Interaction of high mobility group-I (Y) nonhistone proteins with nucleosome core particles.
Mammalian high mobility group (HMG)-I(Y) chromosomal proteins bind with high affinity to the minor groove of A. T-rich sequences of DNA both in vitro and in vivo. Electrophoretic mobility shift assays demonstrate that in vitro both native and recombinant human HMG-I proteins also bind, but with lower affinity, to preferred regions on isolated avian nucleosome core particles containing approximately 146 base pairs of random sequence DNA. Up to four discrete HMG-I core particle complexes can be detected by electrophoretic mobility shift assays when increasing molar ratios of protein are associated with cores. Both protein-DNA and protein-protein interactions are involved in HMG-I binding to cores. The interaction of HMG-I with core DNA is demonstrated by both thermal denaturation and DNase I footprinting experiments. Chemical cross-linking studies employing reversible photoactivatable cross-linkers, combined with one- and two-dimensional electrophoretic analyses, indicate that in vitro HMG-I binds to cores in close proximity to histones H2A and H2B and H3. In situ cross-linking of K562 human erythroleukemia cell nuclei demonstrate that native HMG-I(Y) binds in a similar manner to nucleosomal histones in vivo. Proteolytic removal of the positively charged amino-terminal tails of the octamer histones abolishes binding of HMG-I to core particles. However, core binding is not mediated by the negatively charged carboxyl-terminal tail of the HMG-I protein since an in vitro produced mutant protein lacking this region binds to core particles in a manner similar to full-length HMG-I. Together these results demonstrate that HMG-I, both in vitro and in vivo, binds to preferred regions on the front face of core nucleosomes.
Reeves
R
R
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660.
Nissen
M S
MS
D000818:Animals; D001386:Azides; D001483:Base Sequence; D002645:Chickens; D002843:Chromatin / Q000378:metabolism; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D003850:Deoxyribonuclease I / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000378:metabolism*; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D009707:Nucleosomes / Q000378:metabolism*; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D001386:Azides; D002843:Chromatin; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D006609:High Mobility Group Proteins; D006657:Histones; D009707:Nucleosomes; D011994:Recombinant Proteins; C033169:3-((4-azidophenyl)dithio)propionic N-hydroxysuccinimide; D003850:Deoxyribonuclease I