Chromatin structure of erythroid-specific genes of immature and mature chicken erythrocytes

Abstract

The beta-globin and histone H5 genes are transcriptionally active in immature chicken erythrocytes and potentially active in mature erythrocytes. In both immature and mature erythrocytes, the majority of these erythroid-specific gene sequences are located in two chromatin fractions: the low-salt-insoluble residual nuclear material and the 0.15 M-NaCl-soluble oligo- and poly-nucleosomes. These salt-soluble chromatin fragments are enriched in hyperacetylated species of H4 and H2B, ubiquitinated and polyubiquitinated species of H2A and H2B and are depleted of linker histones H1 and H5. The competent, transcriptionally inactive embryonic epsilon-globin gene, which is part of the DNAase I-sensitive beta-globin domain, is highly enriched in the 0.15 M-NaCl-soluble polynucleosome fraction but not in the insoluble nuclear material. The repressed vitellogenin gene shows no enrichment in either of these fractions. These results suggest that only those genes that are expressed or have the potential for expression are enriched in the low-salt-insoluble nuclear material of immature or mature erythrocytes. The enrichment of active genes in the low-salt-insoluble residual nuclear material of immature erythrocytes is not dependent on on-going transcription, the presence of RNA or changes in the amount of acetylated histone species. Our results are consistent with the hypothesis that active and potentially active genes are insoluble because of the presence of preinitiation transcription complexes.

Histone Acetylation Is Required to Maintain the Unfolded Nucleosome Structure Associated with Transcribing DNA

“…DNA Preparation and Southern Blot Hybridization-DNA was prepared from the different chromatin fractions as described previously (24). For electrophoresis, equal amounts of DNA were dissolved in DNA sample loading buffer, and the samples were loaded onto 1% agarose minigels containing 0.5 g of ethidium bromide/ml.…”

“…Erythrocyte Chromatin Fractionation-The fractionation of chromatin was done as described previously (24). All buffers contained 1 mM phenylmethylsulfonyl fluoride.…”

“…Isolation and Treatments of Immature and Mature Chicken Erythrocytes-Mature and immature erythrocytes were isolated from normal and anemic young adult White Leghorn chickens, respectively, as described previously (24). Immature and mature erythrocytes were collected in 75 mM NaCl and 25 mM EDTA, while cells to be incubated with sodium butyrate or trichostatin A were collected in 75 mM NaCl, 25 mM EDTA, and 30 mM sodium butyrate.…”

“…Cells were incubated at 37°C in Swims S-77 medium (Sigma) with 30 mM sodium butyrate or 100 ng/ml trichostatin A for 90 min to prevent the deacetylation of highly acetylated histones. To inhibit transcription elongation, cells were incubated at 37°C for 90 min with transcription inhibitors actinomycin D (0.04 g/ml) (24), 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB) 2 (75 M) (24,25), or camptothecin (20 M) (26) followed by a 90-min incubation with or without 30 mM sodium butyrate.…”

Structure of active chromatin: isolation and characterization of transcriptionally active chromatin from rat liver

“…The technique for the fractionation of active chromatin in rat liver tissue is highly reproducible, and hence could be used in other tissues. A limitation in earlier nuclear digestions performed to isolate active chromatin was the inclusion of uni-and bi-valent cations at concentrations known to cause rearrangement of histone H1 [20,[25][26][27][28]. In fact, poly-and short oligonucleosomes enriched in active genes isolated by these methods were depleted of between important to note that the active chromatin fragments were easily extracted from nuclei in our studies, whereas these fragments remained selectively associated with the nuclear matrix on MNase digestion and extraction by other workers [21].…”