Nucleosome Assembly Protein 1 Exchanges Histone H2A-H2B Dimers and Assists Nucleosome Sliding

Proteins often exist as ensembles of interconverting states in solution which are often difficult to quantify. In the present manuscript we show that the combination of MS under nondenaturing conditions and AUC-SV (analytical ultracentrifugation sedimentation velocity) unambiguously clarifies a distribution of states and hydrodynamic shapes of assembled oligomers for the NAP-1 (nucleosome assembly protein 1). MS established the number of associated units, which was utilized as input for the numerical analysis of AUC-SV profiles. The AUC-SV analysis revealed that less than 1% of NAP-1 monomer exists at the micromolar concentration range and that the basic assembly unit consists of dimers of yeast or human NAP-1. These dimers interact non-covalently to form even-numbered higher-assembly states, such as tetramers, hexamers, octamers and decamers. MS and AUC-SV consistently showed that the formation of the higher oligomers was suppressed with increasing ionic strength, implicating electrostatic interactions in the formation of higher oligomers. The hydrodynamic shapes of the NAP-1 tetramer estimated from AUC-SV agreed with the previously proposed assembly models built using the known three-dimensional structure of yeast NAP-1. Those of the hexamer and octamer could be represented by new models shown in the present study. Additionally, MS was used to measure the stoichiometry of the interaction between the human NAP-1 dimer and the histone H2A–H2B dimer or H3–H4 tetramer. The present study illustrates a rigorous procedure for the analysis of protein assembly and protein–protein interactions in solution.

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Histone Chaperone Nap1 Is a Major Regulator of Histone H2A-H2B Dynamics at the InducibleGALLocus

Molecular and Cellular Biology ◽

10.1128/mcb.00835-15 ◽

2016 ◽

Vol 36 (8) ◽

pp. 1287-1296 ◽

Cited By ~ 12

Author(s):

Xu Chen ◽

Sheena D'Arcy ◽

Catherine A. Radebaugh ◽

Daniel D. Krzizike ◽

Holli A. Giebler ◽

...

Keyword(s):

Critical Role ◽

Histone Chaperones ◽

Histone H2a ◽

Nucleosome Assembly ◽

Content Type ◽

Nucleosome Assembly Protein ◽

Nucleosome Assembly Protein 1 ◽

Histone Exchange

Histone chaperones, like nucleosome assembly protein 1 (Nap1), play a critical role in the maintenance of chromatin architecture. Here, we use theGALlocus inSaccharomyces cerevisiaeto investigate the influence of Nap1 on chromatin structure and histone dynamics during distinct transcriptional states. When theGALlocus is not expressed, cells lacking Nap1 show an accumulation of histone H2A-H2B but not histone H3-H4 at this locus. Excess H2A-H2B interacts with the linker DNA between nucleosomes, and the interaction is independent of the inherent DNA-binding affinity of H2A-H2B for these particular sequences as measuredin vitro. When theGALlocus is transcribed, excess H2A-H2B is reversed, and levels of all chromatin-bound histones are depleted in cells lacking Nap1. We developed anin vivosystem to measure histone exchange at theGALlocus and observed considerable variability in the rate of exchange across the locus in wild-type cells. We recapitulate this variability within vitronucleosome reconstitutions, which suggests a contribution of DNA sequence to histone dynamics. We also find that Nap1 is required for transcription-dependent H2A-H2B exchange. Altogether, these results indicate that Nap1 is essential for maintaining proper chromatin composition and modulating the exchange of H2A-H2Bin vivo.

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Dual Roles of p300 in Chromatin Assembly and Transcriptional Activation in Cooperation with Nucleosome Assembly Protein 1 In Vitro

Molecular and Cellular Biology ◽

10.1128/mcb.22.9.2974-2983.2002 ◽

2002 ◽

Vol 22 (9) ◽

pp. 2974-2983 ◽

Cited By ~ 58

Author(s):

Hiroshi Asahara ◽

Sophie Tartare-Deckert ◽

Takeya Nakagawa ◽

Tsuyoshi Ikehara ◽

Fumiko Hirose ◽

...

Keyword(s):

Transcriptional Activation ◽

Chromatin Assembly ◽

Micrococcal Nuclease ◽

Histone H2a ◽

Nucleosome Assembly ◽

Nucleosome Assembly Protein ◽

Nucleosome Assembly Protein 1 ◽

Core Histones ◽

Kix Domain

ABSTRACT In a yeast two-hybrid screen to identify proteins that bind to the KIX domain of the coactivator p300, we obtained cDNAs encoding nucleosome assembly protein 1 (NAP-1), a 60-kDa histone H2A-H2B shuttling protein that promotes histone deposition. p300 associates preferentially with the H2A-H2B-bound form of NAP-1 rather than with the unbound form of NAP-1. Formation of NAP-1-p300 complexes was found to increase during S phase, suggesting a potential role for p300 in chromatin assembly. In micrococcal nuclease and supercoiling assays, addition of p300 promoted efficient chromatin assembly in vitro in conjunction with NAP-1 and ATP-utilizing chromatin assembly and remodeling factor; this effect was dependent in part on the intrinsic histone acetyltransferase activity of p300. Surprisingly, NAP-1 potently inhibited acetylation of core histones by p300, suggesting that efficient assembly requires acetylation of either NAP-1 or p300 itself. As p300 acted cooperatively with NAP-1 in stimulating transcription from a chromatin template in vitro, our results suggest a dual role of NAP-1-p300 complexes in promoting chromatin assembly and transcriptional activation.

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