Although a general understanding that methylation of DNA serves to silence gene expression has been accepted, information about how that methylation is regulated and what other functions, if any, it serves is still forthcoming. A genetic approach to those questions has produced a collection of Arabidopsis thaliana mutants (ddm) that contain reduced cytosine methylation. ddm1 loss of function mutations lead to a rapid loss of methylation from most repetitive sequences in the genome, but only a slow loss of methylation from low copy sequences. The DDM1 gene has been recently cloned and found to encode a SW12/SNF2-like protein, implicating chromatin dynamics as an important process in DNA methylation. An experimental plan to test different models of DDM1 action has been created. Specific goals are to 1) test purified recombinant DDM1 and DDM1-associated complexes for DNA-dependent ATPase activity, nucleosome remodeling and chromatin assembly, 2) investigate whether DDM1 associates with cytosine methyltransferases or other chromatin modifying activities, 3) compare the ability of ddm1 and wild-type nuclear extracts to methylate chromatin substrates. A series of experiments to understand the consequences of DDM1 loss of function will be undertaken. Genetic analysis indicates that the ddm1-induced defects are due to the generation of stable variant alleles or epialleles at unlinked loci. The genomic region affected has been localized in one ddm1-induced dwarfing line (bal) to a 147 kb interval containing a leucine-rich repeat (LRR) "disease resistance" gene cluster. A change in gene expression in this cluster has been identified as a possible cause of the bal phenotype. Methylation and chromatin changes associated with bal variant (epi) alleles will be characterized. Transgenic experiments to determine if misexpression of the gene cluster is sufficient to cause the dwarfing phenotype will be done.
Although knowing the DNA sequence is necessary to an understanding of genetic control of life processes, it is not sufficient. The type of information gained from studies of this sort will have critical impact upon both our understanding and genetic engineering of both plants and animals.
