Mitotic misbehavior of a Drosophila melanogaster satellite in ring chromosomes: insights into intragenomic conflict among heterochromatic sequences

Abstract

In eukaryotes, abnormally circularized chromosomes, known as ‘rings,’ can be mitotically unstable. Some rings derived from a compound X-Y chromosome induce mitotic abnormalities during the embryonic cleavage divisions and early death in Drosophila melanogaster, but the underlying basis is poorly understood. We recently demonstrated that a large region of 359-bp satellite DNA, which normally resides on the X chromosome, prevents sister ring chromatids from segregating properly during these divisions. Cytogenetic comparisons among 3 different X-Y rings with varying levels of lethality showed that all 3 contain similar amounts of 359-bp DNA, but the repetitive sequences surrounding the 359-bp DNA differ in each case. This finding suggests that ring misbehavior results from novel heterochromatin position effects on the 359-bp satellite. The purpose of this view is to explore possible explanations for these effects with regard to heterochromatin formation and replication of repetitive sequences. Also discussed are similarities of this system to a satellite-based hybrid incompatibility and potential influences on genome evolution.

Chromosome aberrations can have dire genetic consequences in predictable ways. For instance, large deletions cause haplo-insufficiency in the diploid state, while certain inversions disrupt the expression of important genes located near the breakpoints.¹ However, some chromosome aberrations are more enigmatic. An intriguing example is the abnormal circularization of chromosomes. In their normal state, eukaryotic chromosomes are linear. But they can become abnormally circularized through either improper joining of 2 broken chromosome ends during repair or rare intra-chromosomal recombination.^2-4 Circularized chromosomes – here referred to as ‘rings’ – have been documented in multiple organisms, including insects, rodents, and humans.^4-9 In many cases, individuals are somatically mosaic for the rings, indicating that these chromosomes are unstable during mitotic division.^4,10,11 Rings of the sex chromosomes (X and Y) can even be lethal to the carrying individual,^9,11 although the underlying basis is poorly understood.

In one of the first experimental efforts to study ring instability, Oster (1964) used X-ray mutagenesis to produce a collection of rings derived from a linear, compound X-Y chromosome in Drosophila melanogaster. Interestingly, several of his X-Y rings induced chromosome bridges during the embryonic cleavage divisions, regardless of whether they were paternally or maternally inherited.^9,11 These bridges led to the accumulation of additional mitotic abnormalities and embryonic death.^9,11 Several important questions were borne out of Oster’s observations. First, were the bridges in ring-carrying individuals made solely of ring chromatin, and if so, did specific sequences in the rings cause bridging? Or alternatively, did the rings cause bridging of the other chromosomes? Second, the developmental period in which the bridges formed—the cleavage divisions—occur before the onset of embryonic gene expression. Thus, the bridges and lethality could not be reasonably explained by the loss or disruption of linked coding genes acting zygotically. Thus, were non-coding sequences involved? Additionally, a number of Oster’s X-Y rings were incapable of inducing lethality, clearly demonstrating that chromosome circularity itself does not cause bridging.^9,11 What then was the basis of chromosome bridging caused by rings?

Some years after Oster’s work, Stone (1982) mapped the lethal effect of a particular X-Y ring to a region near the terminus of the Y’s short arm. The ring’s precursor chromosome, B^S Y y⁺, carries on each of its ends a region of translocated heterochromatin from the X’s pericentromeric region, in addition to a visible marker.^1,11 Upon ring formation, both visible markers were lost (which was, in fact, Oster’s means of screening for rare rings) but much of the X-derived heterochromatin remained. Because of this and the fact that the Y chromosome is largely heterochromatic, the lethal-causing region of this X-Y ring promised to be heterochromatic sequences derived from the X chromosome, the Y chromosome, or a combination of the two. These observations further begged the question of how and why the underlying sequences, whatever they might be, misbehaved in the ring context but not in their native chromosomal arrangements.

In addition, Stone demonstrated that the maternal genotype strongly influences whether a given ring is lethal or not. For example, some rings were strongly lethal when crossed into certain maternal genotypes but almost fully viable within other maternal genotypes.¹¹ The interpretation was that factors in the egg’s cytoplasm governed ring instability, but what are these factors and how do they operate?

Our group recently conducted a study to begin addressing these questions.¹² We performed cytological analyses of young embryos carrying 3 different X-Y rings: a highly lethal one known as Y^cF#2 from Oster’s original collection,^9,11 another called R(1;Y)15 that was produced more recently through FLP-FRT recombination¹³ and is moderately lethal, and a third, R(1)2, which is completely non-lethal.¹² We found that bridging occurs largely through the abnormal mitotic behavior of an α-satellite known as the 359-bp repeat, which is present in all examined rings. Our work argues that the misbehavior of this satellite during mitosis depends largely on its heterochromatic sequence environment—that is, the adjacently located, repetitive sequences and their unique chromatin compositions—in addition to the maternal genotype. Thus, this lethal ring system may reveal a new property of heterochromatic sequences – that certain ones can be ‘incompatible’ with others when they are closely juxtaposed. Such intragenomic incompatibilities may disrupt normal chromatin structure, thereby inducing replication defects or breaks that inhibit ring segregation. These possibilities are discussed here, along with similarities of this system to a satellite-based hybrid incompatibility and implications for genome stability and evolution.

359-bp Satellite DNA Prevents Ring Chromatid Segregation

Using fluorescence in situ hybridization (FISH), we screened through a number of repetitive sequences that mark each of the sex and non-sex chromosomes in order to identify the particular sequences comprising the ring-induced bridges. We discovered that a large part of the bridges consisted of 359-bp satellite DNA (Fig. 1).¹² Normally, this satellite exists as hundreds to thousands of tandemly repeated 359-bp monomers located primarily in a single, multi-megabase pair region or ‘block’ within the pericentromeric heterochromatin of the X chromosome.¹⁴ This block spans between the centromere and the bobbed⁺ (rDNA) locus (Fig. 1). However, each of the examined rings carries a 359-bp block that is similar in size to the one present on the X (Fig. 1).¹² During the embryonic cleavage divisions, the ring-linked 359-bp block becomes stretched across the metaphase plate, and comprises the majority of lagging chromatin in the bridges (Fig. 1).¹² In contrast, no sequences from the autosomes or the normal X, including the X-linked 359-bp block, mis-segregate.¹² The bridging effect, therefore, maps uniquely to the ring-linked 359-bp block.

Figure 1. The 359-bp satellite block, normally located on the Drosophila melanogaster X chromosome, causes chromosome bridges when present on some X-Y ring chromosomes. (A) a mitotic cleavage division at telophase with a chromosome bridge. The 359-bp block in its native position on the X segregates normally (white arrows); in contrast, a similarly sized 359-bp block on the highly lethal Y^cF#2 ring forms a chromosome bridge (red arrow). This effect results presumably because sister ring chromatids cannot separate properly at the 359-bp block. Top2 (green) accumulates on the bridge and 359-bp DNA. (B) and (C) mitotic chromosomes from larval neuroblasts. The genotype in (B) includes the Y^cF#2 ring, containing a single block of 359-bp satellite (red arrow) that was formed from the joining of the 2 smaller satellite regions in the precursor chromosome. The genotype in (C) includes the ring precursor, B^S Y y⁺, which contains a region of 359-bp satellite DNA on each of its ends (red arrowheads). In (B) and (C) the white arrows indicate the 359-bp block on the X chromosome, while AATAT satellite is labeled in green.

The fact that 359-bp DNA becomes stretched across the metaphase plate while more centromere-proximal sequences move toward the spindle poles indicates that the sister ring chromatids attempt to separate at anaphase but cannot due to separation failure specifically at the 359-bp block. This separation failure almost certainly reflects a physical connectedness of the 2 sister ring chromatids in this region, perhaps by DNA entanglement or unresolved crossovers. Support for this idea stems from our observation that Topoisomerase 2 (Top2) becomes enriched on the lagging 359-bp DNA (Fig. 1).¹² Top2 is an enzyme that catalyzes DNA catentation-decatenation during S-phase and DNA repair.¹⁵ The enrichment of Top2 on ring bridges likely reflects a response of this enzyme to resolve tangled or joined sequences within the 359-bp block.

Heterochromatin Position Effects on the 359-bp Satellite

An important question is why the 359-bp satellite forms bridges in some ring forms but not in others or its native position on the X chromosome. A major difference among these chromosomes is the local sequence environment in which the 359-bp satellite is located. Thus, the 359-bp satellite may be prone to positional effects that stem from its proximity to certain other repetitive sequence blocks. In support of this idea, we found that the repetitive sequences surrounding the 359-bp block differ among the 3 examined rings.¹² For example, the 359-bp block on the strongly lethal ring is flanked on either end with small regions of rDNA and AATAT satellite. In contrast, the 359-bp block on the moderately lethal ring resides beside rDNA on one end and a small region of unidentified heterochromatin on the other, and the 359-bp block on the non-lethal ring is adjacent to AATAT satellite on one side and unidentified sequences on the other. Thus, in each ring the 359bp block has become positioned next to repetitive sequences that are either not normally located near it, such as Y-derived sequences, or reordered from their normal arrangements within the X pericentromeric region.

Based on these findings, we propose that ring bridging ultimately stems from ‘incompatibilities’ between the 359-bp satellite and certain other repetitive sequences, which normally reside at different chromosomal locations or in different arrangements. When closely juxtaposed or rearranged, however, these sequences interact deleteriously to cause bridging – and the ring structure itself provides a means for bringing these sequences together. Discussed below are some ideas for chromatin-level interactions that could underlie these repetitive sequence incompatibilities and how they lead to sister ring chromatid separation failure.

Potential Causes of Heterochromatin Position Effects

The primary cause of ring bridging may involve heterochromatin structural abnormalities. Heterochromatin, which forms during the cleavage divisions in Drosophila, is characterized by its association with proteins such as Heterochromatin Protein 1 (HP1) and Su(Var)3–9.^16-19 These proteins localize to the pericentromeric regions beginning around mitotic cycle 10.^20,21 However, a recent study demonstrated that mutations in Piwi and Ago2, genes known to play important roles in heterochromatin maintenance in multiple organisms, result in chromosome mis-segregation as early as the first mitotic division.²² Thus, although these defects could result from pleiotropic effects, they may instead reflect that heterochromatin formation begins during the earliest cleavage divisions.

Although all of heterochromatin associates with general factors like HP1, heterochromatin likely exists as different types, each defined by unique combinations of additional proteins and posttranslational histone marks.^23,24 For example, in D. melanogaster, the AT hook motif-containing protein D1 associates primarily with AT-rich satellite repeats including AATAT and the 359-bp repeat.²³ Similarly, the GAGA protein, which functions during interphase as a transcription factor, associates with GA repeat blocks during mitosis.²⁵ In addition, many regions of constitutive heterochromatin contain histone H3 methylated at lysine residue 9, whereas heterochromatin located nearby protein-coding genes often has histone H3 methylated at lysine residue 27.¹⁶ Experimental manipulation of these histone marks can induce heterochromatin structure abnormalities and chromosome mis-segregation.^26-28

Thus, the abnormal behavior of 359-bp in ring form may result from interference between different heterochromatin types, perhaps through the spreading of certain proteins between the 359-bp block and surrounding sequence blocks as heterochromatin forms during cleavage (Fig. 2). The idea of protein spreading among different sequence blocks is supported by previous observations that the D1 protein localizes in a gradient across the X-linked 359-block and adjacent rDNA sequences in larval neuroblasts.²³ D1 abundance is highest within the 359-bp block and tapers away into the rDNA region.²³

Figure 2. Model for heterochromatin position effects on satellite DNA in lethal ring chromosomes. Two linear chromosomes (top), each containing a set of compatible satellite blocks in their pericentric regions (red and gray blocks, or green and gray blocks). Chromosomal rearrangements such as fusions, translocations, and circularization events bring together incompatible satellite blocks (red and green blocks). Satellite incompatibility may result from inappropriate spreading of satellite repeat-specific proteins (green circles) into the adjacent red region, representative of the 359-bp block. This effect causes abnormal heterochromatin structure as heterochromatin begins to form during the early mitotic cleavage divisions. The upshot is incomplete replication and/or formation of abnormal crossovers due to repair of DNA breakage, which tethers together sister ring chromatids and prevents them from separating properly during anaphase. Chromatin-associated factors in the maternal cytoplasm may influence any of these stages, thereby modulating ring-induced mitotic defects and lethality level.

Perturbation of 359-bp chromatin structure may cause sister ring chromatid separation failure by disruption of replication in this satellite block. Under normal circumstances, the replication of satellites occurs later during S-phase than euchromatic sequences,^29,30 a characteristic that may occur at least in part because satellites are highly condensed into heterochromatin³¹ and, in some cases, they can be prone to the formation of hairpin or triple-helical structures when in single strand form within the replication fork, an effect that may induce replication fork stalling.^32,33 Perhaps even more challenging is achieving complete replication of satellite DNA during the rapid cleavage divisions, each cycle containing no G1 or G2 phase and lasting only several minutes.¹ Replication during these rapid divisions may require the firing of additional replication origins and, thus, a greater need for resolving congressing origins.³⁴ Interestingly, there are strong ties between the replication of heterochromatic sequences and the protein components of heterochromatin. For example, previous studies demonstrated genetic and biochemical interactions between replication licensing components, such as ORC2, and heterochromatin proteins HP1 and HOAP, a telomere capping protein.^35-37 RNAi depletion of HP1 results in an accelerated replication rate for heterochromatic sequences in Drosophila Kc cells.³⁸ Thus, the structure of heterochromatin may regulate the firing of replication origins in heterochromatin as it forms during cleavage. Structural alteration of the 359-bp block in lethal rings may disrupt the recognition of replication origins, loading of the replication licensing machinery, or function of the replication apparatus itself, thus resulting in inappropriate joining of sister ring chromatids (Fig. 2).

Alternatively, separation failure of sister ring chromatids may stem from unresolved crossovers in the 359-bp block. Normally, crossovers formed from repair of double strand breaks are resolved or even inhibited during cleavage by the BLM helicase.³⁹ Loss of this enzyme through mutation results in chromosome bridges and other mitotic defects during the cleavage divisions, perhaps because crossovers cannot be properly resolved.³⁹ Structural alterations of the 359-bp block when positioned within a certain new heterochromatin environment may make 359-bp satellite DNA more sensitive to breaks and/or incomplete replication, similar to certain ‘fragile’ chromosome sites in humans.^40,41 DNA breaks also may result from abnormal mobilization of transposable elements (TEs) that become de-repressed because of satellite structural alterations. Indeed, recent studies have found evidence for chromatin proteins that evolve rapidly between Drosophila species in order to suppress mobilization of a wide range of TEs in heterochromatin.^42,43 In either case, the repair machinery may be incapable of resolving crossovers from repair of breaks, thus leading to tethered ring chromatids and bridging.

Influence of the Maternal Genotype on Ring-Induced Lethality

In agreement with observations made by Stone (1982), our experiments demonstrate that the maternal genotype substantially influences the penetrance of ring lethality. For example, the strongly lethal ring shows a wide range of lethality levels when crossed to a panel of females from different wild caught, genetic backgrounds (Table 1; see also ref. 12 for a more extensive panel of crosses). The moderately lethal ring shows proportional levels of lethality to these same maternal lines,¹² strongly suggesting that the same maternal effect(s) are governing instability of both lethal rings. This idea is consistent with the fact that the mitotic cleavage divisions occur under the control of maternally loaded factors.⁴⁴

Table 1. The maternal genotype strongly influences embryonic lethality caused by an X-Y ring chromosome.

Maternal Genotype	F1 males	F1 females	Percent F1 males (ring-bearing progeny)
PN3E	122	161	43.1
Cam27	65	118	35.5
PN1E	44	125	26.0
Cam5	33	172	16.1
Bei68	0	160	0.0

Females in the crosses are from wild caught isolines (courtesy of C. Aquadro). These females were crossed to males carrying the Y^cF#2 highly lethal ring¹¹ and progeny were reared at 25 °C.

What might these ring-interacting factors be and how could they influence ring instability? Females with low ring instability may produce egg cytoplasm containing a higher level of proteins that facilitate better resolution of sister ring chromatids. For example, higher levels of Top2 in certain female lines may lead to more efficient repair of catenated 359-bp DNA between sister ring chromatids, resulting in weaker embryonic lethality. Another possibility is variation in maternally loaded factors that influence heterochromatin structure, such PIWI, AGO2 or small RNAs produced through the piRNA pathway.^22,45-48 Differences in levels of heterochromatin factors in the egg cytoplasm may buffer the effects induced by other repetitive sequences on the structure of the 359-bp block, thereby suppressing sister ring catenation (Fig. 2). Indeed, it is likely that the highly lethal Y^cF#2 ring from Oster’s collection has persisted for decades because it resides in a genetic background that contains these or perhaps other maternal factors that suppress ring bridging.

Parallels with a Hybrid Lethal System Involving the 359-bp Satellite

The cellular phenotypes of the lethal ring system are strikingly similar to those arising in lethal hybrid embryos produced from D. simulans females and D. melanogaster males. Specifically, hybrid embryos receiving an X chromosome from D. melanogaster (i.e., female-destined embryos) undergo widespread chromosome bridges that form solely from mis-segregating X chromatids during the cleavage divisions.⁴⁹ The X chromatids fail to separate at anaphase, and similar to the lethal ring system, chromatid separation failure occurs at the 359-bp block.⁴⁹ Additional similarities include enrichment of Top2 on the lagging 359-bp DNA and a strong influence of the D. simulans maternal genetic background on level of hybrid lethality.⁴⁹ Generally, these characteristics argue that the satellite-based defects in these 2 systems occur through similar mechanisms. The satellite based hybrid embryonic lethality is a clear example of an early, post-zygotic reproductive barrier resulting from distinct evolutionary changes between the genomes of D. melanogaster and D. simulans. We propose that the lethal ring system may serve as an effective intra-specific (i.e., within the D. melanogaster species) model system to explore the role of satellite DNA dysfunction in hybrid embryonic lethality.

An important aspect worthy of mention is that, whereas the 359-bp satellite block in lethal ring chromosomes has been placed into a foreign heterochromatic sequence environment, this satellite block resides in its native location on the X chromosome in lethal hybrids. In the latter case, the primary incompatibility resulting in 359-bp-induced segregation defects stems from factors in the D. simulans egg cytoplasm.^50,51 It remains to be determined if the same maternal factors govern 359-bp behavior in these 2 systems. Indeed, we previously hypothesized that variations or even absence of certain small RNAs or other heterochromatin factors may underlie the influence of the D. simulans maternal cytoplasm on the 359-bp block.^49,52 Another interesting possibility is that the D. simulans DNA replication or repair machinery is directly incompatible with the 359-bp satellite. A variant of this satellite is present in the D. simulans genome, but it is highly diverged from the D. melanogaster sequence and is much less abundant.^49,53 It is possible that the specific structure of the 359-bp satellite repeat, perhaps in combination with its high copy number in one location, poses unique challenges to D. simulans components of DNA replication or repair. In general, the D. simulans genome contains substantially less satellite DNA than D. melanogaster.^14,54 Despite this difference, other large pericentromeric regions from D. melanogaster segregate normally in hybrid embryos, suggesting that they undergo replication normally.⁴⁹ Thus, the 359-bp defects do not result simply from the inability of the D. simulans replication machinery to replicate large quantities of satellite repeats.

The Uniqueness of the 359-bp Satellite and Implications for Genome Evolution

The first few mitotic divisions of the embryo are arguably one of the most critical periods of development, giving rise to the cells that will found the major tissue types. In Drosophila, these cleavage divisions involve packaging the genome’s repetitive sequences into heterochromatin, a process that is important for stable transmission of the hereditary material. Our recent analyses of lethal X-Y rings, along with previous studies on lethal hybrid embryos, together demonstrate a unique sensitivity of the D. melanogaster 359-bp satellite to variations in its local sequence environment and the maternal genotype during these divisions. We propose that these variations can perturb the chromatin structure of the 359-bp block, thereby causing incomplete replication or unresolved crossovers that prevent sister chromatid separation. A number of questions remain to be addressed regarding the cis effect on this satellite. What are the specific molecular interactions that dictate incompatibility between the 359-bp satellite and other heterochromatic sequences in ring form? Are there other heterochromatic sequences that, like the 359-bp satellite, misbehave when placed into new chromosomal environments? And if so, are these sequences restricted to the sex chromosomes?

Misbehavior of satellite DNA clearly has strong and immediate effects on genome stability. This phenomenon also can influence the evolution of the eukaryotic genome. Our previous work demonstrated that 359-bp-induced mitotic defects cause strong reproductive isolation between D. melanogaster and several of its sibling species.⁴⁹ This effect, in turn, blocks gene flow, which may facilitate further genome divergence between the parent species. In addition, deleterious interactions among different heterochromatic sequences may also influence genome evolution within individual species. Previous studies have shown that existing genome organization in a number of different species has arisen in part from large-scale rearrangements that occurred both within and between individual chromosomes.^55-59 Our work suggests that for rearrangements occurring within heterochromatin, only those that involve joining of compatible repetitive sequence blocks will persist. Thus, the misbehavior of the 359-bp block reveals a novel type of intragenomic conflict that likely has helped to shape the heterochromatic sequence organization of eukaryotic chromosomes.