Islands of acetylated histone H4 in polytene chromosomes and their relationship to chromatin packaging and transcriptional activity

The four histones of the nucleosome core particle are all subject to enzyme-catalysed, post-translational acetylation at defined lysine residues in their amino-terminal domains. Much circumstantial evidence suggests a role for this process in modifying chromatin structure and function, but detailed mechanisms have not been defined. To facilitate studies on the functional significance of histone acetylation, we have prepared antibodies specific for the acetylated isoforms of histone H4. Because of the extreme evolutionary conservation of H4, these antisera can be applied to a wide variety of organisms and experimental systems. In the present study we have used polytene chromosomes from the salivary glands of larvae of the midge Chironomus to examine the distribution of acetylated H4 in interphase chromatin. By indirect immunofluorescence, antisera to acetylated H4 labeled the four Chironomus chromosomes with reproducible patterns of sharply defined, fluorescent bands. An antiserum to non-acetylated H4 gave a completely different, more-diffuse labelling pattern. Thus, there are defined regions, or islands, in the interphase genome that are enriched in acetylated H4. Double-labelling experiments with two antisera specific for H4 molecules acetylated at different sites, showed that each antiserum gave the same banding pattern. Immunolabelling patterns were not dependent on the pattern of phase-dense bands characteristic of these chromosomes; strongly labelled regions could correspond to phase-dense bands (i.e. condensed chromatin), to interbands or, frequently, to band-interband junctions. Immunogold electron microscopy confirmed the immunofluorescence results and showed further that regions of relatively high labelling could be either transcriptionally active or quiescent, as judged by the presence or absence of ribonucleoprotein particles. Two rapidly transcribed genes on chromosome 4 of Chironomus form characteristic ‘puffs’, the Balbiani rings BRb and BRc. The antiserum to non-acetylated H4 gave diffuse labelling throughout these puffs, demonstrating the continued presence of this histone in these transcriptionally active regions. Antisera to acetylated H4 strongly labelled the boundaries of BRb and BRc, and revealed clearly defined islands of increased H4 acetylation just within the expanded chromatin of the puffs. Labelling within the central region of each puff was much less intense. A similar pattern was observed in puffs on other chromosomes. Thus, increased H4 acetylation is not found throughout actively transcribed chromatin but occurs only at defined sites, possibly in the non-transcribed flanking regions. H4 acetylation is clearly not required for the passage of RNA polymerase through the nucleosome and we speculate that its role may be to facilitate the binding to DNA of polymerases and other proteins prior to the onset of transcription and possibly replication.