The crwn1 and crwn2 mutations have opposite and additive effects on constitutive heterochromatin

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Components of plant heterochromatin

In eukaryotic cells, the chromosomes are formed by complexes of DNA and proteins that are called chromatin. In 1928, Emil Heitz classified chromatin into 2 types: heterochromatin and euchromatin. Whereas the first remains highly condensed throughout the cell cycle, the latest decondenses during interphase. This binary classification system, which was originally based on cytological observations in mosses, is still widely used to describe chromatin in all eukaryotes. However, it has evolved tremendously in the past 15 years, and central dogmas, such as the inertness and transcriptional inactivity of heterochromatin, have been challenged.
The classification system has been expanded to include molecular and biochemical character-istics, such as symmetric or asymmetric DNA methylation, post-translational histone modifi-cations, nucleosome composition and arrangement, and transcriptional status, as determined by specialized polymerases. Chromatin states at the scale of the nucleus are difficult to de-termine due to limitations in resolution, and only the relatively large-scale heterochromatin compartments of interphase nuclei have been analyzed using cytological approaches.
The main heterochromatic regions of A. thaliana, which are visible by microscopy after DNA counterstaining, occur at the centromeres, pericentromeric regions, telomeres, and nucleolar organizer regions (NORs) (Figures 2.1 and 5.2). These regions are referred to as constitutive heterochromatin, whereas chromatin that occasionally acquires heterochromatin characteristics and is dispersed throughout the genome is known as facultative heterochromatin.
The cytological appearance of plant heterochromatin varies depending on the genome size (ranging from 63–149,000 Mb) [Heslop-Harrison and Schwarzacher (2011)] and chromosomal organization (ranging in dicotyledonous species from 2n = 4, such as in Haplopappus gracilis, to 2n = 640 in Sedum suaveolens; http://www.tropicos.org/Project/IPCN).
Plant heterochromatin is either located in discrete and well-defined subnuclear regions that exhibit intense labeling with DNA stain, also called chromocenters (CCs) in some species, e.g. A. thaliana and Oryza sativa (rice), or it is distributed throughout the genome in less defined substructures as, for instance, in Zea mays (maize).
The heterochromatin fraction of A. thaliana is estimated to account for 7.1 % of the total chromosome length at pachytene ( 330 m) based on a cytological approach [Fransz et al. (1998)], for 10–15 % of the genome based on the genome sequence [Arabidopsis Genome Ini-tiative (2000)], and for 16% of the genome (22 Mb out of the 135 Mb of the genome) based on DNA accessibility analyzed by DNase I chip [Shu et al. (2012)].
The relative heterochromatin fraction (RHF), defined as the ratio between the sum of inten-sities of the chromocenter pixels and the whole nucleus fluorescence intensity in DAPI (4’, 6-diamidino-2-phenylindole) counterstaining, is estimated to be 15 % [Soppe et al. (2002); Schönrock et al. (2006b)], with variations depending on cell type and developmental and envi-ronmental cues. The confidence of this important measure could be put into question due to the intensity variation between acquisitions.
Heterochromatin is rich in repetitive DNA sequences and transposable elements, has few genes, and exhibits little or no transcriptional activity. Furthermore, it presents distinct molecular and biochemical variations according to localization and function. Centromeres are the primary constrictions along mitotic/meiotic chromosomes. The relative location of the centromere differs for each type of chromosome (Figure 2.1) (detailed in Ma et al. (2007)). Centromeres of A. thaliana are composed of arrays of a 178 bp satellite repeat, ranging from 0.4 to 1.4 Mb in different chromosomes [Fransz et al. (1998); Copenhaver et al. (1999); Heslop-Harrison et al.

Arabidopsis model of chromosome organization centered on heterochromatin

In A. thaliana, chromocenters correspond to the coalescence of centromeric and pericentromeric regions of a chromosome and the NOR (if the chromosome bears a NOR). These heterochro-matic structures function as genome organizer centers. Indeed, euchromatic chromosomal re-gions form loops that are 0.2–2 Mb long and are anchored to CCs [Fransz et al. (2002)]. This organization contributes to the overall structure of chromosome territories as described in the chromocenter-loop model [Fransz et al. (2002)], also named the rosette-like model [van Driel and Fransz (2004)]. Furthermore, it was shown that highly repetitive elements and TEs located on euchromatic chromosomal arms, colocalize with CCs and remain associated with CCs despite extensive demethylation of the genome [Soppe et al. (2002)]. This suggests that TEs both anchor the euchromatin loops and organize the pericentromeric regions [Soppe et al. (2002)]. Variations in the number, size and shape of centromeric foci and CCs as well as the cell type-specific organization of heterochromatin have been reported in a number of studies. The nuclei of most cells (e.g. parenchyma cells, epidermal guard cells, and root cells) exhibit a ’classical CC’ pattern, with 4–12 (mean 8-11) conspicuous CCs (Figures 2.2 and 5.2) [Fransz et al. (2002); Andrey et al. (2010)]. The heterochromatin index (HX), defined as the percentage of nuclei showing the classical CC pattern, was thus calculated in numerous studies to quantify heterochromatin distribution [Fransz et al. (2003)]. However, nuclei with uniform DAPI fluorescent nucleoplasms have been reported in some cells such as the diploid interphase tapetal cells of premeiotic anthers [Weiss and Maluszynska (2001); Talbert et al. (2002)]. In the root tip, centromeric foci exist in a vari-ety of shapes, from dots of 0.5 m in diameter to discontinuous strings (1.0–2.0 m in length) of smaller bead-like dots, suggesting that centromeres have a range of compaction ratios [Talbert et al. (2002)]. Given that the root tip is actively dividing, this range in centromeric foci shape might be, at least partially, cell cycle dependent. Interestingly, nuclei of the triploid endosperm tissue also have a peculiar heterochromatin organization, with small CCs and additional hete-rochromatic foci interspersed in euchromatin which is likely linked to parental dosage [Baroux et al. (2007)].

Distribution of heterochromatin in the nuclear domain

In some species, chromosomes exhibit a polarized orientation with all centromeres clustered at one pole of the interphase nucleus and all telomeres at the other. This peculiar interphase nuclear organization, originally observed in salamander cell nuclei, was named the Rabl config-uration [Rabl (1885)]. It has been described in Allium cepa (onion), Hordeum vulgare (barley), Triticum aestivum (wheat), Secale cereale (rye), and Avena sativa (oats) [Stack and Clarke (1974); Schwarzacher et al. (1989); Dong and Jiang (1998); Santos and Shaw (2004); Roberts et al. (2009)]. However this configuration is not present in all species. The Rabl configuration is not displayed in A. thaliana interphase nuclei. Rather, the centromeres are located at CCs which preferentially occupy peripheral positions, and the telomeres are preferentially associated with the nucleolus [Armstrong et al. (2001); Fransz et al. (2002); Schubert et al. (2012)].
Interestingly, it was observed that plants with large genomes, e.g. A. cepa, 149,000 Mb, tend to exhibit the Rabl pattern, whereas those with smaller genomes, e.g. A. thaliana, 135 Mb, tend to exhibit a non-Rabl pattern. These data suggest a correlation between the Rabl configuration and genome size. However, the non-Rabl configuration was also reported in Sorghum bicolor (sorghum) and maize [Dong and Jiang (1998)], two species with quite large genomes. Thus, other determinants of the Rabl configuration may exist.
Even more interesting, species of the same plant with a small genome size can exhibit both spatial patterns, Rabl and non-Rabl configurations, as Brachypodium [Idziak et al. (2015)]. Moreover the non-Rabl configuration appears to be tissue-specific in diploid rice; whereas the Rabl configuration is present in root xylem vessels, it is absent in other root tissues [Prieto et al. (2004)]. Endoreduplication may occur in the large nuclei of vascular tissues and induce these changes in chromatin distribution, in agreement with the previously described correlation, or the large nucleolus of xylem cells might interfer with the redistribution of centromeres and telomeres. The preferential locations of telomeres at the nucleolus and the dispersed peripheral distribution of centromeres were also observed during meiotic interphase in A. thaliana [Arm-strong et al. (2001)]. In meiotic prophase of most species (e.g. A. thaliana and maize), the ends of chromosomes cluster together on the inner surface of the nuclear envelope and form a structure called the ’bouquet’ [Franklin and Cande (1999); Cowan et al. (2001); Tiang et al. (2012)]. Thus, in maize the Rabl configuration is observed prior to the last premeiotic cell division and is lost during the following interphase [Bass et al. (1997)] and a bouquet is formed in meiotic prophase.
These observations demonstrate that the distribution of chromosomes in the nuclear volume is tightly regulated. Many studies using fixed nuclei have reported that A. thaliana centromeres tend to preferentially localize to the nuclear periphery. This centromere distribution was con-firmed by measuring the distances between centromeres and the nuclear envelope in 3D images of various diploid living cells from transgenic A. thaliana plants expressing HTR12-GFP [Fang and Spector (2005)]. This distance measurement is not sufficient to confirm a specific trend of a spatial organization. Considering the definition of peripheral as the fact to be far from the domain center, in a simple spherical domain an object has more probabilities to be located ’at the periphery’ just by the chances derived from this domain zone with larger volume (see Figure 2.10).Besides a statistical analysis would be required to demonstrate this peripheral organization and validate the departure from pure randomness. Live-cell imaging also revealed that centromeres cluster transiently at opposite poles at the end of mitosis in root meristem-atic cells [Fang and Spector (2005)] and in root tip cells [Lindhout et al. (2007)]. Lastly, in A. thaliana and A. lyrata interphase nuclei, CCs from NOR-bearing chromosomes 2 and 4 are more frequently located in close proximity to the nucleolus [Fransz et al. (2002); Berr et al. (2006); Schubert et al. (2012)].
Using spatial statistics, a recent study showed that the 3D intra-nuclear distribution of CCs in leaf cell nuclei was not completely random and that this distribution was more regular than a completely random one [Andrey et al. (2010)]. This finding was observed in both round and elongated nuclei of plant cells which differ in differentiation stage and ploidy level. This regularity trend was evidenced based on the global analysis of the chromocenters population. Therefore, it is not incompatible with some frequent associations of specific CCs, such as CC2s and CC4s. CCs in close proximity (CC clusters) has also been reported by de Nooijer et al. (2009). How-ever, in this study the frequency and the intensity of the phenomenon remain elusive as no quantification was provided.
Thereby CCs could be obeying a global apparent repulsive trend showing some attraction between specific CCs. It remains to be determined whether this regular distribution of CCs can be fully explained by the peripheral positioning or whether additional constraints are present to explain the apparent distancing between CCs. For example, the existence of euchromatin loops anchored at CCs, as proposed by the rosette model of chromosome organization [Fransz et al. (2002)], could prevent CCs from coming into close proximity. Specific proteins may also be involved as recently demonstrated by the clustering of centromeres in CAP-D protein mutants [Schubert et al. (2013)].

Dynamics of the heterochromatin compartment during development

The plant life cycle is characterized by major developmental phase transitions and the reiterative production of plant phytomers but also by diverse adaptations to environmental changes. These events require transcriptional reprogramming events that modulate the expression of specific sets of genes. Recent studies showed that these transcriptional reprogramming events are accompanied by reorganization of heterochromatin compartments, illustrating that the nucleus is highly plastic [Baroux et al. (2011); Schubert and Shaw (2011); van Zanten et al. (2012a)]. Whether this reorganization participates in or is a consequence of gene regulation remains to be elucidated. The female spore mother cell (or megaspore mother cell, MMC) differentiates from somatic cells within ovules and ultimately gives rise to female gametes. Large-scale chromatin repro-gramming occurs during the specification of the MMC, and this probably contributes to the acquisition of the gametophyte fate [Baroux et al. (2011)]. During this nuclear reorganiza-tion, the nucleolus and nucleus expand, the RHF and CC undergo a reduction in number, and the heterochromatin decondenses [She et al. (2013)]. MMC chromatin reprogramming may be divided into 2 distinct phases: an early and rapid phase during which the composition of the nucleosome changes, followed by a late phase during which histone modifications undergo important changes [She et al. (2013)].
In A. thaliana, embryonic development is completed about 10 days after pollination (DAP). After a phase of seed maturation, which involves the accumulation of sufficient reserves and desiccation (from 10–20 DAP), the seed undergoes a period of dormancy. Seed maturation is accompanied by 2 independent processes, nuclear shrinkage and chromatin compaction, which occur between 8 and 12 DAP and precede the major dehydration event of the maturing seed [Mansfield and Briarty (1992); van Zanten et al. (2011); van Zanten et al. (2012b)]. The RHF in embryonic cotyledon nuclei increases sharply during the maturation phase, while the 45S rDNA loci and the centromeric and pericentromeric repeats remain localized to the CCs. Interestingly, the nuclear volume is independent of both the moisture content and dormancy status of the seed but is developmentally controlled. ABSCISIC ACID INSENSITIVE3 (ABI3), a key transcription factor in seed maturation, participates in nuclear shrinkage which is thought to be a general adaptive response to desiccation tolerance [van Zanten et al. (2011)].
During the early events of seed germination (48–72 h after imbibition), the nuclear volume in-creases again, and this increase requires the activity of LITTLE NUCLEI1 (LINC1) and LINC2, 2 lamin-like analogues -recently renamed to CROWDED NUCLEI (CRWN)- [van Zanten et al. (2011); van Zanten et al. (2012b); Ciska et al. (2013)]. Furthermore, chromatin reorganiza-tion accompanies this event. Whereas the 45S rDNA loci remain localized to CCs during germination, the centromeric and pericentromeric repeats are more dispersed at the onset of germination [van Zanten et al. (2012b)]. These CCs are smaller than those present in mature seeds. The classical conspicuous CC pattern reappears later during seedling growth.
During floral transition, which corresponds to the short developmental switch from the vegeta-tive to the reproductive phase, a transient reduction in both RHF and HX was observed in 3 accessions [Landsberg erecta (Ler ), Col-0, Cvi] which was accompanied by the decompaction of pericentromeric regions and 5S rDNA chromatin, followed by their subsequent relocation to CCs 3 days after bolting [Tessadori et al. (2007a)].

Dynamics of the heterochromatin compartment in response to environmental cues

Two recent studies reported a correlation between heterochromatin organization and ambient light intensity; specifically, the RHF and HX increase with a rise in light intensity [Tessadori et al. (2009); van Zanten et al. (2010, 2012a)].
In the first study, Tessadori et al. (2009) analyzed the HX in 21 A. thaliana accessions orig-inating from different geographical habitats and identified a significant correlation between geographical latitude, which determines the photon flux density (light intensity) of the re-gion, and the HX. Interestingly, the HX was found to plateau (at 100 mol m2 s-1 for Col-0 and at 200 mol m2 s-1 for Ler, a widely-used Central-European accession). The lowest HX was observed in the sub-tropical Cvi-0 accession which has smaller and fewer CCs than Ler. Furthermore, the Cvi-0 accession exhibited dispersed 5S rDNA and pericentromeric repeats, and the centromeric and 45S rDNA sequences remained in the reduced CCs. This chromatin arrangement is reminiscent of the one observed during floral transition.
The second study showed that chromatin compaction progressively decreases after a reduction in light intensity from 200 to 15 mol m2 s-1. This heterochromatic event is reversible with return to normal light conditions, and the intensity of the response varies in different accessions (with Col-0 being more sensitive than Ler ) [van Zanten et al. (2010)]. Therefore, chromatin plasticity seems to contribute to the plant’s adaptation to environmental light conditions. Alter natively, the heterochromatin response to low light can be viewed as an abiotic stress response. Upon exposure to another abiotic stress, namely prolonged heat stress, the transcription of centromeric and pericentromeric repeats is reactivated, and these regions exhibit a dispersed pattern in FISH [Pecinka et al. (2010)]. Interestingly, throughout recovery, transcription of centromeric and pericentromeric repeats was progressively silenced, whereas decondensation persisted for up to 1 week. Thus, this is another example showing that chromatin condensation status and gene expression can be uncoupled. Furthermore, such alterations did not occur in meristematic cells or in cells from leaves produced after a period of heat stress. It was proposed that the specific meristematic chromatin response indicates the existence of a safeguard mech-anism that minimizes genome damage in the germline [Pecinka et al. (2010)]. Interestingly, heterochromatin decompaction was not observed after freezing or UV-C treatments [Pecinka et al. (2010)]. Therefore, decondensation of the heterochromatin compartments is either not a general stress response or each type of stress is associated with chromatin reorganization in a specific compartment or with a distinctive timing and amplitude pattern.

Table of contents :

1 General introduction 
2 State of the art 
2.1 Nuclear architecture
2.1.1 Components of plant heterochromatin
2.1.2 Arabidopsis model of chromosome organization centered on heterochromatin
2.1.3 Distribution of heterochromatin in the nuclear domain
2.1.4 Dynamics of the heterochromatin compartment during development
2.1.5 Dynamics of the heterochromatin compartment in response to environmental cues .
2.1.6 Mechanisms involved in the spatial heterochromatin distribution
2.1.7 Chromosome territories
2.1.8 Recapitulation
2.2 Statistical analysis of spatial point patterns
2.2.1 Classical spatial statistics
2.2.2 Summary statistics
2.2.3 Testing spatial configurations
2.3 Spatial analysis approaches on nuclear architecture
2.3.1 Microscopic imaging
2.3.2 Extraction of the nucleus and its compartments in a three-dimensional image .
2.3.3 Quantitative image analysis of nuclear organizations
2.3.4 Analysis of nuclear organizations using spatial statistics
2.4 Motivation of the present work
3 Methods for analyzing spatial object patterns in confined spaces 
3.1 Representation of 3D objects and domains
3.2 Spatial statistical descriptors
3.2.1 F-Function .
3.2.2 G-Function .
3.2.3 H-Function .
3.2.4 B-Function .
3.2.5 C-Function .
3.2.6 Z-Function .
3.2.7 Other new spatial statistical descriptors
3.3 Spatial models to decipher spatial configurations
3.3.1 Completely random point process in a 3D domain
3.3.2 3D hardcore random spatial point model
3.3.3 Orbital spatial point model
3.3.4 Orbital 3D spatial objects model
3.3.5 3D maximum repulsion spatial point process
3.3.6 Hardcore 3D territorial spatial model
3.3.7 Orbital 3D territorial spatial model
3.4 Statistical Distribution Index: a statistical tool to test model goodness-of-fit
3.5 Recapitulation of the chapter
4 Results (Part 1): Numerical investigations on methods 
4.1 Unbiased testing of spatial models using replicated data
4.2 Characterization of spatial models
4.2.1 Orbital 3D spatial objects patterns
4.2.2 Maximum repulsion spatial objects patterns
4.3 Reproducibility of SDI values
4.4 Robustness of spatial analysis to segmentation errors
4.4.1 CSR objects patterns
4.4.2 Real patterns .
4.4.3 Conclusion .
4.5 Inter-group comparison using the SDI
4.5.1 Marginal spatial point process
4.5.2 Thomas spatial point pattern
4.5.3 Conclusion .
5 Results (Part 2): Spatial analyses of nuclear architecture in A. thaliana 
5.1 Image segmentation and quantitative analysis of nuclei and chromocenters
5.1.1 Segmentation of nuclei
5.1.2 Segmentation of chromocenters
5.1.3 Surface extraction
5.1.4 Quantification of nuclear and chromocenters features
5.2 Analysis of A. thaliana wild-type leaf cell nuclei
5.2.1 Analysis of nuclear size and shape
5.2.2 Analysis of heterochromatin features
5.2.3 Testing departure from complete randomness
5.2.4 Analyzing the peripheral organization
5.2.5 Correlation between nuclear features and chromocenters spatial organization
5.2.6 Distinguishing spatial heterogeneity vs interactions between chromocenters
5.2.7 Testing the maximum repulsive organization of chromocenters
5.2.8 Investigating an orbital territorial spatial configuration of chromocenters
5.2.9 Recapitulation
5.3 Characterization of genotypes: crwn and kaku family mutants
5.3.1 Plant materials
5.3.2 The crwn1 and crwn2 mutations have globally similar and additive effects on 3D nuclear size and shape
5.3.3 The crwn1 and crwn2 mutations have opposite and additive effects on constitutive heterochromatin
5.3.4 The crwn mutations impact the relative positioning of chromocenters in the nuclear space .
5.3.5 The crwn mutations impact the distance between the chromocenters and the nuclear periphery
5.3.6 kaku mutations predominantly impact nuclear shape in mesophyll leaf cells
5.3.7 Constitutive heterochromatin is affected in kaku mutants
5.3.8 The spatial distribution of chromocenters is not altered in the kaku mutants
5.3.9 Recapitulation
6 General conclusion and discussion 
6.1 A. thaliana nuclear architecture: chromocenters organization
6.2 The developed spatial statistical approach
6.2.1 Spatial models
6.2.2 Spatial descriptors
6.2.3 Individual vs population tests of spatial models
6.2.4 Comparison of spatial arrangements between nuclei populations
6.2.5 Genericity .
A Appendix 
A.1 Other new spatial statistical descriptors
A.1.1 NN-Function .
A.1.2 LRD-Function .
A.1.3 SRD-Function .
A.2 Technical validation of the complete random models at the population level
A.2.1 Complete random spatial point patterns
A.2.2 Hardcore 3D random spatial objects patterns
A.3 Comparison of A. thaliana isolated nuclei populations
A.3.1 Morphological and size evaluation of A. thaliana Col-0 leaf cell nuclei
A.3.2 Parametrization of the heterochromatin in A. thaliana Col-0 leaf cell nuclei
A.3.3 Analysis of the spatial configuration of chromocenters in A. thaliana Col0 leaf cell nuclei .

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