While maintainers of chromatin states have largely been untangled, the precise sequence of events enabling chromatin state switches from transcriptionally repressive to permissive remains unclear. The SAND domain-containing factor ULTRAPETALA1 (ULT1) activates the expression of floral MADS genes via decrease in repressive H3K27me3 marks, and interacts with the ARABIDOPSIS TRITHORAX LIKE1 (ATX1) chromatin activator (Carles & Fletcher, 2009). In the past period, we have unraveled the genome-wide effect of ULT1 and protein partners on chromatin marks, and the dynamics of chromatin landscapes in the developing flower. By genome-wide studies of chromatin dynamics, we found that changes in active marks may be pioneer for gene regulation, at the launching of developmental programs. We also have, with genetics and genome-wide approaches, positioned the ULT1 transcriptional regulator as a switch from an H3K27me3-repressed state to an active state.

        We further aim at reaching a comprehensive, dynamic view of the epigenetic roadmap for development. While the evolutionary conserved H3K4me3, H3K27me3 and H3K36me3 chromatin marks are recognized since several decades as central to cell fates, their molecular functions in transcription remain unclear, mainly because conventional genetics brought only correlative insights. We will thus assess their causal effect on development and nuclear dynamics, and dissect the molecular mechanisms of switch from one to the other. 
 

Editing epigenetic modifications to rewire plant development

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                   REWIRE PRC, 2019-2022, coord. C. Carles (Coll. M-E. Chabouté/A. Berr, IBMP Strasbourg). 
                   Permanent members: R. Blanvillain, G. Vachon, M. Le Masson. Post-doc: Kateryna Fal. 

Recent data from us and others indicate that editing of histone amino acid residues induce drastic changes in plant architecture: Arabidopsis lines accumulating H3 mutated at K27 (H3K27A) or at K36 (H3K36M) display strong defects in plant size, flowering transition, stem elongation), while those accumulating H3K4A do not. We aim at pushing the strategy further by inducing precise enzymatic editing of histone marks at H3K4, K27 or K36 residues. Our objectives are to discover their precise impact on transcription and chromatin dynamics and to rewire the development of plants to further modify their architecture at will. With epigenome editing coupled to in cyto imaging and in situ molecular analyses, we shall reach cause-effect resolution of histone mark functions.
 

Decrypting ULT1 molecular functions in gene expression switches

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             DRF Impulsion InChroMA, 2019 and PhD fellowships (ENS 2018, CEA 2019), coord. C. Carles (Coll.: J. Kadlec IBS, S. Ravanel LPCV). 

             Permanent members: M. Le Masson, G. Vachon. PhD students: C. Thouly, JB Izquierdo.

We found that ULT1 counters PRC2 at hundreds of target genes and physically interacts with the CLF PRC2 sub-unit. This, together with the discovery that ULT1 activity is independent from the JMJ H3K27me3 demethylases, indicates that ULT1 may directly imped PRC2 activity. We also found that ULT1 activates hundreds of targets in common with the BRAHMA (BRM) nucleosome SWI/SNF remodeler and physically interacts with BRM and its homologue SPAYED (SYD). Such findings, added to previously discovered interaction with the ATX1 H3K4me3 deposer and RNA Pol II, provide a unique case in eukaryotes of molecular connection between 2 antagonistic chromatin functions and put ULT1 as a candidate scaffold for chromatin state switches.

Integrative study of ULT1 as a chromatin modifier antagonist: Combining structural and functional studies, we want to resolve the molecular mechanism through which ULT1 counteracts PRC2 function, with outlooks at the atomic, molecular, cellular and in planta scales. We associate for this with J. Kadlec (IBS), specialist in biochemical, biophysical and structural analyses of chromatin complexes. Via the “Partnership for Structural Biology”, we have access to platforms for high-throughput crystallogenesis, biostructural technologies and biophysics.

Interaction analyses between ULT1 and the BRM/SYD remodelers: We are further characterising the ULT1-BRM/SYD interactions using domain deletions, and analysing ChIP-seq and RNA-seq datasets to identify groups of co-regulated, bound genes. With loss- and gain-of-function lines we want to evaluate the overlap between ULT and BRM/SYD developmental pathways as well as BRM-ULT1 interdependency to bind and regulate chromatin. 

Characterisation of ULT1 protein complexes: In order to better comprehend ULT1 activities, we are currently identifying novel interactors by TAP-MS on cell cultures and IP-MS on inflorescences, as well as proximity labelling (coll. K. Gasser, Univ Regensburg; K. Kaufmann, Univ. Berlin; EDyP). 
All together, these investigations should allow decrypting a unique mechanism for switches in chromatin states.  

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Key words

Chromatin, transcriptional activation, plant development, stem cells, differentiation, flower morphogenesis, Arabidopsis thaliana