MADS dynamics : gene regulation in flower development by changes in chromatin structure and MADS-domain protein binding

Abstract During the life cycle, a plant undergoes a series of developmental phase changes. The first phase change is the transition from the initial juvenile vegetative stage into the adult vegetative phase. During the juvenile phase plants produce leaves and axillary buds, whereas during the adult phase the initiation of reproductive structures occurs. The next developmental change is the switch from vegetative to reproductive growth, when the shoot apical meristem acquires the identity of an inflorescence meristem that will then produce floral meristems. Arabidopsis floral meristems produce four concentric whorls of floral organs: sepals, petals, stamens and carpels. Each developmental change is controlled by coordinated network of regulators, known as gene regulatory networks (GRNs), which determine the transcription of a specific set of genes. The aim of the study presented in this thesis was to understand the dynamics of GRNs during floral organ development in Arabidopsis and correlate the binding of key regulatory MADS domain transcription factors with the accessibility of the chromatin in a genome-wide context. In chapter 1 and 2 we reviewed the current knowledge on the regulation of transcription in the model plant Arabidopsis thaliana. In chapter 1 we mainly focus on how the view of the GRN underling flower development has changed during the last decades, while in chapter 2 we more broadly revised the mechanisms that control developmental switches in plants. The recent introduction of next-generation sequencing and genome-wide approaches has changed our view on gene regulation and GRNs. We moved from linear genetic interactions towards global highly connected gene networks. The high numbers of interactions that were detected in protein-DNA binding profiles revealed a much higher network complexity than previously anticipated and demonstrated that master regulators of development not only control another layer of regulators, but also genes encoding structural proteins, enzymes and signalling proteins. Moreover, most transcription factors bind to their own locus, highlighting that auto-regulatory loops are a common mechanism of regulation. The discovery of interactions between transcriptional master regulators with epigenetic factors provides new insights into general transcriptional regulatory mechanisms. Switches of developmental programmes and cell fates in complex organisms are controlled at the level of gene expression by the combined action of chromatin regulators and transcription factors. Although many master regulators of meristem and organ identities have been identified, it is still not well understood how they act at the molecular level and how they can switch an entire developmental program in which thousands of genes are involved. Using flower development as a model system, in chapters 3 and 4 we investigated general concepts of transcription regulation by analysing the dynamics of protein-DNA binding, chromatin accessibility and gene expression. Using an inducible system for synchronised flower formation, we characterised DNA-binding profiles of two MADS-domain transcription factors, APETALA1 (AP1) and SEPALLATA3 (SEP3), at three stages of flower development. Our study revealed that these MADS-domain proteins, select their binding sites, and thereby their target genes, in a partly stage-specific fashion. By combining the information from DNA-binding and gene expression data, we proposed models of stage-specific GRNs in flower development. Since developmental control of gene expression is tightly linked with dynamic changes in chromatin accessibility, we identified DNase I hypersensitive sites (DHSs, chapter 3) and we characterised nucleosome occupancy (chapter 4) at different stages of flower development. We observed dynamics in chromatin landscape manifested in increasing and decreasing DHSs as well as in changes in nucleosome occupancy and position. Next, we addressed the question how MADS-domain protein stage-specific binding is achieved at the molecular level in a chromatin context. In the nucleus the DNA is wrapped around histone octamers to form nucleosomes, which are then packed into highly dense structures, and hence transcription factor binding sites may not be easily accessible. A result of the combined analysis of MADS-domain binding and chromatin dynamics is that MADS-domain proteins bind prevalently to nucleosome depleted regions, and that binding of AP1 and SEP3 to DNA precedes opening of the chromatin, which suggests that these MADS-domain transcription factors may act as so-called “pioneer factors”. The isolation and analysis of developing flowers of specific stages increased the specificity of our genome-wide experiments, enabling the identification of novel actors in the GRN that regulates flower development. In this thesis we characterised the role of some novel regulators in more detail: in chapter 3 we focussed on the GROWTH REGULATING FACTOR (GRF) family genes; in chapter 5 we investigated the action of STERILE APETALA (SAP); and in chapter 6 we elucidated the regulation and the role of a member of the WUSCHEL-related homeobox (WOX) family, WOX12. GRF family genes are dynamically bound by AP1 and SEP3 at the different stages of flower development. All family members are bound by SEP3, while only a subset of the genes is bound by AP1. The defects in floral organs observed upon down-regulation of these genes highlight their role down-stream of MADS-domain transcription factors. In addition to AP1 and SEP3, SAP is also a target of other MADS-domain proteins, such as APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG). SAP is strongly expressed in meristems and loss of function of SAP causes strong aberrations in flowers, such as a reduction in petal and stamen numbers. We found that SAP interacts with proteins of the SCF ubiquitin ligase complex, suggesting that SAP could act in the ubiquitination pathway. WOX12 down-regulation leads to defects in floral organ identity specification with the formation of stamenoid-petals, while ectopic expression of WOX12 leads to an opposite effect: it causes the formation of petaloid-stamens in the third whorl. WOX12 acts downstream of AP1. Ectopic expression of WOX12 leads to reduction of AG expression, suggesting a role for WOX12 in the antagonistic interplay between the homeotic genes AP1 and AG. In chapter 7 we discuss the findings of this thesis. Taken together, the work performed in this thesis increased our knowledge on the GRN that regulates flower development and on the mode of action of MADS-domain transcription factors. We hypothesise that MADS-domain proteins may act as pioneer factors, proteins that access and remodel condensed chromatin. However, differently from other pioneer factors, MADS-domain transcription factors do not actively deplete nucleosomes, but instead they interact with chromatin remodelers to shape chromatin landscape. Given the important roles of MADS-domain proteins as master regulators of developmental switches, their pioneer behaviour represents an intriguing mode of action. </p

Study of the bacterial community affiliated to Hyalesthes obsoletus, the insect vector of “bois noir” phytoplasma of grape

Grape yellows caused by phytoplasmas afflict several important wine-producing areas of Europe. A grape yellows with increasingincidence in European vineyards is “bois noir” (BN), caused by ‘Candidatus Phytoplasma solani’. Its vector is the planthopperHyalesthes obsoletus Signoret (Hemiptera Cixiidae), occasionally feeding on grapevine. An innovative strategy for reducing thediffusion of the disease could be symbiotic control, exploiting the action of symbiotic microorganisms of the insect host. To investigatethe occurrence of possible microbial candidates for symbiotic control we performed a molecular characterization of thebacteria associated to H. obsoletus. Length heterogeneity PCR was applied for a preliminary population screening. Taxonomicaffiliations of the bacterial species were analyzed by denaturing gradient gel electrophoresis, showing, within the microbial diversity,the intracellular reproductive parasite Wolbachia pipientis and a Bacteroidetes symbiont with 92% nt identity with ‘CandidatusSulcia muelleri’. PCR essays specific for these bacteria showed they co-localize in several organs of H. obsoletus. Fluorescentin situ hybridization was performed to assess the distribution of these microorganisms within the insect body, showing interestinglocalization patterns, particularly in insect gonads and salivary glands. These results could be a starting point for a deeper investigationof functions and relationships between microbial species

Asaia, a versatile acetic acid bacterial symbiont, capable of cross-colonizing insects of phylogenetically-distant genera and orders

Bacterial symbionts of insects have been proposed for blocking transmission of vector-borne pathogens. However, in many vector models the ecology of symbionts and their capability of cross-colonizing different hosts, an important feature in the symbiotic control approach, is poorly known. Here we show that the acetic acid bacterium Asaia, previously found in the malaria mosquito vector Anopheles stephensi, is also present in and capable of cross-colonizing other sugar-feeding insects of phylogenetically distant genera and orders. PCR, real-time PCR and in situ-hybridization experiments showed Asaia in the body of the mosquito Aedes aegypti and the leafhopper Scaphoideus titanus, vectors of human viruses and a grapevine phytoplasma, respectively. Cross colonization patterns of the body of Ae. aegypti, An. stephensi and S. titanus have been documented with Asaia strains isolated from An. stephensi or Ae. aegypti, and labelled with plasmid- or chromosome-encoded fluorescent proteins (Gfp and DsRed, respectively). Fluorescence and confocal microscopy showed that Asaia, administered with the sugar meal, efficiently colonized guts, male and female reproductive systems and the salivary glands. The ability in cross-colonizing insects of phylogenetically distant orders indicates that Asaia adopts body invasion mechanisms independent from the host biological characteristics. This versatility is an important property for the development of symbiont-based therapies of different vector-borne diseases

Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factors in flower development.

BACKGROUND: Development of eukaryotic organisms is controlled by transcription factors that trigger specific and global changes in gene expression programs. In plants, MADS-domain transcription factors act as master regulators of developmental switches and organ specification. However, the mechanisms by which these factors dynamically regulate the expression of their target genes at different developmental stages are still poorly understood. RESULTS: We characterized the relationship of chromatin accessibility, gene expression, and DNA binding of two MADS-domain proteins at different stages of Arabidopsis flower development. Dynamic changes in APETALA1 and SEPALLATA3 DNA binding correlated with changes in gene expression, and many of the target genes could be associated with the developmental stage in which they are transcriptionally controlled. We also observe dynamic changes in chromatin accessibility during flower development. Remarkably, DNA binding of APETALA1 and SEPALLATA3 is largely independent of the accessibility status of their binding regions and it can precede increases in DNA accessibility. These results suggest that APETALA1 and SEPALLATA3 may modulate chromatin accessibility, thereby facilitating access of other transcriptional regulators to their target genes. CONCLUSIONS: Our findings indicate that different homeotic factors regulate partly overlapping, yet also distinctive sets of target genes in a partly stage-specific fashion. By combining the information from DNA-binding and gene expression data, we are able to propose models of stage-specific regulatory interactions, thereby addressing dynamics of regulatory networks throughout flower development. Furthermore, MADS-domain TFs may regulate gene expression by alternative strategies, one of which is modulation of chromatin accessibility