Functional in vitro and in vivo analysis of the Arabidopsis SET domain protein ASHR3

Abstract

ABSTRACT The evolutionary conserved SET-domain is found in proteins encoded by more than 30 genes in Arabidopsis thaliana and is known to be involved in the epigenetic control of gene expression and organization of chromatin structure. Some of these SET-domain proteins are members of the Polycomb group (PcG) and the trithorax group (trxG). PcG and trxG proteins cooperatively regulate gene expression during development by repressing and activating target genes, respectively, and often constitute protein complexes. Here we have studied the functional role of the trxG protein ASHR3 which in addition to its C-terminal SET-domain contains a divergent PHD finger in the N-terminal. YFP fusion constructs of ASHR3 and a deletion construct of ASHR3 lacking the PHD finger both localize to euchromatin, arguing that the PHD finger is not responsible for this localization. To investigate the in vivo function of ASHR3, a line containing a T-DNA insertion in the ASHR3-gene was identified and analyzed. These ashr3 knock-out mutants showed a slight reduction in the number of lateral roots compared to wild type, suggesting that ASHR3 is involved in the regulation of lateral root development. Gene expression profiling of ashr3 mutants identified putative down-stream targets for ASHR3 regulation. Some of these genes are jasmonate responsive genes and are expressed in anthers and roots, which is the organs ASHR3 is most highly expressed. Direct yeast two-hybrid (Y2H) matings between ASHR3 and candidate interacting proteins revealed a weak interaction between ASHR3 and the scaffolding protein RACK1C. Interestingly, RACK1C has also shown to be involved in lateral root development. In addition the Y2H results suggests that the ASHR3 interacting protein ABORTED MICROSPORES (AMS) can work as a transcription factor in yeast. Based on these results, combined with previous publications, we propose that ASHR3 could positively regulate the expression of down-stream target genes involved in anther or root development, like for example the jasmonate responsive genes.