Komplementationsstudien der cpd-Mutante und Analyse der CPD-Interaktionspartner mittels reverser Genetik

Anhand von Komplementationsstudien der cpd-Mutante konnte die Funktionalitaet eines CPD-GFP-Fusionsproteins in planta demonstriert werden. Die Komplementation erfolgte unter Kontrolle des endogenen CPD-Promotors und unter Kontrolle des konstitutiven CaMV-35S-Promotors. Die transgenen Pflanzen wurden zur zellulaeren und gewebespezifischen Lokalisation des CPD-Proteins verwendet. Die Lokalisation des CPD-Proteins an der ER-Membran konnte sowohl ueber die Detektion der GFP-Fluoreszenz des generierten CPD-Fusionsproteins als auch mit einer Westen-Blot-Analyse eines differentiell zentrifugierten Zellextrakts bestaetigt werden. Konditionale Komplementationsstudien der cpd-Mutante verdeutlichen die wichtige Rolle von Brassinosteroiden bei der Pflanzenentwicklung. Durch konditionale, biochemische Komplementationen wurde demonstriert, daß Brassinosteroide kontinuierlich waehrend der Pflanzenentwicklung benoetigt werden. Durch die konditionale genetische Komplementation der cpd-Mutante mit einem Oestradiol-induzierbaren Komplementationskonstrukt und der Verwendung eines unabhaengigen, induzierbaren GUS-Reporterkonstruktes war es moeglich einen Steroidtransport in planta nachzuweisen. Anhand des induziebaren GUS-Konstruktes wurde die inhomogene Oestradiol-Verteilung nach der lokal begrenzten Applikation mit Oestradiol-haltiger Lanolinpaste gezeigt. Verschiedene Studien belegen den Transport des Oestradiols. Die entsprechende lokale Komplementation der cpd-Mutante hat eine ganzheitliche Reaktion der Pflanze gezeigt, was einen Transport des Syntheseendprodukts Brassinolid oder eines nachgeschalteten Signals belegt. Die Analyse der CPD-Interaktionspartner ueber reverse Genetik hat zur Identifikation der iap2-1-Mutante gefuehrt, die eine T-DNA-Insertion in einem, fuer ein Ringfingerprotein-codierendes Gen traegt. Die Mutante weist einen vergleichbaren Phaenotyp wie CPD-Ueberexpressionslinien auf, die auf Kontrollmedium ein verstaerktes Wurzelwachstum entwickeln. Die iap2-1-Mutante zeigt auf Hochsteroidmedium eine hypersensitive Reaktion gegenueber Brassinolid, was auf einen veraenderten, endogenen Steroidgehalt oder eine modulierte Signaltransduktion hindeutet. Diese Ergebnisse stehen im Einklang mit der postulierten Funktion des IAP2-Ringfingerproteins, das als E3-Ubiquitin-Ligase an der Regulation der CPD-Proteinstabilitaet beteiligt sein kann. Es bleibt aber offen, ob IAP2 an der direkten Regulation des CPD-Proteins oder einer anderen Komponente der Brassinosteroidsignaltransduktion beteiligt ist. Die Analyse von T-DNA-Insertionsmutanten in fuenf OBP-Genen hat nicht zur Identifikation eines Steroidphaenotyps gefuehrt. Damit zeichnet sich eine redundante Funktionalitaet innerhalb der OBP-Proteinfamilie aus A. thaliana ab, wie sie z. B. bei den homologen OSH-Proteinen in Hefe beobachtet wurde ab. Analysen von Doppel- und Mehrfachmutanten der CPD-Interaktionspartner und insbesondere der obp-Mutanten werden ein genaueres Bild der IAP und OBP-Funktion geben

Natural epigenetic polymorphisms lead to intraspecific variation in Arabidopsis gene imprinting

Imprinted gene expression occurs during seed development in plants and is associated with differential DNA methylation of parental alleles, particularly at proximal transposable elements (TEs). Imprinting variability could contribute to observed parent-of-origin effects on seed development. We investigated intraspecific variation in imprinting, coupled with analysis of DNA methylation and small RNAs, among three Arabidopsis strains with diverse seed phenotypes. The majority of imprinted genes were parentally biased in the same manner among all strains. However, we identified several examples of allele-specific imprinting correlated with intraspecific epigenetic variation at a TE. We successfully predicted imprinting in additional strains based on methylation variability. We conclude that there is standing variation in imprinting even in recently diverged genotypes due to intraspecific epiallelic variation. Our data demonstrate that epiallelic variation and genomic imprinting intersect to produce novel gene expression patterns in seeds. - See more at: http://elifesciences.org/content/3/e03198#sthash.B3zTCoEp.dpufNational Science Foundation (U.S.) (MCB 1121952)Pew Charitable Trusts (Pew Scholars Program in the Biomedical Sciences)National Science Foundation (U.S.) (Graduate Research Fellowship

Dynamic Regulation of H3K27 Trimethylation during Arabidopsis Differentiation

During growth of multicellular organisms, identities of stem cells and differentiated cells need to be maintained. Cell fate is epigenetically controlled by the conserved Polycomb-group (Pc-G) proteins that repress their target genes by catalyzing histone H3 lysine 27 trimethylation (H3K27me3). Although H3K27me3 is associated with mitotically stable gene repression, a large fraction of H3K27me3 target genes are tissue-specifically activated during differentiation processes. However, in plants it is currently unclear whether H3K27me3 is already present in undifferentiated cells and dynamically regulated to permit tissue-specific gene repression or activation. We used whole-genome tiling arrays to identify the H3K27me3 target genes in undifferentiated cells of the shoot apical meristem and in differentiated leaf cells. Hundreds of genes gain or lose H3K27me3 upon differentiation, demonstrating dynamic regulation of an epigenetic modification in plants. H3K27me3 is correlated with gene repression, and its release preferentially results in tissue-specific gene activation, both during differentiation and in Pc-G mutants. We further reveal meristem- and leaf-specific targeting of individual gene families including known but also likely novel regulators of differentiation and stem cell regulation. Interestingly, H3K27me3 directly represses only specific transcription factor families, but indirectly activates others through H3K27me3-mediated silencing of microRNA genes. Furthermore, H3K27me3 targeting of genes involved in biosynthesis, transport, perception, and signal transduction of the phytohormone auxin demonstrates control of an entire signaling pathway. Based on these and previous analyses, we propose that H3K27me3 is one of the major determinants of tissue-specific expression patterns in plants, which restricts expression of its direct targets and promotes gene expression indirectly by repressing miRNA genes

EMF1 and PRC2 Cooperate to Repress Key Regulators of Arabidopsis Development

EMBRYONIC FLOWER1 (EMF1) is a plant-specific gene crucial to Arabidopsis vegetative development. Loss of function mutants in the EMF1 gene mimic the phenotype caused by mutations in Polycomb Group protein (PcG) genes, which encode epigenetic repressors that regulate many aspects of eukaryotic development. In Arabidopsis, Polycomb Repressor Complex 2 (PRC2), made of PcG proteins, catalyzes trimethylation of lysine 27 on histone H3 (H3K27me3) and PRC1-like proteins catalyze H2AK119 ubiquitination. Despite functional similarity to PcG proteins, EMF1 lacks sequence homology with known PcG proteins; thus, its role in the PcG mechanism is unclear. To study the EMF1 functions and its mechanism of action, we performed genome-wide mapping of EMF1 binding and H3K27me3 modification sites in Arabidopsis seedlings. The EMF1 binding pattern is similar to that of H3K27me3 modification on the chromosomal and genic level. ChIPOTLe peak finding and clustering analyses both show that the highly trimethylated genes also have high enrichment levels of EMF1 binding, termed EMF1_K27 genes. EMF1 interacts with regulatory genes, which are silenced to allow vegetative growth, and with genes specifying cell fates during growth and differentiation. H3K27me3 marks not only these genes but also some genes that are involved in endosperm development and maternal effects. Transcriptome analysis, coupled with the H3K27me3 pattern, of EMF1_K27 genes in emf1 and PRC2 mutants showed that EMF1 represses gene activities via diverse mechanisms and plays a novel role in the PcG mechanism

Kicking against the PRCs - a domesticated transposase antagonises silencing mediated by polycomb group proteins and is an accessory component of polycomb repressive complex 2

The Polycomb group (PcG) and trithorax group (trxG) genes play crucial roles in development by regulating expression of homeotic and other genes controlling cell fate. Both groups catalyse modifications of chromatin, particularly histone methylation, leading to epigenetic changes that affect gene activity. The trxG antagonizes the function of PcG genes by activating PcG target genes, and consequently trxG mutants suppress PcG mutant phenotypes. We previously identified the ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN1 (ALP1) gene as a genetic suppressor of mutants in the Arabidopsis PcG gene LIKE HETEROCHROMATIN PROTEIN1 (LHP1). Here, we show that ALP1 interacts genetically with several other PcG and trxG components and that it antagonizes PcG silencing. Transcriptional profiling reveals that when PcG activity is compromised numerous target genes are hyper-activated in seedlings and that in most cases this requires ALP1. Furthermore, when PcG activity is present ALP1 is needed for full activation of several floral homeotic genes that are repressed by the PcG. Strikingly, ALP1 does not encode a known chromatin protein but rather a protein related to PIF/Harbinger class transposases. Phylogenetic analysis indicates that ALP1 is broadly conserved in land plants and likely lost transposase activity and acquired a novel function during angiosperm evolution. Consistent with this, immunoprecipitation and mass spectrometry (IP-MS) show that ALP1 associates, in vivo, with core components of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a widely conserved PcG protein complex which functions as a H3K27me3 histone methyltransferase. Furthermore, in reciprocal pulldowns using the histone methyltransferase CURLY LEAF (CLF), we identify not only ALP1 and the core PRC2 components but also plant-specific accessory components including EMBRYONIC FLOWER 1 (EMF1), a transcriptional repressor previously associated with PRC1-like complexes. Taken together our data suggest that ALP1 inhibits PcG silencing by blocking the interaction of the core PRC2 with accessory components that promote its HMTase activity or its role in inhibiting transcription. ALP1 is the first example of a domesticated transposase acquiring a novel function as a PcG component. The antagonistic interaction of a modified transposase with the PcG machinery is novel and may have arisen as a means for the cognate transposon to evade host surveillance or for the host to exploit features of the transposition machinery beneficial for epigenetic regulation of gene activity.Fil: Liang, Shih Chieh. University of Edinburgh; Reino UnidoFil: Hartwig, Ben. Max Planck Institute for Plant Breeding Research; AlemaniaFil: Perera, Pumi. University of Edinburgh; Reino UnidoFil: Mora Garcia, Santiago. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; ArgentinaFil: de Leau, Erica. University of Edinburgh; Reino UnidoFil: Thornton, Harry. University of Edinburgh; Reino UnidoFil: Lima de Alves, Flavia. University of Edinburgh; Reino UnidoFil: Rapsilber, Juri. University of Edinburgh; Reino UnidoFil: Yang, Suxin. University of Edinburgh; Reino UnidoFil: James, Geo Velikkakam. Max Planck Institute for Plant Breeding Research; AlemaniaFil: Schneeberger, Korbinian. Max Planck Institute for Plant Breeding Research; AlemaniaFil: Finnegan, E. Jean. University of Edinburgh; Reino UnidoFil: Turck, Franziska. Max Planck Institute for Plant Breeding Research; AlemaniaFil: Goodrich, Justin. Mc Gill University; Canad