Ong MS et al. (2010), Divalent metal- and high mobility group N prote...

XB-ART-42536

J Nucleic Acids 2010 Dec 06;2010:143890. doi: 10.4061/2010/143890.

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Divalent metal- and high mobility group N protein-dependent nucleosome stability and conformation.

Ong MS , Vasudevan D , Davey CA .

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High mobility group N proteins (HMGNs) bind specifically to the nucleosome core and act as chromatin unfolding and activating factors. Using an all-Xenopus system, we found that HMGN1 and HMGN2 binding to nucleosomes results in distinct ion-dependent conformation and stability. HMGN2 association with nucleosome core particle or nucleosomal array in the presence of divalent metal triggers a reversible transition to a species with much reduced electrophoretic mobility, consistent with a less compact state of the nucleosome. Residues outside of the nucleosome binding domain are required for the activity, which is also displayed by an HMGN1 truncation product lacking part of the regulatory domain. In addition, thermal denaturation assays show that the presence of 1 mM Mg(2+)> or Ca(2+) gives a reduction in nucleosome core terminus stability, which is further substantially diminished by the binding of HMGN2 or truncated HMGN1. Our findings emphasize the importance of divalent metals in nucleosome dynamics and suggest that the differential biological activities of HMGNs in chromatin activation may involve different conformational alterations and modulation of nucleosome core stability.

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Species referenced: Xenopus
Genes referenced: hmgn1 hmgn2 mia

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	Figure 1. HMGN binding to the nucleosome core is ion dependent and capable of eliciting a transition. Gel labels indicate HMGN identity (N1t, HMGN1t; N1, HMGN1; N2, HMGN2; n, NCP alone; a, nucleosome array alone), and numbers designate HMGN : nucleosome molar stoichiometry. (a) EMSAs of HMGN binding to two distinct NCP constructs, NCP-601 (left) and NCP147 (right), under three different buffer conditions: low ionic strength (top), near physiological ionic strength (middle), and near physiological ionic strength with 1 mM Mg2+ (bottom). Under noncooperative binding conditions (top), 1 : 1 as well as 2 : 1 HMGN : nucleosome species are observed at low stoichiometry [12]. At higher ionic strength (middle), 2 : 1 species (S1) are the only specific complexes that occur. In the presence of divalent metal, a distinct, slow-migrating species (S2) is also observed for HMGN1t and HMGN2 binding. (b) EMSA showing migration rate for S1 and S2 of NCP-601 (HMGN : nucleosome = 4 : 1) relative to NCP and a 4-nucleosome array. (c) SDS-polyacrylamide gel analysis of HMGN purification products (see Methods). Samples from HMGN1 (left) and HMGN2 (right) overexpression are shown for crude extract (cr) and after gel filtration (gf) and ion-exchange chromatography (N1, N1t, and N2). (d) Xenopus HMGN primary structures. Functional domains are the NBD (blue) and RD (red). Nuclear localization elements are shown in green.
	Figure 2. HMGN association with nucleosomal array is capable of inducing the S2 transition. Gel labels indicate HMGN identity, (N1t, HMGN1t; N1, HMGN1; N2, HMGN2; a, 12-array alone) and numbers designate HMGN : nucleosome molar stoichiometry. (a) HMGN binding to 12-array at low (top) and near physiological ionic strength (bottom). ((b), (c)) HMGN binding to 12-array at near physiological ionic strength with 1 mM Mg2+.
	Figure 3. S2 formation is divalent metal dependent and requires HMGN elements outside of the NBD. EMSA gel labels indicate HMGN identity (N1t, HMGN1t; N1, HMGN1; N2, HMGN2; a, 12-array alone; n, NCP alone), and numbers designate HMGN : nucleosome molar stoichiometry. (a) HMGN binding to 12-array at near physiological ionic strength with 1 mM Ca2+. (b) Binding of HMGN proteins and NBD peptides to NCP-601 at near physiological ionic strength with 1 mM Mg2+. (c) Binding of HMGN NBD peptides to 12-array at low ionic strength (top), near physiological ionic strength (middle), and near physiological ionic strength with 1 mM Mg2+ (bottom).
	Figure 4. Thermal denaturation assays reveal influence of divalent metal and HMGNs on nucleosome stability. ((a)–(h)) Sample buffer was near physiological ionic strength without divalent metal ((a)–(d); 1XTB, default) or with 1 mM Mg2+ or Ca2+ ((a)–(c), (e)–(h); +Mg, +Ca). Assays were conducted on naked NCP DNA fragments alone (a), NCP ((b), (c)), NCP-601 with 3 : 1 HMGN : NCP stoichiometry in the absence of divalent metal (d), NCP-601 with 1 : 1 (e), 2 : 1 (f), or 3 : 1 (g) HMGN : NCP stoichiometry and NCP147 with 2 : 1 HMGN : NCP stoichiometry (h). ((d)–(h)) “NCP” corresponds to an HMGN-free control sample.
	Figure 5. Off-centered NCP undergoes a transition in the presence of divalent metal and associates with HMGNs to form an S2-like state. (a) Crystal structure-based [19] models for off-centered and centered NCP. DNA strands are colored orange and cyan and histone proteins are grey. Off-centered NCP is missing an interaction between one DNA terminus and the N-terminal tail of H3 (MIA). (b) EMSAs of HMGN binding to centered (C; right) versus off-centered (O; left) NCP146b. Gel labels indicate HMGN identity (N1t, HMGN1t; N1, HMGN1; N2, HMGN2; n, NCP alone), and numbers designate HMGN:nucleosome molar stoichiometry. Off-centered samples (left 7 lanes) also contain a fraction of faster migrating centered NCP. Gels were run under three different buffer conditions: low ionic strength (top), near physiological ionic strength (middle), and near physiological ionic strength with 1 mM Mg2+ (bottom). HMGN : nucleosome molar stoichiometry (*) for centered samples (right 3 lanes) is 1 : 1 (top, middle) or 3 : 1 (bottom). Assignments of 1 : 1 and 2 : 1 HMGN : off-centered NCP species are inferred by comparison with those for centered NCP (see text for details). The additional band observed at low HMGN stoichiometry with Mg2+ (arrow) may coincide with a minor fraction of off-centered NCP in a compact state associated with one molecule of HMGN.

References [+] :

Anderson, Sequence and position-dependence of the equilibrium accessibility of nucleosomal DNA target sites. 2000, Pubmed