Heterochromatin in interphase nuclei of Arabidopsis thaliana

The eukaryotic nucleus represents a complex arrangement of heterochromatic and euchromatic domains, each with their specific nuclear functions. Somatic cells of a multicellular organism are genetically identical, yet they may differ completely in nuclear organization and gene expression patterns. Stable changes in gene expression without modifying the sequence are the result of epigenetic changes and include covalent modifications in cytosine residues of DNA and in histone tails giving rise to altered chromatin protein complexes, remodeling of chromatin and changes in chromatin compaction. Large-scale differences in chromatin structure are visible at the microscopic level as euchromatin and heterochromatin. Arabidopsis thaliana chromosomes display a relatively simple distribution of euchromatic and heterochromatic segments overlapping with gene-rich and repeat-rich regions, respectively. Recently, we have shown that Arabidopsis provides a well-defined system to study individual chromosomes and chromatin domains in interphase nuclei as well as the relationship between chromatin condensation and epigenetic mechanisms of gene silencing. This overview focuses on the organization and composition of heterochromatin in Arabidopsis nuclei.     

Methylation of a euchromatin-heterochromatin transition region in Arabidopsis thaliana chromosome 5 left arm

Cytosine methylation was studied at the level of the euchromatin/heterochromatin transition genomic region of the Arabidopsis chromosome 5 left arm. It has been shown using a monoclonal antibody against 5-methylcytosines that the density of DNA methylation increases from the euchromatin towards the heterochromatin. YACs mapped along this region were characterized for their repeated sequences content. Some of them, corresponding to euchromatin, euchromatin/heterochromatin border and heterochromatin regions, were used as probes for a Southern blot analysis of methylation. This revealed that the degree of mCmCGG and GATmC methylation increases significantly from the euchromatin towards the heterochromatin. Moreover, an analysis of cytosine methylation levels (% of 5-methylcytosine) of different DNA fragments, inside the same genomic region, was performed using PCR and/or Southern blot approaches. There is a gradual increase of methylation along the genomic region analyzed: CpG methylation in the euchromatic fraction, CpG and CpNpG methylation at the euchromatin/heterochromatin transition and an additional asymmetrical methylation in the repeated-heterochromatic fraction. The most methylated repeated family at CpG, CpNpG and asymmetrical sites is the 5S ribosomal DNA, highly methylated even though it is transcribed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chiasma formation in Arabidopsis thaliana accession Wassileskija and in two meiotic mutants

Meiotic chiasmata were analysed in metaphase I pollen mother cells (PMCs) of wild-type Arabidopsis thaliana and in two meiotic mutants. Fluorescence in situ hybridisation (FISH) with 45S rDNA and 5S rDNA as probes was used to identify the five chromosome pairs. A wild-type chiasma frequency of 9.24 per cell was found, consistent with estimated genetic recombination values. Individual bivalent chiasma frequencies varied according to chromosome size; chromosome 1 had the highest mean chiasma frequency (2.14) while the short acrocentric chromosomes had the lowest frequencies (1.54 and 1.56). FISH analysis was extended to two meiotic mutants (asy1 and dsy1) having low residual bivalent and chiasma frequencies. Mutant dsy1 gave no indication of chromosome preference for residual bivalent formation; instead it showed a general reduction in bivalent and chiasma frequencies. In asy1, the longest chromosome (1) had the lowest bivalent frequency and chiasma frequency while the short acrocentric chromosome 2 had the highest frequencies. This chromosome pair may be preferentially involved in synapsis and chiasma formation because of their association with the nucleolus. However, other factors may be operating since the other acrocentric chromosome (4), with similar size and structure to chromosome 2, did not share these chiasma properties.     

Chromatin dynamics and Arabidopsis development

The plant life cycle involves a series of developmental phase transitions. These transitions require the regulation and highly co-ordinated expression of many genes. Epigenetic controls have now been shown to be a key element of this mechanism of regulation. In the model plant Arabidopsis, recent genetic and molecular studies on chromatin have begun to dissect the molecular basis of these epigenetic controls. Chromatin dynamics represent the emerging and exciting field of gene regulation notably involved in plant developmental transitions. By comparing plant and animal systems, new insights into the molecular complexes and mechanisms governing development can be delineated. We are now beginning to identify the components of chromatin complexes and their functions.     

High heterochromatin content in somatic chromosomes of two unrelated species of Diplopoda (Myriapoda)

For the first time, a conventional analysis of C-banded karyotypes was carried out in two distantly related diplopod species; this revealed an impressive percentage of heterochromatin in both genomes. In Acanthopetalum sicanum (Order Callipodida) (2n = 12), heterochromatin constitutes about 60% of the total DNA in females and 56% in males, whereas in Enologus oxypygum (Order Julida) (2n = 22) it is about 67% in both sexes. Heterochromatin of the two species was found to be similar in base composition (AT rich) and heterochromatin distribution, indicating that it has accumulated in a species-specific manner. Sex-determining mechanisms of the XY type were detected in both A. sicanum and E. oxypygum. In A. sicanum, the Y presented the lowest heterochromatic content of all chromosomes in the karyotype, whereas the X presented the highest.This revised version was published online in November 2005 with corrections to the Cover Date.     

Various organizations of the complex repeats in vole sex chromosome heterochromatin

Different patterns of the DNA sequences organization were revealed in the vole (Rodentia) sex chromosome heterochromatin using dual-label fluorescence in-situ hybridization on extended DNA fibers with different repetitive DNA sequences as probes. In Microtus rossiaemeridionalis, the basic type represents the homogeneous relatively short tracks consisting of tandemly reiterated monomers of the MS3 family alternating with similar tracks of MS4 monomers and with non-fluorescent spacers. These tracks varied in the length of both repeats, with an average size of 12-22 kb or 3-5 copies. Apart from this, some continuous tracks of both families spanning 100-200 kb were interrupted by short spacers or single signals from the sequences with homology to LINEs. These results, together with that obtained by the analysis of phage clones of the genomic library, unequivocally demonstrate a variable large-scale DNA structural organization in heterochromatin of the M. rossiaemeridionalis sex chromosome. The dominant type of large-scale DNA organization in M. transcaspicus heterochromatin represents the unicolor relatively long tracks consisting of monotonous but not alternating monomers of MS3 or MS4 with sizes ranging from 15 to 40 kb and separated by extended spacers with an average length of 20 kb. Thus, the formation of the vole sex chromosome heterochromatic regions occurred relatively recently during speciation.     

Analysis of NORs and NOR-associated heterochromatin in the mussel Mytilus galloprovincialis Lmk

The chromosomes of the mussel Mytilus galloprovincialis were analysed by means of chromomycin A3 (CMA), distamycin A/DAPI (DA/DAPI), DAPI/actinomycin D (DAPI/AMD) and chromomycin A3/distamycin A/DAPI(CDD) fluorescence banding techniques, C-banding, silver staining, N-banding and in situ hybridization with 18S+28S rDNA and telomere probes. 18S+28S rDNA clusters were located on the telomeres of two pairs of submeta/subtelocentric chromosomes. The nucleolar organizing regions (NORs) were associated with bright CMA fluorescence, dull DAPI fluorescence and C- and N-positive bands, but not all four NOR-associated heterochromatin bands showed bright CMA fluorescence in a given cell; intra- and interindividual variability was found in this character. Additional non-ribosomal C-bands did not show any differential fluorescent behaviour.This revised version was published online in November 2005 with corrections to the Cover Date.     

Epigenomic mapping in <Emphasis Type="Italic">Arabidopsis</Emphasis> using tiling microarrays

In addition to genetic information, chromosomes transmit epigenetic information from cell to cell during division, and sometimes from generation to generation. While genetic information is encoded directly in the DNA sequence, epigenetic information is not, although it is usually associated with specific chromosomal regions. Epigenetic modifications in plants include cytosine methylation as well as modification of histones and other chromosomal proteins. Small interfering RNA play major roles in targeting these modifications to specific regions. Genomic tiling microarrays are powerful tools for analysing epigenetic information, and we review their application in building epigenomic maps in the model plant, Arabidopsis.     

Using Arabidopsis to understand centromere function: Progress and prospects

Arabidopsis thaliana has emerged in recent years as a leading model for understanding the structure and function of higher eukaryotic centromeres. Arabidopsis centromeres, like those of virtually all higher eukaryotes, encompass large DNA domains consisting of a complex combination of unique, dispersed middle repetitive and highly repetitive DNA. For this reason, they have required creative analysis using molecular, genetic, cytological and genomic techniques. This synergy of approaches, reinforced by rapid progress in understanding how proteins interact with the centromere DNA to form a complete functional unit, has made Arabidopsis one the best understood centromere systems. Yet major problems remain to be solved: gaining a complete structural definition of the centromere has been surprisingly difficult, and developing synthetic mini-chromosomes in plants has been even more challenging.     

Characterization of DNA sequences constituting the terminal heterochromatin of the chicken Z chromosome

Two clones, pCZTH5-8 and pCZTH12-8, were isolated from a female chicken genomic library by screening with sequences obtained from genomic libraries which had been constructed from a terminal region of a single Z chromosome of chicken utilizing laser microbeam irradiation and PCR amplification. Fluorescencein situ hybridization to the mitotic Z chromosome and the lampbrush ZW bivalent of chicken demonstrated that both the cloned sequences are located in the heterochromatic region of the Z chromosome at the end opposite to the pairing region with the W chromosome. The sequences pCZTH5-8 and pCZTH12-8 are distributed widely on both the telomeric bow-like loops (TBL) and the region I (short loops region) of the Z lampbrush chromosome. These clones, pCZTH12-8 particularly notably, hybridized also to the TBLs of lampbrush bivalents 1–4 of chicken. Both sequence are transcribed in the lampbrush stage oocytes on the Z chromosome and on other macrobivalents. The subfragment of pCZTH5-8 which hybridizes to the TBLs and the insert of pCZTH12-8 contain regions that are closely similar in sequence. The pCZTH5-8 sequence has no internal repeats and may be part of the 24-kb macrosatellite repeating unit that is evident afterNhel digestion of the genomic DNA. A cloned 24-kb unit, pFN-1, does not show significant DNA curvature, but cytosines of its CpG dinucleotides may be highly methylatedin vivo. This contrasts with the repeat sequences of the W heterochromatin which not only have highly methylated CpG but are also strongly curved. The 24-kb unit is repeated about 830 times in the diploid genome of a female chicken, suggesting that nearly the entire terminal heterochromatin on the Z chromosome consists of this macrosatellite family. Sequences of the greater part of the pCZTH5-8 are restricted to the genusGallus but the sequence of one subregion which hybridizes to TBLs is present in the genomes of the order Galliformes.accepted for publication by M. Schmid     

DNA repair and recombination functions in <Emphasis Type="Italic">Arabidopsis</Emphasis> telomere maintenance

In this review, we discuss recent advances in the knowledge of plant telomere maintenance, focusing on the model plant Arabidopsis thaliana and, in particular, on the roles of proteins involved in DNA repair and recombination. The question of the interrelationships between DNA repair and recombination pathways and proteins with telomere function and maintenance is of increasing interest and has been the subject of a number of recent reviews (Cech 2004, d’Adda di Fagagna et al. 2004, Hande 2004, Harrington 2004, Maser & DePinho 2004). Understanding of telomere biology, DNA repair and recombination in plants has rapidly progressed over the last decade, substantially due to genetic approaches in Arabidopsis, and we feel that this is an appropriate time to review current knowledge in this field. A number of recent reviews have dealt more generally with the subject of plant telomere structure and evolution (Riha et al. 2001, McKnight et al. 2002, Riha & Shippen 2003b, McKnight & Shippen 2004, Fajkus et al. 2005) and we thus focus specifically on plant telomere biology in the context of DNA repair and recombination in Arabidopsis.     

Rice as a model for centromere and heterochromatin research

Rice (Oryza sativa) has become an important model plant species in numerous research projects involving genome, molecular and evolutionary biology. In this review we describe the reasons why rice provides an excellent model system for centromere and heterochromatin research. In most multicellular eukaryotes, centromeres and heterochromatic domains contain long arrays of repetitive DNA elements that are recalcitrant to DNA sequencing. In contrast, three rice centromeres and the majority of the cytologically defined heterochromatin in the rice genome have been sequenced to high quality, providing an unparalleled resource compared to other model multicellular eukaryotes. Most importantly, active genes have been discovered in the functional domains of several rice centromeres. The centromeric genes and sequence resources provide an unprecedented opportunity to study function and evolution of centromeres and centromere-associated genes.     

Retroelements (LINEs and SINEs) in vole genomes: Differential distribution in the constitutive heterochromatin

The chromosomal distribution of mobile genetic elements is scarcely known in Arvicolinae species, but could be of relevance to understand the origin and complex evolution of the sex chromosome heterochromatin. In this work we cloned two retrotransposon sequences, L1 and SINE-B1, from the genome of Chionomys nivalis and investigated their chromosomal distribution on several arvicoline species. Our results demonstrate first that both retroelements are the most abundant repeated DNA sequences in the genome of these species. L1 elements, in most species, are highly accumulated in the sex chromosomes compared to the autosomes. This favoured L1 insertion could have played an important role in the origin of the enlarged heterochromatic blocks existing in the sex chromosomes of some Microtus species. Also, we propose that L1 accumulation on the X heterochromatin could have been the consequence of different, independent and rapid amplification processes acting in each species. SINE elements, however, were completely lacking from the constitutive heterochromatin, either in autosomes or in the heterochromatic blocks of sex chromosomes. These data could indicate that some SINE elements are incompatible with the formation of heterochromatic complexes and hence are necessarily missing from the constitutive heterochromatin.