Minisymposium 6: Epigenetics

Abs # 16005: Discovery of a Genetic Insulator in Arabidopsis thaliana

Presenter:	Gan, Susheng , sg288@cornell.edu
Authors	Yang, Xiaohan  (A)   Xu, Hengping  (B)   Xie, Mingtang  (B)   Gan, Susheng  (A) (B)
Affiliations:	(A): Cornell University (B): University of Kentucky
Web Site:	http://www.hort.cornell.edu/gan/

Genetic insulators are naturally-occurring DNA sequences that buffer against the influence of elements of neighboring gene promoters, define gene and chromatin boundaries, and play an important role in the regulation of gene expression during development. The operational definition of a genetic insulator is that (1) when placed between enhancers and minimal promoter, it blocks the enhancers from activating the minimal promoter, (2) but when placed outside of a promoter, it has no negative effect on the promoter, and (3) when flanking a transgene, it eliminates positional effects. Two dozens insulators have been identified from yeast and animal systems. Some (but not all) of nuclear matrix attachment regions (MARs) have also been shown to block against position effects in animals and plants. These insulators and MARs are generally more than 200 bp in length. Although plant MARs have been identified and used to improve transgene expression, no insulator sequence of plant origin has been identified. Here we report that a 16-bp palindromic DNA sequence, 5'-GAATATATATATATTC-3', in Arabidopsis functions as a genetic insulator. We placed 1x or 2x the palindrome between the 35S enhancers and the P_35Smini, and found that the enhancers could no longer activate the reporter gene GUS that was fused with the P_35Smini, and that the palindromes did not negatively impact the P_35S. The palindromic nature per se was not sufficient for the insulating effect because its transversion mutant M3 (5’-GAATATcgcgATATTC-3’) and the lacO (5'-ATTGTGAGCGCTCACAAT-3'), both of which were perfect palindromes, failed to insulate gene expression. GUS activities in the 35S-GUS transgenic plants varied significantly but the variation was reduced to a very minimal level when the 35S-GUS transgene was flanked by the insulating palindrome but not by its mutant M3. Nuclear run-on assays revealed that the insulator buffered transgene expression at the transcriptional level. Gel mobility shift assays revealed that there were nuclear proteins that specifically bound to the insulator (but not to the M3 mutant and lacO palindromes). This represents not only the first report of a genetic insulator of plant origin, but it is also by far the shortest insulator so far discovered. The insulator may be used in both basic research (e.g., promoter dissection, controlled gene expression) and agricultural biotechnology (e.g., elimination of clonal variations in transgene expression due to position effects).