Association States of Nucleosome Assembly Protein 1 and Its Complexes with Histones

The histone chaperone NAP1 is a carrier of histones during nuclear import, nucleosome assembly, and chromatin remodeling. Analytical ultracentrifugation was used to determine the association states of NAP1 alone and in complexes with core histones. In addition, the concentration dependence of the association was quantified by determining the equilibrium dissociation constant between different NAP1 species. At physiological protein and salt concentrations the prevalent species were the NAP1 dimer and octamer. These were also the association states found to interact with histones in a stoichiometry of one NAP1 monomer per histone. Based on these results a model for a cell cycle-dependent shift of the NAP1 dimer-octamer equilibrium is proposed that reflects different biological functions of NAP1.

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Assembly states of the nucleosome assembly protein 1 (NAP-1) revealed by sedimentation velocity and non-denaturing MS

Biochemical Journal ◽

10.1042/bj20102063 ◽

2011 ◽

Vol 436 (1) ◽

pp. 101-112 ◽

Cited By ~ 15

Author(s):

Masanori Noda ◽

Susumu Uchiyama ◽

Adam R. McKay ◽

Akihiro Morimoto ◽

Shigeki Misawa ◽

...

Keyword(s):

Protein Interactions ◽

Electrostatic Interactions ◽

Analytical Ultracentrifugation ◽

Three Dimensional ◽

Sedimentation Velocity ◽

Dimensional Structure ◽

Histone H2a ◽

Nucleosome Assembly ◽

Nucleosome Assembly Protein ◽

Nucleosome Assembly Protein 1

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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Yeast Chromatin Reconstitution System Using Purified Yeast Core Histones and Yeast Nucleosome Assembly Protein-1

Protein Expression and Purification ◽

10.1006/prep.1996.0716 ◽

1997 ◽

Vol 10 (1) ◽

pp. 132-140 ◽

Cited By ~ 13

Author(s):

John Pilon ◽

Andrea Terrell ◽

Paul J. Laybourn

Keyword(s):

Nucleosome Assembly ◽

Nucleosome Assembly Protein ◽

Nucleosome Assembly Protein 1 ◽

Core Histones

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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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Drosophila NAP-1 is a core histone chaperone that functions in ATP-facilitated assembly of regularly spaced nucleosomal arrays.

Molecular and Cellular Biology ◽

10.1128/mcb.16.6.3112 ◽

1996 ◽

Vol 16 (6) ◽

pp. 3112-3124 ◽

Cited By ~ 175

Author(s):

T Ito ◽

M Bulger ◽

R Kobayashi ◽

J T Kadonaga

Keyword(s):

Chromatin Assembly ◽

Drosophila Embryo ◽

Core Histone ◽

In Vivo Studies ◽

Nucleosome Assembly ◽

Nucleosome Assembly Protein ◽

Nucleosome Assembly Protein 1 ◽

Core Histones ◽

Assembly Factor

We describe the cloning and analysis of Drosophila nucleosome assembly protein 1 (dNAP-1), a core histone-binding protein that functions with other chromatin assembly activities in a Drosophila chromatin assembly factor 1-containing fraction (dCAF-1 fraction) in the ATP-facilitated assembly of regularly spaced nucleosomal arrays from purified core histones and DNA. Purified, recombinant dNAP-1 acts cooperatively with a factor(s) in the dCAF-1 fraction in the efficient and DNA replication-independent assembly of chromatin. In the presence of histone H1, the repeat length of the chromatin is similar to that of native chromatin from Drosophila embryos. By coimmunoprecipitation analysis, dNAP-1 was found to be associated with histones H2A and H2B in a crude whole-embryo extract, which suggests that dNAP-1 is bound to the histones in vivo. Studies of the localization of dNAP-1 in the Drosophila embryo revealed that the factor is present in the nucleus during S phase and is predominantly cytoplasmic during G2 phase. These data suggest that NAP-1 acts as a core histone shuttle which delivers the histones from the cytoplasm to the chromatin assembly machinery in the nucleus. Thus, NAP-1 appears to be one component of a multifactor chromatin assembly machinery that mediates the ATP-facilitated assembly of regularly spaced nucleosomal arrays.

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GW24-e1857 Nucleosome Assembly Protein 1-like 1 Knockdown Promotes Cardiomyocytes Differentiation by Mesoderm Induction through Notch Signalling in Mouse Induced Pluripotent Stem Cells

Heart ◽

10.1136/heartjnl-2013-304613.203 ◽

2013 ◽

Vol 99 (Suppl 3) ◽

pp. A73.2-A74

Author(s):

Gong Hui ◽

Yuan Yan ◽

Yuanyuan Xue ◽

Peipei Yin ◽

Zhiwen Ding ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Notch Signalling ◽

Mesoderm Induction ◽

Nucleosome Assembly ◽

Nucleosome Assembly Protein ◽

Nucleosome Assembly Protein 1 ◽

Induced Pluripotent

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Zygotic nucleosome assembly protein–like 1 has a specific, non–cell autonomous role in hematopoiesis

Blood ◽

10.1182/blood-2005-02-0598 ◽

2005 ◽

Vol 106 (2) ◽

pp. 514-520 ◽

Cited By ~ 9

Author(s):

Anita Abu-Daya ◽

Wendy M. Steer ◽

Alexandra F. Trollope ◽

Christine E. Friedeberg ◽

Roger K. Patient ◽

...

Keyword(s):

Chromatin Remodeling ◽

Neural Cells ◽

Nucleosome Assembly ◽

Globin Mrna ◽

Blood Island ◽

Nucleosome Assembly Protein ◽

Hematopoietic Lineage ◽

Core Histones ◽

Hematopoietic Precursor ◽

Assembly Proteins

Abstract Nucleosome assembly proteins (NAPs) bind core histones, facilitate chromatin remodeling, and can act as transcriptional coactivators. We previously described the isolation of a Xenopus NAP1-like (xNAP1L) cDNA, which encodes a member of this protein family. Its zygotic expression is restricted to neural cells, the outer cells of the ventral blood island (VBIs), and the ectoderm overlying the blood precursors. Here, we report that depletion of zygotic xNAP1L in embryos produces no obvious morphologic phenotype, but ablates α-globin mRNA expression in the VBIs. Transcript levels of the hematopoietic precursor genes SCL and Xaml (Runx-1) are also reduced in the VBIs. SCL expression can be rescued by injection of xNAP1L mRNA into the ectoderm, showing that the effect of xNAP1L can be non–cell autonomous. Fli1 and Hex, genes expressed in hemangioblasts but subsequently endothelial markers, were unaffected, suggesting that xNAP1L is required for the hematopoietic lineage specifically. Our data are consistent with a requirement for xNAP1L upstream of SCL, and injection of SCL mRNA into xNAP1L-depleted embryos rescues α-globin expression. Thus, xNAP1L, which belongs to a family of proteins previously believed to have general roles, has a specific function in hematopoiesis.

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