Gary H. Karpen

Conserved organization of centromeric chromatin in flies and humans

Recent studies have highlighted the importance of centromere-specific histone H3-like (CENP-A) proteins in centromere function. We show that Drosophila CID and human CENP-A appear at metaphase as a three-dimensional structure that lacks histone H3. However, blocks of CID/CENP-A and H3 nucleosomes are linearly interspersed on extended chromatin fibers, and CID is close to H3 nucleosomes in polynucleosomal preparations. When CID is depleted by RNAi, it is replaced by H3, demonstrating flexibility of centromeric chromatin organization. Finally, contrary to models proposing that H3 and CID/CENP-A nucleosomes are replicated at different times in S phase, we show that interspersed H3 and CID/CENP-A chromatin are replicated concurrently during S phase in humans and flies. We propose that the unique structural arrangement of CID/CENP-A and H3 nucleosomes presents centromeric chromatin to the poleward face of the condensing mitotic chromosome.

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.

Centromeric chromatin exhibits a histone modification pattern that is distinct from both euchromatin and heterochromatin

Post-translational histone modifications regulate epigenetic switching between different chromatin states. Distinct histone modifications, such as acetylation, methylation and phosphorylation, define different functional chromatin domains, and often do so in a combinatorial fashion. The centromere is a unique chromosomal locus that mediates multiple segregation functions, including kinetochore formation, spindle-mediated movements, sister cohesion and a mitotic checkpoint. Centromeric (CEN) chromatin is embedded in heterochromatin and contains blocks of histone H3 nucleosomes interspersed with blocks of CENP-A nucleosomes, the histone H3 variant that provides a structural and functional foundation for the kinetochore. Here, we demonstrate that the spectrum of histone modifications present in human and Drosophila melanogaster CEN chromatin is distinct from that of both euchromatin and flanking heterochromatin. We speculate that this distinct modification pattern contributes to the unique domain organization and three-dimensional structure of centromeric regions, and/or to the epigenetic information that determines centromere identity.

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.

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.

Molecular Structure of a Functional Drosophila Centromere

Centromeres play a critical role in chromosome inheritance but are among the most difficult genomic components to analyze in multicellular eukaryotes. Here, we present a highly detailed molecular structure of a functional centromere in a multicellular organism. The centromere of the Drosophila minichromosome Dp1187 is contained within a 420 kb region of centric heterochromatin. We have used a new approach to characterize the detailed structure of this centromere and found that it is primarily composed of satellites and single, complete transposable elements. In the rest of the Drosophila genome, these satellites and transposable elements are neither unique to the centromeres nor present at all centromeres. We discuss the impact of these results on our understanding of heterochromatin structure and on the determinants of centromere identity and function.

Heterochromatic sequences in a Drosophila whole-genome shotgun assembly

BackgroundMost eukaryotic genomes include a substantial repeat-rich fraction termed heterochromatin, which is concentrated in centric and telomeric regions. The repetitive nature of heterochromatic sequence makes it difficult to assemble and analyze. To better understand the heterochromatic component of the Drosophila melanogaster genome, we characterized and annotated portions of a whole-genome shotgun sequence assembly.ResultsWGS3, an improved whole-genome shotgun assembly, includes 20.7 Mb of draft-quality sequence not represented in the Release 3 sequence spanning the euchromatin. We annotated this sequence using the methods employed in the re-annotation of the Release 3 euchromatic sequence. This analysis predicted 297 protein-coding genes and six non-protein-coding genes, including known heterochromatic genes, and regions of similarity to known transposable elements. Bacterial artificial chromosome (BAC)-based fluorescence in situ hybridization analysis was used to correlate the genomic sequence with the cytogenetic map in order to refine the genomic definition of the centric heterochromatin; on the basis of our cytological definition, the annotated Release 3 euchromatic sequence extends into the centric heterochromatin on each chromosome arm.ConclusionsWhole-genome shotgun assembly produced a reliable draft-quality sequence of a significant part of the Drosophila heterochromatin. Annotation of this sequence defined the intron-exon structures of 30 known protein-coding genes and 267 protein-coding gene models. The cytogenetic mapping suggests that an additional 150 predicted genes are located in heterochromatin at the base of the Release 3 euchromatic sequence. Our analysis suggests strategies for improving the sequence and annotation of the heterochromatic portions of the Drosophila and other complex genomes.

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.

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.