Structure of the Chromosome VII Centromere Region in Neurospora crassa: Degenerate Transposons and Simple Repeats

Centromeres are regions of chromosomes that direct formation of the kinetochore and its subsequent attachment to the spindle, enabling the faithful segregation of the genetic material during cell division. This chromosomal domain, found in all eukaryotes, is functionally conserved but structurally quite divergent between organisms. The overall size and sequence complexity of centromeres generally appears to parallel the developmental complexity of the organism. In Saccharomyces cerevisiae, for example, the centromere is small, consisting of only approximately 200 bp for full function. In higher eukaryotes, however, centromeric regions of the genome not only are much larger (up to 5 Mb in length [see reference 55 for review]) but show no apparent sequence conservation and have been referred to as “regional” centromeres. Despite recent advances in the development of higher eukaryotic experimental systems, relatively little is known about the sequence constituents of centromeres and centric heterochromatin in complex organisms. For Drosophila, the centromere of a minichromosome has been mapped by deletion analysis, and the minimal sequences required for function are now being identified (37, 67). The sequence components necessary for full centromere function appear to include transposable elements of several types as well as low-complexity satellite DNAs (67). The apparent absence of a defined sequence that is responsible for kinetochore formation, as is seen in S. cerevisiae, suggests a redundant, nonsequence-specific initiation event leading to centromere and/or kinetochore formation. There is also evidence in vertebrates for similar redundancy, i.e., Indian muntjac centromeres can be fractionated into multiple individual kinetochore-like units (76). Indirect evidence from humans suggests that megabase arrays of α-satellite or alphoid DNAs, which are a family of A+T-rich 171-bp tandem repeats, are associated with active centromeres (46) and may be sufficient for centromere function (23, 68). Centromere activity can be observed, however, in activated human neocentromeres lacking alphoid repeats (19). The large size of regional centromeres may be important for the additional functions that have been attributed to centromeres of higher eukaryotes, including chromosome adhesion in achiasmate disjunction (32), as well as providing domains of specialized chromatin structure (heterochromatin) in which euchromatic gene transcription and recombination are both repressed. The characteristics of regional centromeric DNA may also reflect an underlying mechanism by which chromatin structure nucleates kinetochore formation. Several regional centromeres display an epigenetic control phenomenon called centromere activation. In the fission yeast Schizosaccharomyces pombe, formation of the centromere into an active state can require multiple cell divisions after introduction of naked minichromosome DNA (66). In other organisms, aberrant chromosomes can form neocentromeres in locations differing from the original centromere, resulting in a mixture of cells containing a chromosome with one site or the other acting as the centromere (1, 6, 69). The regional heterochromatic character of centromeres in complex organisms, however, may result from the accumulation of repeated sequence elements. One consequence of recombinational repression in heterochromatic regions may be the accumulation of mobile genetic elements (13). The prevailing model explaining the observed excess of transposons in centric heterochromatin holds that chromosome rearrangements due to ectopic recombination between similar transposons at different sites in the genome may lead to decreased fitness and selection against such ectopic insertions (12). Alternatively, the insertion of transposons into genic regions may have negative effects on the fitness of individuals in the population, resulting in fewer elements in euchromatic regions of the genome (28). It is possible, however, that repeated sequence elements in some circumstances may have positive effects on fitness rather than just neutral or negative effects. Like centromeric domains, telomeric and subtelomeric regions of most organisms are also regions of specialized chromatin structure and are home to numerous repeated elements (58, 70, 75). The telomerase-elongated simple DNA sequence repeats at the end of the chromosomes of most eukaryotes are essential elements for continued chromosomal end replication and for segregation of the genome. Remarkably, telomeric sequences can function in place of centromeric heterochromatin in Drosophila in the formation of neocentromeres (2, 6, 56, 73). In Drosophila, telomere functions are probably served by the HeT-A and Tart retrotransposons that are found at all chromosomal termini (14, 43). The cell apparently has taken advantage of such elements to serve an essential function. In multicellular fungi, little is known about centromere structure. The chromosomes of the filamentous fungus Neurospora crassa show regions of heavy, intense staining (heterochromatin) in meiosis (44, 64). Centola and Carbon (10) cloned and partially characterized a contiguous set of artificial yeast chromosomes (YACs) containing DNA that spanned the centromere region of linkage group (LG) VII of Neurospora. This region, approximately 450 kb in length, was found to be both A+T rich and recombination deficient. In addition, they identified a centromere-specific repeated DNA sequence. Comparison of the sequence of this centromere-specific clone to homologous DNAs from elsewhere in the genome suggested that they had undergone repeat-induced point mutation (RIP), a process that scans the genome of Neurospora for repeated DNAs during the sexual cycle and induces GC to AT transition mutations, and often DNA methylation, specific to the duplicated sequences (61). In this study, we have further characterized the centromere region of LG VII of Neurospora and have discovered a nested cluster of putative transposable elements and simple sequence repeats. A repeated DNA sequence previously found to map to centromere-linked regions of the Neurospora genome (10) is now shown to be a copia-like element (named Tcen) which is novel in that it is the only known transposon to be shown to map exclusively to centromere regions. In addition, the region contains the degenerate remains of several other transposons, as well as three different low-complexity DNAs organized in a tightly nested arrangement. Although these features have yet to be associated with kinetochore formation, the structural similarity of the Neurospora centromere VII region to the centromere of the Drosophila Dp1187 minichromosome (37, 67) suggests that Neurospora kinetochore-forming regions may be similarly redundant and nonspecific.