Microarray analysis of chromatin-immunoprecipitated DNA identifies specific regions of tobacco genes associated with acetylated histones

The acetylation states of histones present on the upstream, promoter, coding or intronic regions of 88 tobacco genes were examined with chromatin immunoprecipitation (ChIP) experiments using antibodies that recognised acetylated histone H4. The DNA sequences enriched in the immunoprecipitates were amplified by ligation-mediated PCR, labelled with Cy-dUTP and hybridised to DNA microarrays. In green tobacco shoots, histone H4 acetylation was localised to 300-600-bp sequences in the promoters or coding regions of 31 genes, or occurred extensively over several kilobase-pair regions containing the upstream, promoter and/or coding regions of 25 genes. Genes associated with high histone H4 acetylation levels at promoters were actively expressed, whereas genes depleted in acetylated histone H4 were non-transcribed or expressed at very low levels, suggesting a correlation between histone H4 acetylation and gene activity. Trichostatin A (TA), an inhibitor of histone deacetylases (HDAs), did not alter histone H4 acetylation states globally but increased acetylation levels at specific tobacco sequences, suggesting that HDAs are targeted to particular nucleosomes. Genes that were upregulated by TA were associated with increased histone H4 acetylation at promoter or coding regions, indicating that acetylation of histones on coding regions may activate transcription. Increased histone H4 acetylation leading to elevated expression was observed on genes with diverse functions, suggesting that histone H4 acetylation is involved in regulation of many plant processes.