Alternative splicing is affected by changes in DNA and histone methylation

Abstract

Alternative splicing (AS) increases transcript and proteomic diversity of intron containing genes and has emerged as a pervasive property of eukaryotic genes. DNA methylation is a regulator of gene expression which is present in many eukaryotes. Recently, chromatin structure and histone modifications have been shown to regulate AS in humans (1), but it is unknown if this also occurs in plants. Here, I investigated the regulatory role that DNA and histone methylation have on AS in Arabidopsis thaliana. I utilized the SR protein gene family as a model system to examine changes in isoform abundance in response to chemical inhibition of DNA and histone methylation using the histone deacetylase inhibitor Tricostatin A (TSA) and the DNA demethylating agent 5-Azadeoxycytidine (Azad). RT-PCR revealed that 12 SR genes had isoforms that had altered abundance in response to TSA and Azad-C. Antagonistic effects were found when both drugs were applied since changes in splicing were only seen for 10 of the SR genes. AS patterns of the SR genes are known to change according to organ and developmental stage (2) and DNA methylation is also known to change over the lifespan of the plant (3). I investigated if organ type and developmental patterns of AS are disrupted in a DNA hypermethylation mutant. Splicing patterns of the SR genes displayed tissue and developmental specific changes in the DNA hypermethylation mutant. Computational analysis of three different DNA methylation mutants was performed using a publically available Illumina dataset (4). Widespread changes in AS was detected in each of the mutants across all types of AS. Changes in methylcytosine content within the coding region of the gene did not account for a large proportion of the novel AS events detected. Splice site sequence analysis of introns uniquely retained in each of the mutant genotypes uncovered sequence changes around the functionally important sequence elements. The results of my thesis indicate for the first time that AS in plants is regulated in part by changes in DNA methylation and histone modifications.