Stability of the Higher-Order Structure of Chicken-Erythrocyte Chromatin in Solution

Abstract

A detailed hydrodynamic study of chicken erythrocyte chromatin as a function of ionic strength, and comparison with results reported earlier for rat liver chromatin [Butler, P. J. G. and Thomas, J. 0. (1980) J . Mol. Bid. 140,529; Thomas, J. 0. and Butler, P. J. G. (1980) J . Mol. Biol. 144,[89][90][91][92][93] shows similar behaviour in the two cases, but quantitative differences indicate a greater stability of the chicken erythrocyte chromatin. The hydrodynamic method may thus provide a general assay for the stability in solution of the higher-order structure of chromatin -a helical coiling into a solenoid.When sedimentation coefficients of nucleosoine oligomers of various lengths from chicken erythrocytes are measured as a function of ionic strength, two main changes in behaviour are observed. The first is between pentamer and hexamer as in rat liver chromatin, suggesting that the solenoid has 5 -6 nucleosomes per turn. The longer repeat length of chicken erythrocyte chromatin (212 base pairs compared with 200 base pairs for rat liver chromatin) is thus accommodated into the solenoid without obvious change of structure.The second change in behaviour is a jump of about 9 % in the sedimentation coefficient at ionic strength 50 mM for nucleosome oligomers above a critical size, which we attribute to the relative instability of highcrorder structure in long oligomers at lower ionic strength. In chicken erythrocyte chromatin this critical size is about 60 nucleosomes, compared with 50 nucleosomes in rat liver chromatin. The increased axial stability in the solenoid that this indicates possibly derives from the erythrocyte-specific histone H5, and would be expected in this chromatin which is virtually inactive in transcription.During erythropoiesis in nucleated erythrocytes, pronounced structural and functional changes occur in the chromatin. The appearance [I] and dephosphorylation [2,3] of the tissue-specific histone H5 is accompanied by a reduction in nuclear activity, and the nucleosome repeat length increases from about 200 to 212 base pairs [1,4]. In the mature erythrocyte transcription is barely detectable [5], and H I has been replaced by [2,6], or supplemented with [I], substantial amounts of H5, which has a higher arginine content than H I but is sufficiently homologous to be regarded as an extreme H I variant [7,8]. It might be expected that such profound modification would be reflected in the physical properties of the chromatin compared with that from a biologically active nucleus such as rat liver.We have previously examined the higher-order structure of rat liver chromatin using measurement of the sedimentation coefficient of a range of nucleosome oligomers to monitor salt-induced conformational changes, and two salt-dependent structural transitions were observed [9,10]. One, occurring between nucleosome pentamers and hexamers, was attributed to the appearance of the simplest element of higherorder structure, a single stable turn of a helix. The second, evident only in oligomers of more than 50 nuc...

The higher-order structure of chromatin: evidence for a helical ribbon arrangement.

“…This suggests that some new state of compaction is possible with the larger polynucleosomes, consistent, for example, with the completion of one turn of a simple superhelix or solenoid. Similar sedimentation results were obtained with chicken erythrocyte chromatin (2) . In an attempt to understand this effect, we have examined the structure of tetra-to heptanucleosomes in different NaCl concentrations using the micareplica technique.…”

Higher order structure in a short repeat length chromatin.

“…In these respects the neuronal chromatin was similar in appearance to erythrocyte higher order fibers (Fig. 7, b and c), (6,24), and to the fibers that have been described for other chromatins under similar conditions (36). Chromatin that had been prepared by reconstitution of depleted neuronal polynucleosomes with calf thymus H 1 presented similar structures to those seen in the native samples (Fig.…”

“…Previous work has revealed that H 1 is located on a nucleosome, or chromatosome (33), at that point where the DNA both leaves and enters the nucleosome (2,6,33) . The characteristic protection from nucleases of a DNA fragment 168 base pairs long occurs as a direct result of this interaction (27).…”

“…In both cases the removal of linker histone from the native material caused a substantial reduction in the sedimentation velocity of the resulting chromatin . For chicken erythrocyte chromatin, it has been demonstrated that in the presence of linker histone the polynucleosome fiber adopts a regular higher order structure in buffers containing 80 mM NaCI, and it is for this reason that native erythrocyte polynucleosomes sediment more rapidly than depleted polynucleosomes (3,6). The fact that neuronal polynucleosomes behave in a similar manner suggests that this material also adopts a highly folded conformation in the presence of H 1 .…”

Dissection of the role of MHC class II A and E genes in autoimmune susceptibility in murine lupus models with intragenic recombination

“…Chromatin was prepared as described in ref. 26. Briefly, nucleosomes were isolated by solubilizing chromatin from purified chicken erythrocyte nuclei with micrococcal nuclease.…”