The H3K27me3 epigenetic mark is crucial for callus cell identity and for the acquisition of new fate during root and shoot regeneration

Abstract

We combined molecular, genomic and genetic approaches to study the molecular mechanisms underlying cell totipotency and competency to regenerate in Arabidopsis.By performing comparative analysis of mRNA-seq and chromatin landscapes between leaf differentiated cells and callus totipotent cells and between WT callus and calli derived from the emf2 mutant, exhibiting impaired regenerative capacity we revealed the following:1. That callus cells express numerous genes of many developmental pathways such as root, leaf, embryo, shoot, meristem and seed. This suggests a mechanism to allow rapid response to a signal by maintaining genes of all potential developmental pathways active, without the needs to release transcriptional silencing and to go through the intricate multistep process of transcription. 2. That key transcription factors that are sufficient to derive differentiation or organogenesis are silenced and marked by the H3K27me3. 3. That callus derived from the emf2 mutant which is impaired in setting the H3K27methylation, lost the capacity to regenerate and that 78 transcription factors from which 18 regulate flower development, where up-regulated compared with WT callus.Altogether our results suggest that competency to regenerate is achieved by keeping the chromatin of developmental genes active, and that upon a signal for cell fate switch, a mechanism to repress those genes is required to allow the one desired developmental pathway to dominate. When this mechanism is impaired the capacity to regenerate is decline.

Lysine 27 of histone H3.3 is a fine modulator of developmental gene expression and stands as an epigenetic checkpoint for lignin biosynthesis in Arabidopsis

“…1h). Since the PRC2 mutant emf2 callus displays impaired capacity to regenerate shoot (Mandel et al ., 2022), we assessed this capacity for 30-day-old H3.3 K27A calli, by culturing them on Shoot Inducing Medium (SIM). After 9 days of incubation, the control H3.3 line showed dark green foci characteristic of de novo shoot organogenesis initials, whereas many root hairs appeared on H3.3 K27A line calli.…”

“…Out of the 1764 up-regulated genes in H3.3 K27A , 534 (30 %) were identified as H3K27me3-marked genes in WT calli grown under the same conditions (Mandel et al ., 2022) (Representation factor (RF)=2.1, p-value<8.495e-71 for significant overlap). The up-regulation of these 534 genes is therefore likely a direct consequence of the K27A substitution and the resulting decrease in H3K27me3 (Fig.…”

“…3b, Table S3). Furthermore, among the 534 genes up-regulated in H3.3 K27A , only 195 are part of the 370 emf2 up-regulated genes (compared with WT, log2FC≥1) (Mandel et al ., 2022). This indicates that our approach results in a widespread H3K27me3 decrease that goes beyond that of the PRC2 mutant emf2 , whose smaller population of mis-regulated genes likely reflects redundancy between PRC2 components.…”

“…S4) and further validates our RNA-seq approach on calli. Interestingly, SOC1 gene is covered by H3K27me3 but not found among the emf2 up-regulated genes (Mandel et al ., 2022).…”

“…The role of H3.3K27 is revealed by the mis-regulation of more than 3000 genes, out of which more than 1700 are up-regulated, and 620 (>36%) are direct targets of H3K27me3 or of PRC2. In general, studies on PRC2 mutants such as clf, swn or emf2 do not show such a good overlap even when considering PRC2-bound genes (Shu et al ., 2019) (Mandel et al ., 2022). Comparing our RNA-seq data with dataset obtained on emf2 calli, we found that there are many more up-regulated genes in the H3.3 K27A calli than in the PRC2 loss-of-function mutant.…”