A microscopic analysis of Arabidopsis chromatin
Genetic information of eukaryotic organisms is stored as DNA in the nuclei of their cells. Nuclear DNA is associated with several proteins, which together form chromatin. The most abundant chromatin proteins arehistones,they arrange the initial packaging step of the DNA. DNA staining reveals two cytogenetically different versions of chromatin; lightly stained euchromatin and intensely stained heterochromatin. Heterochromatinization is used to keep DNA elements selectively repressed. Several modifications on histones and/or DNA are used to distinguish eu- from heterochromatin. The histone modifications are collectively called the histone code. HP1 is one of the proteins that can bind histone modifications; it has a bi-partite organization to allow simultaneous binding to histones as well as to other proteins. Arabidopsis thaliana , the model plant to investigate chromatin organization, has a small genome and a simple distribution of eu- and heterochromatin allowing an easy distinction between the two forms of chromatin by light microscopy. The fixed division pattern of the Arabidopsis root allows monitoring of the chromatin organization through developmental progression. The dynamic chromatin organization in Arabidopsis is investigated in this thesis.Chapter 2 describes a semi-automated method to quantify fluorescence intensity in intact organs and tissues, composed of several cell layers. The method has been developed andtestedon whole mount preparations of Arabidopsis root tips containing Propidium Iodide stained nuclei. With a diameter of less than 150 mm the root tip is thin enough for standard confocal 3D microscopy, which makes the organ very suitable for whole mount imaging. Advantages of the root as model system for such a study are the lack of chlorophyll and the presence of transparent cell walls with only little background fluorescence. In addition the technique enables structural and quantitative analyses of stereotype tissue patterns and cell position, thus providing important information about the developmental history of every cell. With our method we now can measure DNA amounts in spatially reconstructed nuclei of a complete root tip. In our novel averaging 3D method we calculate the mean of the summed fluorescence intensities of all nuclear sections of one nucleus and interpolate the missingsections, thereby avoiding small detection problems with accuracy comparable with the existing 3D methods. The quantification showed that vascular tissue cells endoreduplicate after the first cell division from the stem cell. Furthermore, cortical and endodermal cells progress through the cell cycle at comparable velocity as mother and daughter cells, as visualized by groups of cells containing increased amounts of DNA. The organizational changes in chromatin during development led us to investigate mobility of principal parts of chromatin like histones and histone binding proteins.Histones are the proteins organizing the first step of folding DNA into chromatin fibers. These organizing proteins need to be flexibly positioned on the DNA to allow accession to the DNA for several processes. By making use of H2B-YFP expressing plants in a wt and in a DNA methylation mutant (ddm1) background, the mobility of the core histone H2B was analysed using FRAP (Chapter 3). In both transgenic plants the heterochromatic sequences appeared, in the majority of the nuclei, as distinct spots which allowed us to determine the mobility of H2B in euchromatic, heterochromatic, and centromeric regions. The mobility of H2B was measured on a time scale of about half an hour in living cells of intact roots and three distinguishable fractions of H2B were found; a euchromatic mobile fraction, a less mobile heterochromatic fraction and an immobile fraction. The half time of recovery in euchromatin was aboud 80 seconds, therefore the binding time of a H2B protein inside a nucleosome is around 2 minutes in these regions. Heterochromatic mobility of H2B was slower with a half time of recovery of aboud 7 minutes. The centromeric H2B was shown not to be mobile at all (immobile fraction 95%). Since histones seem to be reasonably fixed in position especially in heterochromatin we wondered about the dynamics of histone binding proteins.AtLHP1 the HP1 homologue in Arabidopsis was shown to be located in foci in the euchromatic area of interphase root cell nuclei in which relatively high levels of H3K9m3 and/or H3K27m3 occur (Chapter 4), and by using FRET-FLIM, to closely interact with DNA. The interaction with DNA was quite loose as FRAP data shows an average binding time of 1.2 seconds. The mobility of AtLHP1 did not differ between foci and interfoci, and also no distinction in DNA binding efficiency was observed. The AtLHP1 containing foci most likely represent chromatin complexes controlling the expression of genes present in these foci.Using mutated versions of AtLHP1 from which the Chromodomain (CD), Chromoshadow domain (CSD), the Acidic domain (AD), or the conserved part of the hinge (H) were deleted we assessed the functions of these separate domains (Chapter 5). Localization studies showed that the CD and H are essential for foci formation, whereas no influence of deletions of the CSD or AD were observed. FRET-FLIM data showed that the CSD and AD are essential for DNA binding anywhere in the nucleus, while deletion of the CD did not have any effect on the DNA binding capacity of AtLHP1. FRAP data indicated that the AD is essential to keep AtLHP1 mobile, whereas the CD, and CSD are essential for binding. Based on these results a hypothetical model of AtLHP1 functioning is presented (Chapter 5).In Chapter 6 the results obtained in the preceding chapters are discussed. Limits of the microscopical techniques used in this thesis are evaluated. Furthermore the histone displacement caused by transcription is discussed.
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| Format: | Doctoral thesis biblioteca |
| Language: | English |
| Subjects: | arabidopsis, biological techniques, chromatin, dna, fluorescence, histones, microscopy, root tips, biologische technieken, chromatine, fluorescentie, histonen, microscopie, worteltopjes, |
| Online Access: | https://research.wur.nl/en/publications/a-microscopic-analysis-of-arabidopsis-chromatin |
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