Control of Flowering and Cell Fate by LIF2, an RNA Binding Partner of the Polycomb Complex Component LHP1

Polycomb Repressive Complexes (PRC) modulate the epigenetic status of key cell fate and developmental regulators in eukaryotes. The chromo domain protein LIKE HETEROCHROMATIN PROTEIN1 (LHP1) is a subunit of a plant PRC1-like complex in Arabidopsis thaliana and recognizes histone H3 lysine 27 trimethylation, a silencing epigenetic mark deposited by the PRC2 complex. We have identified and studied an LHP1-Interacting Factor2 (LIF2). LIF2 protein has RNA recognition motifs and belongs to the large hnRNP protein family, which is involved in RNA processing. LIF2 interacts in vivo, in the cell nucleus, with the LHP1 chromo shadow domain. Expression of LIF2 was detected predominantly in vascular and meristematic tissues. Loss-of-function of LIF2 modifies flowering time, floral developmental homeostasis and gynoecium growth determination. lif2 ovaries have indeterminate growth and produce ectopic inflorescences with severely affected flowers showing proliferation of ectopic stigmatic papillae and ovules in short-day conditions. To look at how LIF2 acts relative to LHP1, we conducted transcriptome analyses in lif2 and lhp1 and identified a common set of deregulated genes, which showed significant enrichment in stress-response genes. By comparing expression of LHP1 targets in lif2, lhp1 and lif2 lhp1 mutants we showed that LIF2 can either antagonize or act with LHP1. Interestingly, repression of the FLC floral transcriptional regulator in lif2 mutant is accompanied by an increase in H3K27 trimethylation at the locus, without any change in LHP1 binding, suggesting that LHP1 is targeted independently from LIF2 and that LHP1 binding does not strictly correlate with gene expression. LIF2, involved in cell identity and cell fate decision, may modulate the activity of LHP1 at specific loci, during specific developmental windows or in response to environmental cues that control cell fate determination. These results highlight a novel link between plant RNA processing and Polycomb regulation

Etude de quelques partenaires de la protéine chromatinienne LHP1 chez arabidopsis thaliana

Les mécanismes épigénétiques permettent le maintien de l'intégrité des génomes eucaryotes et sont impliqués dans le contrôle du développement ainsi que dans les réponses des organismes à l environnement, principalement par le biais de la régulation de l expression des génomes. Les protéines de la famille HP1 (Heterochromatin Protein 1) sont des composants de la chromatine impliqués dans la répression et l activation transcriptionnelles, qui reconnaissent des lysines methylés des histones avec différentes spécificités selon les membres. Elles permettent le recrutement d un grand nombre de protéines, en servant de plateforme dynamique à de nombreux partenaires protéiques. Chez Arabidopsis thaliana, LHP1, homologue de HP1 reconnaît, par son chromodomaine, la méthylation de la lysine 27 de l histone H3 (H3K27me) et représente le premier membre d un complexe PRC1 végétal. Pour caractériser d autres membres des complexes impliquant LHP1, nous avons identifié des partenaires de LHP1 et nous avons choisi d approfondir l étude de l interaction avec LIF11, un facteur de transcription ERF, et LIF4, une protéine HMGB. Nous avons montré que ces partenaires interagissent avec LHP1 via son chromo-shadow domain. Nous avons validé l interaction entre LIF4 et LHP1 in vivo. L analyse du phénotype des mutants lif4 a révélé une floraison tardive de certains allèles alors que les mutants lif11 présentent une floraison précoce. Nous avons montré que LIF4 joue un rôle dans la réponse au stress. Nous avons tenté d établir un modèle de formation du complexe LIF11/LHP1 en réponse à un stress pour comprendre les modifications de la chromatine et leur impact sur l expression des gènes.Epigenetic mechanisms allow the maintenance of eukaryotic genome integrity and are involved in organism responses to environment, mainly in regulating gene expression. The HP1 (Heterochromatin Protein 1) family proteins are essential chromatin components involved in transcriptional repression and activation of the two chromatin compartments : euchromatin and heterochromatin. HP1 proteins recognize histone methylated lysines with different specificities depending on the family members. They enable the recruitment of other proteins, thus serving as a dynamic platform. In Arabidopsis thaliana LHP1, which is HP1 plant homolog, recognizes histone H3 lysine 27 methylation (H3K27me) via its chromodomain, and so represents the first member of plants PRC1-like complex. In order to characterize other LHP1 chromatin complex members, we identified LHP1 partner proteins and we chose to study their interactions with an ERF transcription factor, LIF11, and a HMGB protein, LIF4. We determined LHP1 s interaction domains with these partners. Interaction between LHP1 and LIF4 in vivo was validated. Phenotypic analysis of lif4 mutants revealed late flowering for some specific alleles and an early flowering phenotype for lif11 mutants. We showed that LIF4 is involved in stress response. We also tried to establish a model for the LHP1 / LIF11 complex formation during stress response to understand chromatin modifications and their impact on gene expression.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

The Arabidopsis GAGA-Binding Factor BASIC PENTACYSTEINE6 Recruits the POLYCOMB-REPRESSIVE COMPLEX1 Component LIKE HETEROCHROMATIN PROTEIN1 to GAGA DNA Motifs

Polycomb-repressive complexes (PRCs) play key roles in development by repressing a large number of genes involved in various functions. Much, however, remains to be discovered about PRC-silencing mechanisms as well as their targeting to specific genomic regions. Besides other mechanisms, GAGA-binding factors in animals can guide PRC members in a sequence-specific manner to Polycomb-responsive DNA elements. Here, we show that the Arabidopsis (Arabidopsis thaliana) GAGA-motif binding factor protein BASIC PENTACYSTEINE6 (BPC6) interacts with LIKE HETEROCHROMATIN PROTEIN1 (LHP1), a PRC1 component, and associates with VERNALIZATION2 (VRN2), a PRC2 component, in vivo. By using a modified DNA-protein interaction enzyme-linked immunosorbant assay, we could show that BPC6 was required and sufficient to recruit LHP1 to GAGA motif-containing DNA probes in vitro. We also found that LHP1 interacts with VRN2 and, therefore, can function as a possible scaffold between BPC6 and VRN2. The lhp1-4 bpc4 bpc6 triple mutant displayed a pleiotropic phenotype, extreme dwarfism and early flowering, which disclosed synergistic functions of LHP1 and group II plant BPC members. Transcriptome analyses supported this synergy and suggested a possible function in the concerted repression of homeotic genes, probably through histone H3 lysine-27 trimethylation. Hence, our findings suggest striking similarities between animal and plant GAGA-binding factors in the recruitment of PRC1 and PRC2 components to Polycomb-responsive DNA element-like GAGA motifs, which must have evolved through convergent evolution