Refine
Has Fulltext
- yes (4)
Document Type
- Doctoral Thesis (4) (remove)
Language
- English (4)
Is part of the Bibliography
- yes (4)
Keywords
- transcription factor (4) (remove)
Institute
- Institut für Biochemie und Biologie (4) (remove)
The NAC transcription factor (TF) JUNGBRUNNEN1 (JUB1) is an important negative regulator of plant senescence, as well as of gibberellic acid (GA) and brassinosteroid (BR) biosynthesis in Arabidopsis thaliana. Overexpression of JUB1 promotes longevity and enhances tolerance to drought and other abiotic stresses. A similar role of JUB1 has been observed in other plant species, including tomato and banana. Our data show that JUB1 overexpressors (JUB1-OXs) accumulate higher levels of proline than WT plants under control conditions, during the onset of drought stress, and thereafter. We identified that overexpression of JUB1 induces key proline biosynthesis and suppresses key proline degradation genes. Furthermore, bZIP63, the transcription factor involved in proline metabolism, was identified as a novel downstream target of JUB1 by Yeast One-Hybrid (Y1H) analysis and Chromatin immunoprecipitation (ChIP). However, based on Electrophoretic Mobility Shift Assay (EMSA), direct binding of JUB1 to bZIP63 could not be confirmed. Our data indicate that JUB1-OX plants exhibit reduced stomatal conductance under control conditions. However, selective overexpression of JUB1 in guard cells did not improve drought stress tolerance in Arabidopsis. Moreover, the drought-tolerant phenotype of JUB1 overexpressors does not solely depend on the transcriptional control of the DREB2A gene. Thus, our data suggest that JUB1 confers tolerance to drought stress by regulating multiple components. Until today, none of the previous studies on JUB1´s regulatory network focused on identifying protein-protein interactions. We, therefore, performed a yeast two-hybrid screen (Y2H) which identified several protein interactors of JUB1, two of which are the calcium-binding proteins CaM1 and CaM4. Both proteins interact with JUB1 in the nucleus of Arabidopsis protoplasts. Moreover, JUB1 is expressed with CaM1 and CaM4 under the same conditions. Since CaM1.1 and CaM4.1 encode proteins with identical amino acid sequences, all further experiments were performed with constructs involving the CaM4 coding sequence. Our data show that JUB1 harbors multiple CaM-binding sites, which are localized in both the N-terminal and C-terminal regions of the protein. One of the CaM-binding sites, localized in the DNA-binding domain of JUB1, was identified as a functional CaM-binding site since its mutation strongly reduced the binding of CaM4 to JUB1. Furthermore, JUB1 transactivates expression of the stress-related gene DREB2A in mesophyll cells; this effect is significantly reduced when the calcium-binding protein CaM4 is expressed as well. Overexpression of both genes in Arabidopsis results in early senescence observed through lower chlorophyll content and an enhanced expression of senescence-associated genes (SAGs) when compared with single JUB1 overexpressors. Our data also show that JUB1 and CaM4 proteins interact in senescent leaves, which have increased Ca2+ levels when compared to young leaves. Collectively, our data indicate that JUB1 activity towards its downstream targets is fine-tuned by calcium-binding proteins during leaf senescence.
With Saccharomyces cerevisiae being a commonly used host organism for synthetic biology and biotechnology approaches, the work presented here aims at the development of novel tools to improve and facilitate pathway engineering and heterologous protein production in yeast. Initially, the multi-part assembly strategy AssemblX was established, which allows the fast, user-friendly and highly efficient construction of up to 25 units, e.g. genes, into a single DNA construct. To speed up complex assembly projects, starting from sub-gene fragments and resulting in mini-chromosome sized constructs, AssemblX follows a level-based approach: Level 0 stands for the assembly of genes from multiple sub-gene fragments; Level 1 for the combination of up to five Level 0 units into one Level 1 module; Level 2 for linkages of up to five Level 1 modules into one Level 2 module. This way, all Level 0 and subsequently all Level 1 assemblies can be carried out simultaneously. Individually planned, overlap-based Level 0 assemblies enable scar-free and sequence-independent assemblies of transcriptional units, without limitations in fragment number, size or content. Level 1 and Level 2 assemblies, which are carried out via predefined, computationally optimized homology regions, follow a standardized, highly efficient and PCR-free scheme. AssemblX follows a virtually sequence-independent scheme with no need for time-consuming domestication of assembly parts. To minimize the risk of human error and to facilitate the planning of assembly projects, especially for individually designed Level 0 constructs, the whole AssemblX process is accompanied by a user-friendly webtool. This webtool provides the user with an easy-to-use operating surface and returns a bench-protocol including all cloning steps. The efficiency of the assembly process is further boosted through the implementation of different features, e.g. ccdB counter selection and marker switching/reconstitution. Due to the design of homology regions and vector backbones the user can flexibly choose between various overlap-based cloning methods, enabling cost-efficient assemblies which can be carried out either in E. coli or yeast. Protein production in yeast is additionally supported by a characterized library of 40 constitutive promoters, fully integrated into the AssemblX toolbox. This provides the user with a starting point for protein balancing and pathway engineering. Furthermore, the final assembly cassette can be subcloned into any vector, giving the user the flexibility to transfer the individual construct into any host organism different from yeast.
As successful production of heterologous compounds generally requires a precise adjustment of protein levels or even manipulation of the host genome to e.g. inhibit unwanted feedback regulations, the optogenetic transcriptional regulation tool PhiReX was designed. In recent years, light induction was reported to enable easy, reversible, fast, non-toxic and nearly gratuitous regulation, thereby providing manifold advantages compared to conventional chemical inducers. The optogenetic interface established in this study is based on the photoreceptor PhyB and its interacting protein PIF3. Both proteins, derived from Arabidopsis thaliana, dimerize in a red/far-red light-responsive manner. This interaction depends on a chromophore, naturally not available in yeast. By fusing split proteins to both components of the optical dimerizer, active enzymes can be reconstituted in a light-dependent manner. For the construction of the red/far-red light sensing gene expression system PhiReX, a customizable synTALE-DNA binding domain was fused to PhyB, and a VP64 activation domain to PIF3. The synTALE-based transcription factor allows programmable targeting of any desired promoter region. The first, plasmid-based PhiReX version mediates chromophore- and light-dependent expression of the reporter gene, but required further optimization regarding its robustness, basal expression and maximum output. This was achieved by genome-integration of the optical regulator pair, by cloning the reporter cassette on a high-copy plasmid and by additional molecular modifications of the fusion proteins regarding their cellular localization. In combination, this results in a robust and efficient activation of cells over an incubation time of at least 48 h. Finally, to boost the potential of PhiReX for biotechnological applications, yeast was engineered to produce the chromophore. This overcomes the need to supply the expensive and photo-labile compound exogenously. The expression output mediated through PhiReX is comparable to the strong constitutive yeast TDH3 promoter and - in the experiments described here - clearly exceeds the commonly used galactose inducible GAL1 promoter.
The fast-developing field of synthetic biology enables the construction of complete synthetic genomes. The upcoming Synthetic Yeast Sc2.0 Project is currently underway to redesign and synthesize the S. cerevisiae genome. As a prerequisite for the so-called “SCRaMbLE” system, all Sc2.0 chromosomes incorporate symmetrical target sites for Cre recombinase (loxPsym sites), enabling rearrangement of the yeast genome after induction of Cre with the toxic hormonal substance beta-estradiol. To overcome the safety concern linked to the use of beta-estradiol, a red light-inducible Cre recombinase, dubbed L-SCRaMbLE, was established in this study. L-SCRaMbLE was demonstrated to allow a time- and chromophore-dependent recombination with reliable off-states when applied to a plasmid containing four genes of the beta-carotene pathway, each flanked with loxPsym sites. When directly compared to the original induction system, L-SCRaMbLE generates a larger variety of recombination events and lower basal activity. In conclusion, L-SCRaMbLE provides a promising and powerful tool for genome rearrangement.
The three tools developed in this study provide so far unmatched possibilities to tackle complex synthetic biology projects in yeast by addressing three different stages: fast and reliable biosynthetic pathway assembly; highly specific, orthogonal gene regulation; and tightly controlled synthetic evolution of loxPsym-containing DNA constructs.
Leaf senescence is an active process required for plant survival, and it is flexibly controlled, allowing plant adaptation to environmental conditions. Although senescence is largely an age-dependent process, it can be triggered by environmental signals and stresses. Leaf senescence coordinates the breakdown and turnover of many cellular components, allowing a massive remobilization and recycling of nutrients from senescing tissues to other organs (e.g., young leaves, roots, and seeds), thus enhancing the fitness of the plant. Such metabolic coordination requires a tight regulation of gene expression. One important mechanism for the regulation of gene expression is at the transcriptional level via transcription factors (TFs). The NAC TF family (NAM, ATAF, CUC) includes various members that show elevated expression during senescence, including ORE1 (ANAC092/AtNAC2) among others. ORE1 was first reported in a screen for mutants with delayed senescence (oresara1, 2, 3, and 11). It was named after the Korean word “oresara,” meaning “long-living,” and abbreviated to ORE1, 2, 3, and 11, respectively. Although the pivotal role of ORE1 in controlling leaf senescence has recently been demonstrated, the underlying molecular mechanisms and the pathways it regulates are still poorly understood. To unravel the signaling cascade through which ORE1 exerts its function, we analyzed particular features of regulatory pathways up-stream and down-stream of ORE1. We identified characteristic spatial and temporal expression patterns of ORE1 that are conserved in Arabidopsis thaliana and Nicotiana tabacum and that link ORE1 expression to senescence as well as to salt stress. We proved that ORE1 positively regulates natural and dark-induced senescence. Molecular characterization of the ORE1 promoter in silico and experimentally suggested a role of the 5’UTR in mediating ORE1 expression. ORE1 is a putative substrate of a calcium-dependent protein kinase named CKOR (unpublished data). Promising data revealed a positive regulation of putative ORE1 targets by CKOR, suggesting the phosphorylation of ORE1 as a requirement for its regulation. Additionally, as part of the ORE1 up-stream regulatory pathway, we identified the NAC TF ATAF1 which was able to transactivate the ORE1 promoter in vivo. Expression studies using chemically inducible ORE1 overexpression lines and transactivation assays employing leaf mesophyll cell protoplasts provided information on target genes whose expression was rapidly induced upon ORE1 induction. First, a set of target genes was established and referred to as early responding in the ORE1 regulatory network. The consensus binding site (BS) of ORE1 was characterized. Analysis of some putative targets revealed the presence of ORE1 BSs in their promoters and the in vitro and in vivo binding of ORE1 to their promoters. Among these putative target genes, BIFUNCTIONAL NUCLEASE I (BFN1) and VND-Interacting2 (VNI2) were further characterized. The expression of BFN1 was found to be dependent on the presence of ORE1. Our results provide convincing data which support a role for BFN1 as a direct target of ORE1. Characterization of VNI2 in age-dependent and stress-induced senescence revealed ORE1 as a key up-stream regulator since it can bind and activate VNI2 expression in vivo and in vitro. Furthermore, VNI2 was able to promote or delay senescence depending on the presence of an activation domain located in its C-terminal region. The plasticity of this gene might include alternative splicing (AS) to regulate its function in different organs and at different developmental stages, particularly during senescence. A model is proposed on the molecular mechanism governing the dual role of VNI2 during senescence.
A phagocyte-specific Irf8 gene enhancer establishes early conventional dendritic cell commitment
(2011)
Haematopoietic development is a complex process that is strictly hierarchically organized. Here, the phagocyte lineages are a very heterogeneous cell compartment with specialized functions in innate immunity and induction of adaptive immune responses. Their generation from a common precursor must be tightly controlled. Interference within lineage formation programs for example by mutation or change in expression levels of transcription factors (TF) is causative to leukaemia. However, the molecular mechanisms driving specification into distinct phagocytes remain poorly understood. In the present study I identify the transcription factor Interferon Regulatory Factor 8 (IRF8) as the specification factor of dendritic cell (DC) commitment in early phagocyte precursors. Employing an IRF8 reporter mouse, I showed the distinct Irf8 expression in haematopoietic lineage diversification and isolated a novel bone marrow resident progenitor which selectively differentiates into CD8α+ conventional dendritic cells (cDCs) in vivo. This progenitor strictly depends on Irf8 expression to properly establish its transcriptional DC program while suppressing a lineage-inappropriate neutrophile program. Moreover, I demonstrated that Irf8 expression during this cDC commitment-step depends on a newly discovered myeloid-specific cis-enhancer which is controlled by the haematopoietic transcription factors PU.1 and RUNX1. Interference with their binding leads to abrogation of Irf8 expression, subsequently to disturbed cell fate decisions, demonstrating the importance of these factors for proper phagocyte cell development. Collectively, these data delineate a transcriptional program establishing cDC fate choice with IRF8 in its center.