Loosened nucleosome linker folding in transcriptionally active chromatin of chicken embryo erythrocyte nuclei

Abstract

We have investigated the mechanism of the electrophoresis-driven chromatin aggregation which had been described by Weintraub (1984, Cell 38, 17-27) as a putative mean for propagation of genetic repression in eukaryotes. We show that the oligonucleosome aggregates are assembled de novo at the starting zone of DNP electrophoresis. A new system of native two-dimensional DNP electrophoresis has been worked out to separate the oligonucleosome aggregates ('A' particles) and the freely-migrating oligonucleosomes ('B' particles). The 'B' particle fraction which is derived from transcriptionally-active chromatin regions undergoes an extensive nuclease degradation of its DNA termini during the nuclease digestion. This fraction is partially depleted of histones H1 and H5 and is enriched in HMG nonhistone proteins. 'A' particles comprise the repressed chromatin DNA fragments which are about 60 b.p. longer than the corresponding DNA oligomers of 'B' particles. An oligonucleosome preparation containing the elongated DNA oligomers has been also isolated by means of sucrose gradient ultracentrifugation. Exonuclease III mapping reveals that the two chromatin fractions differ by an extent of terminal linker DNA trimming during the Micrococcal nuclease digestion rather than by the nucleosome repeat length. The complex character of nuclease digestion is not observed when the chromatin is digested in solution after the nuclear lysis. We argue that the protection of terminal oligonucleosome linkers is due to selective condensation of inactive chromatin in chicken erythrocyte nuclei and that the terminal DNA tails together with linker histones bound to them mediate the aggregation of repressed chromatin fragments.

Drosophila H1 Regulates the Genetic Activity of Heterochromatin by Recruitment of Su(var)3-9

“…H1 is thought to be nearly ubiquitous in the genome, but several studies report its consistently higher abundance in heterochromatin (22–24). We propose that higher concentrations of H1, equal in stoichiometry to nucleosomes, along extended chromatin domains may be essential to achieve its optimal function as a repressor, whereas substoichiometric or local deposition may only allow for a limited ability to repress genetic activity in euchromatin (Fig.…”

Higher-order folding of heterochromatin: Protein bridges span the nucleosome arrays

“…Besides being developmentally regulated, histone H5 shows a preferential association with repressed and condensed chromatin as demonstrated by both biochemical fractionation (Ridsdale and Davie 1987;Grigoryev et al 1990) and CHIP (Kamakaka and Thomas 1990;Postnikov et al 1991). The area of histone H5 depletion over the active chicken β-globin cluster coincides with increased histone acetylation and DNase I sensitivity, thus suggesting a common molecular mechanism regulating spreading of the heterochromatin-associated structural changes at the boundaries of active and repressed chromosomal domains (Verreault and Thomas 1993).…”

“…10 mM monovalent cation. However, upon the gradual increase of ionic strength to 50-80 mM, oligonucleosomes undergo an increasing self-association giving rise to soluble selfassociated chromatin (SAC) particles notably bigger in size than the individual polynucleosome chains (Weintraub 1984;Jose et al 1987;Grigoryev et al 1990;Kamakaka and Thomas 1990). Upon further increase in salt concentration, the progressing self-association culminates in aggregation and precipitation of chromatin.…”

“…To determine if chromatin selfassociation is specific for certain chromatin regions or occurs at random, the SAC structures isolated from several types of terminally differentiated cells were tested by Southern-blot hybridization with radioactive probes corresponding with actively transcribed and silent genetic loci. Using two different approaches for SAC fractionation, 2D electrophoresis, and sucrose gradient ultracentrifugation, it has been shown that chromatin containing transcribed genes (e.g., βglobin, actin, and thymidine kinase in nucleated chicken erythrocytes; albumin in rat liver), and transcriptionally poised genes (ρ-globin in erythrocytes, α-fetoprotein in adult rat liver) were selectively excluded from the self-associated fractions, while repressed genes (such as ovalbumin and vitellogenin in erythrocytes, β-globin in brain, and γ-casein gene in rat liver) were concentrated within the SAC structures (Weintraub 1984;Jose et al 1987;Grigoryev et al 1990;Kamakaka and Thomas 1990). Furthermore, J. Davie and his group have noticed that the transcriptionally active chromatin tends to resist the complete aggregation and precipitation observed in physiological ionic strength buffers.…”

Nucleosome positioning in Arabidopsis

“…To compare Arabidopsis linker lengths with those from other taxa, linker lengths were collated from the literature (Table 4. Previous studies have demonstrated differences in linker length between cells types exhibiting different transcriptional activity (Grigoryev et al, 1990). As the data presented here are generated from many cell types (within an organ), it is likely that multiple chromatin conformations are represented.…”