Chromatin native

The native form of chromatin in cells assumes a higher order stmcture called the 30-nm filament, which adopts a solenoidal stmcture where the 10-nm filament is arranged in a left-handed cod (Fig. 5). The negative supercoiling of the DNA is manifested by writhing the hehcal axis around the nucleosomes. Chromatin stmcture is an example of toroidal winding whereas eukaryotic chromosomes are linear, the chromatin stmctures, attached to a nuclear matrix, define separate closed-circular topological domains. 

Similar results were obtained from reconstitution experiments with DNA and a non-cross-linked octamer (Thomas and Butler, 1978). Nucleosome-like particles were observed in the EM and a pattern of histone cross-linking comparable to that of native chromatin was obtained. However, only 140-base-pair repeats were obtained upon micrococcal nuclease digestion instead of 200-base-pair repeats obtained for native rat liver chromatin (Noll and Komberg, 1977). This indicates that, in the absence of HI, only core particles can be reconstituted. Nevertheless, these studies with both cross-linked and reassembled un-cross-linked histones demonstrate that the octamer is a complete biological functional unit retaining the information for folding the DNA around the histone core. 

At low ionic strength, chromatin has the appearance of beads-on-a-string as observed in the EM (Olins and Olins, 1974 Woodcock, 1973). However, it is more compact in its native environment in the nucleus (for references, see Chambon, 1978). Several levels of compaction beyond the nucleosome are required to account both for the... 

Antibiotic Mg complex induced alteration in the ultrastructural changes in the native and HI depleted chromatin were monitored by thermal melting analysis, polyacrylamide gel mobility assay, dynamic light scattering experiments and transmission electron microscopic studies. Micrococcal nuclease digestion is the biochemical probe to assess the accessibility of the antibiotic Mg + complexes to nucleosomal DNA. 

Depletion of histone HI after covalent modification from chromatin is a key step in eukaryotic transcription (Lee et al, 1993 Juan et al, 1994 Rice and Allis, 2001). A comparison of the association of the antibiotic Mg + complexes with the normal and HI depleted chromatin suggests that smaller ligands, like anticancer drugs, have better accessibility for HI depleted chromatin compared to native chromatin. HI depleted chromatin is also more prone to aggregation upon association with the complex I of the antibiotic Mg + complexes. It is also less accessible to micrococcal nuclease. We propose that HI depleted chromatin is a better target of these antibiotics compared to native chromatin. This observation is particularly significant in case of neoplastic cells where most of the cell nuclei are transcriptionally active, and, therefore, contain HI depleted chromatin. 

C. L. Woodcock, Chromatin fibers observed in situ in frozen hydrated sections. Native fiber diameter is not correlated with nucleosome repeat length. J. Cell Biol. 125, 11-19 (1994). 

C. L. Woodcock, S. A. Grigoryev, R. A. Horowitz, and N. Whitaker, A chromatin folding model that incorporates linker variabihly generates fibers resembling the native structures. Prvc. Natl. 

This problem first emerged from the necessity to reconcile topological and structural data of nucleosomes and chromatin. As soon as a minichromosome could be reconstituted from pure DNA and histones, the total reduction of the DNA linking number (Lk) was found to be equal to the number of nucleosomes, which was also true for the native Hl-bearing SV40 minichromosome [12]. On the other hand, the first low-resolution crystal of the core particle showed that DNA was wrapped with 1 3/4 turns of a left-handed superhelix. Assuming linker DNAs... 

Fig. 4. Nucleosome relaxation, and influence of histone N-terminal tails. Example of nucleosomes on 356 bp ALk= —2.9 topoisomer from the pBR DNA minicircle series [28]. (a) Mononucleosomes (Mo) were reconstituted with control (Control) or acetylated (Acetyl) histones, incubated at 37 °C in Tris buffer [T 50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 50 mM KCl, 5 mM MgC, and 0.5 mM dithiothreitol] or phosphate buffer [P same as Tris buffer with 50 mM potassium phosphate (pH 7.5) instead of 50 mM Tris-HCl] in the absence (Topo I —) or presence (Topo I +) of topoisomerase I, and electrophoresed in a native polyacrylamide gel at room temperature. Note the splitting of nucleosome relaxation products in two bands. TE starting chromatin in TE buffer, (b) Gel slices (brackets) were cut out, and eluted DNAs were electrophoresed in a chloroquine-containing native polyacrylamide gel, together with control naked topoisomers (C1-C4). Lanes were numbered as in the (a) gel. Autoradiograms are shown, (c) Radioactivity profiles of lanes 2 and 5 in the (b) gel. Topoisomers are indicated by their ALk values. (Adapted from Fig. 2 in Ref. [28].)...

Mg (but not Na" ") results in a structure that is equivalent to the 30-nm compact fiber in the extent of condensation [49]. Finally, the independent and critical function of core histone N-termini in chromatin condensation was demonstrated by showing that nucleosomal filaments reconstituted from core histones lacking their N-terminal domains are unable to condense into folded structures upon an increase of Mg " ", despite the presence of properly bound histone H5 ([50,51], see also Ref. [52] for the discussion of the special role of H3 and H4 tails). Thus, the presence of HI is not a sine-qua-non condition for salt-induced chromatin folding, which can proceed in Hi s absence and is an intrinsic property of filaments consisting of spaced core particles. A key question is how many of the features of the native 30-nm compact fiber are due to the presence of histone HI From the available data it seems that HI may influence the intrinsic folding pathway of the chromatin filament by stabilizing a single ordered conformation. This property can have much to do with the cooperativity of HI interactions within chromatin but also with the way HI is bound to the nucleosome and with the efifect it exerts on the path of linker DNA. 

Studies with native and reconstituted chromatin show that phosphorylated HI destabilize chromatin structure [62,63]. Phosphorylation of HI increases the protein s mobility in the nucleus and weakens its interaction with chromatin [64,65]. In studies with avian fibroblasts transfected with H5 (an HI variant), H5 was shown to inhibit proliferation in normal fibroblasts but not in transformed cells, in which H5 was phosphorylated. Aubert et al. proposed that phosphorylated H5 lacked the ability to condense chromatin [66]. The greater affinity of the dephosphorylated relative to phosphorylated HI for chromatin may interfere with the performance of chromatin remodeling agents. For example, dephosphorylated, but not phosphorylated, HI inhibited chromatin remodeling by SWI/SNF [67]. Thus, these and other studies provide support for the idea that an increase in the phosphorylation of HI leads to destabilization of the chromatin [24]. 

Woodcock, C.L., Grigoryev, S.A., Horowitz, R.A., and Whitaker, N. (1993) A chromatin folding model that incorporates linker variability generates fiber resembling the native structures. Proc. Natl. Acad. Sci. 1 90(19), 9021-9025. 

Unfixed or glutaraldehyde-fixed CE chromatin fibers native or LH-depleted... 

Earlier attempts to use the AFM for mechanically stretching chromatin fibers have run into a rather unexpected artifact. Long native chromatin fibers isolated from chicken erythrocytes, or fibers assembled in vitro from purified histones and relatively short, tandemly repeated DNA sequences were deposited on mica or glass surfaces and pulled with the AFM tip [69,70]. In such stretching experiments the scanning of the sample in the x- and y-direction used for imaging was disabled, and the cantilever-mounted tip was allowed to move only in the z-direction, i.e., upwards and downwards, away and towards the surface. When the AFM tip is pushed into the sample, it may attach to the sample by non-specific adsorption upon retraction it stretches the sample and force-extension curves are recorded (see Fig. lb for an explanation of a typical force curve). 

The experimental approach used to mechanically stretch a chromatin fiber with the AFM is depicted schematically in Fig. 8a, and some example curves obtained with native chicken erythrocyte chromatin fibers are presented in Fig. 8b. These curves exhibited a saw-tooth pattern, similar to the patterns obtained upon stretching of multi-domain proteins like titin [71] or tenascin [72] (Fig. 8c). Each of... 

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