Editing epigenetic modifications to rewire plant development – REWIRE

WP 1: Development of tools to enzymatically edit histone marks in plants (P1 and P2)

WP1.1: Chromatibody(Cb)-based edition tool for genome-wide modification of histone residues (P1)

WP 1.2: CRISPR/dCas9-based edition tool for histone modification at specific genes (P1 and P2)

WP 1.3: Assessment of the tools (P1 and P2)

We have constructed the majority of the aimed editing tools to modify marks at specific Lysine (K) residues on Histone 3 (H3), genome-wide genome-wide, using a Chromatibody, or at target loci, using the CRISPR/dCas9 tool in combination with a Lysine Modifying Enzyme (LME) (Figure 1). We developed the tools to drive mark editing at positions K4, K27 or K36 of H3, in a constitutive and constant manner, or in an inducible manner thanks to a Glucocorticoïd Receptor (GR) tag - Dexamethasone (DEX) system.

WP1.1:
We developed Cb-LME constructions aimed at being introduced in plants in order to induce global increase of me3 on H3K4 or H3K36 residues, or decrease of me3 on H3K27 residue. For this we chose well characterised LMEs: SDG2 for trimethylation of H3K4, SETD2 for trimethylation of H3K36, and REF6 and ELF6 for demethylation of H3K27me3. Thanks to careful in-depth bibliographic searches and structural modelisation analyses, we chose regions narrowed to the enzyme catalytic domain rather than the full-length protein for better precision and efficiency of the intended chromatin modifications.
In a first round of constructs, we use the UBQ10 promoter to ectopically and uniformly express the activities in the plant; these constructs are currently being introduced into Arabidopsis thaliana plants. We will also prepare constructs to express Cb-LME in a tissue-specific manner (in the shoot apex using the ULT1 promoter, in young floral primordia using the LFY promoter), to further refine our characterisation of the effects of marks rewiring on development.

WP 1.2:
We use the same LME catalytic domains as presented in WP1.1, taking good care to cut the LME proteins down to their catalytic domains (e.g. JmJ domain for REF6, in order to avoid any binding bias to other loci in the chromatin via its N-terminal zinc finger domain). We designed a couple of sgRNAs for each target (the flowering repressors FLC and FWA, the CUC3 regulator of boundaries between flower organs, the MADS family genes AG, AP3 and SEP3).

WP1.3:
Due to the Covid-19 sanitary crisis, this sub-WP got delayed and we are planning to start it in September-October, on the first obtained lines from WP 1.1 and WP 1.2.

Deliverables
1.1. Cb-LME:
ectopic constructs UBQ::Cb-LMEx --> obtained; plants currently transformed
contextual constructs --> in elaboration
1.2. dCas9-LME:
constructs UBQ::dCasFWA-LME UBQ::dCasCUC3-LME --> obtained; soon introduced in plants
constructs UBQ::dCasFLC-LME, UBQ::dCasAG-LME, UBQ::dCasAP3-LME, UBQ::dCasSEP3-LME --> in preparation
1.3. Validation of lines for genome-wide or locus-specific mark editing: delayed due to Covid-19.

Publication:
C. Thouly, M. Le Masson, X. Lai, C. C. Carles, G. Vachon. Unwinding BRAHMA functions in plants (2020). Genova, 11(1):90.

In addition to the work carried out and results achieved over the period for WP1 :
1) Extensive phenotypic and molecular studies on transgenic Arabidopsis lines expressing mutated versions of H3, which constituted the 'proof of concept' for the ANR REWIRE project. The discovery of new developmental defects (floral transition, stem elongation, over-proliferation of xylem tissues, aberrant cell types), and of hormonal regulatory pathways sensitive to mutations in H3, will be enhanced by the preparation of a first publication to be submitted in the year 2020. The data to be published have been obtained by the two ANR partners, in association with a long-term collaborator from P1 (Leor Eshed-Williams, Tel Aviv University).
2) Invitation of C. Carles (P1) to be editor for a special issue in the journal Plants (MDPI) to be co-coordinated by A. Berr (P2) and L. Eshed-Williams. Entitled «Chromatin Dynamics for Developmental Transitions in Plants«.
3) Preparation, for the special issue of a review by the partners of the REWIRE project on strategies and technologies for epigenetic editing of chromatin markers, among which the use of nanobodies and CRISPR-dCas9 are at the heart of the REWIRE RRA (submission planned for September 2020).