Chromatin in vitro

Furuyama T, Dalai Y, Henikoff S (2006) Chaperone-mediated assembly of centromeric chromatin in vitro. Proc Natl Acad Sci U S A 103 6172-6177... 

Widom J (1998) Structure, dynamics, and function of chromatin in vitro. Annu Rev Biophys Biomol Struct 27 285-327... 

Several studies have reported on ellipticine- or elliptinium-DNA interactions. The effects of elliptinium on chromatin in vitro or in the nuclei are an unfolding of the overall structure and a disorganization of the partial structure of the core, leading to an unwrapping of the DNA from the histone core 181). The kinetics and thermodynamics of ellipticine and ellipticinium (protonated ellipticine) binding to calf thymus DNA have been carefully investigated 182). It was... 

Belmont, A. S., Braunfeld, M. B., Sedat, J. W., and Agard, D. A. (1989). Large scale chromatin structural domains within mitotic and interphase chromatin in vitro and in vivo. Chromosoma 98, 129-143. 

Pfaller, R., Smythe, C. and Newport, J. W. (1991). Assembly/disassembly of the nuclear envelope membrane Cell-cycle dependent binding of nuclear membrane vesicles to chromatin in vitro. Cell... 

An advantage of cell-free systems is the potential to evaluate independently cytosolic and membrane vesicle (MV) contributions to nuclear development. Membrane-free cytosol is obtained after ultracentrifugation of crude lysates and MVs can be recovered from the pellets. Both cytosolic extracts and MVs can be stored frozen without detectable loss of envelope assembly activity. They can also be manipulated easily by chemical or enzymatic treatments. Such manipulations have enabled the identification of distinct steps of male pronuclear formation and of factors required for each of these steps, notably in Xenopus (Lohka and Masui, 1984 Wilson and Newport, 1988 Vigers and Lohka, 1 1 Boman et al., 1992) and the sea urchin (Cameron and Poccia, 1994 Collas and Poccia, 1995a,b Collas etal., 1995). Studies in the sea urchin and surf clam have indicated that decondensation of sperm chromatin in vitro meets several criteria established by microinjection of sperm nuclei into living eggs (Cothren and Poccia, 1993) and by electron microscopy observations of normal pronuclear formation in vivo (Longo and Anderson, 19( 1970). 

Modulation of chromatin structure by poly(ADP-ribosyl)ation effect of poly(ADP-ribosyl)ation on chromatin superstructure. We have shown that poly(ADP-ribosyl)ation of native chromatin in vitro from endogenous and exogenous activity of the enzyme led to decondensation of the 30 nm chromatin fiber (3, 5, 10). This decondensation was associated with h5 rmodified forms of histone HI at high NAD concentrations. We have thus studied the stmctural effect of poly(ADP-ribosyl)ation at a high NAD level on HI-depleted chromatin where modification of core histones was observed (7). This specific poly(ADP-ribosyl)ation prevented the recondensation of polynucleosomes reconstituted with native histone HI (Fig. 1). 

Another group of non-histone proteins have been identified as essential components for the formation of the condensed chromosome (Table 1). Topoisomerase II (topo II) localizes in the scaffold/matrix fraction of the interphase nuclear (Berrios et al., 1985) and the mitotic chromosome (Maeshima and Laemmli, 2003) (see section 3.1). Topo II forms a ring-shaped homodimer (Berger et al, 1996 Nettikadan et al, 1998) and catalyzes the decatenation and relaxation of DNA double strand (Wang, 2002). In fission yeast, chromosomes cannot be condensed without functional topo II (Uemura et al, 1987). In addition, in in vitro experiment, mitotic extracts containing topo II induce chromatin condensation in the isolated nuclei from HeLa and chicken erythrocyte cells (Adachi et al., 1991). 

The effect of the superhelical strain of the DNA template on the nucleosome structure can be investigated from the in vitro chromatin reconstitution system (for the detail of in vitro chromatin reconstitution, see sections 2.1 and 2.3). Interestingly, the efficiency of the reconstitution becomes higher as the lengths of the DNA used are longer (Hizume et al, 2004) (Fig. 3a-c). In the 3 kb reconstituted chromatin, one nucleosome could be formed in every 826 bp DNA on average, while in the 106 kb chromatin fibers, one nucleosome can be formed in every 260 bp of DNA. The chromatin reconstituted on the any length of linearized plasmid, the efficiency of the reconstitution becomes one nucleosome per 800 bp DNA. The treatment of the... 

In order to investigate the structural and functional characteristics of the chromatin fiber, several methods for in vitro nucleosome reconstitution have been developed (Lusser and Kadonga, 2004). Among them, the salt-dialysis method is the simplest... 

Figure 4. In vitro reconstituted 30 nm chromatin fiber. Dynamic structural changes in the chromatin fiber in the absence (top) or presence (bottom) of linker histone HI with different NaCl concentration were observed by AFM. Nucleosomes were reconstituted on the 106 kb plasmid and then fixed in the buffer containing 50 mM (top left) or 100 mM NaCl (top right). Nucleosomes were well-spread in 50 mM NaCl but attached each other and partially aggregated in 100 mM NaCl. After the addition of histone HI, the thicker fibers were formed. The width of the fibers is 20nm in 50mM NaCl (bottom left) or 30 nm in lOOmM NaCl (bottom right)...

Karymov MA, Tomschik M, Leuba SH, Caiafa P, Zlatanova J (2001) DNA methylation-dependent chromatin fiber compaction in vivo and in vitro requirement for hnker histone. Faseb J 15 2631—2641 Kaszas E, Cande WZ (2000) Phosphorylation of histone H3 is correlated with changes in the maintenance of sister chromatid cohesion during meiosis in maize, rather than the condensation of the chromatin. J Cell Sci 113(Pt 18) 3217-3226... 

Figure 4. Visualization of PARP-1-mediated chromatin compaction by atomic force microscopy. Chromatin assembled in vitro on a circular 10.5kb plasmid DNA was purified, incubated widi or without recombinant human PARP-1, and imaged by atomic force microscopy. Two types of images are shown scan probe oscillation amplitude (top) and topography (bottom height scale is indicated). The length scale is indicated. (See Colour Plate 6.)...

As expected, in vitro transcription assays involving PARP-1, NAD, and PARC illustrate these predicted outcomes (Kim et al, 2004). Even when driven by a transcriptional activator, such as estradiol-bound estrogen receptor, transcription is repressed when PARP-1 is added to chromatin templates. The repression is reversed by NAD+, and the NAD+-dependent effects are reversed by PARC (Kim et al, 2004). This system for transcriptional control shifts new importance onto the enzymes responsible for synthesis of NAD+ in the nucleus, such as nicotinamide mononucleotide adenylyltransferase-1 (Magni et al, 2004). Because NAD+ facilitates the decompaction of chromatin and the derepression of transcription, nuclear NAD+ biosynthetic enzymes may play critical roles as cofactors. 

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