machinery
Only recently it was suggested that auxin responsive genes are regulated by an antagonistically acting system of histone acetylation and deacetylation (Long et al., 2006; Szemenyei et al., 2008). In this respect it was demonstrated, that the repressive function of the AUX/IAA-TF, IAA12 on the expression of auxin responsive genes is dependent on its interaction with members of the so-called TOPLESS (TPL) co-repressor protein family (Szemenyei et al., 2008). As a positive genetic interaction between TPL1 and the Histone De-Acetylase 19 (HDA19) was identified and the Histone Acetyl-Transferase (HAT) GCN5 was shown to be hypostatic towards TPL1 mediated responses, it was suggested that the AUX/IAA repressor proteins as well as TPL1 and HDA19 are located in a common repressive signalling pathway, whereas the HAT GCN5 is positioned in a counteracting, expression promoting system. By this means the respective proteins might provide a dynamic regulatory mechanism to rapidly adjust auxin responsive gene expression (Long et al., 2006; Szemenyei et al., 2008).
In order to verify this hypothesis and to address if the bZIP induced auxin responsive genes are regulated by histone acetylation or deacetylation respectively, a GCN5 family specific HAT- or a broad-spectrum HDAC inhibitor was applied (Chapter 3). In fact the results from this pharmacological approaches, revealed that the auxin-induced expression of the group S1 AtbZIP target genes; AtGH3.3, AtAUX/IAA3, AtAUX/IAA7
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and AtPIN4 was indeed dependent on a functional GCN5 specific histone acetylation system and was controlled by a counteracting deacetylation machinery (Chapter 3).
The HAT GCN5 was demonstrated to be a conserved co-activator of bZIP-TFs in yeast and plants (Topalidou et al., 2003; Locatelli et al., 2009). In order to determine if the bZIP-TF mediated induction of auxin-responsive target genes is at least partially attributed to the recruitment of a histone remodelling complex, further reverse genetic and biochemical approaches were conducted (Chapter 3). By this means it could be demonstrated that mutants of diverse HAT genes, including the gcn5 and the GCN5 related hag4 and hag5 mutants exhibited a significant, in part HAT specific reduction of the bZIP auxin-responsive target genes (Chapter 3). According to the concept of HAT regulated auxin responses, mutations in these genes should consequently result in auxin insensitive phenotypes. In fact, it was presented that gcn5 mutant plants display pleiotropic auxin-related phenotypic alterations such as severe dwarfism, loss of apical dominance, aberrant meristem function, abnormal root and leaf development and reduced petal and stamen growth (Vlachonasios et al., 2003, Bertrand et al., 2003; Long et al., 2006; Kornet and Scheres, 2009). Similar observations were reported for mutants of the highly homologous, apparently functional redundant HAG4 and HAG5 genes.
Whereas homozygous double mutants were not viable, sesqui-mutant plants (HAG4/hag4 – hag5/hag5) displayed severe disorders in the auxin dependent pollen development (Latrasse et al., 2008; Cecchetti et al., 2008; Iven et al., 2010).
However all these HAGs seem to be involved in modulating auxin-regulated transcription, the observed differences in the HAG mutant phenotypes and their individual ability to affect auxin-regulated gene expression (Chapter 3) indicate that the GCN5 enzyme and the two HATs of the related MYST gene family (HAG4 and HAG5) might implement in part distinct auxin-mediated responses.
The HAT GCN5 was described to be in general associated in large protein complexes.
Similar to the yeast GCN5, the Arabidopsis and maize homologous enzymes are incorporated in a SAGA-like histone remodelling complex (Vlachonasios et al., 2003;
Bhat et al., 2004).
In maize the bZIP-TF O2 was demonstrated to regulate seed storage genes by recruiting the SAGA complex to its target promoters via a direct interaction with the complex adapter protein ADA2 (Locatelli et al., 2009; Bhat et al., 2004)
Analogously, it was tested if group S1 and C AtbZIPs might also be able to recruit this complex upon binding to the ZmADA2 homologous Arabidopsis AtADA2a and AtADA2b complex components. In fact, particularly the group S1 AtbZIP2, -11 and -44 TFs exhibited a strong capacity to bind both AtADA2 adapter proteins, whereas the O2 orthologues AtbZIP10 and AtbZIP25 (Alonso et al., 2009; Jakoby et al., 2002) showed a significant binding exclusively to AtADA2b (Chapter 3). This indicates that diverse bZIP-TFs are in principle able to recruit the SAGA complex to regulate their diverse target genes. However further work is necessary to validate this assumption and to define the individually involved SAGA complex HAT and adapter components, which besides the promoter bound TFs apparently also confer some specificity to the regulatory system.
Based on the acidic nature of the N-terminus of the group S1 AtbZIP-TFs (Chapter 3), and the fact that a similar structured region was already suggested to be the potential ADA2 interaction surface within the O2 protein (Bhat et al., 2004), the activation and ADA2 binding properties of N-terminally truncated AtbZIP11 and AtbZIP44 derivatives were analysed in transient protoplast transfection assays and in stable transgenic plants. The obtained results demonstrated that ADA2 binding was mediated by the bZIP’s N-terminus, which acted as an activation domain and was crucial to induce the expression of the bZIP auxin responsive target genes and for the manifestation of bZIP mediated auxin-related plant growth responses (Chapter 3).
As variants of the observed polar, acidic activation and ADA2 binding domain are apparently conserved within certain homologous bZIP-TF classes from diverse species, these results could explain the reported, in part dominant negative property of the Arabidopsis group C and maize O2 homologous tobacco NtBZI-1 protein on the expression of the auxin responsive NtGH3 gene and on auxin mediated plant responses (Heinekamp et al., 2004).
In order to finally clarify, if the activation potential of the AtbZIP11 and -44 TFs was mediated by the recruitment of a chromatin remodelling, GCN5 histone acetylation complex to the auxin responsive target promoters, CHIP analyses were performed (Chapter 3). By this means it could be demonstrated that upon enhanced AtbZIP11 or -44 promoter binding, the rate of GCN5 specific histone acetylation in the GRE rich AtGH3.3 promoter region and the assembly of the RNA polymerase II near the
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transcriptional start site of the GH3.3 gene was significantly increased (Chapter 3). As this went along with a bZIP mediated enhanced transcription of the AtGH3.3 gene it can be assumed that in particular the AtbZIP11-related TFs are capable to induce their auxin responsive target genes, by consulting an Arabidopsis SAGA-like HAT complex to their target promoters and thereby represent a counteracting system to the repressive action of the AUX/IAA-TFs, which are thought to control target gene expression by histone deacetylation. As the cis-regulatory GRE-AuxRE module was found to be frequently distributed in the promoters of auxin responsive genes (Chapter 1) the bZIP/GRE regulatory system likely constitutes an expression modulating system, which enables a rapid and highly dynamic regulation of the bZIP auxin responsive target genes and their associated responses (Figure 2).
Figure 2: BZIP transcription factors recruit the Arabidopsis SAGA complex to their auxin responsive target genes. A detailed description of the model can be found in the discussion in Chapter 3.
In this work it was demonstrated that group S1 AtbZIP11 related TFs are able to recruit a GCN5/ADA2 composed histone-remodelling complex to their auxin responsive genes, to induce their expression. However besides these TFs also further group S1 and C AtbZIPs (Chapter3) and also group D AtbZIP-TFs (unpublished results) are able to bind the adapter protein AtADA2b, in in vivo approaches. Therefore it is conceivable that these and likely other bZIP-TFs, which exhibit a polar, acidic ADA2 interaction
domain, recruit the SAGA complex to their target promoters and that this might constitute a general regulatory mechanism by which bZIPs are able to rapidly and dynamically regulate their target genes. However prospective work is required to verify this assumption and to define the relevance of the SAGA complex recruitment in diverse bZIP controlled responses.