Histone dynamics and roles of histone acetyltransferases during cold-induced gene regulation in Arabidopsis.

In Arabidopsis, CBF transcription factors bind to and activate certain cold-regulated (COR) gene promoters during cold acclimation. Consistent with the prevailing model that histone acetylation and nucleosomal depletion correspond with transcriptionally active genes, we now report that H3 acetylation increases and nucleosome occupancy decreases at COR gene promoters upon cold acclimation. Overexpression of CBF1 resulted in a constitutive increase in H3 acetylation and decrease in nucleosome occupancy, consistent with the constitutive activation of COR gene expression. Overexpression of a truncated form of CBF2 lacking its transcriptional activation domain resulted in a cold-stimulated increase in H3 acetylation, but no change in nucleosomal occupancy or COR gene expression, indicating that histone acetylation is congruent with but not sufficient for cold-activation of COR gene expression. Plants homozygous for T-DNA disruption alleles of GCN5 (encoding a histone acetyltransferase) or ADA2b (a GCN5-interacting protein) show diminished expression of COR genes during cold acclimation. Contrary to expectations, H3 acetylation at COR gene promoters was stimulated upon cold acclimation in ada2b and gcn5 plants as in wild type plants, but the decrease in nucleosome occupancy was diminished. Thus, GCN5 is not the HAT responsible for histone acetylation at COR gene promoters during cold acclimation. Several other HAT mutant plants were also tested; although some do affect COR gene expression, none affected histone acetylation. Therefore, H3 acetylation at the COR gene promoters is not solely dependent on any of the HATs tested.

Two Arabidopsis orthologs of the transcriptional coactivator ADA2 have distinct biological functions.

Histone acetylation is an example of covalent modification of chromatin structure that has the potential to regulate gene expression. Gcn5 is a prototypical histone acetyltransferase that associates with the transcriptional coactivator Ada2. In Arabidopsis, two genes encode proteins that resemble yeast ADA2 and share approximately 45% amino acid sequence identity. We previously reported that plants harboring a T-DNA insertion in the ADA2b gene display a dwarf phenotype with developmental defects in several organs. Here we describe T-DNA insertion alleles in the ADA2a gene, which result in no dramatic growth or developmental phenotype. Both ADA2a and ADA2b are expressed in a variety of plant tissues; moreover, expression of ADA2a from a constitutive promoter fails to complement the ada2b-1 mutant phenotype, consistent with the hypothesis that the two proteins have distinct biochemical roles. To further probe the cellular roles of ADA2a and ADA2b, we studied the response of the transcriptional coactivator mutants to abiotic stress. Although ada2b seedlings display hypersensitivity to salt and abscisic acid and altered responses to low temperature stress, the responses of ada2a seedlings to abiotic stress generally parallel those of wildtype plants. Intriguingly, ada2a;ada2b double mutant plants display an intermediate, gcn5-like phenotype, suggesting that ADA2a and ADA2b each work independently with GCN5 to affect genome function in Arabidopsis.

Physical and functional interactions of Arabidopsis ADA2 transcriptional coactivator proteins with the acetyltransferase GCN5 and with the cold-induced transcription factor CBF1.

The Arabidopsis GCN5, ADA2a and ADA2b proteins are homologs of components of several yeast and animal transcriptional coactivator complexes. Previous work has implicated these plant coactivator proteins in the stimulation of cold-regulated gene expression by the transcriptional activator protein CBF1. Surprisingly, protein interaction studies demonstrate that the DNA-binding domain of CBF1 (and of a related protein, TINY), rather than its transcriptional activation domain, can bind directly to the Arabidopsis ADA2 proteins. The ADA2a and ADA2b proteins can also bind directly to GCN5 through their N-terminal regions (comparable to a region previously defined in yeast Ada2) and through previously unmapped regions in the middle of the ADA2 proteins, which bind to the HAT domain of GCN5. The ADA2 proteins enhance the ability of GCN5 to acetylate histones in vitro and enable GCN5 to acetylate nucleosomal histones. Moreover, GCN5 can acetylate the ADA2 proteins at a motif unique to the plant homologs and absent from fungal and animal homologs. We speculate that this modification may represent a novel autoregulatory mechanism for the plant SAGA-like transcriptional coactivator complexes.