Gene Body Methylation in Plants: Mechanisms, Functions, and Important Implications for Understanding Evolutionary Processes

Abstract

Gene body methylation (gbM) is an epigenetic mark where gene exons are methylated in the CG context only, as opposed to CHG and CHH contexts (where H stands for A, C, or T). CG methylation is transmitted transgenerationally in plants, opening the possibility that gbM may be shaped by adaptation. This presupposes, however, that gbM has a function that affects phenotype, which has been a topic of debate in the literature. Here, we review our current knowledge of gbM in plants. We start by presenting the well-elucidated mechanisms of plant gbM establishment and maintenance. We then review more controversial topics: the evolution of gbM and the potential selective pressures that act on it. Finally, we discuss the potential functions of gbM that may affect organismal phenotypes: gene expression stabilization and upregulation, inhibition of aberrant transcription (reverse and internal), prevention of aberrant intron retention, and protection against TE insertions. To bolster the review of these topics, we include novel analyses to assess the effect of gbM on transcripts. Overall, a growing body of literature finds that gbM correlates with levels and patterns of gene expression. It is not clear, however, if this is a causal relationship. Altogether, functional work suggests that the effects of gbM, if any, must be relatively small, but there is nonetheless evidence that it is shaped by natural selection. We conclude by discussing the potential adaptive character of gbM and its implications for an updated view of the mechanisms of adaptation in plants.

Keywords: DNA methylation; epigenetics; gene expression; population epigenomics; transcription.

Fig. 1.

The establishment and maintenance of gbM in plants. The DNA is represented as a line coiled around nucleosomes. Red dots indicate methylated H3K9 tails. CG, CHG, and CHH DNA methylation are drawn as black, gray, and white lollipops, respectively. (a and b) CMT3 induces de novo methylation at CHG sites of genes associated with inaccessible chromatin marks and heterochromatin histone variants (Papareddy et al. 2021). (b and c) CHG-H3K9me2 self-reinforcing feedback loop is then established. (c and d) CMT3 preferentially de novo methylates CWG sites but to a lesser extent also methylates other contexts, such as CG. (d and e) Demethylation of H3K9 by IBM1 is coupled to gene transcription. (f) After a few cell divisions, only CG methylation (mCG) remains due to MET1 maintenance.

Fig. 2.

Potential gbM functions and evolutionary consequences. Unmethylated genes, represented on the left column, are compared to gbM genes on the right. TSS stands for the transcription start site, TTS for the transcription termination site, and TE for the transposable element. (a) gbM is hypothesized to upregulate gene expression. The number of mRNA molecules, represented by wavy lines, illustrates the gene expression level. (b) gbM may stabilize gene expression by triggering consistent expression levels among the cells of a tissue. (c) gbM may stabilize gene expression, as seen by the more constant and conserved expression levels observed among species. (d) gbM could prevent aberrant internal and reverse transcription by silencing alternative promoters within genes. gbM might also inhibit aberrant TTS. These hypotheses are coherent with the typical depletion of CG methylation observed around the TSS and TTS of genes. (e) gbM is hypothesized to facilitate correct splicing and prevent aberrant intron retention. (f) Some TEs preferentially insert into genes; however, gbM may protect against deleterious insertions within genes.

Fig. 3.

Novel analyses to assess the effect of gbM on transcripts. (a) Proportion of full-length Isoseq reads with conventional TSS in gbM and UM genes in maize and A. thaliana. Isoseq reads that started after the start of exon 1 were considered as nonconventional. (b) RNA-seq read coverage ratio between exon 3 and exon 1 for gbM and UM genes in A. thaliana. Internal transcription starts happening between exon 1 and exon 3 and is expected to increase the ratio of exon 3 to exon 1 coverage. (c) RNA-seq read coverage of introns (in RPKM) for gbM and UM genes in A. thaliana. Pools of gbM genes are drawn in red, and those of UM genes are drawn in turquoise. In data set 1, WT controls were compared to met1-3 mutants. In data set 2, other WT controls were compared to met1,sdg7–8 triple mutants. The boxplots show the median, the hinges are the first and third quartiles (the 25th and 75th percentiles), and the whiskers extend from the hinge to the largest or smallest value no further than 1.5 times the interquartile range (distance between the first and third quartiles).

Fig. 4.

Tempo of epigenetic versus genetic change. The rates of change come from a series of sources (Jelesko et al. 2004; Ossowski et al. 2010; Gaut et al. 2011; van der Graaf et al. 2015).