Project description:This study identified direct targets of TFL1 and FD and showed that TFL1 is recruited to the chromatin by FD. We performed ChIP-seq using anti-GFP antibodies in gTFL1-GFP tfl1-1, gTFL1-GFP fd-1 and controls (nontransgenic plants). We used anti-GUS antibodies for gFD-GUS fd-1. ChIP was conducted on dissected, non-bolted, inflorescences from 42-day-old short-day-grown Arabidopsis thaliana.
Project description:This study identified genes that re-upregulated in far-red enriched photoperiod in the wild type or tfl1-1, but not in ft-10 (FT-dependent photoinduction). We performed RNA seq on tfl1-1, wild type and ft-10 grown for 42 days in short-day conditions and treated for 24 hours with far-red enriched photoinduction (24 hr FRP) or grown for 43 days in short-day conditions (no FRP). RNA was isolated from trimmed and dissected non-bolted inflorescences.
Project description:The protein hormone florigen is a universal systemic inducer of flowering and a generic growth terminator across meristems. To understand the developmental rational for its pleiotropic functions and to uncover the deep cellular systems mobilized by florigen beyond flowering we explored the radial expansion of tomato stems. RNAseq, genetic analysis and histological validations show that endogenous, mobile, or induced florigen accelerate secondary cell wall biogenesis (SCWB), and hence vascular maturation, in coordination with but independently of flowering. Conversely, a systemic FT-like florigen antagonist from Ginkgo biloba, arrests SCWB. Downstream of florigen, RNAseq identified MADS/FUL and MIF genes, that similarly impact SCWB independent from flowering. Florigen we show is remarkably stable and distributed to all organs regardless of pre-existing endogenous levels. By accelerating SCWB, florigen triggers a global redistribution of resources, signals and mechanical loads, thereby harmonizing vascular maturation with the reproductive development it had originally set into motion.
Project description:We used mutant analysis, protein–protein interaction assays and DNA affinity purification (DAP) sequencing coupled to in silico prediction of binding syntaxes to study several bZIP proteins that assemble into florigen activation/repressor complexes (FACs/FRCs).
Project description:Validation of physiologic miRNA targets has been met with significant challenges. We employed HITS-CLIP to identify which miRNAs participate in liver regeneration, and to identify their target mRNAs. miRNA recruitment to the RISC is highly dynamic, changing more than five-fold for several miRNAs. miRNA recruitment to the RISC did not correlate with changes in overall miRNA expression for these dynamically recruited miRNAs, emphasizing the necessity to determine miRNA recruitment to the RISC in order to fully assess the impact of miRNA regulation. We incorporated RNAseq quantification of total mRNA to identify expression-weighted Ago footprints, and developed a microRNA regulatory element (MRE) prediction algorithm that represents a greater than 20-fold refinement over computational methods. These high confidence MREs were used to generate candidate competing endogenous RNA (ceRNA) networks. Conclusion: HITS-CLIP analysis provide novel insights into global miRNA:mRNA relationships in the regenerating liver .
Project description:Most eukaryotic transcription factors (TFs) are part of large protein families, with members of the same family (i.e. paralogous TFs) recognizing similar DNA-binding motifs but performing different regulatory functions. Many TF paralogs are co-expressed in the cell, and thus can compete for target sites across the genome. Here, we show that direct competition for DNA binding between TF paralogs is a major determinant of their genomic binding patterns. Using yeast proteins Cbf1 and Pho4 as our model system, we designed a high-throughput quantitative assay to capture the genomic binding profiles of competing TFs in a cell-free system. Our data shows that Cbf1 and Pho4 greatly influence each other’s occupancy by competing for their common putative genomic binding sites. The competition is different at different genomic sites, as dictated by the TFs' expression levels and their divergence in DNA-binding specificity and affinity. Analyses of ChIP-seq data show that the biophysical rules that dictate the competitive TF binding patterns in vitro are also followed in vivo, in the complex cellular environment. Furthermore, the Cbf1-Pho4 competition for genomic sites, as characterized in vitro using our new assay, plays a critical role in the specific activation of their target genes in the cell. Overall, our study highlights the importance of direct TF-TF competition for genomic binding and gene regulation by TF paralogs, and proposes an approach for studying this competition in a quantitative and high-throughput manner.