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Functional genomics of Arabidopsis

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Titel Functional genomics of Arabidopsis
Looptijd 02 / 2004 - 12 / 2013
Status Lopend
Dissertatie Ja
Onderzoeknummer OND1309150

Samenvatting (EN)

Overall aim and key objectives. The overall aim of his program is to elucidate the role of all Arabidopsis chromatin remodelling genes in (1) the nuclear architecture, notably heterochromatin formation, and (2) the epigenetic regulation of gene expression in development. Recent data from various biological systems indicate that the higher-order genome organization in terms of position in the nucleus and degree of (hetero)chromatinization, has a major role in epigenetic control of gene expression during growth and development. An example are the Arabidopsis fas (encoding subunits of CAP 1) mutants in which e.g. the cellular organization of the root meristem, the overall heterochromatin formation as well as SCARECROW (a gene that plays a key role in root patterning) expression are disturbed. Genes encoding chromatin-remodelling proteins that influence chromosome dynamics and heterochromatin formation are therefore the most prominent candidates to be involved in the regulation of his level of gene activity. In Arabidopsis, heterochromatic and euchromatic regions are distributed in a rather simple manner. Heterochromatic regions occur around the centromeres of all 5 Arabidopsis chromosomes, whereas the remaining parts of the chromosomes are in general euchromatic. Therefore interface nuclei contain few distinct heterochromatic compartments or chromocentres. Each chromocentre accommodates a (peri)centromeric heterochromatic region, while the euchromatin segments form 0.2 -2 Mb p loops that emanate from such chromocentre, resulting in a chromocenter-loop arrangement. We propose that the recruitment of "euchromatic" parts into heterochromatic chromocenters is used to regulate certain genes that play a key role in the differentiation of meristematic initial cells. In other words, genes like SCARECROW are located on a euchromatic arm of a chromosome, but by recruiting it into a heterochromatic chromocenter the gene is silenced in cells were it should not be active. This chromocentre loop arrangement of interphase chromosomes provides a well-defined and relatively simple framework to investigate microscopically and compare on a large scale chromatin organization in nuclei of cells at subsequent developmental stages in a developing organ in different mutant backgrounds. Further, it will be possible by FISH (fluorescence in situ hybridization) to determine the position of key genes with respect to the heterochromatic chromocenters. The relative simplicity of the Arabidopsis nucleus (compared to for example the human nucleus) makes Arabidopsis a very suitable model to perform such studies on nuclear architecture and positioning of genesin relation to development. This research will have an impact that is likely to go beyond plant systems only. The overall aim of the program will be accomplished by a combination of interrelated concrete key objectives: (1) A collection of activation tagged Arabidopsis mutants will be generated in chromatin remodelling genes to complete the set of knock-out mutants obtained from international collaborations and facilities. (2) Chromatin remodelling mutants will be analysed (i) for their nuclear architecture. (heterochromatin distribution) in defined cell types. (ii) Further, it will be tested whether the well characterized epigenetically silenced HTP gene will be activated. (3) The mutants with the most dramatic nuclear phenotype aberrations will be studied in detail by a variety of methods. It is anticipated that these more detailed studies will be limited to about 10 genes. For these, a semi-high throughput whole-mount FISH technology will be developed to allow the determination of the nuclear organization of each individual cell in a developing organ. These studies will be focused on the root meristem. In particular, the position of key regulatory genes such as SCARECROW will be determined. (4) A genome-wide expression analysis will be performed using the CA TMA facilities. (5) To develop technology to determine the causative relationship between chromatin organization and regulation of key genes. (6) To develop DAM methylation based technology by which genome-wide DNA sequences can be identified that are targets of chromatin remodelling proteins. - b. Approach. 1. The knockout mutants in chromatin remodelling genes will be obtained from international collaborations and facilities. Activation tagged mutants in the same genes will be identified by long-range PCR approaches in a library of 20,000 Arabidopsis lines carrying the four-times repeated enhancer of the CaMV promoter. 2. The nuclear architecture of all the mutants will be analysed: the morphology of the chromocenters will be described, the level of heterochromatinization will be quantified and by hybridisation with (peri)centromeric probes it will be shown which sequences are present in the heterochromatin. The nuclear architecture will be studied in root meristems and, as a reference, in young leafs. An Arabidopisis line is available that contains a well characterised epigenetically silenced hygromycin resistance gene (HTP). It is known that this gene can be activated by a mutation in the chromatin "remodelling" gene DDMI. This line can be used in a fast assay to determine the effect of a mutation(s) in epigenetic gene silencing. 3. and 4. Of the mutants with the most dramatic aberrations, the nuclear phenotype and genome activity will be established in detail by a variety of established methods and innovative microscopic approaches. A genome-wide microarray expression analysis will be performed using the NWO CATMA facilities to describe the impact of the mutation investigated on the activity of the whole genome. In addition, an innovative semi-high throughput whole mount FISH microscopic technology will be developed to be able to determine the nuclear architecture of each individual cell in the developing root. With this, the nuclear position of selected key regulatory genes such as SCARECROW will be determined. Further, these analyses will be combined with (whole mount) genome-wide (microscopic) analyses of the methylation and acetylation status of the nuclear DNA by immuno-labelling. 5. Our working hypothesis implies a direct causal relationship between nuclear architecteture and precise regulation of genes like SCARECOW. However, it is also possible that the changes in nuclear architecture have several pleiotropic effects which affect in an indirect manner the expression of a key gene. Therefore methodology has to be developed to demonstrate a direct causal relationship. With the available Jas mutants the causal relationship between F AS/nuclear architecture and gene expression can already be tested by exploiting the technology developed in the Scheres group to create specific clonal regions with mutated cells. This should lead to a more general method to determine the causal relationship between nuclear architecture and fine-tuned regulation of key genes. 6. To study in the future mechanisms by which chromatin remodelling proteins affect nuclear architecture and development it is essential to develop methodology for Arabidopsis by which target sequences can be identified. A chromatin profiling method using DNA adenine methyltransferase DAM) and cDNA microarrays will be developed for Arabidopsis and allow the genome-wide mapping of target sites of chromatin remodelling proteins in different developmental stages and cells. - c. Elements of innovation. The program is focused on the introduction of innovative cytogenetics approaches to the analysis of mutants in chromatin remodelling genes. The novel microscopical approach of whole mount FISH will broaden and deepen the toolkit of functional genomics and will allow for the first time to zoom in on the relation between nuclear architecture and activity of genes in all cells of a developing organ. The approaches taken will generate a virtually limitless collection of novel activation tag mutants in genes involved in chromatin remodelling to complement intentional effort to obtain knockout mutants. In addition, two new technologies will be developed for the future study and validation of the targets and role of chromatin organisation in (plant) development. - d. Relevance for NWO Genomics program/importance for Dutch genomics infrastructure. The programs will establish the functional relation between the set of chromatin remodelling genes and nuclear architecture and genome activity, notably in plant cell development by implementing innovative microscopy/cytogenetics. It will probably reveal new molecular mechanisms that contribute to cell differentiation, pattern formation and morphogenesis in plants and presumably other eukaryotes as well. The program builds on and extends existing expertise in the participating groups from Amsterdam, Utrecht and Wageningen. The cytogenetic expertise of the Wageningen and Amsterdam groups is unique. The Wageningen group is also reputed for its transposon tagging activities and the Utrecht group has an excellent reputation in Arabidopsis developmental genetics. This provides the optimal setting to develop the aimed-for link between nuclear organization and the molecular mechanism controlling development. The joint efforts in this program may result in the establishment of a (virtual) Center of Excellence for innovative light microscopy and genomic analysis.

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Classificatie

A21000 Landbouw en tuinbouw
C10000 Biotechnologie
D21400 Genetica
D22500 Plantkunde

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