Maintenance of CG methylation (mCG) patterns is essential for chromatin-mediated epigenetic regulation of transcription in plants and mammals. Yet, transgenerational epigenetic inheritance of mCG has been defined by the study of relatively few loci. Further, it is not clear to what extent epigenetic variation impacts on phenotypic variation. Thus, the establishment, maintenance, and effects of epigenetic modifications on regulating gene expression and/or chromatin stability are my current research interests.

My Ph.D. has focused on the functional links between transgenerational inheritance of DNA methylation and phenotypic variation using successive generations of an Arabidopsis thaliana mutant deficient in maintaining mCG, met1-3, and within a population of recombinant inbred lines with epigenetically mosaic chromosomes consisting of wild-type and CG methylation-depleted segments (epiRILs). Within the met1-3 mutant epigenome following progressive inbreeding in the absence of mCG, new and aberrant epigenetic patterns could be detected. These patterns were consistently detected using conventional bisulfite sequencing, the 5-methylcytosine  immunoprecipitation (mCIP) technique, and by using BiMP (for Bisulfite Methylation Profiling), a genome-wide DNA methylation profiling technology we developed using a novel amplification step for DNA subjected to bisulfite-mediated cytosine conversion.

Despite phenotypic selection, transgenerational inheritance of plant fitness was highly unstable in met1-3 mutant plants; supporting the belief that mCG is a central coordinator of epigenetic memory important for stable transgenerational inheritance in plants.

I next investigated the functional links between transgenerational inheritance of DNA methylation and phenotypic variation following the restoration of wild type MET1. In the epiRIL population, significant phenotypic variation was detected for a broad spectrum of traits, suggesting epigenetic variation was possibly affecting the inheritance of phenotypic variation in this population. Using BiMP and Southern blot analyses, recombined parental chromosomal segments interspersed with unexpectedly high frequencies of non-parental methylation polymorphism were also identified within three profiled epiRILs. Hence, epigenetic inheritance in hybrids derived from parents with divergent epigenomes permits long-lasting epi-allelic interactions that violate Mendelian expectations. Such persistently “unstable” epigenetic states complicate linkage-based epigenomic mapping. Additionally, CACTA DNA transposons, that were immobile in the parental lines, displayed stochastic movement in 28% of the epiRILs.

Therefore the assumption that all of the phenotypic variation was solely attributable to epigenetic variation was violated and genetic instabilities should be expected following the loss of mCG. Further, since a wide range of inheritance modes can be detected for DNA methylation, ranging from stable and to highly unstable, new strategies to either diminish or account for metastable epigenetic variation, which can rapidly change between generations and occur independently of meiotic recombination, should be considered to facilitate identifying epialleles affecting phenotypic variation.