NF-Y associates with H3-H4 tetramers and octamers by multiple mechanisms.
NF-Y is a CCAAT-binding trimer with two histonic subunits, NF-YB and NF-YC, resembling H2A-H2B. We previously showed that the short conserved domains of NF-Y efficiently bind to the major histocompatibility complex class II Ea Y box in DNA nucleosomized with purified chicken histones. Using wild-type NF-Y and recombinant histones, we find that NF-Y associates with H3-H4 early during nucleosome assembly, under conditions in which binding to naked DNA is not observed. In such assays, the NF-YB-NF-YC dimer forms complexes with H3-H4, for whose formation the CCAAT box is not required. We investigated whether they represent octamer-like structures, using DNase I, micrococcal nuclease, and exonuclease III, and found a highly positioned nucleosome on Ea, whose boundaries were mapped; addition of NF-YB-NF-YC does not lead to the formation of octameric structures, but changes in the digestion patterns are observed. NF-YA can bind to such preformed DNA complexes in a CCAAT-dependent way. In the absence of DNA, NF-YB-NF-YC subunits bind to H3-H4, but not to H2A-H2B, through the NF-YB histone fold. These results indicate that (i) the NF-Y histone fold dimer can efficiently associate DNA during nucleosome formation; (ii) it has an intrinsic affinity for H3-H4 but does not form octamers; and (iii) the interactions between NF-YA, NF-YB-NF-YC, and H3-H4 or nucleosomes are not mutually exclusive. Thus, NF-Y can intervene at different steps during nucleosome formation, and this scenario might be paradigmatic for other histone fold proteins involved in gene regulation.
Caretti
G
G
Dipartimento di Genetica e Biologia dei Microrganismi, Università di Milano, 20133 Milan, Italy.
Motta
M C
MC
Mantovani
R
R
D000818:Animals; D001156:Artemia; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D002645:Chickens; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D019281:Dimerization; D005090:Exodeoxyribonucleases / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D008969:Molecular Sequence Data; D009707:Nucleosomes / Q000378:metabolism; D011485:Protein Binding; D012996:Solutions; D014982:Xenopus laevis
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D006657:Histones; D009707:Nucleosomes; D012996:Solutions; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D003850:Deoxyribonuclease I