Composition and formation of heterochromatin in Arabidopsis thaliana

The term heterochromatin has been applied to both large-scale, microscopically visible chromocentres and small-scale, silent genes located outside chromocentres. This may cause confusion in the interpretation of epigenetic marks for both features. The model plant Arabidopsis thaliana provides an excellent system to investigate composition and function of chromatin states at different levels of organization. In this review we will discuss recent developments in molecular networks underlying gene silencing and the relationship with visible heterochromatin in Arabidopsis.     

Analysis of heterochromatic epigenetic markers in the holocentric chromosomes of the aphid <Emphasis Type="Italic">Acyrthosiphon pisum</Emphasis>

Monomethylated-K9 H3 histones (Me9H3) and heterochromatin protein 1 (HP1) are reported as heterochromatin markers in several eukaryotes possessing monocentric chromosomes. In order to confirm that these epigenetic markers are evolutionarily conserved, we sequenced the HP1 cDNA and verified the distribution of Me9H3 histones and HP1 in the holocentric chromosomes of the aphid Acyrthosiphon pisum. Sequencing indicates that A. pisum HP1 cDNA (called ApHP1) is 1623 bp long, including a 170 bp long 5′UTR and a 688 bp long 3′UTR. The ApHP1 protein consists of 254 amino acidic residues, has a predicted molecular mass of 28 kDa and a net negative charge. At the structural level, it shows an N terminal chromo domain and a chromo shadow domain at the C terminus linked by a short hinge region. At the cytogenetic level, ApHP1 is located exclusively in the heterochromatic regions of the chromosomes. The same heterochromatic regions were labelled after immuno-staining with antibodies against Me9H3 histones, confirming that Hp1 and Me9H3 co-localize at heterochromatic chromosomal areas. Surprisingly, aphid heterochromatin lacks DNA methylation and methylated cytosine residues were mainly spread at euchromatic regions. Finally, the absence of DNA methylation is observed also in aphid rDNA genes that have been repeatedly described as mosaic of methylated and unmethylated units in vertebrates.     

Histone modification and the control of heterochromatic gene silencing in Drosophila

Covalent modifications of histones index structurally and functionally distinct chromatin domains in eukaryotic nuclei. Drosophila with its polytene chromosomes and developed genetics allows detailed cytological as well as functional analysis of epigenetic histone modifications involved in the control of gene expression pattern during development. All H3K9 mono- and dimethylation together with all H3K27 methylation states and H4K20 trimethylation are predominant marks of pericentric heterochromatin. In euchromatin, bands and interbands are differentially indexed. H3K4 and H3K36 methylation together with H3S10 phosphorylation are predominant marks of interband regions whereas in bands different H3K27 and H4K20 methylation states are combined with acetylation of H3K9 and H3K14. Genetic dissection of heterochromatic gene silencing in position-effect variegation (PEV) by Su(var) and E(var) mutations allowed identification and functional analysis of key factors controlling the formation of heterochromatin. SU(VAR)3-9 association with heterochromatic sequences followed by H3K9 methylation initiates the establishment of repressive SU(VAR)3-9/HP1/SU(VAR)3-7 protein complexes. Differential enzymatic activities of novel point mutants demonstrate that the silencing potential of SU(VAR)3-9 is mainly determined by the kinetic properties of the HMTase reaction. In Su(var)3-9^ptn a significantly enhanced enzymatic activity results in H3K9 hypermethylation, enhanced gene silencing and extensive chromatin compaction. Mutations in factors controlling active histone modification marks revealed the dynamic balance between euchromatin and heterochromatin. Further analysis and definition of Su(var) and E(var) genes in Drosophila will increase our understanding of the molecular hierarchy of processes controlling higher-order structures in chromatin.     

The paracentric inversion In(2Rh)PL alters the centromeric organization of chromosome 2 in Drosophila melanogaster

Centromeres are complex structures involved in an evolutionarily conserved function, the correct segregation of chromosomes and chromatids during meiosis and mitosis. The centromere is determined by epigenetic processes that result in a particular nucleosome organization (CEN chromatin) that differs from the rest of the chromatin including the heterochromatin that normally surrounds the centromere in higher organisms. Many of the current models of centromere origin and organization rely on the molecular and cytological characterization of minichromosomes and their derivatives, and on studies on the origin and maintenance of neocentromeres. Here, we describe the peculiar centromere organization observed in In(2Rh)PL, a paracentric D. melanogaster inversion in which the centromere is maintained in its natural context but is directly flanked by a euchromatic domain as a result of the rearrangement. We have identified the breakpoints of the inversion and show that the proximal one is within the centromere region. The data presented suggest that, notwithstanding the loss of all the pericentric 2Rh heterochromatin, the centromere of the In(2Rh)PL chromosome is still active but presents a nucleosomal organization quite different from the organization usually observed in the centromeric region.     

Epigenetics of eu- and heterochromatin in inverted and conventional nuclei from mouse retina

To improve light propagation through the retina, the rod nuclei of nocturnal mammals are uniquely changed compared to the nuclei of other cells. In particular, the main classes of chromatin are segregated in them and form regular concentric shells in order; inverted in comparison to conventional nuclei. A broad study of the epigenetic landscape of the inverted and conventional mouse retinal nuclei indicated several differences between them and several features of general interest for the organization of the mammalian nuclei. In difference to nuclei with conventional architecture, the packing density of pericentromeric satellites and LINE-rich chromatin is similar in inverted rod nuclei; euchromatin has a lower packing density in both cases. A high global chromatin condensation in rod nuclei minimizes the structural difference between active and inactive X chromosome homologues. DNA methylation is observed primarily in the chromocenter, Dnmt1 is primarily associated with the euchromatic shell. Heterochromatin proteins HP1-alpha and HP1-beta localize in heterochromatic shells, whereas HP1-gamma is associated with euchromatin. For most of the 25 studied histone modifications, we observed predominant colocalization with a certain main chromatin class. Both inversions in rod nuclei and maintenance of peripheral heterochromatin in conventional nuclei are not affected by a loss or depletion of the major silencing core histone modifications in respective knock-out mice, but for different reasons. Maintenance of peripheral heterochromatin appears to be ensured by redundancy both at the level of enzymes setting the epigenetic code (writers) and the code itself, whereas inversion in rods rely on the absence of the peripheral heterochromatin tethers (absence of code readers).     

X chromosome painting in <Emphasis Type="Italic">Microtus</Emphasis>: Origin and evolution of the giant sex chromosomes

Sex chromosomes in species of the genus Microtus present some characteristic features that make them a very interesting group to study sex chromosome composition and evolution. M. cabrerae and M. agrestis have enlarged sex chromosomes (known as ‘giant sex chromosomes’) due to the presence of large heterochromatic blocks. By chromosome microdissection, we have generated probes from the X chromosome of both species and hybridized on chromosomes from six Microtus and one Arvicola species. Our results demonstrated that euchromatic regions of X chromosomes in Microtus are highly conserved, as occurs in other mammalian groups. The sex chromosomes heterochromatic blocks are probably originated by fast amplification of different sequences, each with an independent origin and evolution in each species. For this reason, the sex heterochromatin in Microtus species is highly heterogeneous within species (with different composition for the Y and X heterochromatic regions in M. cabrerae) and between species (as the composition of M. agrestis and M. cabrerae sex heterochromatin is different). In addition, the X chromosome painting results on autosomes of several species suggest that, during karyotypic evolution of the genus Microtus, some rearrangements have probably occurred between sex chromosomes and autosomes.     

Heterochromatin establishment at pericentromeres depends on nuclear position

Mammalian development begins with fertilization of an oocyte by the sperm followed by genome-wide epigenetic reprogramming. This involves de novo establishment of chromatin domains, including the formation of pericentric heterochromatin. We dissected the spatiotemporal kinetics of the first acquisition of heterochromatic signatures of pericentromeric chromatin and found that the heterochromatic marks follow a temporal order that depends on a specific nuclear localization. We addressed whether nuclear localization of pericentric chromatin is required for silencing by tethering it to the nuclear periphery and show that this results in defective silencing and impaired development. Our results indicate that reprogramming of pericentromeric heterochromatin is functionally linked to its nuclear localization.     

Association of adipogenic genes with SC-35 domains during porcine adipogenesis

Spatial organization of the genome within interphase nuclei is non-random. It has been shown that not only whole chromosomes but also individual genes occupy specific nuclear locations and these locations can be changed during different processes like differentiation or disease. Using a porcine in vitro adipogenesis stem cell differentiation system as a model to study nuclear organization, it was demonstrated that nuclear position of selected genes involved in porcine adipogenesis was altered with the up-regulation of gene expression, correlating with these genes becoming more internally located within nuclei, without whole territory relocation. Here, we investigated whether the gene relocation observed during porcine adipogenesis is related to spatial co-association with SC-35 domains. These domains are nuclear speckles enriched in numerous splicing and RNA metabolic factors. Using a DNA immuno-FISH approach we investigated the localisation of three adipogenic genes (PPARG, SREBF1, and FABP4) with SC-35 domains in porcine mesenchymal stem cells and after they were differentiated into adipocytes. We found that the location of these genes relative to SC-35 domains was non-random and correlated with the up-regulation of gene expression. In addition, we observed more frequent clustering of the studied genes located on different chromosomes around the same nuclear speckle in differentiated adipocytes than in mesenchymal stem cells. However, the choice of the domain was more random. This study adds to the evidence that SC-35 domains are hubs of gene activity and gene-domain association may be considered as a common mechanism to enhance gene expression.     

Differential replication of heterochromatin in polytene chromosomes of the Old World screw-worm fly,Chrysomya bezziana (Diptera: Calliphoridae), analysed by fluorescence staining

The distribution and replication of heterochromatin in polytene trichogen chromosomes of the Old World screw-worm fly,Chrysomya bezziana, were studied using fluorescent staining techniques. Quinacrine and distamycin-DAPI, which selectively stain AT-rich DNA, and chromomycin, specific for GC-rich sequences, were used. Bright quinacrine and DA-DAPI fluorescence was found in the sex chromosome body and in all autosomal centromere regions. Chromomycin (CMA) staining results in very little bright fluorescence of the sex chromosome body and autosomal centromeric regions, but many bright bands of varying morphology are distributed in autosomal arms. The expected negative CMA staining of quinacrine and DA-DAPI bright regions was not found. The lack of reciprocal staining patterns may result from changes in the higher order chromatin structure of polytene chromosomes, or intercalation of divergent heterochromatic sequences. Comparison of the different staining techniques in mitotic and polytene cells shows that heterochromatin is differentially under-replicated, so that the proportions of the distinct fluorescent-specific chromatin changes during polytenization. CMA staining within autosomal arms suggests that repeated sequences intercalated in euchromatin are co-replicated during polytenization. The numerous fluorescent markers described also provide further morphological features for use in comparative cytological analysis ofC. bezziana.     

5-Azacytidine produces differential undercondensation of alpha,beta and classical human satellite DNAs

Fluorescencein situ hybridization employing human alphoid, beta and classical satellite DNA probes was performed on 5-azacytidine treated and untreated chromosomes obtained from human lymphocytes. The individual used in this study presented a polymorphism of constitutive heterochromatin of chromosomes 1 and 9 as revealed byin situ digestion with the restriction endonucleaseAlul. Neither the alphoid nor the beta satellite DNA domains were susceptible to condensation-inhibition by 5-azacytidine. Only the classical satellite localized on chromosome 9 was affected. The constitutive heterochromatin size polymorphism was shown to depend mainly on variations of the classical satellite DNA domain. Therefore, condensation-inhibition, as a phenomenon which may modify the natural folding of the chromatin fibre, regionally affects human constitutive heterochromatin and seems to be dependent on the heterochromatic family.     

Nuclear organization of centromeric domains is not perturbed by inhibition of histone deacetylases

It is well established that modification of lysines in histone molecules correlates with gene expression and chromatin structure. It is not known whether this operates entirely at a local level, e.g. through the recruitment of specific proteins, or whether histone modifications might impact on more long-range aspects of chromatin organization. There is a distinctive organization of chromatin within the nucleus and the chromatin at the nuclear periphery of mammalian cells appears to be hypoacetylated. Previously it had been suggested that inhibition of histone deacetylases by TSA causes a gross remodeling of nuclear structure, specifically the recruitment of centromeric heterochromatin to the nuclear periphery. Here, we have quantified the nuclear organization of histone modifications and the localization of centromeric domains in human cells before and after TSA treatment. TSA alters the nuclear distribution of histone acetylation, but not that of histone methylation. TSA elevates levels of histone acetylation at the nuclear periphery but we see no alteration in the position of centromeric domains in the nuclei of treated cells. We conclude that the distinctive nuclear localization of centromeric domains is independent of histone acetylation.     

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.     

Nature of B chromosomes in the harvest mouse Reithrodontomys megalotis by fluorescence in situ hybridization (FISH)

Using fluorescence in situ hybridization, we examined the characteristics of two types of B chromosomes in harvest mice of the genus Reithrodontomys. B chromosomes were interrogated with rDNA, telomeric repeat, LINE element and centromeric heterochromatin probes. The two types of B chromosomes share the following features: (a) telomeres present on the ends of both arms; (b) hybridization to LINE probes; (c) absence of hybridization to the ribosomal gene probes; (d) C-band-positive centromeric regions; and (e) euchromatic arms. They differ as follows: (a) the larger B element hybridizes to the centromeric heterochromatin (pMeg-1) probe whereas the smaller B element does not; (b) the amount of C-band-positive material is reduced in the smaller B chromosome relative to that present on the larger B chromosome; and (c) the smaller element is reduced in size by about a third. It is concluded that the larger B chromosome arose as a leftover centromere from centric fusion, whereas the smaller element has a different origin — perhaps as an intact fragment or as an amplified region from the A chromosomes. The presence of euchromatic regions on B chromosomes may account for their survival in the karyotype.This revised version was published online in November 2005 with corrections to the Cover Date.     

FISH mapping and molecular organization of the major repetitive sequences of tomato

This paper presents a bird’s-eye view of the major repeats and chromatin types of tomato. Using fluorescence in-situ hybridization (FISH) with Cot-1, Cot-10 and Cot-100 DNA as probes we mapped repetitive sequences of different complexity on pachytene complements. Cot-100 was found to cover all heterochromatin regions, and could be used to identify repeat-rich clones in BAC filter hybridization. Next we established the chromosomal locations of the tandem and dispersed repeats with respect to euchromatin, nucleolar organizer regions (NORs), heterochromatin, and centromeres. The tomato genomic repeats TGRII and TGRIII appeared to be major components of the pericentromeres, whereas the newly discovered TGRIV repeat was found mainly in the structural centromeres. The highly methylated NOR of chromosome 2 is rich in [GACA]₄, a microsatellite that also forms part of the pericentromeres, together with [GA]₈, [GATA]₄ and Ty1-copia. Based on the morphology of pachytene chromosomes and the distribution of repeats studied so far, we now propose six different chromatin classes for tomato: (1) euchromatin, (2) chromomeres, (3) distal heterochromatin and interstitial heterochromatic knobs, (4) pericentromere heterochromatin, (5) functional centromere heterochromatin and (6) nucleolar organizer region.