Intragenic methylation repatterning is associated with alternative splicing and unique epigenetic phenotypes

The role of intragenic cytosine methylation in shaping phenotypes has been contentious. Recent studies show association between stress and alternative splicing of transcripts, but without functional genome-wide or single-position analysis. We utilized the msh1 experimental system in Arabidopsis as a model of reproducible epigenetic states with stress-responsive phenotypes, including commitment to heritable memory for at least seven generations. We mapped the methylome to single-cytosine resolution with signal-detection, verified by machine learning. Differentially methylated genes were overlapped with msh1-derived transcript isoforms to show that different patterns of exonic methylation led to different levels of isoform expression. Alternatively spliced and differentially methylated genes were enriched in key regulators of growth and development and spliceosome components. Genes targeted for differential methylation also contained a known CTT motif. These results demonstrate a direct relationship in plants between environmentally responsive differential methylation and alternative splicing behavior leading to phenotype changes.