The activation of a neocentromere in Drosophila requires proximity to an endogenous centromere.

The centromere is essential for proper segregation and inheritance of genetic information. Centromeres are generally regulated to occur exactly once per chromosome; failure to do so leads to chromosome loss or damage and loss of linked genetic material. The mechanism for faithful regulation of centromere activity and number is unknown. The presence of ectopic centromeres (neocentromeres) has allowed us to probe the requirements and characteristics of centromere activation, maintenance, and structure. We utilized chromosome derivatives that placed a 290-kilobase "test segment" in three different contexts within the Drosophila melanogaster genome--immediately adjacent to (1) centromeric chromatin, (2) centric heterochromatin, or (3) euchromatin. Using irradiation mutagenesis, we freed this test segment from the source chromosome and genetically assayed whether the liberated "test fragment" exhibited centromere activity. We observed that this test fragment behaved differently with respect to centromere activity when liberated from different chromosomal contexts, despite an apparent sequence identity. Test segments juxtaposed to an active centromere produced fragments with neocentromere activity, whereas test segments far from centromeres did not. Once established, neocentromere activity was stable. The imposition of neocentromere activity on juxtaposed DNA supports the hypothesis that centromere activity and identity is capable of spreading and is regulated epigenetically.

The role of Drosophila CID in kinetochore formation, cell-cycle progression and heterochromatin interactions.

Centromere function requires the coordination of many processes including kinetochore assembly, sister chromatid cohesion, spindle attachment and chromosome movement. Here we show that CID, the Drosophila homologue of the CENP-A centromere-specific H3-like proteins, colocalizes with molecular-genetically defined functional centromeres in minichromosomes. Injection of CID antibodies into early embryos, as well as RNA interference in tissue-culture cells, showed that CID is required for several mitotic processes. Deconvolution fluorescence microscopy showed that CID chromatin is physically separate from proteins involved in sister cohesion (MEI-S332), centric condensation (PROD), kinetochore function (ROD, ZW10 and BUB1) and heterochromatin structure (HP1). CID localization is unaffected by mutations in mei-S332, Su(var)2-5 (HP1), prod or polo. Furthermore, the localization of POLO, CENP-meta, ROD, BUB1 and MEI-S332, but not PROD or HP1, depends on the presence of functional CID. We conclude that the centromere and flanking heterochromatin are physically and functionally separable protein domains that are required for different inheritance functions, and that CID is required for normal kinetochore formation and function, as well as cell-cycle progression.

Determining centromere identity: cyclical stories and forking paths.

The centromere is the genetic locus required for chromosome segregation. It is the site of spindle attachment to the chromosomes and is crucial for the transfer of genetic information between cell and organismal generations. Although the centromere was first recognized more than 120 years ago, little is known about what determines its site(s) of activity, and how it contributes to kinetochore formation and spindle attachment. Recent work in this field has supported the hypothesis that most eukaryotic centromeres are determined epigenetically rather than by primary DNA sequence. Here, we review recent studies that have elucidated the organization and functions of centromeric chromatin, and evaluate present-day models for how centromere identity and propagation are determined.

The Drosophila Su(var)2-10 locus regulates chromosome structure and function and encodes a member of the PIAS protein family.

The conserved heterochromatic location of centromeres in higher eukaryotes suggests that intrinsic properties of heterochromatin are important for chromosome inheritance. Based on this hypothesis, mutations in Drosophila melanogaster that alter heterochromatin-induced gene silencing were tested for effects on chromosome inheritance. Here we describe the characterization of the Su(var)2-10 locus, initially identified as a Suppressor of Position-Effect Variegation. Su(var)2-10 is required for viability, and mutations cause both minichromosome and endogenous chromosome inheritance defects. Mitotic chromosomes are improperly condensed in mutants, and polytene chromosomes are structurally abnormal and disorganized in the nucleus. Su(var)2-10 encodes a member of the PIAS protein family, a group of highly conserved proteins that control diverse functions. SU(VAR)2-10 proteins colocalize with nuclear lamin in interphase, and little to no SU(VAR)2-10 is found on condensed mitotic chromosomes. SU(VAR)2-10 is present at some polytene chromosome telomeres, and FISH analyses in mutant polytene nuclei revealed defects in telomere clustering and telomere-nuclear-lamina associations. We propose that Su(var2-10 controls multiple aspects of chromosome structure and function by establishing/maintaining chromosome organization in interphase nuclei.

Identification of chromosome inheritance modifiers in Drosophila melanogaster.

Faithful chromosome inheritance is a fundamental biological activity and errors contribute to birth defects and cancer progression. We have performed a P-element screen in Drosophila melanogaster with the aim of identifying novel candidate genes involved in inheritance. We used a "sensitized" minichromosome substrate (J21A) to screen approximately 3,000 new P-element lines for dominant effects on chromosome inheritance and recovered 78 Sensitized chromosome inheritance modifiers (Scim). Of these, 69 decreased minichromosome inheritance while 9 increased minichromosome inheritance. Fourteen mutations are lethal or semilethal when homozygous and all exhibit dramatic mitotic defects. Inverse PCR combined with genomic analyses identified P insertions within or close to genes with previously described inheritance functions, including wings apart-like (wapl), centrosomin (cnn), and pavarotti (pav). Further, lethal insertions in replication factor complex 4 (rfc4) and GTPase-activating protein 1 (Gap1) exhibit specific mitotic chromosome defects, discovering previously unknown roles for these proteins in chromosome inheritance. The majority of the lines represent mutations in previously uncharacterized loci, many of which have human homologs, and we anticipate that this collection will provide a rich source of mutations in new genes required for chromosome inheritance in metazoans.

Sister-chromatid cohesion via MEI-S332 and kinetochore assembly are separable functions of the Drosophila centromere.

Attachment, or cohesion, between sister chromatids is essential for their proper segregation in mitosis and meiosis [1,2]. Sister chromatids are tightly apposed at their centromeric regions, but it is not known whether this is due to cohesion at the functional centromere or at flanking centric heterochromatin. The Drosophila MEI-S332 protein maintains sister-chromatid cohesion at the centromeric region [3]. By analyzing MEI-S332's localization requirements at the centromere on a set of minichromosome derivatives [4], we tested the role of heterochromatin and the relationship between cohesion and kinetochore formation in a complex centromere of a higher eukaryote. The frequency of MEI-S332 localization is decreased on minichromosomes with compromised inheritance, despite the consistent presence of two kinetochore proteins. Furthermore, MEI-S332 localization is not coincident with kinetochore outer-plate proteins, suggesting that it is located near the DNA. We conclude that MEI-S332 localization is driven by the functional centromeric chromatin, and binding of MEI-S332 is regulated independently of kinetochore formation. These results suggest that in higher eukaryotes cohesion is controlled by the functional centromere, and that, in contrast to yeast [5], the requirements for cohesion are separable from those for kinetochore assembly.

Centromere proteins and chromosome inheritance: a complex affair.

Centromeres and the associated kinetochores are involved in essential aspects of chromosome transmission. Recent advances have included the identification and understanding of proteins that have a pivotal role in centromere structure, kinetochore formation, and the coordination of chromosome inheritance with the cell cycle in several organisms. A picture is beginning to emerge of the centromere-kinetechore as a complex and dynamic structure with conservation of function at the protein level across diverse species.

Neocentromere activity of structurally acentric mini-chromosomes in Drosophila.

Chromosome fragments that lack centromeric DNA (structurally acentric chromosomes) are usually not inherited in mitosis and meiosis. We previously described the isolation, after irradiation of a Drosophila melanogaster mini-chromosome, of structurally acentric mini-chromosomes that display efficient mitotic and meiotic transmission despite their small size (under 300 kb) and lack of centromeric DNA. Here we report that these acentric mini-chromosomes bind the centromere-specific protein ZW10 and associate with the spindle poles in anaphase. The sequences in these acentric mini-chromosomes were derived from the tip of the X chromosome, which does not display centromere activity or localize ZW10, even when separated from the rest of the X. We conclude that the normally non-centromeric DNAs present in these acentric mini-chromosomes have acquired centromere function, and suggest that this example of 'neocentromere' formation involves appropriation of a self-propagating centromeric chromatin structure. The potential relevance of these observations to the identity, propagation and function of normal centromeres is discussed.

Identification of trans-acting genes necessary for centromere function in Drosophila melanogaster using centromere-defective minichromosomes.

Deletions in the Drosophila minichromosome Dp1187 were used to investigate the genetic interactions of trans-acting genes with the centromere. Mutations in several genes known to have a role in chromosome inheritance were shown to have dominant effects on the stability of minichromosomes with partially defective centromeres. Heterozygous mutations in the ncd and klp3A kinesin-like protein genes strongly reduced the transmission of minichromosomes missing portions of the genetically defined centromere but had little effect on the transmission of minichromosomes with intact centromeres. Using this approach, ncd and klp3A were shown to require only the centromeric region of the chromosome for their roles in chromosome segregation. Increased gene dosage also affected minichromosome transmission and was used to demonstrate that the nod kinesin-like protein gene interacts genetically with the centro mere, in addition to interacting with extracentromeric regions as demonstrated previously. The results presented in this study strongly suggest that dominant genetic interactions between mutations and centromere-defective minichromosomes could be used effectively to identify novel genes necessary for centromere function.

Trans-suppression of terminal deficiency-associated position effect variegation in a Drosophila minichromosome.

Position effect variegation (PEV) is the clonal inactivation of euchromatic or heterochromatic genes that are abnormally positioned within a chromosome. PEV can be influenced by modifiers in trans, including single gene mutations and the total amount of heterochromatin present in the genome. Terminal deletions of a Drosophila minichromosome (Dp1187) dramatically increase PEV of a yellow+ body-color gene located in cis, even when the terminal break is > 100 kb distal to the yellow gene. Here we demonstrate that terminal deficiency-associated PEV can be suppressed by the presence of a second minichromosome, a novel phenomenon termed "trans-suppression." The chromosomal elements responsible for trans-suppression were investigated using a series of minichromosomes with molecularly characterized deletions and inversions. The data suggest that trans-suppression does not involve communication between transcriptional regulatory elements on the homologues, a type of transvection known to act at the yellow locus. Furthermore, trans-suppression is not accomplished by titration through the addition of extra centric heterochromatin, a general mechanism for PEV suppression. We demonstrate that trans-suppression is disrupted by significant changes in the structure of the suppressing minichromosome, including deletions of the yellow region and centric heterochromatin, and large inversions of the centric heterochromatin. We conclude that chromosome pairing plays an important role in trans-suppression and discuss the possibility that terminal deficiency-associated PEV and trans-suppression reflect changes in nuclear positioning of the chromosomes and the gene, and/or the activity and distribution of telomere-binding proteins.

The case for epigenetic effects on centromere identity and function.

The centromere is required to ensure the equal distribution of replicated chromosomes to daughter nuclei. Centromeres are frequently associated with heterochromatin, an enigmatic nuclear component that causes the epigenetic transcriptional repression of nearby marker genes (position-effect variegation or silencing). The process of chromosome segregation by movement along microtubules to spindle poles is highly conserved, yet the putative cis-acting centromeric DNA sequences bear little or no similarity across species. Recently, studies in several systems have revealed that the centromere itself might be epigenetically regulated and that the higher-order structure of the underlying heterochromatin contributes to centromere function and kinetochore assembly.

Centric heterochromatin and the efficiency of achiasmate disjunction in Drosophila female meiosis.

The chromosomal requirements for achiasmate (nonexchange) homolog disjunction in Drosophila female meiosis I have been identified with the use of a series of molecularly defined minichromosome deletion derivatives. Efficient disjunction requires 1000 kilobases of overlap in the centric heterochromatin and is not affected by homologous euchromatin or overall size differences. Disjunction efficiency decreases linearly as heterochromatic overlap is reduced from 1000 to 430 kilobases of overlap. Further observations, including rescue experiments with nod kinesin-like protein transgenes, demonstrate that heterochromatin does not act solely to promote chromosome movement or spindle attachment. Thus, it is proposed that centric heterochromatin contains multiple pairing elements that act additively to initiate or maintain the proper alignment of achiasmate chromosomes in meiosis I. How heterochromatin could act to promote chromosome pairing is discussed here.

Islands of complex DNA are widespread in Drosophila centric heterochromatin.

Heterochromatin is a ubiquitous yet poorly understood component of multicellular eukaryotic genomes. Major gaps exist in our knowledge of the nature and overall organization of DNA sequences present in heterochromatin. We have investigated the molecular structure of the 1 Mb of centric heterochromatin in the Drosophila minichromosome Dp1187. A genetic screen of irradiated minichromosomes yielded rearranged derivatives of Dp1187 whose structures were determined by pulsed-field Southern analysis and PCR. Three Dp1187 deletion derivatives and an inversion had one breakpoint in the euchromatin and one in the heterochromatin, providing direct molecular access to previously inaccessible parts of the heterochromatin. End-probed pulsed-field restriction mapping revealed the presence of at least three "islands" of complex DNA, Tahiti, Moorea, and Bora Bora, constituting approximately one half of the Dp1187 heterochromatin. Pulsed-field Southern analysis demonstrated that Drosophila heterochromatin in general is composed of alternating blocks of complex DNA and simple satellite DNA. Cloning and sequencing of a small part of one island, Tahiti, demonstrated the presence of a retroposon. The implications of these findings to heterochromatin structure and function are discussed.

Localization of centromere function in a Drosophila minichromosome.

The DNA elements responsible for centromere activity in a metazoan have been localized using the Drosophila minichromosome Dp1187. Deleted minichromosomes were generated by irradiation mutagenesis, and their molecular structures were determined by pulsed-field Southern blot analysis. Analyses of the transmission behavior of Dp1187 derivatives localized sequences necessary for chromosome inheritance within the centric heterochromatin. The essential core of the centromere is contained within a 220 kb region that includes significant amounts of complex DNA. Completely normal inheritance also requires approximately 200 kb on either side of the essential core. This flanking DNA predominantly contains highly repeated sequences, and the amount required for normal transmission differs among division types and between the sexes. We propose that the essential core is the site of kinetochore formation and that flanking DNA provides two functions: sister chromatid cohesion and indirect assistance in kinetochore formation or function.

Interactions between the nod+ kinesin-like gene and extracentromeric sequences are required for transmission of a Drosophila minichromosome.

In this study, we demonstrate a role for extracentromeric sequences in chromosome inheritance. Genetic analyses indicate that transmission of the Drosophila minichromosome Dp1187 is sensitive to the dosage of nod+, a kinesin-like gene required for the meiotic transmission of achiasmate chromosomes. Minichromosome deletions displayed increased loss rates in females heterozygous for a loss-of-function allele of nod (nod/+). We have analyzed the structures of nod-sensitive deletions and conclude that multiple regions of Dp1187 interact genetically with nod+ to promote normal chromosome transmission. Most nod+ interactions are observed with regions that are not essential for centromere function. We propose that normal chromosome transmission requires forces generated outside the kinetochore, perhaps to maintain tension on kinetochore microtubules and stabilize the attachment of achiasmate chromosomes to the metaphase spindle.

A transposable element can drive the concerted evolution of tandemly repetitious DNA.

Recombination and conversion have been proposed to drive the concerted evolution of tandemly repeated DNA sequences. However, specific correction events within the repeated genes of multicellular organisms have not been observed directly, so their nature has remained speculative. We investigated whether the excision of transposable P elements from tandemly repeated sequences would induce unequal gene conversion. Genetically marked elements located in a subtelomeric repeat were mobilized, and the structure of the region was analyzed in progeny. We observed that the number of repeats was frequently altered. Decreases were more common than increases, and this bias probably resulted from intrinsic mechanisms governing P element-induced double-strand break repair. Our results suggest that transposable elements play an important role in the evolution of repetitious DNA.

A rosy future for heterochromatin.

The demonstration by Zhang and Spradling (1) of efficient P element transposition into heterochromatic regions will aid ongoing studies of heterochromatin structure and function. P element insertions will provide entry points for further molecular analysis of heterochromatin and will allow the isolation of small and large heterochromatic deficiencies. The generation of heterochromatic P insertions also will aid the study of heterochromatic genes. Of the heterochromatic insertions isolated by Zhang and Spradling, five were homozygous lethal, and one of these defined a lethal locus not previously uncovered by heterochromatic deficiencies. P elements have previously been used to mutagenize and clone specific heterochromatic genes (14, 19, 26). New methods, like those described here (1, 32), should allow the efficient identification and molecular isolation of other single-copy heterochromatic genes. Furthermore, since position-effect suppression allowed the recovery of heterochromatic P insertions, it may also allow the recovery of insertions in euchromatic regions previously refractory to P mutagenesis. Studies of position-effect variegation show that genes normally found in heterochromatin require a heterochromatic context for normal expression and that heterochromatin is inhibitory to euchromatic gene expression (16). The physical basis of these related phenomena--chromatin assembly, nuclear positioning, and/or heterochromatin elimination--can be resolved only with a more thorough understanding of heterochromatin structure and functions. Analyzing heterochromatin also will help define the chromosomal components responsible for inheritance processes such as chromosome pairing, sister chromatid adhesion, and centromere function. These efforts will be facilitated by the effective use of P elements combined with other current molecular-genetic approaches.

Position-effect variegation and the new biology of heterochromatin.

The phenomenon of position-effect variegation has long been used as evidence for the importance of chromosome position to gene expression in eukaryotes. Investigations published within the past few years demonstrate that position-effect variegation is caused by multiple mechanisms, and that direct tests of hypotheses are possible with numerous model systems.