Two complexes that contain histones are required for nucleosome assembly in vitro: Role of nucleoplasmin and N1 in Xenopus egg extracts

Dilworth, S. J.; Black, Susan J.; Laskey, Ronald A.

doi:10.1016/0092-8674(87)90587-3

Cited by 260 publications

(186 citation statements)

References 27 publications

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“…Our ability to do this is likely facilitated by the presence of ATP-independent nucleosome assembly factors in the egg extracts, which may greatly reduce barriers to assembly (and therefore to disassembly) of nucleosomes onto DNA. In Xenopus egg extracts, these factors are known: the protein nucleoplasmin is known to act primarily as a chaperone for histones H2A/B, proteins N1 and N2 are associated with H3/4 (Dilworth et al, 1987;Kleinschmidt et al, 1990;Laskey et al, 1993), whereas the protein NAP-1 is associated with the linker histone B4 the embryonic variant of H1 (Shintomi et al, 2005). Because they are ATP independent, these factors must mediate both assembly and disassembly processes: for example, yeast NAP-1 facilitates lateral "sliding" of nucleosomes along DNA through transient removal and replacement of H2A/B (Park et al, 2005), and it is known to play a role in formation of regularly spaced nucleosome arrays in more complex chromatin assembly reactions (Ito et al, 1996).…”

Section: Stall Force Of Assembly Reaction Indicates Nucleosome Assembmentioning

confidence: 99%

“…These extracts contain a mixture of enzymes competent to assemble not only nucleosomes but also active chromatin. Despite the high complexity of extract reactions, drugs and antibodies have been used to identify and study the contribution of specific proteins to chromatin structure and function (Dilworth et al, 1987;Kleinschmidt et al, 1990;Hirano and Mitchison, 1994;MacCallum et al, 2002;Maresca et al, 2005). Extracts diluted from 2.5-to 400-fold have been used to assemble chromatin onto single DNAs against low piconewton forces (Ladoux et al, 2000;Wagner et al, 2005).…”

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confidence: 99%

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Micromanipulation Studies of Chromatin Fibers in Xenopus Egg Extracts Reveal ATP-dependent Chromatin Assembly Dynamics

Yan

Maresca

Skoko

et al. 2007

MBoC

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We have studied assembly of chromatin using Xenopus egg extracts and single DNA molecules held at constant tension by using magnetic tweezers. In the absence of ATP, interphase extracts were able to assemble chromatin against DNA tensions of up to 3.5 piconewtons (pN). We observed force-induced disassembly and opening-closing fluctuations, indicating our experiments were in mechanochemical equilibrium. Roughly 50-nm (150-base pair) lengthening events dominated force-driven disassembly, suggesting that the assembled fibers are chiefly composed of nucleosomes. The ATP-depleted reaction was able to do mechanical work of 27 kcal/mol per 50 nm step, which provides an estimate of the free energy difference between core histone octamers on and off DNA. Addition of ATP led to highly dynamic behavior with time courses exhibiting processive runs of assembly and disassembly not observed in the ATP-depleted case. With ATP present, application of forces of 2 pN led to nearly complete fiber disassembly. Our study suggests that ATP hydrolysis plays a major role in nucleosome rearrangement and removal and that chromatin in vivo may be subject to highly dynamic assembly and disassembly processes that are modulated by DNA tension. INTRODUCTIONTranscription, replication, and other in vivo DNA processing in eukaryotes take place in the context of chromatin. The processive nature of these activities, and the necessity to disrupt histone-DNA contacts to accomplish them, suggests that chromatin must be dynamic in its structure, with actively transcribing genes perhaps in a continual state of structural rearrangement. The simplest example of chromatin rearrangement that would allow base pair access is displacement or dissociation of part or all of the histone octamer (Felsenfeld, 1996).Chromosome visualization in vivo gives insight into chromatin dynamics at large length scales (Belmont, 2003;Levi et al., 2005) but is as yet unable to reveal events at the scale of individual nucleosome displacements. A complementary approach is to study individual chromatin fibers by using micromanipulation (Cui and Bustamante, 2000;Ladoux et al., 2000;Bennink et al., 2001;Brower-Toland et al., 2002;Leuba et al., 2003;Claudet et al., 2005;Gemmen et al., 2005;Bancaud et al., 2006). A major objective of such experiments has been the study of mechanically triggered changes in protein-DNA contacts, with an emphasis on force-driven opening of nucleosomes.However, biophysical micromanipulation experiments offer possibilities beyond simply disassembling chromatin by force; experiments in "active" solutions containing chromatin-organizing or chromatin-processing enzymes permit direct observation of chromatin dynamics, and they can reveal details of structure and mechanism concerning compaction of DNA into chromatin, chromatin remodeling, gene expression in chromatin, mitotic chromosome condensation, and how such processes are affected by DNA tension. DNA tension is physiologically relevant because pulling of chromatin is likely to occur in vivo, given the larg...

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Section: Stall Force Of Assembly Reaction Indicates Nucleosome Assembmentioning

confidence: 99%

mentioning

confidence: 99%

Micromanipulation Studies of Chromatin Fibers in Xenopus Egg Extracts Reveal ATP-dependent Chromatin Assembly Dynamics

Yan

Maresca

Skoko

et al. 2007

MBoC

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“…In Xenopus eggs, the core histones H2A-H2B and H3-H4 stored in the cytoplasm are associated with histone chaperones nucleoplasmin and N1͞N2, respectively (11,12). To investigate whether B4, the linker histone of Xenopus eggs (13)(14)(15), is found complexed to other proteins, egg extract was fractionated by gel filtration (Superose 12), and fractions were probed with Abs to B4.…”

Section: Incorporation Of Linker Histones Into Dinucleosomesmentioning

confidence: 99%

“…In this study, we examine linker histone-binding proteins in Xenopus eggs, of which chaperones for the core histones have been identified; the core histones H2A-H2B and H3-H4 stored in the egg cytoplasm are associated with histone chaperone nucleoplasmin and N1͞N2, respectively (11,12). By using egg extracts, we found that B4 (ϭH1X or H1M), the linker histone of Xenopus eggs (13)(14)(15), is associated with nucleosome assembly protein-1 (NAP-1), first identified in mammalian cells as a protein that facilitates the assembly of nucleosome cores in vitro, and has been found in a wide variety of organisms, including yeast, soybean, Drosophila, and humans (16)(17)(18)(19).…”

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confidence: 99%

Nucleosome assembly protein-1 is a linker histone chaperone in Xenopus eggs

Shintomi

Iwabuchi²,

Saeki³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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In eukaryotic cells, genomic DNA is primarily packaged into nucleosomes through sequential ordered binding of the core and linker histone proteins. The acidic proteins termed histone chaperones are known to bind to core histones to neutralize their positive charges, thereby facilitating their proper deposition onto DNA to assemble the core of nucleosomes. For linker histones, however, little has been known about the regulatory mechanism for deposition of linker histones onto the linker DNA. Here we report that, in Xenopus eggs, the linker histone is associated with the Xenopus homologue of nucleosome assembly protein-1 (NAP-1), which is known to be a chaperone for the core histones H2A and Xenopus laevis ͉ chromatosome assembly ͉ cell-free system

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“…Both mouse (12) and sea urchin oocytes (13) also store histones in a non-chromatin-bound form. Other mammalian histone-binding proteins, for example the nucleosome assembly factor NAP-1 (14 -17), the chromatin assembly factor CAF-1 (18 -21), and the transcription proteins HIRA-HIRIP3 (22), not only bind histones but appear to be important for the assembly of chromatin (19,(23)(24)(25)(26). Consequently NASP may be important not only for both the storage and transport of histones but also for the assembly of chromatin.…”

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confidence: 99%

Characterization of the Histone H1-binding Protein, NASP, as a Cell Cycle-regulated Somatic Protein

Richardson¹,

Batova²,

Widgren³

et al. 2000

Journal of Biological Chemistry

135

160

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Nuclear autoantigenic sperm protein (NASP), initially described as a highly autoimmunogenic testis and sperm-specific protein, is a histone-binding protein that is a homologue of the N1/N2 gene expressed in oocytes of Xenopus laevis. Here, we report a somatic form of NASP (sNASP) present in all mitotic cells examined, including mouse embryonic cells and several mouse and human tissue culture cell lines. Affinity chromatography and histone isolation demonstrate that NASP from myeloma cells is complexed only with H1, linker histones. Somatic NASP is a shorter version of testicular NASP (tNASP) with two deletions in the coding region arising from alternative splicing and differs from tNASP in its 5 untranslated regions. We examined the relationship between NASP mRNA expression and the cell cycle and report that in cultures of synchronized mouse 3T3 cells and HeLa cells sNASP mRNA levels increase during Sphase and decline in G 2 , concomitant with histone mRNA levels. NASP protein levels remain stable in these cells but become undetectable in confluent cultures of nondividing CV-1 cells and in nonmitotic cells in various body tissues. Expression of sNASP mRNA is regulated during the cell cycle and, consistent with a role as a histone transport protein, NASP mRNA expression parallels histone mRNA expression. NASP, 1 initially described as a highly autoimmunogenic testis and sperm-specific protein, is present in the nucleus of spermatozoa and spermatogenic cells (1-3), hence the name nuclear autoantigenic sperm protein. Previous studies (4) have demonstrated that human NASP contains three functional histone binding sites: site I (amino acids 116 -127), site II, (amino acids 469 -512), and site III (amino acids 211-244). In vitro, recombinant NASP will bind both linker and core histones (4). 2As a histone-binding protein (4, 5), NASP appears to be a homologue of the N1/N2 gene expressed in oocytes of Xenopus laevis (6 -9) because, in addition to the conserved coding regions, there is an almost 60% identity between the 3Ј untranslated regions of rabbit NASP and Xenopus N1/N2 mRNAs (2). In Xenopus oocytes, the non-chromatin-bound core histones H3 and H4 are associated in a complex with N1/N2 (10, 11), providing a mechanism for the storage of histones for DNA replication in the early embryo. Both mouse (12) and sea urchin oocytes (13) also store histones in a non-chromatin-bound form. Other mammalian histone-binding proteins, for example the nucleosome assembly factor NAP-1 (14 -17), the chromatin assembly factor CAF-1 (18 -21), and the transcription proteins HIRA-HIRIP3 (22), not only bind histones but appear to be important for the assembly of chromatin (19,(23)(24)(25)(26). Consequently NASP may be important not only for both the storage and transport of histones but also for the assembly of chromatin.Here, we report a somatic form of NASP (sNASP) present in almost all tissues examined, including mouse embryonic cells and several mouse and human tissue culture cell lines. Interestingly, an analysis of the histones comp...

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Two complexes that contain histones are required for nucleosome assembly in vitro: Role of nucleoplasmin and N1 in Xenopus egg extracts

Cited by 260 publications

References 27 publications

Micromanipulation Studies of Chromatin Fibers in Xenopus Egg Extracts Reveal ATP-dependent Chromatin Assembly Dynamics

Micromanipulation Studies of Chromatin Fibers in Xenopus Egg Extracts Reveal ATP-dependent Chromatin Assembly Dynamics

Nucleosome assembly protein-1 is a linker histone chaperone in Xenopus eggs

Characterization of the Histone H1-binding Protein, NASP, as a Cell Cycle-regulated Somatic Protein

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