The Arabidopsis SET-domain protein ASHR3 is involved in stamen development and interacts with the bHLH transcription factor ABORTED MICROSPORES (AMS).

The Arabidopsis thaliana genome contains more than 30 genes encoding SET-domain proteins that are thought to be epigenetic regulators of gene expression and chromatin structure. SET-domain proteins can be divided into subgroups, and members of the Polycomb group (PcG) and trithorax group (trxG) have been shown to be important regulators of development. Both in animals and plants some of these proteins are components of multimeric protein complexes. Here, we have analyzed the Arabidopsis trxG protein ASHR3 which has a SET domain and pre- and post-SET domains similar to that of Ash1 in Drosophila. In addition to the SET domain, a divergent PHD finger is found in the N-terminus of the ASHR3 protein. As expected from SET-domain proteins involved in transcriptional activation, ASHR3 (coupled to GFP) localizes to euchromatin. A yeast two-hybrid screening revealed that the ASHR3 protein interacts with the putative basic helix-loop-helix (bHLH) transcription factor ABORTED MICROSPORES (AMS), which is involved in anther and stamen development in Arabidopsis. Deletion mapping indicated that both the PHD finger and the SET domain mediate the interaction between the two proteins. Overexpression of ASHR3 led in general to growth arrest, and specifically to degenerated anthers and male sterility. Expression analyses demonstrated that ASHR3 like AMS is expressed in the anther and in stamen filaments. We therefore propose that AMS can target ASHR3 to chromatin and regulate genes involved in stamen development and function.

An inverted repeat transgene with a structure that cannot generate double-stranded RNA, suffers silencing independent of DNA methylation.

Transgene silencing in plants is most often dependent on homologous sequences, e.g. tandemly repeated T-DNAs. We have identified an Arabidopsis line (ex2-4 line 4) displaying silencing of the T-DNA-born nptII gene. This line contains a truncated copy of the T-DNA encompassing the nptII gene with its nos promoter adjacent to an intact T-DNA copy. The orientation of the intact and the truncated copies preclude the generation of a double-stranded nptII transcript. Therefore, we have investigated the genomic landscape surrounding T-DNA insertion in the silenced ex2-4 line 4 and five single-copy ex2-4 lines without silencing in search of features that might explain the silencing phenomenon. GC content, putative matrix-attachment regions and transcriptional interference from neighbouring genes could all be ruled out as major causes of silencing. Bisulphite sequencing revealed de novo methylation of the nos promoter both in non-silenced and silenced plants of this line, thus silencing was not correlated to DNA methylation level. Also, the methylation pattern deviated from that characteristic for RNA-mediated DNA methylation and silencing. Our data therefore suggest that ex2-4 line 4 represents a case where silencing is due to DNA-DNA pairing, i.e. pairing between the intact T-DNA and the adjacent truncated, inverted T-DNA copy.

Analyses of single-copy Arabidopsis T-DNA-transformed lines show that the presence of vector backbone sequences, short inverted repeats and DNA methylation is not sufficient or necessary for the induction of transgene silencing.

In genetically transformed plants, transgene silencing has been correlated with multiple and complex insertions of foreign DNA, e.g. T-DNA and vector backbone sequences. Occasionally, single-copy transgenes also suffer transgene silencing. We have compared integration patterns and T-DNA/plant DNA junctions in a collection of 37 single-copy T-DNA-transformed Arabidopsis lines, of which 13 displayed silencing. Vector sequences were found integrated in five lines, but only one of these displayed silencing. Truncated T-DNA copies, positioned in inverse orientation to an intact T-DNA copy, were discovered in three lines. The whole nptII gene with pnos promoter was present in the truncated copy of one such line in which heavy silencing has been observed. In the two other lines no silencing has been observed over five generations. Thus, vector sequences and short additional T-DNA sequences are not sufficient or necessary to induce transgene silencing. DNA methylation of selected restriction endonuclease sites could not be correlated with silencing. Our collection of T-DNA/plant DNA junctions has also been used to evaluate current models of T-DNA integration. Data for some of our lines are compatible with T-DNA integration in double-strand breaks, while for others initial invasion of plant DNA by the left or by the right T-DNA end seem important.