The influence of chromatin on immediate-early gene expression has been studied in a model of Egr1 induction in intact mouse cells. ChIP analysis of factor and RNA polymerase binding reveals that the gene is constitutively poised for transcription in nonstimulated cells, but a repressing chromatin structure hampers productive transcription. Stimulation with phorbol esters results in a transient activation, which starts at 5 min and peaks at 30 min. Quantitative mapping of promoter occupancy by the different factors shows for the first time that no direct competition between SP1 and EGR1 occurs. The phosphorylation of ELK1 and CREB, which involves both the cascades of MEK1/2 and p38 kinases, is required for gene expression, which ceases following the binding of NAB1 and NAB2 to the promoter. The changes in histone acetylation and the differential recruitment of histonemodifying complexes further show the role of chromatin in the activation of this immediate-early gene.

At least seven isoforms (PsABI3-1 to PsABI3-7) of a putative, pea ABI3-like factor, originated by alternative splicing, have been identified after cDNA cloning. A similar variability had previously only been described for monocot genes. The full-length isoform, PsABI3-1, contains the typical N-terminal acidic domains and C-terminal basic subdomains, B1 to B3. Reverse transcriptase-PCR analysis revealed that the gene is expressed just in seeds, starting at middle embryogenesis; no gene products are observed in embryo axes after 18 h post-imbibition although they are more persistent in cotyledons. The activity of the isoforms was studied by yeast one-hybrid assays. When yeast was transformed with the isoforms fused to the DNA binding domain of Gal4p, only the polypeptides PsABI3-2 and PsABI3-7 failed to complement the activity of Gal4p. Acidic domains A1 and A2 exhibit transactivating activity, but the former requires a small C-terminal extension to be active. Yeast two-hybrid analysis showed that PsABI3 is able to heterodimerize with Arabidopsis thaliana ABI5, thus proving that PsABI3 is functionally active. The minimum requirement for the interaction PsABI3–AtABI5 is the presence of the subdomain B1 with an extension, 81 amino acids long, at their C-terminal side. Finally, a transient onion transformation assay showed that both the active PsABI3-1 and the inactive PsABI3-2 isoforms are localized to nuclei. Considering that the major isoforms remain approximately constant in developing seeds although their relative proportion varied, the possible role of splicing in the regulatory network of ABA signalling is discussed.

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Summary• Aspects of transcriptional regulation in plants, such as the order in which transcriptional factors and the preinitiation complex are assembled, are obscure because studies carried out under conditions in which native chromatin structure is preserved are still few in comparison with those carried out under other conditions.• In vivo chromatin immunoprecipitation (ChIP) experiments were used here to study the regulation of Pisum sativum psp54, which codes for the precursor of a chromatin-associated protein in dry seeds.• Antibodies against PsSNF5, a component of the SWI ⁄ SNF remodelling complex, and against the transcriptional factor Pisum sativum abscisic acid insensitive 3 (PsABI3) were raised and used for ChIP experiments, which showed that both factors are bound to the psp54 promoter only when the gene is actively expressed during seed maturation and germination. However, RNA polymerase II appeared to be bound to the inactive promoter, which was poised for transcription, before the assembly of factors. Micrococcal nuclease protection assays showed that chromatin conformation at the proximal psp54 promoter changes in shifting from the active to inactive state.• The changes in the promoter chromatin of psp54 are discussed. Stalled polymerase is described for the first time at the promoter of a non-heat-shock plant gene.

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