Transcriptional gene silencing mutants in "Arabidopsis thaliana" and their impact on nuclear architecture and heterochromatin organization

The epigenetic regulation of gene expression is defined by covalent modifications of DNA and histone tails as well as by chromatin structure and nuclear architecture. Maintenance of epigenetic states therefore requires proteins engaged in chromatin remodeling, histone modifications, DNA replication and methylation. Using Fluorescence In Situ Hybridization, immunolocalization, genetic as well as molecular biology techniques I investigated chromatin structure and properties in Arabidopsis mutants impaired in the maintenance of transcriptional gene silencing (TGS). I showed that silencing of a multicopy transgenic locus results in neo-heterochromatin formation, accompanied by hypermethylation of DNA and of histone H3K9, two modifications enriched also in constitutive heterochromatin. Loss of the SWI2/SNF2 chromatin-remodeling factor DDM1 involved in maintenance of DNA methylation patterns, but also lack of functional protein MOM1 release silencing from specific repetitive targets. While the reactivation in ddm1 mutants is accompanied by a significant decondensation of heterochromatin and changes in histone modification patterns, mom1 allows transcription within a heterochromatic environment without disturbing DNA and histone methylation. An analysis of epistasis revealed that the nuclear MOM1 protein is part of a novel, methylation-independent pathway. Therefore decondensation of heterochromatin may accompany transcriptional activation, but it is not an obligate prerequisite. However, both pathways may act synergistically, as shown by the additive effects of reactivation, chromatin and phenotype aberrations in mom1/ddm1 double mutants. Within the methylation-based pathway, the different chromatin modifications are interrelated since the correct setting of H3K9 methylation marks depends on CpG methylation. In the complete absence of CpG methylation, histone H3 methylated at K9 becomes redistributed away from the chromocenters. Heterochromatin structure at the chromocenters, however, can be maintained even in the absence of both modifications previously assumed to be essential for heterochromatin formation and maintenance. In addition to the factors influencing chromatin properties globally, other components seem to have more specific targets, as loss of functional histone de-acetylaseresults in hyperacetylation and DNA methylation changes preferentially at rDNA loci. In general, the work presented here revealed several connections between chromatin shape and modifications at transgenic and endogenous parts of the genome and added to our insight into the complexity of epigenetic transcriptional regulation in Arabidopsis.