Binding mode of nucleosome‐assembly protein (AP‐I) and histones

Abstract

Studies were made on the binding mode of the nucleosome-assembly protein AP-I with histones H2A + H2B and/or H3 + H4. Histones H2A + H2B bound with AP-I to form a 7-S complex which contained twice as much AP-I (by weight) as histones. Histone H3 + H4 formed an 8-S complex with AP-I. The 7-S and 8-S complexes did not form a new complex when mixed, but significant amounts of two histone pairs were assembled into a 12-S complex on mixing the (H2A + H2B)-AP-I complex (7-S) with free H3 + H4. In contrast, when the (H3 + H4) -AP-I complex ( 8 4 was incubated with free H2A + H2B, almost no assembly occurred, but the 7-S complex of H2A + H2B was newly formed. Binding studies by enzyme-linked immunosorbent assay showed that AP-I bound with H2A + H2B faster than with H3 + H4.From these results, it is suggested that AP-I has a higher binding affinity for histone H2A + H2B than for H3 + H4, and that the 7-S complex is an intermediate in formation of the 12-S octamer complex (H2A + H2BIn eukaryotic cells, DNA is bound with histones and organized in the nucleosome structure [I]. The four core histones are present in an octamer complex (H2A + H2B + H3 + H4)2 [2] around which 145 -165 base pairs of DNA are wrapped. In 2 M NaCl at neutral pH, the hstone octamers separate from DNA and are stable in these conditions [3]. When the salt concentration is decreased to 0.6M, the octamer dissociates into two H2A+H2B dimers and a (H3 + H4)2 tetramer. At lower ionic strength or low pH, these complexes dissociate further into histone monomers [3].Nucleosomes can be reconstituted in vitro in physiological ionic conditions by several methods. They can be assembled by direct mixing of histone and DNA under suitable conditions [4, 51, but several factors are known to facilitate their assemly [6, 71. Nucleoplasmin (29 kDa), which was first found in Xenopus eggs by Laskey et al. [S], facilitates nucleosome assembly in vitro by its interaction with core histones. Recently, Kleinschmidt et al. [9] demonstrated the existence of a histone-nucleoplasmin complex in nuclei of Xenopus oocytes. They also discovered another histone complex containing H3 and/or H4 tightly bound to one of a pair of very acidic peptides, designated as N1 and N2.We have purified a novel protein (AP-I, 53 kDa) from mammalian cells that facilitates nucleosome assembly. AP-I binds with core histones forming 6 -7-S and 12-S complexes [lo]. The 6 -7-S complex contains histone H2A + H2B and the 12-S complex consists of one histone octamer bound with a stoichiometric amount of AP-I. Nucleosomes could be formed by mixing the 12-S complex with DNA. In experiments using monoclonal antibodies against AP-I, we found Correspondence to F. Hanaoka, Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo, Tokyo, Japan 113Abbreviations. AP-I, nucleosome-assembly protein; SDS, sodium dodecyl sulfate ; ELISA, enzyme-linked immunosorbent assay.that AP-I exists as a 58-kDa peptide in vivo, not as a 53-kDa peptide, and that it is localized in the nucl...

Herpes simplex virus type 1 tegument protein VP22 interacts with TAF-I proteins and inhibits nucleosome assembly but not regulation of histone acetylation by INHAT

“…8), which forms a short a-helix and contacts the Cterminal tail of histone H4 in the nucleosome. Interestingly, NAP-1, a close homologue of TAF-I, which also appears to function as a chaperone and facilitates deposition of histones onto DNA (Ishimi & Kikuchi, 1991;Ishimi et al, 1987;McQuibban et al, 1998), has been found to be associated with histone H2A in co-immunoprecipitation analysis (Chang et al, 1997). Analysis of interactions with deletion mutants of VP22 should help define whether this region is important in the TAF-I interaction.…”

NAP‐I is a functional homologue of TAF‐I that is required for replication and transcription of the adenovirus genome in a chromatin‐like structure

“…Nucleosome assembly protein (NAP-I), which contains three negatively charged regions, is found in various eukaryotic cells and has been shown to interact with cellular histones and to facilitate nucleosome formation (Fujii-Nakata et al 1992;Ishimi et al 1987). Here we examined whether NAP-I also has similar stimulatory activity in the transcription assay as in the replication assay (Fig.…”

“…NAP-I facilitates the formation of nucleosomes by interacting with cellular histones and transferring them to DNA (Ishimi et al 1987;Ishimi & Kikuchi 1991;Fujii-Nakata et al 1992). Since NAP-I can substitute for TAF-I in the replication and transcription ( Fig.…”

“…TAF-I shows significant homology with nucleosome assembly protein-I (NAP-I) von Lindern et al 1992a), which contains tripartite negatively charged regions (Fujii-Nakata et al 1992;Ishimi & Kikuchi 1991). NAP-I transfers histones to DNA and facilitates the association of histones with DNA to form nucleosomes (Ishimi et al 1987).…”

Gene silencing of TSPYL5 mediated by aberrant promoter methylation in gastric cancers

“…[16][17][18]20,21 In humans, the NAP family members include NAP1, NAP1-like protein (NAP1L), SE translocation (SET), testis-specific protein, Y-encoded (TSPY), and testis-specific protein, Y-encoded like (TSPYL). 17,20,[22][23][24][25][26] It is known that proteins in the NAP family participate in chromatin structure and diverse cellular events, and may be involved in regulating gene expression as a result of histone accessibility. 17 NAP1, which is the best characterized member of the NAP family, plays a role in the exchange process of histone H2A and H2B dimers from assembled nucleosomes in DNA replication as a histone chaperone, and NAP1 may assist nucleosome sliding.…”