The molecular mechanism underling plant cell competency to regenerate

Certain plants cells retain plasticity and are capable - under the appropriate stimuli, e.g. after wounding or following hormone stimulation - to switch their developmental program, re-enter the cell cycle and form a mass of less-differentiated totipotent cells, termed callus. Those cells restore a “stem cell” status and acquire a competence to respond rapidly to diverse stimuli taking on new fate accordingly and regenerate. The acquisition of new fate is characterized by massive reprogramming of gene expression for example switching on or off hundreds of developmental genes. In eukaryotes, gene expression is the outcome of transcription by RNA polymerase II (Pol II) machinery, acting on the highly dynamic platform of the chromatin and regulated by combinatorial epigenetic modifications. Two of the histone modifications, the active mark H3K4me3, catalyzed by the Trithorax group (TrxG) and the repressive mark H3K27me3 which is catalyzed by the Polycomb Repressive Complex 2 (PRC2) and their interplay can set the gene in an active, repressive or poised for activation transcriptional state.

 To elucidate the molecular mechanism underlying plant cell competency to regenerate we are examining the capacity of callus derived from various mutant of the PRC2 and TrxG to response to external cues and to regenerate. Using ChIP-seq analysis we are characterizing the H3k4me3 and H3K27me3 landscapes and mapping the PolII occupation genome wide and relate it to the transcription levels in WT and in various mutants. Our goal is to determine the combinatorial code that sets the transcriptional state of each gene into active, repressed or poised for activation in the totipotent callus and to compare it with a non-totipotent leaf cells and the various mutant.

Numerous genes in the emf2 mutant callus exhibit H3K4me3 acquisition and high expression level. Expression, H3K27me3 and H3K4me3 patterns on individual genes in WT and in emf2 mutant calli. WT and emf2 calli generated from young cotyledons were subjected to mRNA-seq and ChIP-seq analysis with specific antibodies for H3K27me3 and H3K4me3 in three biological replicates. (A) An example for a gene that in WT exhibits no H3K27me3, a peak of H3K4me3 and high expression level. The emf2 callus shows the same pattern. (B-D) example of genes that in WT exhibit H3K27me3 marks, no H3K4me3 and no transcripts. In the emf2 mutant these genes acquire H3K4me3 mark and become transcriptionally active.
                                                                                                                                                                                                       We have identified in callus 800 putative bivalent genes (see below), in which the genes are marked by both H3K27me3 and H3K4me3 and in WT no mRNA. This can serve as a mechanism to set the genes poised for activation. In the emf2 mutant, which is impaired in setting the H3K27me3 mark, those gene have the  H3K4me3 mark and the genes are expressed.

 Numerous genes in the WT callus are potentially marked by both H3K27me3 and H3K4me3 and serve as candidates for bivalent genesExamples for genes that in WT callus marked by both H3K27me3 and H3K4me3 and exhibit no transcripts. In the emf2 mutant these genes are marked by H3K4me3 and show high expression level.