Natural depletion of H1 in sex cells causes DNA demethylation, heterochromatin decondensation and transposon activation

Transposable elements (TEs) are largely inactive. Interestingly, a subset of TEs are naturally expressed in the vegetative cell (VC) of the male gametophyte, pollen in Arabidopsis. However, the extent and mechanism of such TE activation were unknown. Through RNA-seq, we identified pollen-activated TEs and annotated the transcriptional start sites (TSSs) and transcriptional termination sites (TTSs) using a program called Mikado. We have previously shown that H1 is expressed in sperm but not in VC, which prompted us to ectopically express H1 in VC for studying TE regulation through RNA-seq and bisulfite-seq. Our RNA-seq data shows that H1 expression in VC represses some of pollen-activated TEs. Furthermore, bisulfite-seq data from the pollen nuclei isolated via fluorescence-activated cell sorting (FACS) shows that H1 expression in VC increases DNA methylation of some of H1-repressed TEs around their TSSs, while leaving other H1-repressed TEs unchanged in DNA methylation. Our results indicate that H1 represses TE expression through both DNA methylation-dependent and -independent mechanisms and that natural depletion of H1 in VC allows TEs to be activated. Overall design: To identify pollen-activated TEs, we did RNA-seq using pollen grains from WT and four replicates were included. To find out if H1 expression in VC can repress TE expression, we performed RNA-seq using pollen grains from WT or pVC::H1 with three replicates being done for each genotype. Bisulfite-seq was performed using sperm nuclei (SN) and vegetative nuclei (VN) from WT as well as VN from pVC::H1. By comparing WT SN with WT VN, we want to show if pollen-activated TEs are regulated by DEMETER-mediated DNA demethylation and if H1 represses TE expression through DNA methylation regulation.