Nucleosomes associated with transcribing chromatin of mammalian cells have an unfolded structure in which the normally buried cysteinyl-thiol group of histone H3 is exposed. In this study we analyzed transcriptionally active/competent DNA-enriched chromatin fractions from chicken mature and immature erythrocytes for the presence of thiol-reactive nucleosomes using organomercury-agarose column chromatography and hydroxylapatite dissociation chromatography of chromatin fractions labeled with [ 3 H]iodoacetate. In mature and immature erythrocytes, the active DNA-enriched chromatin fractions are associated with histones that are rapidly highly acetylated and rapidly deacetylated. When histone deacetylation was prevented by incubating cells with histone deacetylase inhibitors, sodium butyrate or trichostatin A, thiol-reactive H3 of unfolded nucleosomes was detected in the soluble chromatin and nuclear skeleton-associated chromatin of immature, but not mature, erythrocytes. We did not find thiol-reactive nucleosomes in active DNA-enriched chromatin fractions of untreated immature erythrocytes that had low levels of highly acetylated histones H3 and H4 or in chromatin of immature cells incubated with inhibitors of transcription elongation. This study shows that transcription elongation is required to form, and histone acetylation is needed to maintain, the unfolded structure of transcribing nucleosomes.Acetylation of the core histones (H2A, H2B, H3, and H4) is a dynamic process catalyzed by histone acetyltransferases and histone deacetylases (1, 2). In chicken immature erythrocytes, 4% of the modifiable lysine sites participate in dynamic histone acetylation. These core histones are rapidly acetylated (t1 ⁄2 ϭ 12 min for monoacetylated H4) and rapidly deacetylated (t1 ⁄2 ϭ 5 min for the tetraacetylated isoform of H4) (3, 4). Histones undergoing rapid acetylation and deacetylation are associated with transcriptionally active chromatin (5-7). The recent findings that histone acetyltransferases and deacetylases are transcriptional coactivators and corepressors have increased our understanding of how the process of dynamic histone acetylation is established on transcriptionally active chromatin (2, 8).Transcriptionally active chromatin has a soluble and insoluble nature (9). Transcribed DNA is found in chromatin fragments that are soluble in 0.15 M NaCl and/or 2 mM MgCl 2 and in chromatin fragments associated with the low salt-insoluble residual nuclear material (nuclear skeletons) (for review, see Davie (10)). Chromatin engaged in transcription is thought to be retained by the nuclear skeleton by multiple dynamic attachments between the nuclear matrix and transcribed chromatin; hence rendering the transcribing chromatin insoluble (11, 12). As histone acetyltransferase and deacetylase activities are associated with the nuclear matrix (7, 13), we proposed that these nuclear matrix-bound enzymes may mediate some of the dynamic attachments between active chromatin and nuclear matrix (13,14). Most information on the structure ...