Ctf4-related protein recruits LHP1-PRC2 to maintain H3K27me3 levels in dividing cells in Arabidopsis thaliana

Abstract

Polycomb Repressive Complex (PRC) 2 catalyzes the H3K27me3 modification that warrants inheritance of a repressive chromatin structure during cell division, thereby assuring stable target gene repression in differentiated cells. It is still under investigation how H3K27me3 is passed on from maternal to filial strands during DNA replication; however, cell division can reinforce H3K27me3 coverage at target regions. To identify novel factors involved in the Polycomb pathway in plants, we performed a forward genetic screen for enhancers of the like heterochromatin protein 1 (lhp1) mutant, which shows relatively mild phenotypic alterations compared with other plant PRC mutants. We mapped enhancer of lhp1 (eol) 1 to a gene related to yeast Chromosome transmission fidelity 4 (Ctf4) based on phylogenetic analysis, structural similarities, physical interaction with the CMG helicase component SLD5, and an expression pattern confined to actively dividing cells. A combination of eol1 with the curly leaf (clf) allele, carrying a mutation in the catalytic core of PRC2, strongly enhanced the clf phenotype; furthermore, H3K27me3 coverage at target genes was strongly reduced in eol1 clf double mutants compared with clf single mutants. EOL1 physically interacted with CLF, its partially redundant paralog SWINGER (SWN), and LHP1. We propose that EOL1 interacts with LHP1-PRC2 complexes during replication and thereby participates in maintaining the H3K27me3 mark at target genes.Ctf4 | epigenetic inheritance | ENHANCER OF LHP1 1 | Polycomb repressive complex 2 | replication

Histone H2A‐H2B binding by Pol α in the eukaryotic replisome contributes to the maintenance of repressive chromatin

“…In the future, it will be of particular interest to study organisms with more complex epigenetic regulation of gene expression than Saccharomyces cerevisiae, since such studies might reveal more complex phenotypes upon mutation of the replisome's histonebinding activities. For example, we note that a recent study of the plant orthologue of Ctf4 (Zhou et al, 2017) found that it was required to preserve gene silencing that is dependent upon trimethylation of histone H3K27-an epigenetic mark not found in budding yeast. Moreover, previous work showed that mutations in fission yeast Pol alpha also lead to defects in gene silencing, reflecting displacement from chromatin of the Swi6 chromodomain protein that binds to H3K9Me-another mark that is absent in S. cerevisiae (Nakayama et al, 2001).…”

Disruption of an RNA-binding hinge region abolishes LHP1-mediated epigenetic repression

“…It is commonly regarded as a "reader" of H3K27me3 acting downstream from PRC2 activity in the silencing mechanism (Turck et al 2007). LHP1 has also been found recently to interact with a component of the DNA replication machinery (Zhou et al 2017). While the role of LHP1 in Polycomb silencing is in contrast to the traditional role of HP1 proteins in the maintenance of heterochromatin, it has been recognized more recently that many eukaryotes contain multiple HP1 paralogs, some of which have euchromatic functions (Canzio et al 2014).…”

A RETINOBLASTOMA-RELATED transcription factor network governs egg cell differentiation and stress response inArabidopsis

“…A minimum of five independent transgenic lines were randomly chosen for genetic analysis of marker selection and seed set phenotyping; two representative lines were chosen for further analysis. Transient transformation of tobacco leaf mesophyll cells was achieved by Agrobacterium -mediated infiltration, as explained previously [59].…”

“…Due to technical limitations in large-scale isolation of single egg cells required for H3K27me3 ChIP experiments, it was not possible to disentangle egg cell histone methylation patterns from the surrounding reproductive sporophytic tissues; therefore, we used mature unfertilized gynoecia. ChIP experiments were performed accordingly to the X-ChIP protocol as described in [59] using anti-GFP (Abcam, ab290), anti-RFP (AbCam, ab62341), anti-H3K27me3 (Millipore, #07-449) and anti-IgG (Abcam, ab6703) antibody.…”