Abstract
The histone domain of macro-H2A, which constitutes the N-terminal one third of this histone variant, is only 64% identical to major H2A. We have shown previously that the main structural differences in a nucleosome in which both H2A moieties have been replaced by macro-H2A reside in the only point of contact between the two histone dimers, the L1-L1 interface of macro-H2A. Here we show that the L1 loop of macro-H2A is responsible for the increased salt-dependent stability of the histone octamer, with implications for the nucleosome assembly pathway. It is unknown whether only one or both of the H2A-H2B dimers within a nucleosome are replaced with H2A variant containing nucleosomes in vivo. We demonstrate that macro-H2A preferentially forms hybrid nucleosomes containing one chain each of major H2A and macro-HA in vitro. The 2.9-A crystal structure of such a hybrid nucleosome shows significant structural differences in the L1-L1 interface when comparing with homotypic major H2A- and macro-H2A-containing nucleosomes. Both homotypic and hybrid macro-nucleosome core particles (NCPs) are resistant to chaperone-assisted H2A-H2B dimer exchange. Together, our findings suggest that the histone domain of macro-H2A modifies the dynamic properties of the nucleosome. We propose that the possibility of forming hybrid macro-NCP adds yet another level of complexity to variant nucleosome structure and function.
Highlights
Since we do not see any crystal contacts in the structure of canonical nucleosomes that could potentially favor a particular orientation of the hybrid nucleosome in the crystal lattice, and since it was unlikely that the His-tag that is attached to the disordered N-terminal tail of H2A would contribute to crystal packing, we expected the density for the H2A moiety in the hybrid nucleosome to be a convolution between major H2A and macro-H2A (Fig. 4A)
One important way of locally or globally altering chromatin structure and dynamics is the incorporation of histone variants into nucleosomes
We have demonstrated that the histone domain of macro-H2A has a significant effect on in vitro nucleosome assembly and dynamics due to amino acid differences in a four-amino acid stretch of macro-H2A that forms the only point of contact between the two H2A-H2B dimers
Summary
Expression and Purification of Histone Proteins; Reconstitution of Nucleosomes—The expression vector for the histone domain of macro-H2A was a kind gift from Dr Saadi Khochbin. The histone domain of macro-H2A (macro-H2AHD) and X. laevis H2B was refolded into a dimer, as was His-tagged mouse H2A with mouse H2B. Macro-H2A-H2B dimer, His-tagged H2A-H2B dimer, (H3-H4)[2] tetramer, and a 146-bp palindromic fragment of DNA derived from human ␣-satellite regions (␣-sat DNA) (1) were mixed in a 1:1:1:1 molar ratio for nucleosome reconstitution by salt gradient dialysis. NCPs reconstituted with only macro-H2A-H2B dimers or only His-tagged H2A-H2B dimers (tetramer:dimer:DNA ϭ 1:2:1) were prepared as controls. Yeast Nucleosome Assembly Protein 1 (yNAP-1)-dependent NCP Reconstitution—Glutathione S-transferase-tagged yNAP-1 was purified as described (25). Mutants were expressed, purified, and reconstituted into nucleosomes along with the other core histones from X. laevis as described above, resulting in mL1-NCPs and mDD-NCP, respectively. Incorporation of fluorescently labeled H2A-H2B dimer into unlabeled nucleosomes was monitored on a non-denaturing 5% PAGE gel at 302 nm
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