Control of flowering and cell fate by LIF2, an RNA binding partner of the polycomb complex component LHP1.

Polycomb Repressive Complexes (PRC) modulate the epigenetic status of key cell fate and developmental regulators in eukaryotes. The chromo domain protein like heterochromatin protein1 (LHP1) is a subunit of a plant PRC1-like complex in Arabidopsis thaliana and recognizes histone H3 lysine 27 trimethylation, a silencing epigenetic mark deposited by the PRC2 complex. We have identified and studied an LHP1-Interacting Factor2 (LIF2). LIF2 protein has RNA recognition motifs and belongs to the large hnRNP protein family, which is involved in RNA processing. LIF2 interacts in vivo, in the cell nucleus, with the LHP1 chromo shadow domain. Expression of LIF2 was detected predominantly in vascular and meristematic tissues. Loss-of-function of LIF2 modifies flowering time, floral developmental homeostasis and gynoecium growth determination. lif2 ovaries have indeterminate growth and produce ectopic inflorescences with severely affected flowers showing proliferation of ectopic stigmatic papillae and ovules in short-day conditions. To look at how LIF2 acts relative to LHP1, we conducted transcriptome analyses in lif2 and lhp1 and identified a common set of deregulated genes, which showed significant enrichment in stress-response genes. By comparing expression of LHP1 targets in lif2, lhp1 and lif2 lhp1 mutants we showed that LIF2 can either antagonize or act with LHP1. Interestingly, repression of the FLC floral transcriptional regulator in lif2 mutant is accompanied by an increase in H3K27 trimethylation at the locus, without any change in LHP1 binding, suggesting that LHP1 is targeted independently from LIF2 and that LHP1 binding does not strictly correlate with gene expression. LIF2, involved in cell identity and cell fate decision, may modulate the activity of LHP1 at specific loci, during specific developmental windows or in response to environmental cues that control cell fate determination. These results highlight a novel link between plant RNA processing and Polycomb regulation.

DamID, a new tool for studying plant chromatin profiling in vivo, and its use to identify putative LHP1 target loci.

We show here that the in vivo methylation-based tagging technique DamID (DNA adenine methyltransferase identification) can be used for studies of DNA-protein interactions or chromatin profiling in plants. We have demonstrated the feasibility, reproducibility and sensitivity of the method in Arabidopsis thaliana, using the well-known yeast GAL4 transcription factor, for which DNA-binding sites (UAS(G)) were introduced into the plant genome. We monitored the methylation resulting from the activity of DNA adenine methyltransferase fused to the protein of interest, by combining digestion with methylation-sensitive restriction enzymes and quantitative PCR. We then used DamID to identify genomic targets of LHP1, a protein mostly associated with euchromatin. We showed that LHP1 was targeted to the promoter and transcribed regions of four genes: AG, AP3, FT and PI. Our data also demonstrate that LHP1, like its animal homologues, has a high binding affinity for A/T-rich regions, binding particularly strongly to the large regulatory introns of AG and PI. We identified three major characteristics of LHP1 binding, highlighting the similarities between plant LHP1 and animal HP1 proteins.