Conventional genetic approaches, although effective in characterizing factors involved in changes in gene activity, reach their limits when it deals with the direct impact of
epigenetic marks* on transcription and plant development; this is due, among other things, to factors with redundant and multivalent activities. In an attempt to circumvent this limitation, researchers from the Chromatin Dynamics and Developmental Transitions (ChromDev) team at
CEA-Irig/LPCV used a new epigenetic editing approach based on the
dCas9 (dead Cas9)
technology*, allowing to reveal the direct and real functions of epigenetic marks.
The epigenetic mark H3K27me3, a chromatin modification conserved in multicellular eukaryotes, is strongly associated with the repression of developmental genes. The study demonstrates its exact function in plants for the targeted recruitment, via dCas9, of enzymatic activity on the developmental gene
CUP SHAPED COTYLEDON 3 (CUC3) in
Arabidopsis thaliana, using an epigenetic editing tool. Removal of the H3K27me3 mark induces more extensive transcription of CUC3 in plant tissues, leading to altered leaf morphology and the production of bifid inflorescences.
Figure : Demonstration of the effectiveness of an epigenetic editing tool on a specific target: the CUC3 border gene of Arabidopsis thaliana, for which removal of the H3K27me3 mark leads to the extension of its expression domain, followed by developmental changes in the plant (e.g., the size and shape of rosette leaves, as illustrated here).
© CEA-Irig/LPCV/ChromDev/C. Carles
Epigenetic editing promises to be a powerful approach for dissecting the functional impacts of epigenetic marks on transcription and morphogenesis. The implementation of inducible editing systems will make it possible to monitor their effects in real time.
Epigenetic editing*: targeted molecular modification by the action of an enzyme that alters an epigenetic mark, without changing the DNA sequence. This editing aims to reprogram the activity of the targeted gene or genomic region.
Epigenetic/chromatin mark*: chemical group attached to DNA or to the histone proteins associated with it. This mark can influence gene accessibility and modulate gene transcription. The chromatin mark edited in this study is trimethylation of lysine at position 27 of histone H3 (H3K27me3); with our work, we observe its repressive role on transcription.
dCas9 technology*: a variant of the CRISPR-Cas9 technology. The latter uses a short RNA (guide RNA) to bring the Cas9 enzyme to a specific DNA sequence to make a cut, triggering a repair mechanism and thus modifying the corresponding gene. dCas9 (dead Cas9) is rendered inactive for cutting, but can still bind to a specific location in the DNA thanks to the guide RNA. When coupled with an enzyme that modifies an epigenetic mark, dCas9 brings it to a specific region of DNA to change that mark (here, to remove H3K27me3).
Fundings :
Agence Nationale de la Recherche (ANR-18-CE20-0011-01, PRC projet REWIRE coordonné par Christel Carles), Labex Gral (Grenoble Alliance for Cell and Structural Biology, ANR-10-LABX-49-01), et Graduate School CBH de l'UGA (ANR-17-EURE-0003).
Collaboration :
IBMP (Institut de Biologie Moléculaire des Plantes), Dr. Alexandre Berr.