Current , 2002, 3, 563-576 563 Nucleolar Dominance: A ‘David and Goliath’ Imprinting Process

W. Viegas1,*, N. Neves1,2, M. Silva1, A. Caperta1,2, and L. Morais-Cecílio1

1Centro de Botânica Aplicada à Agricultura, Secção de Genética/DBEB, Instituto Superior de Agronomia, 1349-017 Lisboa, 2Departamento de Ciências Biológicas e Naturais, Universidade Lusófona de Humanidades e Tecnologias, Campo Grande 376, 1749-042 Lisboa, Portugal

Abstract: Nucleolar dominance is an enigma. The puzzle of differential amphiplasty has remained unresolved since it was first recognised and described in Crepis hybrids by Navashin in 1934. Here we review the body of knowledge that has grown out of the many models that have tried to find the genetic basis for differential rRNA gene expression in hybrids, and present a new interpretation. We propose and discuss a chromatin imprinting model which re-interprets differential amphiplasty in terms of two of differing size occupying a common space within the nucleus, and with as a key player in the scenario. Difference in size between two parental genomes induces an inherited epigenetic mark in the that allows patterns of chromatin organization to have positional effects on the neighbouring domains. This chromatin imprinting model can be also used to explain complex genomic interactions which transcend nucleolar dominance and which can account for the overall characteristics of hybrids. Gene expression in hybrids, relative to parentage, is seen as being based on the nuclear location of the sequences concerned within their genomic environment, and where the presence of particular repetitive DNA sequences are ‘sensed’, and render silent the adjacent information.

THE EARLY DAYS the secondary constrictions observed in metaphase cells are clearly associated with the formation of nucleoli during A differential gene expression phenomenon that results in interphase, since nucleoli were shown to form at or near nucleolus formation by only one parental set of rRNA gene these constrictions. clusters is widely found in hybrids. The process was first observed about 50 years ago by Navashin [1], after crossing Another important contribution to the understanding of several different from the genus Crepis, and later this phenomenon came from the work of Barbara termed ‘Nucleolar Dominance’ [2]. Navashin clearly McClintock. Linking her own results on differential rRNA demonstrated that after hybridization the from gene expression patterns in maize with Navashin’s results, parental species undergo morphological modifications in the McClintock reasoned that species can be arranged in a hybrid. Changes that affected all of the chromosomes from hierarchy of dominance in their capacity to produce nucleoli, one parent were termed “amphiplasty” or “neutral in hybrids (e.g. Crepis) or in re-arranged lines amphiplasty”, whereas those that targeted only particular (e.g. maize). Moreover, McClintock was the first geneticist chromosomes he named “differential amphiplasty”. This to use the term “nucleolar organiser” (or Nucleolar Organiser latter case resulted from Navashin’s observations on a Region, NOR) to describe the chromosomal loci at which particular chromosome group of the genus Crepis - the D nucleoli form [4]. In maize chromosome six the NOR chromosomes - that usually display a distal small segment includes a -proximal heterochromatic knob (a (the satellite) connected to the remainder of the chromosome large chromomere) followed by the secondary constriction by a thin strand of chromatin (the secondary constriction) at and a distal satellite region. By analysing nucleolus forma- metaphase. In interspecific hybrids, only one D-chromosome tion in wild-type and translocation lines, McClintock showed this characteristic morphology, while the other failed suggested that the NOR must bear redundant genetic infor- to display its secondary constriction. Furthermore this mation [4]. Later it was shown that the NORs correspond to differential behaviour always belonged to the chromosome clusters of multiple copies of rRNA genes [5, 6]. from the same parental species in reciprocal crosses (Fig. 1). In backcrosses of the hybrid to its appropriate under- At this point, it became clear that nucleolar dominance dominant parental species, Navashin detected the restoration was a process observed in hybrids which leads to nucleolus of the secondary constriction and the satellite structures, formation mediated by the NORs of only one parental thereby showing that these changes are reversible and not species. Discrimination between transcriptional and post- due to any permanent loss and/or damage of these chromatin transcriptional levels of regulation of NOR activity was regions. In parallel, Heitz [3] proved that the satellite and addressed shortly afterwards through studies in the frog Xenopus by Honjo and Reeder [2]. They used a biochemical approach to determine the presence and origin of rRNA *Address correspondence to this author at the Centro de Botânica Aplicada transcripts during the embryogenesis of X. laevis x X. à Agricultura, Secção de Genética/DBEB, Instituto Superior de Agronomia, borealis hybrids; and showed that the only nucleoli present Tapada da Ajuda, 1349-017 Lisboa, Portugal; Tel: +351 21 365 32 81; when nucleolar dominance was detected were those Fax: +351 21 363 50 31; E-mail: [email protected]

1389-2029/02 $35.00+.00 © 2002 Bentham Science Publishers Ltd. 564 Current Genomics, 2002, Vol. 3, No. 6 Viegas et al.

Fig. (1). Diagram representing one pair of chromosomes carrying NORs (secondary constrictions) and terminal satellites in two parental species and their F1 hybrid, as first described in Crepis spp by Navashin [1]. The dominant species retains its visible NOR in the hybrid, and the rRNA gene cluster in the under-dominant species becomes ‘silenced’. Silencing is accompanied by condensed chromatin and the NOR is no longer seen. In backcrosses of the hybrid to its appropriate under-dominant parental species the secondary constriction and the satellite structures are restored showing that these changes are epigenetic and reversible. originated from the NORs and transcripts of X. laevis, repetitive DNA elements [7]. These repeat-DNA spacer indicating that the process occurs at the level of gene sequences were proven to act as pol I transcription transcription. enhancers, since transcription was stimulated strongly when they were cloned and located adjacent to a ribosomal gene The next logical steps were straight-forward: (i) to promoter, and injected into oocytes or young embryos [8, 9]. determine if the process results from selective activation of Nevertheless, when these same sequences were co-injected rRNA genes or from silencing of the under-dominant rDNA; with a second transporting a promoter, transcription (ii) to find out whether the process is achieved ‘gene-by- was severely inhibited by the original promoter [9], gene” or through a single large-scale NOR-targeted suggesting that enhancers bind to only one transcription mechanism; and (iii), to evaluate the molecular mechanisms factor that interacts with the gene promoter. The importance involved in establishing dominance and enforcing the of the DNA repetitive sequences within the rDNA intergenic process throughout cell cycle. Although considerable efforts spacer was further supported by the observation that mini- have been made to answer these fundamental questions, genes, with a X. laevis promoter attached to X. laevis spacer several possibilities remain, and no general rule has yet been sequences, are preferentially transcribed over identical discovered. As we will see, the most likely answer seems to constructs with a borealis rRNA gene spacer [10]. These and be firmly based on the original amphiplasty phenomenon other findings prompted the hypothesis that differences in first described by Navashin. sequence or number of regulatory elements in the intergenic spacers create the conditions to sequester transcription factors in limiting concentration. MECHANISMS FOR ESTABLISHMENT AND ENFORCEMENT: RULES AND EXCEPTIONS Further support favouring this model was gained by the analysis of nucleolar dominance in bread wheat. Bread wheat The Enhancer Imbalance Model (Triticum aestivum) is an allohexaploid originating from three different ancestral diploid species with multiple NOR The first clue about how nucleolar dominance operates loci [11]. Of these, the NORs located on chromosomes 1B came from the early studies of McClintock [4], when she and 6B are responsible for 90% of the total nucleolar volume launched the enhancer imbalance hypothesis. In maize in wheat cells, and hence are called the wheat major NORs translocation lines under-dominant DNA loci in somatic cells [12]. Several studies indicate that nucleolar volumes (as a produce large (normal) nucleoli when they are the only ones sensitive measure of nucleolar activity) have more to do with present (as in haploid pollen grains), and McClintock the number of active rRNA genes at a NOR than with the suggested that this may imply a competition for a factor total number of ribosomal genes per NOR locus [12-15]. The (“positive substance”) essential for nucleolus formation that 1B loci account for 60% of nucleolar mass followed by 6B is present in limiting amounts, more efficiently tritated by NORs (30%) and wheat minor NORs (10%); however, 1B the dominant rRNA genes. This popular hypothesis was NORs bear half the number of rRNA genes present in 6B settled by molecular studies using Xenopus as a model. In the loci [16, 17]. These authors clearly demonstrated not only hybrid X. laevis x X. borealis, the dominant X. laevis rRNA that rRNA gene number is not the primary determinant genes have larger intergenic spacer sequences upstream of responsible for NOR activity, but also that 1B NOR units the gene promoter and are composed of higher number of have longer intergenic spacers than the 6B rDNA units [14, Nucleolar Dominance: A ‘David and Goliath’ Chromatin Imprinting Process Current Genomics, 2002, Vol. 3, No. 6 565

17]. These results suggested a process similar to that independent of the number of rRNA genes located at a NOR. enunciated for the Xenopus model, where the largest However, this simple and apparently effective rule cannot be intergenic spacers are the more effective in attracting factors extended to other systems, namely and in limiting concentration which are essential for rRNA gene Arabidopsis. By detecting the genomic origin of rRNA transcription [16, 17]. Although this case cannot be transcripts in Brassica hybrids it was possible to ascribe a accommodated within a strict view of nucleolar dominance nucleolar dominance relationship between several Brassica (as both NORs belong to the same ancestral B species, where the sequence is B. nigra > B. rapa > B. donator), the enhancer imbalance model still seems plausible oleracea [26]. Sequencing of intergenic rDNA spacers from in interspecific genomic interactions in cereal hybrids. In this the three species allowed a direct comparison of the length of case, a preferential suppression of wheat rDNA loci occurs the repetitive regions and the number of repetitive elements. in plants resulting from the cross of Aegilops umbellulata (a Unexpectedly, B. oleracea has the longest intergenic rRNA wheat wild relative) and wheat [15]. These results can be gene spacers, yet these genes are inactivated in the presence explained under this hypothesis, as A. umbellulata rDNA of B. napus or B. nigra NORs. Another clear-cut exception units bear longer spacers than the largest wheat rRNA gene to the enhancer imbalance model has been reported in spacers [16, 17]. Arabidopsis, based on modifications. In A. suecica, the natural amphiploid between A. thaliana and A. arenosa, Nucleolar dominance was also observed in wheat x rye the A. arenosa NORs are dominant, and the A. thaliana hybrids, and in the artificial amphiploid triticale. In this case, rRNA genes are transcriptionally inactive [27]; even though wheat dominates over rye in the expression of rDNA loci, the A. arenosa spacer is shorter than that of A. thaliana. resulting in an almost total inactivation of rRNA genes of rye Furthermore, this dominance can be altered following origin (Fig. 2); [18-22]. Comparison of rDNA spacers from genome ratio modifications. By appropriate backcrosses, wheat and rye showed that rye spacers are shorter, displaying where recombinant lines were produced with a 3:1 A. several deletions amongst the repetitive DNA sequences thaliana:A. arenosa ratio, the A. arenosa rDNA becomes [23]. Once more it is tempting to reason that rye rRNA genes transcriptionally silent. This intriguing result also argues are less effective in attracting transcription factors leading to against the hypothesis of straightforward competition the preferential expression of wheat NORs. between rDNA intergenic spacers for limiting transcription factors: if less dominant rRNA genes are present, then more As previously pointed out [24, 25], the attractiveness of transcription factors may become available to allow under- the enhancer imbalance hypothesis resides in its simplicity: dominant rDNA transcription, presumably leading to a co- rRNA genes from distinct genomic origins being dominance situation. However, the observation of silencing differentially expressed on a biochemical basis. As of the normally dominant genes (dominance reversal) cannot presented, this mechanism could clearly explain the be predicted or explained by the enhancer imbalance establishment of McClintock’s hierarchical NOR expression hypothesis [24, 25, 28]. relationships, regardless of maternal or paternal effects and

Fig. (2). Nucleolar dominance in triticale (wheat-rye amphiploid), as shown by sequential silver staining and in situ hybridization techniques. The silver staining procedure (Ag-staining) reveals only the metaphase rDNA loci that were transcribed during the preceeding interphase, and DNA:DNA in situ hybridization reveals DNA sequences in the genome regardless of their expression patterns. Most triticale metaphase cells show only two pairs of silver-stained NORs (2a, black arrows), and in situ detection of the rye chromosomes (2b, in red) and rDNA sites (2b, in green) reveals the two pairs of NORs previously detected by Ag-staining on the wheat genome and two additional rDNA loci on rye chromosomes that were not detected by Ag-staining (2b,white arrows). 566 Current Genomics, 2002, Vol. 3, No. 6 Viegas et al.

The wheat-rye system, already referred to as an example (species-specific) transcription factor could be the supporting the enhancer imbalance hypothesis, can also be explanation for the dominance of mouse rDNA over human recruited to disprove the simple involvement of rDNA rRNA genes. In plants several results account to this intergenic size in attracting positive transcription factors, or hypothesis: a tomato rRNA gene promoter is not recognized other factors that prevent binding of silencing mediators. In when transfected into Arabidopsis protoplasts [40], and the wheat addition lines for the rye nucleolar chromosomes, rye same happens when a tobacco promoter is introduced into a NORs are expressed (Fig. 3); if not less, then at least the bean cell-free transcription extract [41]. same amount of transcription factors is present as in triticale (the artificial wheat-rye amphiploid), and both plant As pointed out by Reeder [42] the results achieved in materials have the same complement of wheat chromosomes evolutionary wide crosses may not be relevant in hybrids [29]. Even higher levels of rye NOR expression are formed by normal reproductive processes. Several moreover observed in wheat lines with the translocation of observations support this view, tending to rule out this the short arm of rye nucleolar chromosomes (Fig. 4). If the hypothesis as the major mechanism responsible for nucleolar dominance process was based only in the enhancer establishing nucleolar dominance. Clear examples of this are imbalance hypothesis then rye NORs should remain silent in found in interspecific hybrids in Brassica and Arabidopsis. the addition and translocation wheat lines. It seems clear In Brassica species, rRNA gene promoters were found to be therefore that the process of nucleolar dominance must be functional across species limits in transient expression assays established by other mechanisms. [43], although these species can be arranged in a nucleolar dominance relationship [26, 44]. Moreover, in these species both dominant and under-dominant rRNA genes were show to be equally transcribed in vivo after transfection into protoplasts of either the dominant or amphiploid species [45]. It seems clear that dominant and under-dominant rRNA genes of closely related species or genera may use the same transcription factors when together in the same nucleus [24, 25, 27]. In fact, an Arabidopsis rRNA gene promoter was found to be active in a cell-free system of a Brassica species [46]. Differential rRNA gene expression was also observed within a species, as in maize translocation and recombinant lines [4, 47], and between homologous rDNA loci in diploid rye [48]. Thus, the discrimination between active and silenced rDNA must be attributable to other mechanisms.

The Chromatin Modification Model: DNA Methylation and Acetylation Fig. (3). Rye NORs are usually silent in wheat x rye hybrids and triticale, but can be reactivated in other wheat-rye genomic During the last ten years another model for nucleolar combinations. In wheat lines carrying rye nucleolar chromosomes dominance has been proposed based on chromatin as the only source of alien chromatin, wheat and rye rDNA loci are modification related mechanisms, such as DNA methylation expressed in the majority of cells. Hence, in most metaphase cells, and histone acetylation which are responsible for alternative two pairs of rRNA gene clusters from wheat-origin ( ) and chromatin states of transcriptionally inactive and active one pair of rye-origin rDNA loci ( ) are revealed by silver- rRNA gene loci. Nucleolar dominance, as with other staining, indicating their previous transcription. epigenetic phenomena (such as the inactivation in mammalian females, gametic imprinting, paramutation events, homology-dependent gene-silencing, The Species-Specific Transcription Factor Model and transposon activity), has been shown to be modulated by DNA methylation [49]. Several reports in cereals, such as An alternative hypothesis emerged from the fact that hexaploid wheat, have shown that transcribed NOR loci are although the DNA sequences specifying the 18S, 5.8S and less methylated than ones that are untranscribed [16, 17]. In 25/26S rRNAs are highly conserved in higher wheat x rye F1 hybrids, and in triticale, the under-dominant [30-32], intergenic rDNA sequences have diverged rye-origin NORs are more methylated within the rDNA substantially during evolution [23-25]. In parallel, RNA pol I intergenic spacer sequences [50, 51]. In addition, after transcription factors would have also co-evolved bringing treatment with the hypomethylating agent 5-aza-2’- rRNA gene transcription to a species-specific condition, deoxycytosine (5-AC) [52], strong reactivation of rye NORs where the rRNA genes of one species could only be in the wheat-rye system was observed [50, 53]. The same transcribed if the appropriate species-specific transcription result was reported for under-dominant rRNA gene sets in factors were present. Evidence for this hypothesis came from Arabidopsis [27] and Brassica [44]. The direct involvement the use of a cell-free transcription system in mouse-human of methylation in the nucleolar dominance process has been hybrid cell lines. It was shown that a mouse extract can be primarily inferred from other studies that show a relation re-programmed to promote a human rRNA gene between methylated bases and point changes in DNA transcription event adding a specific human transcription topology that either prevent the binding of transcription factor [33-39]. Therefore, the absence of an adequate Nucleolar Dominance: A ‘David and Goliath’ Chromatin Imprinting Process Current Genomics, 2002, Vol. 3, No. 6 567

Fig. (4). In the wheat-rye system, rye-origin NORs can be expressed at various levels, according to the wheat-rye genomic combination. Whereas in the wheat-rye amphiploid triticale the rye NORs are clearly under-dominant (revealed by silver staining in only 26% of metaphase cells), their expression increases in the addition line (61%), and increases even more in the wheat-rye translocation lines (88%). These results, obtained in the same system, show that the enhancer-imbalance hypothesis along with the potential need of species-specific transcription factors are not applicable to this case. activators or allow the binding of methyl-chromoproteins genes, usually silent in the amphiploid triticale [21, 22, 53, that inhibits gene transcription. 55], are more heavily methylated [50, 51]. However, in the wheat addition line for the rye nucleolar chromosomes a However, the association of DNA methylation patterns significant increase in rye rRNA gene expression is observed with broader differential ribosomal gene expression [29], the rye rDNA being methylated at levels equivalent to processes has several unclear and some clear but the ones observed in triticale where the same genes are contradictory aspects in its general appliance. Firstly, it is highly suppressed [51]. Furthermore, where nucleolar difficult to reason how DNA methylation may account for dominance has been described in interspecific hybrids in early discrimination between rRNA gene sets in hybrids, Drosophila [56], only very diminutive amounts of methyla- leading to the preferential silencing of one parental set. In ted cytosine residues have been recently discovered in these this context, this phenomenon could be due to differences in species, showing no particular distribution patterns through- rDNA intergenic length and/or spacer sequences, in terms of out the genome, nor any variation during development [57]. competition for either methylation preventing molecules (that would preferentially attach to the dominant NORs) or Another mechanism usually related to chromatin pro-methylating agents (that would selectively bind to the alterations and gene activity is histone acetylation [58]. To under-dominant rDNA loci). However, mechanisms that are our knowledge, there are few studies proving the only based in the intergenic rRNA gene spacers seem involvement of histone acetylation in nucleolar dominance unlikely to account for such a discriminatory process, as no processes, and only in Brassica hybrids it was proven that clear rule concerning size and/or type of intergenic treatments with hyper-acetylating agents cause activation of sequences seem applicable. Aside from these theoretical normally non-transcribed rRNA genes [45]. However, the considerations, the experimental results are not in agreement direct relation found between histone acetylation at the gene with the general validity of this relationship. For example, in level and transcriptional activity mediated by RNA pol II and Brassica hybrids, rRNA transcripts from normally under- III [59, 60] may not extend to all rRNA genes. In the field dominant rDNA loci in untreated plants are clearly detected bean (Vicia faba) it seems that the NOR transcribed by the following seed germination in the presence of 5-AC [44]; RNA pol I complex does not follow this general rule [61]. In and both normal and methylated under-dominant rRNA this species, as in barley [62], direct immunolabelling of minigenes were shown to have equal abilities for in vitro acetylated and non-acetylated showed that the transcription [45]. Several hypothesis can be discussed, such overall acetylation patterns of the rDNA are not strictly as: (i) minor changes of methylation density in particular correlated with their transcriptional activity. These patterns sequences at the rDNA, (ii) other (regulatory) loci may be are inversely correlated with their replication patterns, as the responsible for the activation of previously silent ribosomal strongest histone acetylation at the NOR occurs during genes [45], or (iii) methylation might help in establishing a when the rRNA genes are being neither transcribed repressive chromatin state preventing gene promoter nor replicated. The absence of both acetylated and non- accessibility to the transcription machinery [24, 25]. Another acetylated histone forms within the nucleolus may be related intriguing result came from studies in Xenopus hybrids with the fact that transcriptional activation of the rDNA where there was no apparent difference in the methylation requires disruption of preformed [63]. In fact, state of dominant and under-dominant rRNA genes [54]. In active rRNA genes within nucleoli have been shown to be the wheat-rye system, it was already shown that rye rRNA devoid of complete core particles [61]. 568 Current Genomics, 2002, Vol. 3, No. 6 Viegas et al.

Nevertheless, this feature does not seem to be a particular transcription of rRNA genes, determine the form and characteristic of active rRNA genes, as transcriptionally function of NORs. The key to our understanding of nucleolar silent rDNA copies at condensed perinucleolar knobs are dominance may therefore reside in the chromatin structure of also free of acetylated histones [61]. Interestingly, induced rRNA gene loci. DNA hypomethylation and histone hyperacetylation do not have an additive effect on reactivating usually silent rDNA We also note that this type of genomic interaction, loci [45]. This suggests that both elements may play a part in causing preferential expression of genes according to their chromatin remodelling mechanisms that may be responsible parental origin, does not seem to be of general appliance. for chromatin topological states that can either promote or There are only a few examples of redundant prevent transcription [24, 25]. in hybrids and polyploids [71-73]. Several studies based on isozyme analysis clearly suggest that most gene loci Collectively these models for explaining nucleolar inherited from two or more progenitor species are dominance suggest that at the level of chromatin organiza- codominant [74-76] (Pontes and Viegas, unpublished data). tion is where most of the establishment and enforcement Using AFLP-cDNA to perform a genome screen for mechanisms reside. Hence the necessity of finding a broader orthologous genes silenced in Arabidopsis polyploids it was and more unifying explanation for this process, probably shown that only 2,5% of genes are subjected to differential associated with chromatin topology. expression [77]. Thus, these results indicate that nucleolar dominance may not be part of a broader genome silencing process as a result of a large-scale genomic interaction [78]. A CHROMATIN IMPRINTING MODEL: A RE- One apparent difference between the rRNA genes exhibiting INTERPRETATION OF NAVASHIN’S WORK differential amphiplasty and other genes that display codominance is the chromatin structure that underlies the Dominance and Codominance in Hybrids: States of two types of genetic information. While most genes exist in Chromatin Organization? the genome as single sequences in euchromatic domains, rRNA genes are present as large tandem arrays of repetitive We have alluded to, and ruled out, certain contenders for rDNA units arranged in clusters that clearly resemble the a role in provisioning nucleolar dominance, namely gene heterochromatic fraction. In fact, since McClintock’s promoter sequences, species-specific transcription factors, observation of the chromocentre associated with maize DNA methylation and histone acetylation. A consideration rDNA loci, it was shown that heterochromatin domains are of these options has led us to the view that DNA methylation frequently present in NORs [79]. and histone acetylation are more likely to be involved in maintaining a transcriptionally silent state for the under- In passing, we may also note that when genomes are dominant rRNA genes; and that the actual process of challenged by hybridity there are sometimes other responses discriminating between parental rDNA sets occupying the of a more ‘violent’ nature, involving the exclusion of an same nucleus is a matter of understanding the topology of entire genome as in haploidisation following wide species the rDNA chromatin. crosses [80], or even more significant events where somatic recombination/translocation accompanies the genome exclu- Wallace and Langridge [64] stated that the centromere- sion [81, 82]. These matters, however, are beyond the scope proximal portion of the NOR chromatin (the chromomere) is of this review. composed of rRNA genes that are in a highly condensed state (hence not being transcribed), while the secondary constriction is composed of a subset of rRNA genes that, by Size Matters: Smaller Genomes have More Penetrance virtue of having been previously transcribed remain decondensed even at metaphase. These authors also noted, Heterochromatin, a term introduced by Heitz [83], was by analyzing nucleoli formation in maize translocation lines, initially used to describe that part of a genome which is more that genes packaged in the chromomere can be reactivated compact than the . This chromatin fraction is following translocation events. Furthermore, several studies composed of highly repetitive DNA sequences which based on DNase I accessibility showed that the more active establish close relations with several heterochromatin- rRNA gene loci have more DNase I hypersensitive sites. specific proteins [79, 84, 85]. Heterochromatin was initially These sites are also less methylated in wheat and maize [47, distinguished by analysis, and was described as 65]; and in Xenopus hybrids dominant rRNA genes are also the fraction of the genome that maintained its overall more sensitive to DNase I treatments, but without detectable condensation throughout the cell cycle. However, repetitive changes in DNA methylation [54]. These results also DNA sequences are not only present in large/conspicuous indicate that actively transcribed ribosomal loci are in a more heterochromatic clusters. They can be dispersed in the decondensed chromatin state. In situ hybridization with genome, organized into smaller heterochromatic islands rRNA gene probes showed that rDNA chromatin within the which may pass the cytogeneticist analysis, even during nucleolus appears in general as diffuse chromatin fibers. The nucleus division [86, 87]. In fact, recent results have non-transcribed fraction of the NOR is visualized as a specified the minimum size/mass for C-band positive condensed chromatin block that adopts a perinucleolar staining [88, 89]. position [61, 66-69]. Based on cytogenetic evidences it has also been shown that rDNA decondensation is strictly Besides being present at the NORs, some protein coding necessary for rRNA gene transcription [70]. Thus, it seems genes are also found in the heterochromatin, as for example that the state of organization of rDNA chromatin, and the in Drosophila [90-92]. But in general heterochromatin Nucleolar Dominance: A ‘David and Goliath’ Chromatin Imprinting Process Current Genomics, 2002, Vol. 3, No. 6 569 domains are a gene-poor fraction of the genome [93]. Hence, prove that differences in heterochromatin contents between the initial status attributed to heterochromatin classed this closely related species, or even individuals of the same fraction as “ignorant”, “selfish” or “junk” DNA [93], a species, may account for several phenotypic effects [103, dispensable or parasitic portion of most genomes [94]. John 104], clearly bringing heterochromatin into the field of [95] pointed to heterochromatin as an “evolutionary relic”, . This view is strengthened by Brenner’s maintained in the genome only as a “by-product of molecular studies on vertebrate genome evolution, which suggest that events”. Lately however, studies on evolutionary even the smallest ones have some repetitive (heterochro- along with modern molecular approaches has led to a matic) sequences, sufficient for them to function properly. conceptual shift towards heterochromatin as a source of The basic vertebrate genome, may then have accumulated increasing variability and function [96-99]. It has been other repetitive sequences during evolution leading to high shown that this genome fraction can play important roles in order [105, 106]. Hence, the generic rule that can the modulation of gene expression, in chromosome structure, be drawn from these findings is that larger genomes usually in speciation and in evolution (review in [79]). Particularly reflect higher amounts of heterochromatin [79]. interesting are the studies that reveal a strong contribution of heterochromatin to the broad range of genome sizes To return to differential amphiplasty, and to join up the throughout biological taxa, that is to variation in nuclear thinking, we will now argue the case that the differential DNA amounts which are not correlated with organism expression of rRNA loci is related to genome size, as the complexity (review in [79]). Several approaches indicate that compilation of data in Table 1, from a number of well the variability in genome size in eukaryotes is consistently established cases, clearly indicates. While various achieved through a quantitative variability in copy numbers mechanisms have been found as contributing factors for of repetitive DNA sequences, usually organized in establishing nucleolar dominance, a unifying feature of all heterochromatic domains [79, 100-102]. Studies in the examples given is the difference in DNA amount mammals, cryptonomads and Drosophila (amongst others) between the parental species concerned. It seems clear that

Table 1. The Relation Between Nucleolar Dominance in Hybrids and Genome Size of Parental Species

Genome size (Mpb/1C) Hybrid Dominant sp. References Dominant sp. Under-dominant sp.

Crepis capillaris x C. alpina C. capillaris [1] 2058 [110] 2940 [110]

C. capillaris x C. dioscoridis C. capillaris [1] 2058 [110] 6003 [110]

C. capillaris x C. neglecta C. capillaris [1] 2058 [110] 2842 [110]

C. capillaris x C. tectorum C. capillaris [1] 2058 [110] 3308 [110]

C. setosa x C. tectorum C. setosa [1] 2279 [110] 3308 [110]

Xenopus leavis x X. borealis X. leavis [2] 3100 [111] 3480 [111]

D. melanogaster x D. virilis D. melanogaster [56] 116 [113] 340 [112]

D. melanogaster x D. simulans D. melanogaster [56] 116 [113] 116 [113]

Triticum aestivum x Aegilops umbellulata A. umbellulata [15] 4949 [110] 16979* [110]

Hordeum vulgare x Secale africanum H. vulgare [107] 4873 [110] 7277 [110]

H. bulbosum x H. vulgare H. vulgare [108] 5500 [110] 5500 [110]

T. aestivum x Secale cereale T. aestivum [19] 16979* [110] 8110 [110]

T. durum x Secale cereale T. durum [19] 11785* [110] 8110 [110]

Aegilops squarrosa x S. cereale Ae. squarrosa [109] 4998 [110] 8110 [110]

Brassica nigra x B. rapa B. nigra [26] [44] 473 [114] 512 [114]

B. nigra x B. oleracea B. nigra [26] [44] 473 [114] 633 [114]

B. rapa x B. oleracea B. rapa [26] [44] 512 [114] 633 [114]

* are poliploid species: T. aestivum = AABBDD and T. durum = AABB. 570 Current Genomics, 2002, Vol. 3, No. 6 Viegas et al. the under-dominant rRNA genes in the hybrid always reside heterochromatin content, and what is the rational underlying in the parental species with larger genome (Fig. 5). The the gene silencing process that it mediates. The configuration exceptions to this rule are the hybrids Drosophila of the repetitive DNA in heterochromatin, i.e. its dense melanogaster x D. simulans and Hordeum bulbosum x H. chromatin packaging, appears to correlate strongly with the vulgare, where the parental species have similar nuclear potent transcription silencing of this section of the genome DNA contents. Interestingly, where both parental species do (reviews in [79, 118-120]). Several examples in Drosophila have equivalent amounts of DNA, a small amount of and yeast [120] show that by juxtaposing an euchromatic sequence elimination occurs after hybridization [115, 116], gene with heterochromatin (by translocation or transgene and this may impose an imbalance in genome size which is insertion) a mosaic pattern of expression of the gene is sufficient to induce nucleolar dominance in the hybrid. It observed. This position effect variegation (PEV) is extensive should also be stressed that the differences in DNA contents in mammals, where transgene silencing occurs at a high level listed in the table are much larger than any potential DNA if the transgene is integrated as a high copy-number array, loss observed after hybridization. In hybrids involving but normal expression is allowed for low copy number polyploids, such as Triticum aestivum x Secale cereale and insertions [121]. Moreover, this cis-acting mode for Triticum durum x Secale cereale the species with larger heterochromatin mediated gene silencing is not the only way genome dominates over the smaller one. This apparent for its suppressive effects on gene expression, since nuclear exception is accounted for if DNA contents are compared per compartmentalization and chromosome pairing are also haploid genome. influenced by long-range silencing mechanisms [122, 123]. Both the cis- and trans-inactivation modes were clearly Completing the circle, by drawing in the relationship described for different genes in Drosophila [124-126]. previously established between DNA content and the amount Inactivation of gene expression following specific sequence of heterochromatin, it is plausible to argue that preferential duplication has been also observed in plants [127]. An rDNA transcription in relation to nucleolar dominance in interesting result was obtained in Arabidopsis hybrids, where hybrids occurs from the parental genome with the least parental dominance, examined for 20 candidate genes, was amount of heterochromatin. Besides the examples presented only found at three loci that are composed of repetitive in Table 1, further support for the case can be gained by DNA, the same sequences that build up heterochromatin looking to other cases of differential rRNA gene expression. [128]. Other studies in Drosophila also provide evidence that For example, in maize translocation lines larger nucleoli heterochromatic regions can play a role in interspecific form from recombinant NORs displaying the smaller rDNA nucleolar dominance; such as D. melanogaster NORs heterochromatic knobs [4]; and in wheat 1B NORs, with a (located on chromosomes X and Y) which dominate over the lower level of associated heterochromatin, organize nucleoli D. simulans NOR (located on chromosome X) [56]. double the size of the 6B rDNA loci [11, 117]. However, rearrangement of the heterochromatin that flanks D. melanogaster NORs causes activation of the D. simulans rDNA locus, as well as allowing normal transcription of the Functional Genomics of Heterochromatin: the Basis for D. melanogaster rRNA genes [129]. Furthermore, the role of Nucleolar Dominance heterochromatin domains in establishing repressive effects on gene transcription has been clarified by studies proving To sustain the argument it now has to be reasoned how the nuclear co-localization of the silenced genes and the differential rRNA gene expression is related to heterochromatic domains [79, 118]. It is intellectually

Fig. (5). Diagram illustrating the relationship between genome size of parental species and nucleolar dominance in Crepis hybrids. The under-dominant rDNA loci always belong to the larger genome (data taken from Table 1 in the text). Nucleolar Dominance: A ‘David and Goliath’ Chromatin Imprinting Process Current Genomics, 2002, Vol. 3, No. 6 571 inevitable to propose that the same mechanism underlies recognise and regulate parental gene sets, and to provide for nucleolar dominance. amphiplasty, remains unanswered. Old theories and new results converge and integrate to finally close the circle. In support of the argument one may look into the parallel Bringing together two entire or partial genomes in the same behavior of heterochromatin dynamics and rDNA expression nucleus can be regarded as adjusting each parental genotype patterns throughout development, and examples of the role to a common space, the hybrid nucleus. Nuclear volumes of of heterochromatin in modulating rDNA transcription. In polyploids do not usually reflect the addition of the parental Drosophila the strength of heterochromatin mediated gene nuclear volumes. By determining the nuclear volumes of silencing decreases during development [130]. In addition, meristematic root-tip cells (by confocal microscopy) in rye, C-bands cannot be distinguished in metaphase chromosomes wheat and its amphiploid triticale, this lack of additivity can during early embryogenesis, only acquiring this chromatin be easily observed: the amphiploid volume is significantly state of C-band manifestation from the blastoderm onwards smaller than the added rye and wheat volumes (Silva and [131]. More recently the action of some protein groups has Viegas, unpublished results). It is also known that rye and been related to pattern formation during Drosophila embryo- wheat genomes do not intermix throughout the cell cycle and genesis, through the establishment of repressive chromatin that at interphase both genomes are organized as discrete states [132, 133]. In plants, the number and size of interphase chromatin domains in triticale, revealing a closer association heterochromatin domains is known to be related to the of parental-origin chromatin [55, 137]. This non-random functional activity of the nucleus and to tissue differentiation distribution finds parallel in the heterochromatin disposition [134], and nucleolar activity seems to be correlated with cell patterns, since heterochromatin domains are not randomized type and metabolic rates [135]. Nucleolar dominance also throughout the nuclear territory [79]. If a genome has less appears to be developmentally regulated: the usually silent available nuclear space in the hybrid, relative to its diploid under-dominant rRNA genes becoming active in the condition, chromatin association is more likely to occur, and transition to the floral meristem (e.g. Brassica hybrids, [26]), to a greater extent in the larger genome, establishing a higher or following (e.g. wheat x rye amphiploid, [22]). In level of het-het interactions (Fig. 6). Considering the co- wheat, the largest nucleoli are formed in the haploid micro- localization of enriched heterochromatin domains, as spore [12]. Interestingly, modification in other epigenetic observed for the rDNA, and silent-mating type markers, such as DNA methylation patterns, have also been repetitive DNA regions in yeast [135, 138], and the proven observed during these reproductive related events [136]. capacity of heterochromatin to propagate silent conformations in neighboring chromatin domains, we can begin to develop a model for how parental DNA sets are Seventy Years of Nucleolar Dominance: Closing the discriminated and nucleolar dominance established. The Circle under-dominant genes will belong to the largest genome, and Notwithstanding the commentary given above, the hence to the genome enriched in repetitive DNA organized fundamental question of how discrimination operates to in heterochromatin domains. Intuitively we can now imagine

Fig. (6). Nucleolar dominance: a chromatin imprinting model. Parental genomes are represented as nuclei with large (top left) and with small genomes (top, right). Differences in genome sizes are shown as being due to distinct amounts of repetitive DNA sequences that build up heterochromatin domains. The amphiploid nucleus (below) has a smaller nuclear volume than the sum of the nuclear volumes of the parental species. In this hybrid nucleus, parental chromatin behaves differently in comparison to its behaviour in its own genomic/nuclear environment, where the larger genome adjusts to a smaller volume, triggering a more intimate association between heterochromatic domains. Genes or gene clusters in the under-dominat DNA will undergo a chromatin imprinting process responsible for their silencing, by virtue of being positioned adjacent to repetitive DNA sequences. 572 Current Genomics, 2002, Vol. 3, No. 6 Viegas et al. how the occupancy of a common space will cause the hybrids, allowing for the identification of repetitive DNA heterochromatin-rich under-dominant NORs to be silenced, sequences linked to parental silenced genes. and to assume a quiescent state that is maintained throughout successive cell divisions. Chromatin modeling mechanisms, such as DNA methylation, histone hypoacetylation, and ACKNOWLEDGEMENTS histone methylation (reviewed in [139]) can be enlisted to police this silent mode. This model can also explain why The authors are most grateful to Prof. Neil Jones for the both in Brassica and Arabidopsis hybrids, under-dominant enlightening discussions and critical review of the manu- rRNA minigenes (cloned in plasmid vectors) are expressed script. This work results from research projects supported by in hybrid protoplasts whereas the chromosomal copies of the Fundação para a Ciência e Tecnologia, Portugal (PRAXIS/ under-dominant rDNA units remain silent in the same C/AGR/11171/1998; POCTI/AGR/34000/2000; POCTI/BCI/ protoplasts [27, 45]. Some hitherto unclear results on 38557/2001). nucleolar dominance in the wheat-rye system can also be sustained by the current hypothesis. In wheat addition lines of rye nucleolar chromosomes, the larger expression of the REFERENCES rDNA loci of rye origin, relative to their behaviour in the [1] Navashin, M. 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