Nucleosome assembly protein-1 is a linker histone chaperone in
            <i>Xenopus</i>
            eggs

Shintomi, Keishi; Iwabuchi, Masaki; Saeki, Hideaki; Ura, Kiyoe; Kishimoto, Takeo; Ohsumi, Keita

doi:10.1073/pnas.0500822102

Cited by 78 publications

(93 citation statements)

References 36 publications

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“…We find that we are able to use this system to incorporate H1 isolated from mammalian T-cells. Interestingly, NAP-1 was recently identified as a linker histone chaperone in Xenopus, as well, depositing H1 onto the linker DNA at physiological pH and ionic strengths (22), which supports a role for dNAP-1 as an H1 chaperone in our system.…”

Section: Discussionsupporting

confidence: 82%

Biochemical analyses of transcriptional regulatory mechanisms in a chromatin context

Konesky

Laybourn

2007

Methods

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We have optimized a recombinant chromatin assembly system that properly incorporates core histones and histone H1 into a chromatin template containing a natural promoter sequence. This article provides a step-by-step procedure for expression and purification of the proteins required for assembling well-defined chromatin templates. We describe how the degree of chromatin assembly in the absence and presence of histone H1 is measured using topological analysis and the use of micrococcal nuclease digestion performed to confirm H1 incorporation and determine the quality of in vitro chromatin templates. Further we describe the use sucrose gradient ultracentrifugation to verify that no unincorporated H1 remains as a second means for deciding on the proper H1 to core histone ratio during assembly. Additionally, we discuss the use of both yeast and Drosophila NAP-1 (yNAP-1 and dNAP-1, respectively) in the assembly of H1-containing chromatin. Finally, we provide detailed description of functional assays for investigating the mechanism of transcriptional regulation in a chromatin context (transcription, histone acetyltransferase activity, and protein association with promoter-bound complexes using immobilized chromatin templates).

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Section: Discussionsupporting

confidence: 82%

Biochemical analyses of transcriptional regulatory mechanisms in a chromatin context

Konesky

Laybourn

2007

Methods

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“…To identify a mammalian linker histone chaperone, we first examined the in vivo interaction between histone H1 and putative linker histone chaperone candidates, NAP-1 and NASP (Shintomi et al, 2005;Alekseev et al, 2005). We also examined the binding activity to linker histones of well-characterized histone chaperones for histone H2A and histone H3.…”

Section: Taf-i Binds To Histone H1 In the Mammalian Somatic Cell Nucleusmentioning

confidence: 99%

“…To verify that the H1.1-NCP complex forms proper chromatosome structures, micrococcal nuclease (MNase) digestion assays were performed essentially as described (Shintomi et al, 2005). The MNase digestion of nucleosomes assembled on plasmid DNA generates DNA fragments with a repeat length of about 147 bp.…”

Section: Taf-i Has Linker Histone Chaperone Activitymentioning

confidence: 99%

“…More importantly, the identity of a histone chaperone(s) specific for histone H1 is not known. It has been reported that NAP-1 functions as a linker histone H1 chaperone in Xenopus egg extracts (Shintomi et al, 2005). In mammalian cells, nuclear autoantigenic sperm protein (NASP) has been reported as a linker histone chaperone candidate (Alekseev et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

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Role of Template Activating Factor-I as a chaperone in linker histone dynamics

Kato

Okuwaki

Nagata

2011

Journal of Cell Science

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SummaryLinker histone H1 is a fundamental chromosomal protein involved in the maintenance of higher-ordered chromatin organization. The exchange dynamics of histone H1 correlates well with chromatin plasticity. A variety of core histone chaperones involved in core histone dynamics has been identified, but the identity of the linker histone chaperone in the somatic cell nucleus has been a longstanding unanswered question. Here we show that Template Activating Factor-I (TAF-I, also known as protein SET) is involved in histone H1 dynamics as a linker histone chaperone. Among previously identified core histone chaperones and linker histone chaperone candidates, only TAF-I was found to be associated specifically with histone H1 in mammalian somatic cell nuclei. TAF-I showed linker histone chaperone activity in vitro. Fluorescence recovery after photobleaching analyses revealed that TAF-I is involved in the regulation of histone H1 dynamics in the nucleus. Therefore, we propose that TAF-I is a key molecule that regulates linker histonemediated chromatin assembly and disassembly.

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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%

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

Cited by 78 publications

References 36 publications

Biochemical analyses of transcriptional regulatory mechanisms in a chromatin context

Biochemical analyses of transcriptional regulatory mechanisms in a chromatin context

Role of Template Activating Factor-I as a chaperone in linker histone dynamics

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

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