Danielle Vermaak

The budding yeast Ipl1/Aurora protein kinase regulates mitotic spindle disassembly.

Ipl1p is the budding yeast member of the Aurora family of protein kinases, critical regulators of genomic stability that are required for chromosome segregation, the spindle checkpoint, and cytokinesis. Using time-lapse microscopy, we found that Ipl1p also has a function in mitotic spindle disassembly that is separable from its previously identified roles. Ipl1–GFP localizes to kinetochores from G1 to metaphase, transfers to the spindle after metaphase, and accumulates at the spindle midzone late in anaphase. Ipl1p kinase activity increases at anaphase, and ipl1 mutants can stabilize fragile spindles. As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules. Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly. We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.

Maintenance of chromatin states: an open-and-shut case.

The traditional view of chromatin envisions two states: one is active and accessible to nucleases, whereas the other is silent and relatively inaccessible. Recent evidence that combinations of diverse histone tail modifications represent a spectrum of chromatin states challenges this simple view. Here, we examine inter-relationships between chromatin remodeling, histone modification, DNA methylation, RNA interference, and nucleosome assembly activities. We find that the two-state view can accommodate these new findings, and that nucleosome assembly pathways may ultimately maintain euchromatic and heterochromatic states.

Structure, dynamics, and evolution of centromeric nucleosomes

Centromeres are defining features of eukaryotic chromosomes, providing sites of attachment for segregation during mitosis and meiosis. The fundamental unit of centromere structure is the centromeric nucleosome, which differs from the conventional nucleosome by the presence of a centromere-specific histone variant (CenH3) in place of canonical H3. We have shown that the CenH3 nucleosome core found in interphase Drosophila cells is a heterotypic tetramer, a “hemisome” consisting of one molecule each of CenH3, H4, H2A, and H2B, rather than the octamer of canonical histones that is found in bulk nucleosomes. The surprising discovery of hemisomes at centromeres calls for a reevaluation of evidence that has long been interpreted in terms of a more conventional nucleosome. We describe how the hemisome structure of centromeric nucleosomes can account for enigmatic properties of centromeres, including kinetochore accessibility, epigenetic inheritance, rapid turnover of misincorporated CenH3, and transcriptional quiescence of pericentric heterochromatin. Structural differences mediated by loop 1 are proposed to account for the formation of stable tetramers containing CenH3 rather than stable octamers containing H3. Asymmetric CenH3 hemisomes might interrupt the global condensation of octameric H3 arrays and present an asymmetric surface for kinetochore formation. We suggest that this simple mechanism for differentiation between centromeric and packaging nucleosomes evolved from an archaea-like ancestor at the dawn of eukaryotic evolution.

Multiple Roles for Heterochromatin Protein 1 Genes in Drosophila

Heterochromatin is the gene-poor, transposon-rich, late-replicating chromatin compartment that was first cytologically defined more than 70 years ago. The identification of heterochromatin protein 1 (HP1) paved the way for a molecular dissection of this important component of complex eukaryotic genomes. Although initial studies revealed HP1s key role in heterochromatin maintenance and function, more recent studies have discovered a role for HP1 in numerous processes including, surprisingly, euchromatic gene expression. Drosophila genomes possess at least five HP1 paralogs that have significantly different roles, ranging from canonical heterochromatic function at pericentric and telomeric regions to exclusive localization and regulation of euchromatic genes. They also possess paralogs exclusively involved in defending the germline against mobile elements. Pursuing a survey of recent genetic and evolutionary findings, we highlight how Drosophila genomes represent the best opportunity to dissect the diversity and incredible versatility of HP1 proteins in organizing and protecting eukaryotic genomes.

Recurrent evolution of DNA-binding motifs in the Drosophila centromeric histone

All eukaryotes contain centromere-specific histone H3 variants (CenH3s), which replace H3 in centromeric chromatin. We have previously documented the adaptive evolution of the Drosophila CenH3 (Cid) in comparisons of Drosophila melanogaster and Drosophila simulans, a divergence of ≈2.5 million years. We have proposed that rapidly changing centromeric DNA may be driving CenH3s altered DNA-binding specificity. Here, we compare Cid sequences from a phylogenetically broader group of Drosophila species to suggest that Cid has been evolving adaptively for at least 25 million years. Our analysis also reveals conserved blocks not only in the histone-fold domain but also in the N-terminal tail. In several lineages, the N-terminal tail of Cid is characterized by subgroup-specific oligopeptide expansions. These expansions resemble minor groove DNA binding motifs found in various histone tails. Remarkably, similar oligopeptides are also found in N-terminal tails of human and mouse CenH3 (Cenp-A). The recurrent evolution of these motifs in CenH3 suggests a packaging function for the N-terminal tail, which results in a unique chromatin organization at the primary constriction, the cytological marker of centromeres.

Stepwise Evolution of Essential Centromere Function in a Drosophila Neogene

Essential Novelty The evolution of essential function for newly originated genes presents a conundrum, in that prior to the genes origin either the essential function was absent or else performed by another gene or set of genes. In order to better understand how new genes acquire essential function, Ross et al. (p. 1211) investigated the origin of the Drosophila gene Umbrea. Umbrea became an essential protein in certain Drosophila species through the gain of localization at the centromere and a role in chromosome segregation. How does a recently evolved gene come to encode an essential function? Evolutionarily young genes that serve essential functions represent a paradox; they must perform a function that either was not required until after their birth or was redundant with another gene. How young genes rapidly acquire essential function is largely unknown. We traced the evolutionary steps by which the Drosophila gene Umbrea acquired an essential role in chromosome segregation in D. melanogaster since the genes origin less than 15 million years ago. Umbrea neofunctionalization occurred via loss of an ancestral heterochromatin-localizing domain, followed by alterations that rewired its protein interaction network and led to species-specific centromere localization. Our evolutionary cell biology approach provides temporal and mechanistic detail about how young genes gain essential function. Such innovations may constantly alter the repertoire of centromeric proteins in eukaryotes.

Species-specific positive selection of the male-specific lethal complex that participates in dosage compensation in Drosophila

In many taxa, males and females have unequal ratios of sex chromosomes to autosomes, which has resulted in the invention of diverse mechanisms to equilibrate gene expression between the sexes (dosage compensation). Failure to compensate for sex chromosome dosage results in male lethality in Drosophila. In Drosophila, a male-specific lethal (MSL) complex of proteins and noncoding RNAs binds to hundreds of sites on the single male X chromosome and up-regulates gene expression. Here we use population genetics of two closely related Drosophila species to show that adaptive evolution has occurred in all five protein-coding genes of the MSL complex. This positive selection is asymmetric between closely related species, with a very strong signature apparent in Drosophila melanogaster but not in Drosophila simulans. In particular, the MSL1 and MSL2 proteins have undergone dramatic positive selection in D. melanogaster, in domains previously shown to be responsible for their specific targeting to the X chromosome. This signature of positive selection at an essential protein–DNA interface of the complex is unexpected and suggests that X chromosomal MSL-binding DNA segments may themselves be changing rapidly. This highly asymmetric, rapid evolution of the MSL genes further suggests that misregulated dosage compensation may represent one of the underlying causes of male hybrid inviability in Drosophila, wherein the fate of hybrid males depends on which species X chromosome is inherited.

Phylogenomic Analysis Reveals Dynamic Evolutionary History of the Drosophila Heterochromatin Protein 1 (HP1) Gene Family

Heterochromatin is the gene-poor, satellite-rich eukaryotic genome compartment that supports many essential cellular processes. The functional diversity of proteins that bind and often epigenetically define heterochromatic DNA sequence reflects the diverse functions supported by this enigmatic genome compartment. Moreover, heterogeneous signatures of selection at chromosomal proteins often mirror the heterogeneity of evolutionary forces that act on heterochromatic DNA. To identify new such surrogates for dissecting heterochromatin function and evolution, we conducted a comprehensive phylogenomic analysis of the Heterochromatin Protein 1 gene family across 40 million years of Drosophila evolution. Our study expands this gene family from 5 genes to at least 26 genes, including several uncharacterized genes in Drosophila melanogaster. The 21 newly defined HP1s introduce unprecedented structural diversity, lineage-restriction, and germline-biased expression patterns into the HP1 family. We find little evidence of positive selection at these HP1 genes in both population genetic and molecular evolution analyses. Instead, we find that dynamic evolution occurs via prolific gene gains and losses. Despite this dynamic gene turnover, the number of HP1 genes is relatively constant across species. We propose that karyotype evolution drives at least some HP1 gene turnover. For example, the loss of the male germline-restricted HP1E in the obscura group coincides with one episode of dramatic karyotypic evolution, including the gain of a neo-Y in this lineage. This expanded compendium of ovary- and testis-restricted HP1 genes revealed by our study, together with correlated gain/loss dynamics and chromosome fission/fusion events, will guide functional analyses of novel roles supported by germline chromatin.

Bugs on Drugs Go GAGAA

Close examination of the P31-fed bwD survivors provided intriguing evidence for multiple homeotic transformations (Janssen et al. 2000axJanssen, S, Cuvier, O, Muller, M, and Laemmli, U.K. Mol. Cell. 2000; 6: 1013–1024Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesJanssen et al. 2000a). Extra bristles revealed a transformation of the sixth abdominal segment into the fifth, fewer sex combs revealed a Sex-combs-reduced-like transformation, and larger halteres in combination with an Ultrabithorax gain-of-function mutation revealed enhanced transformation of the third thoracic segment into the second. This unusual combination of phenotypes had been seen before: partial loss-of-function mutations in the gene encoding GAF cause the same syndrome (Farkas et al. 1994xFarkas, G, Gausz, J, Galloni, M, Reuter, G, Gyurkovics, H, and Karch, F. Nature. 1994; 371: 806–808Crossref | PubMed | Scopus (284)See all ReferencesFarkas et al. 1994). So the question became, does P31 interact with bwD in such a way that GAF levels are reduced?An unexpected connection between bwD and GAF had previously been reported (Platero et al. 1998xPlatero, J.S, Csink, A.K, Quintanilla, A, and Henikoff, S. J. Cell Biol. 1998; 140: 1297–1306Crossref | PubMed | Scopus (106)See all ReferencesPlatero et al. 1998). GAF appears to bind exclusively to GA-rich satellites, including bwD, during mitosis (Figure 2AFigure 2A), but does not bind at all during interphase. The absence of GAF from interphase heterochromatin is seen most clearly in polytene chromosomes, where thousands of euchromatic sites have GAF, yet the heterochromatic chromocenter and bwD are completely devoid of GAF (Figure 2BFigure 2B). Therefore, the GA-rich satellites provide a potential binding reservoir for GAF that fills up only at the onset of mitosis, during which time the euchromatin condenses and loses all detectable GAF. These observations provide the connection between bwD and GAF that can potentially explain the P31-bwD syndrome. Perhaps the binding of P31 to bwD opens it up, resulting in the unscheduled transfer of GAF from euchromatic sites to bwD at interphase, a process that normally occurs only during mitosis.Figure 2Relocalization of GAF from Euchromatin to bwD(A) Cycling of GAF from dispersed sites in euchromatin to GA-rich satellites in pericentric heterochromatin and to bwD, which is located near the distal tip of chromosome arm 2R. (B) GAF is found at numerous sites throughout euchromatin, but is undetectable at bwD during interphase. After treatment with P31, but not P9, GAF relocalizes to bwD in polytene nuclei.View Large Image | View Hi-Res Image | Download PowerPoint SlideA critical test of this scenario is to treat with P31 and look for transfer of GAF from euchromatic sites to bwD by staining of polytene chromosomes with anti-GAF antibody. Laemmlis group performed this test, and the result was stunning (Figure 2BFigure 2B). bwD normally shows no anti-GAF staining, whereas nearly all detectable GAF in P31-treated nuclei was found at bwD. Concomitantly, anti-GAF staining of euchromatic bands was nearly abolished (Janssen et al. 2000axJanssen, S, Cuvier, O, Muller, M, and Laemmli, U.K. Mol. Cell. 2000; 6: 1013–1024Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesJanssen et al. 2000a). Thus, it appears that P31-induced opening of bwD provides a sink for GAF, which leaves its normal sites. Consistent with this interpretation, the authors note that P31 binding and GAF binding to GAGAA repeats are not mutually exclusive. Furthermore, normal GAF binding sites, which are typically GAGAG, are not targeted by P31, so that P31 is expected to open up bwD preferentially to euchromatic GAF binding sites. Unscheduled GAF binding to bwD after P31 treatment provides compelling evidence for Laemmlis model that minor-groove binding exerts its effect on chromatin by opening it up.GAF is not the only mitosis-specific satellite binding protein. Drosophila Prod protein, which binds to the AT-rich AATAAGATAC decameric satellite, shows similar cycling behavior (Platero et al. 1998xPlatero, J.S, Csink, A.K, Quintanilla, A, and Henikoff, S. J. Cell Biol. 1998; 140: 1297–1306Crossref | PubMed | Scopus (106)See all ReferencesPlatero et al. 1998), and reservoirs for other proteins might exist in heterochromatin. We wonder if titration by a drug-opened satellite sink had occurred for the AT binders, resulting in PEV suppression. AT-rich satellites are especially abundant in the Drosophila genome, and so it seems reasonable to suspect that if these satellites open up, they would become massive sinks for proteins needed for heterochromatic silencing. Thus, titration of silencing factors by satellites could have led to derepression of the white reporter gene in wm4. This explanation may have precedent in the well-known PEV suppressing effect of extra Y chromosomes, which are thought to derepress wm4 and other PEV mutations by titrating out heterochromatin-specific factors, reducing their availability at sites subject to PEV (Dimitri and Pisano 1989xDimitri, P and Pisano, C. Genetics. 1989; 122: 793–800PubMedSee all ReferencesDimitri and Pisano 1989).The human genome also has satellites in abundance. Indeed, the Ikaros regulatory protein binds to human gamma satellites, which have been proposed to function as Ikaros storage sites in lymphocytes (Cobb et al. 2000xCobb, B.S, Morales-Alcelay, S, Kleiger, G, Brown, K.E, Fisher, A.G, and Smale, S.T. Genes Dev. 2000; 14: 2146–2160Crossref | PubMed | Scopus (173)See all ReferencesCobb et al. 2000). It might not be too far-fetched to imagine that human satellite sinks will someday provide therapeutic drug targets. When converted to dye-conjugated compounds, minor-groove binders have an added advantage, as demonstrated by Laemmlis group, in that they illuminate the DNA target that they open up. Potentially, a therapeutic drug can reveal its DNA target, analogous to illumination of proteins linked to GFP in living organisms. Perhaps future decongestants will not only clear your nose, but also light it up!*To whom correspondence should be addressed (e-mail: steveh@fhcrc.org).

A Surrogate Approach to Study the Evolution of Noncoding DNA Elements That Organize Eukaryotic Genomes

Comparative genomics provides a facile way to address issues of evolutionary constraint acting on different elements of the genome. However, several important DNA elements have not reaped the benefits of this new approach. Some have proved intractable to current day sequencing technology. These include centromeric and heterochromatic DNA, which are essential for chromosome segregation as well as gene regulation, but the highly repetitive nature of the DNA sequences in these regions make them difficult to assemble into longer contigs. Other sequences, like dosage compensation X chromosomal sites, origins of DNA replication, or heterochromatic sequences that encode piwi-associated RNAs, have proved difficult to study because they do not have recognizable DNA features that allow them to be described functionally or computationally. We have employed an alternate approach to the direct study of these DNA elements. By using proteins that specifically bind these noncoding DNAs as surrogates, we can indirectly assay the evolutionary constraints acting on these important DNA elements. We review the impact that such surrogate strategies have had on our understanding of the evolutionary constraints shaping centromeres, origins of DNA replication, and dosage compensation X chromosomal sites. These have begun to reveal that in contrast to the view that such structural DNA elements are either highly constrained (under purifying selection) or free to drift (under neutral evolution), some of them may instead be shaped by adaptive evolution and genetic conflicts (these are not mutually exclusive). These insights also help to explain why the same elements (e.g., centromeres and replication origins), which are so complex in some eukaryotic genomes, can be simple and well defined in other where similar conflicts do not exist.