB (3 nM) and 0, 0.1, 0.4, 2, 10, 50 and 100 nM HMGN1 (top) or unmodified H1 (bottom). Samples were irradiated as indicated (UV). Gels were dried and phosphorimaged.
	Figure 5. HMGN2 binding does not alter specific interactions of the H1 globular domain (GH1) with nucleosomal DNA. (A) Top: colocalization of HMGN2 and H1 on nucleosomes detected by super-shift assay. Lane 1, naked DNA, lanes 2–8 contain nucleosomes (2 nM) in the presence of WT H1 (lanes 3 and 4), H1 S66C-APB (lanes 5 and 6) or H1 G101C-APB (lanes 7 and 8), in the absence (lanes 3, 5 and 7) or presence of 20 nM HMGN2 (lanes 4, 6 and 8, respectively). Bottom: denaturing agarose-SDS gel showing H1 crosslinking to 217 bp nucleosomes in the presence or absence of HMGN2, as indicated. (B) Sequencing gel analysis of H1–nucleosomal DNA crosslinks from samples in A mapped by piperidine base elimination cleavage of DNA. Nucleotides crosslinked to H1 S66C-APB and G101C-APB (detected as enhanced band intensity) are indicated by red and green arrowheads, respectively. (C) Model of GH1 interactions with nucleosomal linker DNA, positions of modified residues and crosslinked nucleotides mapped in B indicated by red and green filled circles and arrowheads, respectively.
	Figure 6. HMGN1 increases condensation of the H1 COOH terminal domain when bound to nucleosomes. (A) Schematic showing sites of Cy3 and Cy5 labeling within H1 G101C K195C. N, G and C denote N-terminal, Globular, and C-terminal domains, respectively. (B) Emission spectra upon excitation at 515 nm for Cy3/Cy5 labeled H1 G101C K195C in the absence of nucleosome (black line), in a 1:1 complex with nucleosomes alone (blue line) or incubated with 1 (red line) or 2 (green lines) HMGN1 per nucleosome. (C) Graph of Ratio(a) measure of FRET efficiency versus HMGN1 per nucleosome. Error bars represent ±1 standard deviation, N = 3.
	Figure 7. Specific interactions between the H3 N-terminal tail domain and nucleosome DNA are altered by HMGN1 and HMGN2. (A) Nucleosomes containing H3 T6C-APB were irradiated with UV light and the sites of crosslinking to the radiolabeled DNA determined by crosslink-specific cleavage of the DNA and analysis of products on denaturing ‘sequencing’ gels. Lane 1, no UV; lanes 2–5, nucleosomes irradiated with UV light in TE (lane 2), 50 mM NaCl binding buffer (lane 3) or binding buffer with HMGN1 or HMGN2 (lanes 4 and 5, respectively). Shown is a phosphorimage of the gel. Sites of crosslinking are indicated by arrows and numbers, according to distance from dyad base (0), as in (5); sites unaffected or affected by HMGN binding are indicated in red and cyan arrows, respectively. The effect of HMGNs on crosslinking was confirmed by band quantification (Supplementary Table S1). The vertical bar indicates DNA within the nucleosome core region, with the dyad marked by an open triangle. (B) Model of H3 N-terminal tail domain crosslinking sites within the nucleosome, showing H3 (yellow) and H3 N-tail residues 1–37 colored green, with site of crosslinker attachment (T6C) indicated by space filling residues and green arrows. All other core histones are omitted for clarity. Sites of crosslinking on the DNA are indicated by space-filling bases, with sites for which crosslinking is unaffected by HMGN association shown in red, while those for which crosslinking is reduced are cyan. Black italic numerals indicate superhelix location (SHL) for the top wrap of DNA in the nucleosome.
	Figure 8. HMGN1 alters crosslinking of an interior position within the H3 tail domain to DNA within the nucleosome. Crosslinking was carried out as in Figure 7 except that the site of APB attachment was located at residue 15 within the H3 tail domain. (A) Sequencing gel showing sites of crosslinking for nucleosomes containing H3 A15C-APB. Lane 1, -UV irradiation, lanes 2 and 3, +UV in the absence or presence of HMGN1, respectively. Sites of crosslinking are indicated by arrows; those unaffected by HMGN1 binding are red, those affected by HMGN1 binding are cyan. (B and C) Models showing top (B) and oblique views (C) of crosslinked bases, with sites unaffected or affected by HMGN1 indicated by red or cyan arrows, respectively. H3 is yellow, with H3 N-tail residues 1–37 colored green and other histones omitted for clarity. Sites of APB attachment indicated by space-filling green residues and green arrows. Other core histones are omitted for clarity.
	Figure 9. HMGN1 alters crosslinking of the H4 tail domain to DNA within the nucleosome. Crosslinking was carried out as in Figure 7 except that the site of APB attachment was within H4 tail domain. (A) Denaturing sequencing gel showing sites of crosslinking for nucleosomes containing H4 L10C-APB. Lane 1, −UV irradiation, lanes 2 and 3, +UV in the absence or presence of HMGN1, respectively. (B) Model of nucleosome with view down superhelical axis, with H4 (yellow), H4 N-tail residues 1–21 colored green and site of crosslinker attachment (L10C) indicated by the space-filling residue in the tail domain. Other histones are omitted for clarity. Nucleosome dyad is indicated as a dashed line. Model adapted from 1KX5 structure (46), in which the H4 are fully modeled and have distinct conformations, indicated here as H4 and H4’ (black arrows). Nucleosome dyad is indicated by dashed line; SHL for top turn of DNA indicated by italic numerals. (C and D) The 90°-rotated views showing crosslinking sites and conformations of the H4 and H4’ tails, respectively. Crosslinked sites unaffected or affected by HMGN1 indicated by red or cyan, respectively, site of crosslinker attachment (L10C) indicated by the space-filling residue in the tail domain and green arrow.
	Figure 10. Model of HMGN1/2 and H1 interaction with the nucleosome. Model shows core histones (gray cartoons), DNA (orange ribbon representation), HMGN2 nucleosome binding domain (magenta space-fill), HMGN2 regulatory domain (broken magenta line), GH1 (Bednar et al., (8)) (blue space fill) and the H1 CTD (blue transparent oval with dotted line). The H3 tail domain is colored green, show as in Figure 7, and sites of H3 tail crosslinking summarized from Figures 7 and 8. Those diminished by HMGN1/2 binding are indicated by cyan space-filling bases, those unaffected by HMGNs are red space-filling bases. The direction of reorientation of the H3 tail is indicated.

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