Della protein function during differential growth processes in arabidopsis

The plant hormones gibberellins (GAs) regulate multiple processes of plant development. Most of this regulation occurs at the transcriptional level, through the activity of the DELLAs, which are nuclear-localized proteins subjected to GA-mediated proteolitic degradation. DELLAs are encoded by five genes, and genetic studies show that each DELLA displays specific, but also partially overlapping roles with respect to their paralogs. In this Thesis, we have addressed two issues: (1) the contribution of DELLA multiplication to the diversification of functions controlled by GAs; and (2) the identification of direct targets regulated by DELLAs in etiolated seedlings with special attention to those involved in differential growth processes. Using combinations of mutants and transgenic lines expressing two phylogenetically distant DELLA genes (RGA and RGL2), we have found that these two DELLA proteins can perform each other's role as long as they are expressed under the reciprocal promoters, indicating that DELLA subfunctionalization relies mainly on their differential expression patterns. To identify direct DELLA targets, we have performed transcriptomic analyses of dark-grown seedlings expressing an inducible version of gai-1, a stable, dominant allele of a DELLA gene. This approach rendered a list of over 150 genes differentially expressed after induction of gai-1. The presence of several auxin-related genes among the primary targets of DELLA proteins has allowed us to establish a new role for GAs in the modulation of hypocotyl gravitropism through the repression of IAA19/MASSUGU2 expression by DELLAs. Moreover, the repression of HOOKLESS1 and the auxin efflux carriers PIN3 and PIN7 by DELLAs, is proposed as the molecular mechanism to explain the already known physiological regulation of apical hook development by GAs.Gallego Bartolomé, J. (2011). Della protein function during differential growth processes in arabidopsis [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/11403Palanci

Targeted DNA demethylation of the Arabidopsis genome using the human TET1 catalytic domain.

DNA methylation is an important epigenetic modification involved in gene regulation and transposable element silencing. Changes in DNA methylation can be heritable and, thus, can lead to the formation of stable epialleles. A well-characterized example of a stable epiallele in plants is fwa, which consists of the loss of DNA cytosine methylation (5mC) in the promoter of the FLOWERING WAGENINGEN (FWA) gene, causing up-regulation of FWA and a heritable late-flowering phenotype. Here we demonstrate that a fusion between the catalytic domain of the human demethylase TEN-ELEVEN TRANSLOCATION1 (TET1cd) and an artificial zinc finger (ZF) designed to target the FWA promoter can cause highly efficient targeted demethylation, FWA up-regulation, and a heritable late-flowering phenotype. Additional ZF-TET1cd fusions designed to target methylated regions of the CACTA1 transposon also caused targeted demethylation and changes in expression. Finally, we have developed a CRISPR/dCas9-based targeted demethylation system using the TET1cd and a modified SunTag system. Similar to the ZF-TET1cd fusions, the SunTag-TET1cd system is able to target demethylation and activate gene expression when directed to the FWA or CACTA1 loci. Our study provides tools for targeted removal of 5mC at specific loci in the genome with high specificity and minimal off-target effects. These tools provide the opportunity to develop new epialleles for traits of interest, and to reactivate expression of previously silenced genes, transgenes, or transposons

Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense

[EN] The mechanisms underlying induction and suppression of RNA silencing in the ongoing plant-virus arms race are poorly understood. We show here that virus-derived small RNAs produced by Arabidopsis Dicer-like 4 (DCL4) program an effector complex conferring antiviral immunity. Inhibition of DCL4 by a viral-encoded suppressor revealed the subordinate antiviral activity of DCL2. Accordingly, inactivating both DCL2 and DCL4 was necessary and sufficient to restore systemic infection of a suppressor-deficient virus. The effects of DCL2 were overcome by increasing viral dosage in inoculated leaves, but this could not surmount additional, non - cell autonomous effects of DCL4 specifically preventing viral unloading from the vasculature. These findings define a molecular framework for studying antiviral silencing and defense in plants.We thank members of the Voinnet laboratory for discussions and Z. Xie for dcl seeds. Funded by CNRS grant to A.D.; NSF grant MCB-0209836, NIH grant AI43288, and U.S. Department of Agriculture grant NRI 2005-35319-15280 to J.C.; and Pao Schloarship (Zhejiang University, China) to J.B. This work is dedicated to the memory of M. and G. Voinnet.Deleris, A.; Gallego Bartolomé, J.; Bao, J.; Kasschau, KD.; Carrinton, JC.; Voinnet, O. (2006). Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense. Science. 313(5783):68-71. https://doi.org/10.1126/science.11282146871313578

Co-targeting RNA Polymerases IV and V Promotes Efficient De Novo DNA Methylation in Arabidopsis.

The RNA-directed DNA methylation (RdDM) pathway in plants controls gene expression via cytosine DNA methylation. The ability to manipulate RdDM would shed light on the mechanisms and applications of DNA methylation to control gene expression. Here, we identified diverse RdDM proteins that are capable of targeting methylation and silencing in Arabidopsis when tethered to an artificial zinc finger (ZF-RdDM). We studied their order of action within the RdDM pathway by testing their ability to target methylation in different mutants. We also evaluated ectopic siRNA biogenesis, RNA polymerase V (Pol V) recruitment, targeted DNA methylation, and gene-expression changes at thousands of ZF-RdDM targets. We found that co-targeting both arms of the RdDM pathway, siRNA biogenesis and Pol V recruitment, dramatically enhanced targeted methylation. This work defines how RdDM components establish DNA methylation and enables new strategies for epigenetic gene regulation via targeted DNA methylation

The MOM1 complex recruits the RdDM machinery via MORC6 to establish de novo DNA methylation

MORPHEUS' MOLECULE1 (MOM1) is an Arabidopsis factor previously shown to mediate transcriptional silencing independent of major DNA methylation changes. Here we find that MOM1 localizes with sites of RNA-directed DNA methylation (RdDM). Tethering MOM1 with an artificial zinc finger to an unmethylated FWA promoter leads to establishment of DNA methylation and FWA silencing. This process is blocked by mutations in components of the Pol V arm of the RdDM machinery, as well as by mutation of MICRORCHIDIA 6 (MORC6). We find that at some endogenous RdDM sites, MOM1 is required to maintain DNA methylation and a closed chromatin state. In addition, efficient silencing of newly introduced FWA transgenes is impaired in the mom1 mutant. In addition to RdDM sites, we identify a group of MOM1 peaks at active chromatin near genes that colocalized with MORC6. These findings demonstrate a multifaceted role of MOM1 in genome regulation

Arabidopsis MORC proteins function in the efficient establishment of RNA directed DNA methylation.

The Microrchidia (MORC) family of ATPases are required for transposable element (TE) silencing and heterochromatin condensation in plants and animals, and C. elegans MORC-1 has been shown to topologically entrap and condense DNA. In Arabidopsis thaliana, mutation of MORCs has been shown to reactivate silent methylated genes and transposons and to decondense heterochromatic chromocenters, despite only minor changes in the maintenance of DNA methylation. Here we provide the first evidence localizing Arabidopsis MORC proteins to specific regions of chromatin and find that MORC4 and MORC7 are closely co-localized with sites of RNA-directed DNA methylation (RdDM). We further show that MORC7, when tethered to DNA by an artificial zinc finger, can facilitate the establishment of RdDM. Finally, we show that MORCs are required for the efficient RdDM mediated establishment of DNA methylation and silencing of a newly integrated FWA transgene, even though morc mutations have no effect on the maintenance of preexisting methylation at the endogenous FWA gene. We propose that MORCs function as a molecular tether in RdDM complexes to reinforce RdDM activity for methylation establishment. These findings have implications for MORC protein function in a variety of other eukaryotic organisms

A DNA methylation reader complex that enhances gene transcription

[EN] DNA methylation generally functions as a repressive transcriptional signal, but it is also known to activate gene expression. In either case, the downstream factors remain largely unknown. By using comparative interactomics, we isolated proteins in Arabidopsis thaliana that associate with methylated DNA. Two SU(VAR)3-9 homologs, the transcriptional antisilencing factor SUVH1, and SUVH3, were among the methyl reader candidates. SUVH1 and SUVH3 bound methylated DNA in vitro, were associated with euchromatic methylation in vivo, and formed a complex with two DNAJ domain-containing homologs, DNAJ1 and DNAJ2. Ectopic recruitment of DNAJ1 enhanced gene transcription in plants, yeast, and mammals. Thus, the SUVH proteins bind to methylated DNA and recruit the DNAJ proteins to enhance proximal gene expression, thereby counteracting the repressive effects of transposon insertion near genes.This work was supported by grants NIH R01 GM60398 (to S.E.J.), NIH R01G M089778 (to J.A.W.) and NIH R35 GM124736 (to S.B.R), by an EMBO Long-Term Fellowship (ALTF 1138-2014) (to C.J.H), and by a Ruth L. Kirschstein National Research Service Award (GM007185) (to L.Y.). S.E.J. is an investigator of the Howard Hughes Medical Institute.Harris, CJ.; Scheibe, M.; Wongpalee, SP.; Liu, W.; Cornett, EM.; Vaughan, RM.; Li, X.... (2018). A DNA methylation reader complex that enhances gene transcription. Science. 362(6419):1182-1186. https://doi.org/10.1126/science.aar785411821186362641