Abstract
Salt-dependent oligomerization of nucleosomal arrays is related to fiber-fiber interactions and global chromosome structure. Previous studies have shown that the H2A/H2B and H3/H4 N-terminal domain (NTD) pairs are able to mediate array oligomerization. However, because of technical barriers, the function(s) of the individual core histone NTDs have not been investigated. To address this question, all possible combinations of "tailless" nucleosomal arrays were assembled from native and NTD-deleted recombinant Xenopus core histones and tandemly repeated 5 S rDNA. The recombinant arrays were characterized by differential centrifugation over the range of 0-50 mm MgCl2 to determine how each NTD affects salt-dependent oligomerization. Results indicate that all core histone NTDs participate in the oligomerization process and that the NTDs function additively and independently. These observations provide direct biochemical evidence linking all four core histone NTDs to the assembly and maintenance of global chromatin structures.
Highlights
Eukaryotic genomes are partitioned into intricate nucleoprotein assemblages called chromosomes
Recent studies using recombinant core mutants have shown that interaction between the H4 N-terminal domain (NTD)3 and a surface-exposed H2A region on neighboring nucleosome is required for assembly of folded 30-nm secondary structures [11,12,13]
Nucleosomal arrays lacking all core histone NTDs are unable to fold into the 30-nm secondary chromatin structures (14 –17), even in the presence of bound linker histones [18]
Summary
208 –12 DNA Purification—208 –12 DNA was purified from DH5␣ cells transformed with the pPOL208 –12 plasmid [22]. Plasmid DNA was grown as previously described [19]. Pure plasmid DNA was digested with HhaI as previously described [19, 22]. The precipitated DNA was rehydrated in 10 mM Tris, pH 7.6, 0.25 mM EDTA (TE) to a concentration of ϳ2 mg/ml and applied to a 2.5 ϫ 90-cm Sephacryl 1000SF (Amersham Biosciences) column. Two ml of the HhaI digest was gently layered onto the column resin bed and the flow rate adjusted to 0.6 ml/min. Fractions (3 ml) were collected beginning 3 h after column loading. Fractions containing pure 208 –12 DNA were pooled and precipitated with ethanol.
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