Reconstitution of nucleosomes with histone macroH2A1.2.
MacroH2A histones have an unusual hybrid structure, consisting of an N-terminal domain that is approximately 65% identical to a full-length histone H2A and a large C-terminal nonhistone domain. To develop an in vitro approach for investigating the effects of macroH2A proteins on chromatin structure and function, we reconstituted nucleosomes with recombinant macroH2A1.2, substituting for conventional H2A. Recombinant macroH2A1.2 was able to efficiently replace both of the conventional H2As in reconstituted nucleosomes. The substitution of macroH2A1.2 for H2A did not appear to grossly perturb the basic structure of the nucleosome core, as assessed by sedimentation and by digestion with micrococcal nuclease or DNase I. However, two differences were observed. First, the region around the midpoint of the nucleosomal core DNA was more resistant to digestion by DNase I in nucleosome core particles reconstituted with macroH2A1.2. Second, preparations of core particles reconstituted with macroH2A1.2 had a greater amount of material that sedimented more rapidly than mononucleosomes, suggesting that macroH2A1.2 may promote interactions between nucleosomes. Recombinant macroH2A proteins should be valuable tools for examining the effects of macroH2A on nucleosome and chromatin structure.
Changolkar
Lakshmi N
LN
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Pehrson
John R
JR
D000818:Animals; D002645:Chickens; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004912:Erythrocytes / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D008297:Male; D009707:Nucleosomes / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012474:Salmon / Q000235:genetics; D013094:Spermatozoa / Q000737:chemistry
D006657:Histones; D009707:Nucleosomes; D011994:Recombinant Proteins; C120756:macroH2A histone; D004247:DNA; D003850:Deoxyribonuclease I