Ping-Hung Hsieh, Shengbo He, Toby Buttress, Hongbo Gao, Matthew Couchman, Robert L. Fischer, Daniel Zilberman, and Xiaoqi Feng

Significance

Cytosine methylation is a mechanism of epigenetic inheritance—the transmission across generations of information that does not reside in DNA sequence. This transmission is mediated by enzymes that copy methylation states following DNA replication. Only a small group of plant cells—gametes and their progenitors—participates in inheritance, yet methylation is usually studied in other cell types, in which cytosine methylation within CG dinucleotides appears to be too low for stable maintenance. Here, we examine methylation in the pollen grains of Arabidopsis thaliana plants with methyltransferase mutations and show that although methylation is maintained by similar mechanisms in pollen and somatic cells, maintenance of CG methylation is more efficient in pollen, explaining how methylation can be faithfully inherited across generations.

Abstract

Cytosine DNA methylation regulates the expression of eukaryotic genes and transposons. Methylation is copied by methyltransferases after DNA replication, which results in faithful transmission of methylation patterns during cell division and, at least in flowering plants, across generations. Transgenerational inheritance is mediated by a small group of cells that includes gametes and their progenitors. However, methylation is usually analyzed in somatic tissues that do not contribute to the next generation, and the mechanisms of transgenerational inheritance are inferred from such studies. To gain a better understanding of how DNA methylation is inherited, we analyzed purified Arabidopsis thaliana sperm and vegetative cells—the cell types that comprise pollen—with mutations in the DRM, CMT2, and CMT3 methyltransferases. We find that DNA methylation dependency on these enzymes is similar in sperm, vegetative cells, and somatic tissues, although DRM activity extends into heterochromatin in vegetative cells, likely reflecting transcription of heterochromatic transposons in this cell type. We also show that lack of histone H1, which elevates heterochromatic DNA methylation in somatic tissues, does not have this effect in pollen. Instead, levels of CG methylation in wild-type sperm and vegetative cells, as well as in wild-type microspores from which both pollen cell types originate, are substantially higher than in wild-type somatic tissues and similar to those of H1-depleted roots. Our results demonstrate that the mechanisms of methylation maintenance are similar between pollen and somatic cells, but the efficiency of CG methylation is higher in pollen, allowing methylation patterns to be accurately inherited across generations.

See: http://www.pnas.org/content/113/52/15132.abstract.html?etoc

PNAS December 27, 2016; vol.113, no.52: 15132–15137

Fig. 4.

Lack of H1 does not increase heterochromatic methylation in pollen. (A–C) Box plots show DNA methylation levels in 50-bp windows within heterochromatic TEs in WT and h1 mutant root, sperm (Spm), and vegetative cell (Veg). Only windows with at least 10 informative sequenced cytosines and methylation of at least 30% for CG and 10% for CHG and CHH are included. (D) Snapshots of CG methylation in root, sperm, and vegetative cell of WT and h1 mutants. Note how DNA methylation levels compare with the horizontal dashed line that denotes 100% methylation. Underlined regions bordering heterochromatic TEs are subject to active DNA demethylation in the vegetative cell