Identification of genes involved in transgene silencing in Arabidopsis

In plant genetic engineering and in human gene therapy, transgenes often become silenced. The loss of transgene expression is often associated with covalent DNA cytosine hypermethylation and histone deacetylation. One early model for gene silencing suggests a linear repression pathway in which DNA methylation brings about histone deacetylation in order to cause gene silencing. Whether this hypothesis is generally true for endogenous plant genes is not clear. By monitoring changes in the expression of approximately 7,800 Arabidopsis thaliana genes affected by global inhibition of DNA methylation and/or histone deacetylation, we show that the majority of responsive genes exhibit complex, non-redundant responses to blocking DNA methylation and/or histone deacetylation. In the course of studying transgene silencing, we have identified two A. thaliana transgenic lines possessing clustered GFP transgenes in which the transgenes are silenced at the transcriptional level. The silencing involves DNA methylation. To identify novel genes that are involved in the regulation of transgene silencing, two independent genetic approaches, an activation-tagging screen and a chemical mutagenesis with EMS were applied to identify suppressors that can derepress the normally silent GFP transgenes. One recessive and four dominant mutants were recovered. One dominant suppressive mutant, sost1-D, (s&barbelow;uppressor o&barbelow;f s&barbelow;ilenced t&barbelow;ransgene 1-d&barbelow;ominant), was identified in the activation-tagging screen. The SOST1 gene encodes a small proline-rich protein that is a member of a family of five genes in A. thaliana. Transient and stable overexpression of SOST1 in the transgenic line bearing the silenced GFP transgenes derepresses the silencing. Surprisingly, sost1-D also reactivates a posttranscriptionally silenced GUS gene in A. thaliana transgenic line L1, a well-characterized line for PTGS. Experimental evidence indicates that overexpression of SOST1 prevents RNA-dependent DNA methylation of the GUS transgene. In summary, these results suggest that SOST1 plays an important role in both transcriptional and post-transcriptional gene silencing. Dominant suppression of transgene silencing may prove useful for stabilizing transgene expression in plant and mammalian cells.