Nucleosome in vitro

The histone variants of H2A form the largest family of identified histone variants (Redon et al, 2002 Sarma and Reinberg, 2005). This could be associated with both the strategic position that has the histone H2A within the histone octamer and the less stable interaction of the H2A-H2B dimmer with both DNA and the (H3-H4)2 tetramer within the nucleosome (Luger et al, 1997). Most of the histone H2A variants exhibit a unique property in addition to the N-terminal tail domain, they also posses an unstructured C-terminal tail. To date four variants of histone H2A have been discovered. These include, H2AZ, H2A.X, macroH2A and H2A.Bbd. The highest differences in the primary structure of these H2A variants are observed in their C-terminal portion. Each of these variants could be efficiently incorporated in the nucleosome in vitro and in vivo. The presence of these variants alter the structural and functional properties of the nucleosome distinctly. 

Tremethick, D.J. and Drew, H.R. (1993) High mobility group proteins 14 and 17 can space nucleosomes in vitro. J Biol. Chem. 268, 11389-11393. 

A general role for HU/HTa proteins in DNA packaging is not clear these proteins are not related to eukaryotic histones in terms of amino-acid sequence similarity (see Fig. 2) and they form pseudo-nucleosomes in vitro whose structure is still controversial [40-42]. HTa tetramers condense the DNA into small particles of 5.5 nm, which is only half... 

However, nuclear proteins that bind histones and assemble them with DNA into nucleosomes In vitro have been characterized. Proteins of this type are thought to assemble histones and newly replicated DNA Into nucleosomes in vivo as well. 

Furthermore, it was found that stimulated human neutrophils are able to produce 5-chloro-2 -deoxycytidine and that the myeloperoxidase system generates just the same levels of 5-chlorocytosine in DNA and RNA in vitro (Reaction (4), Figure 28.3). It is possible that myeloperoxidase-generated chlorinated products may modify nuclear acids of pathogens and nuclear acids in host cells during inflammation. Hawkins et al. [48] suggested that DNA oxidation may be initiated by protein chloramines formed in the reaction of HOCl with histones in the nucleosome. 

A low-energy in vitro form of nucleosome packing was observed in nucleosome core particle crystals (Finch et al., 1977). Two variants of these crystals occurred, (a) Wavy columns of nucleosomes stacked one on top of each other with an axial repeat of 340 A were obtained upon crystallization of nucleosomes containing proteolytically cleaved histones (Finch et al., 1977). (b) Straight columns of closely packed nucleosomes, 110 A in diameter, were obtained upon crystallization of nucleosomes with intact histones (Finch and Klug, 1978). In both these structures histone-histone contacts between nucleosomes are implied. Similar face-to-face packing of nucleosomes in arcs and helical patterns was observed in the EM by Dubochet and Noll (1978). 

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... 

The structure of the nucleosome is affected by ionic environment. The low ion concentration makes a nucleosomal array well spread whereas 100 mM NaCl induces nucleosome-nucleosome interaction (Hizume et al, 2005 Nakai et al, 2005 Olins and Olins, 1972 Thoma et al, 1979). In vitro transcription system has been applied to understand the relationship between the nucleosome assembly and transcription activity. The results demonstrated that a positive super-coiling introduced during the progress of RNA polymerase makes the nucleosomes ahead of the polymerase unstable (Pfaffle et al, 1990). 

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)...

Peterson CL, Laniel MA (2004) Histones and histone modifications. Curr Biol 14, R546-551 Pfaffle P, Gerlach V, Bunzel L, Jackson V (1990) In vitro evidence that transcription-induced stress causes nucleosome dissolution and regeneration. J Biol Chem 265 16830-16840 Poch O, Winsor B (1997) Who s who among the Saccharomyces cerevisiae actin-related proteins ... 

Yoda K, Ando S, Morishita S, Houmura K, Hashimoto K, Takeyasu K, Okazaki T (2000) Human centromere protein A (CENP-A) can replace histone H3 in nucleosome reconstitution in vitro. Proc Natl Acad Sci U S A 97 7266-7271... 

Kleinschmidt J.A, Seiter A Zentgraf H (1990) Nucleosome assembly in vitro separate histone transfer and synergistic interaction of native histone complexes purified from nuclei of Xenopus laevis oocytes. EMBO J 9 1309-1318... 

Figure 1. Nucleolin acts as a histone chaperone and boosts the activity of chromatin remodeler SWI/SNF. (a) In vitro histone deposition assay shows the histone chaperone activity of nucleolin. Recombinant core histones were incubated with 5S 147 bp DNA in presence or absence of nucleolin. Note the disappearance of aggregates in the wells and the formation of nucleosomal particles in presence of increasing amount of nucleolin. (b) Sliding assay performed on centrally positioned 601 nucleosomes (250bp). The amount of SWI/SNF used here is insufficient to slide the nucleosomes. Note that in presence of nucleolin even the suboptimal quantity of SWI/SNF is able to slide the nucleosomes...

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