Stefan Heckmann

Arabidopsis KINETOCHORE NULL2 Is an Upstream Component for Centromeric Histone H3 Variant cenH3 Deposition at Centromeres

This work finds that Arabidopsis KINETOCHORE NULL2 (KNL2) colocalizes with the centromere histone variant cenH3. Characterization of knl2 mutants showed reduction of cenH3 deposition at centromeres, abnormalities of mitosis and meiosis, seed abortion, and alterations in DNA methylation. The centromeric histone H3 variant cenH3 is an essential centromeric protein required for assembly, maintenance, and proper function of kinetochores during mitosis and meiosis. We identified a KINETOCHORE NULL2 (KNL2) homolog in Arabidopsis thaliana and uncovered features of its role in cenH3 loading at centromeres. We show that Arabidopsis KNL2 colocalizes with cenH3 and is associated with centromeres during all stages of the mitotic cell cycle, except from metaphase to mid-anaphase. KNL2 is regulated by the proteasome degradation pathway. The KNL2 promoter is mainly active in meristematic tissues, similar to the cenH3 promoter. A knockout mutant for KNL2 shows a reduced level of cenH3 expression and reduced amount of cenH3 protein at chromocenters of meristematic nuclei, anaphase bridges during mitosis, micronuclei in pollen tetrads, and 30% seed abortion. Moreover, knl2 mutant plants display reduced expression of suppressor of variegation 3-9 homologs2, 4, and 9 and reduced DNA methylation, suggesting an impact of KNL2 on the epigenetic environment for centromere maintenance.

Holocentric Chromosomes of Luzula elegans Are Characterized by a Longitudinal Centromere Groove, Chromosome Bending, and a Terminal Nucleolus Organizer Region

The structure of holocentric chromosomes was analyzed in mitotic cells of Luzula elegans. Light and scanning electron microscopy observations provided evidence for the existence of a longitudinal groove along each sister chromatid. The centromere-specific histone H3 variant, CENH3, colocalized with this groove and with microtubule attachment sites. The terminal chromosomal regions were CENH3-negative. During metaphase to anaphase transition, L. elegans chromosomes typically curved to a sickle-like shape, a process that is likely to be influenced by the pulling forces of microtubules along the holocentric axis towards the corresponding microtubule organizing regions. A single pair of 45S rDNA sites, situated distal to Arabidopsis-telomere repeats, was observed at the terminal region of one chromosome pair. We suggest that the 45S rDNA position in distal centromere-free regions could be required to ensure chromosome stability.

Alternative meiotic chromatid segregation in the holocentric plant Luzula elegans

Holocentric chromosomes occur in a number of independent eukaryotic lineages. They form holokinetic kinetochores along the entire poleward chromatid surfaces, and owing to this alternative chromosome structure, species with holocentric chromosomes cannot use the two-step loss of cohesion during meiosis typical for monocentric chromosomes. Here we show that the plant Luzula elegans maintains a holocentric chromosome architecture and behaviour throughout meiosis, and in contrast to monopolar sister centromere orientation, the unfused holokinetic sister centromeres behave as two distinct functional units during meiosis I, resulting in sister chromatid separation. Homologous non-sister chromatids remain terminally linked after metaphase I, by satellite DNA-enriched chromatin threads, until metaphase II. They then separate at anaphase II. Thus, an inverted sequence of meiotic sister chromatid segregation occurs. This alternative meiotic process is most likely one possible adaptation to handle a holocentric chromosome architecture and behaviour during meiosis.

Point mutation impairs centromeric CENH3 loading and induces haploid plants

Significance The generation of haploids is the most powerful means to accelerate the plant-breeding process. We elucidated whether point mutations in the centromere-specific histone H3 variant CENH3 could be harnessed for the induction of haploids. We identified plants with impaired centromere loading caused by a mutation in the centromere-targeting domain (CATD). The same mutation results in reduced loading of CENH3 in transgenic Arabidopsis and sugar beet. Arabidopsis plants carrying this single point mutation in wild-type CENH3 were used as haploid inducers. Because the identified mutation site is highly conserved and because point mutations can be generated by mutagenesis or genome editing, the described method offers opportunities for application in a wide range of crop species. The chromosomal position of the centromere-specific histone H3 variant CENH3 (also called “CENP-A”) is the assembly site for the kinetochore complex of active centromeres. Any error in transcription, translation, modification, or incorporation can affect the ability to assemble intact CENH3 chromatin and can cause centromere inactivation [Allshire RC, Karpen GH (2008) Nat Rev Genet 9 (12):923–937]. Here we show that a single-point amino acid exchange in the centromere-targeting domain of CENH3 leads to reduced centromere loading of CENH3 in barley, sugar beet, and Arabidopsis thaliana. Haploids were obtained after cenh3 L130F-complemented cenh3-null mutant plants were crossed with wild-type A. thaliana. In contrast, in a noncompeting situation (i.e., centromeres possessing only mutated or only wild-type CENH3), no uniparental chromosome elimination occurs during early embryogenesis. The high degree of evolutionary conservation of the identified mutation site offers promising opportunities for application in a wide range of crop species in which haploid technology is of interest.

Characterization of Eu- and Heterochromatin of Citrus with a Focus on the Condensation Behavior of 45S rDNA Chromatin

To characterize the properties of eu- and heterochromatic regions in Citrus species, the chromosomal distribution of different histone H3 marks, DNA methylation sites (5mC) and 45S ribosomal DNA sites were determined for C. clementina, C. paradisi, C. sinensis, and for the hybrid Ortanique C. reticulata × C. sinensis. Our data show that in the relatively small genomes of investigated Citrus species (genome size ranges from 378–400 Mbp) the euchromatin is characterized by histone H3 lysine 4 mono-, di- and trimethylation (H3K4me1/ 2/3) and histone H3 lysine 9 trimethylation (H3K9me3). In contrast, histone H3 lysine 9 mono- and dimethylation (H3K9me1/2), histone H3 lysine 27 mono-, di- and trimethylation (H3K27me1/2/3) as well as 5-methylcytosine (5mC) were enriched at certain heterochromatin fractions. Whereas H3K9me1/2 and H3K27me1 were preferentially enriched at the chromomycin A3-bright (CMA+) heterochromatin, H3K27me2/3 showed a higher accumulation at the DAPI brightly-stained heterochromatin. 5mC signals were associated with most of the CMA+ areas as well as with the DAPI strongly-stained heterochromatin fraction. Therefore, extensive methylation of DNA as well as of H3K9me1/2 and H3K27me1/2/3, and depletion of H3K4me1/2/3 and H3K9me3 appear to be specific features of heterochromatin in Citrus. Transcriptionally active decondensed 45S rDNA sites were found DNA hypomethylated, while the silenced condensed sites were strongly 5mC methylated. Although the number of chromosomal 45S rDNA sites differed between the species, the number of transcriptionally active rDNA sites remains constant.

Anti-Phosphorylated Histone H2AThr120: A Universal Microscopic Marker for Centromeric Chromatin of Mono- and Holocentric Plant Species

Based on the analysis of 20 different monocot and eudicot species, we propose that the centromeric distribution of the phosphorylated histone H2AThr120 is evolutionary highly conserved across species with mono- and holocentric chromosomes. Therefore, antibodies recognizing the phosphorylated threonine 120 of the histone H2A can serve as a universal marker for the cytological detection of centromeres of mono- and holokinetic plant species. In addition, super resolution microscopy of signals specific to the centromere-specific histone H3 variant CENH3 and to H2AThr120ph revealed that these histone variants are incorporated into different nucleosomes, which form distinct, partly intermingled chromatin domains. This specific arrangement of both histone variants suggests different centromeric functions during the cell cycle.

Atypical centromeres in plants—what they can tell us

The centromere, visible as the primary constriction of condensed metaphase chromosomes, is a defined chromosomal locus essential for genome stability. It mediates transient assembly of a multi-protein complex, the kinetochore, which enables interaction with spindle fibers and thus faithful segregation of the genetic information during nuclear divisions. Centromeric DNA varies in extent and sequence composition among organisms, but a common feature of almost all active eukaryotic centromeres is the presence of the centromeric histone H3 variant cenH3 (a.k.a. CENP-A). These typical centromere features apply to most studied species. However, a number of species display “atypical” centromeres, such as holocentromeres (centromere extension along almost the entire chromatid length) or neocentromeres (ectopic centromere activity). In this review, we provide an overview of different atypical centromere types found in plants including holocentromeres, de novo formed centromeres and terminal neocentromeres as well as di-, tri- and metapolycentromeres (more than one centromere per chromosomes). We discuss their specific and common features and compare them to centromere types found in other eukaryotic species. We also highlight new insights into centromere biology gained in plants with atypical centromeres such as distinct mechanisms to define a holocentromere, specific adaptations in species with holocentromeres during meiosis or various scenarios leading to neocentromere formation.

Holocentric plant meiosis: first sisters, then homologues.

Meiosis is a crucial process of sexual reproduction by forming haploid gametes from diploid precursor cells. It involves 2 subsequent divisions (meiosis I and meiosis II) after one initial round of DNA replication. Homologous monocentric chromosomes are separated during the first and sister chromatids during the second meiotic division. The faithful segregation of monocentric chromosomes is realized by mono-orientation of fused sister kinetochores at metaphase I and by bi-orientation of sister kinetochores at metaphase II. Conventionally this depends on a 2-step loss of cohesion, along chromosome arms during meiosis I and at sister centromeres during meiosis II.

Affinity proteomics reveals extensive phosphorylation of the Brassica chromosome axis protein ASY1 and a network of associated proteins at prophase I of meiosis

Summary During meiosis, the formation of crossovers (COs) generates genetic variation and provides physical links that are essential for accurate chromosome segregation. COs occur in the context of a proteinaceous chromosome axis. The transcriptomes and proteomes of anthers and meiocytes comprise several thousand genes and proteins, but because of the level of complexity relatively few have been functionally characterized. Our understanding of the physical and functional interactions between meiotic proteins is also limited. Here we use affinity proteomics to analyse the proteins that are associated with the meiotic chromosome axis protein, ASY1, in Brassica oleracea anthers and meiocytes. We show that during prophase I ASY1 and its interacting partner, ASY3, are extensively phosphorylated, and we precisely assign phosphorylation sites. We identify 589 proteins that co‐immunoprecipitate with ASY1. These correspond to 492 Arabidopsis orthologues, over 90% of which form a coherent protein–protein interaction (PPI) network containing known and candidate meiotic proteins, including proteins more usually associated with other cellular processes such as DNA replication and proteolysis. Mutant analysis confirms that affinity proteomics is a viable strategy for revealing previously unknown meiotic proteins, and we show how the PPI network can be used to prioritise candidates for analysis. Finally, we identify another axis‐associated protein with a role in meiotic recombination. Data are available via ProteomeXchange with identifier PXD006042.

Identification of ASYNAPTIC4, a component of the meiotic chromosome axis

ASYNAPTIC4 is required for normal meiotic recombination and synapsis in Arabidopsis thaliana. During the leptotene stage of prophase I of meiosis, chromatids become organized into a linear looped array via a protein axis that forms along the loop bases. Establishment of the axis is essential for the subsequent synapsis of the homologous chromosome pairs and the progression of recombination to form genetic crossovers. Here, we describe ASYNAPTIC4 (ASY4), a meiotic axis protein in Arabidopsis (Arabidopsis thaliana). ASY4 is a small coiled-coil protein that exhibits limited sequence similarity with the carboxyl-terminal region of the axis protein ASY3. We used enhanced yellow fluorescent protein-tagged ASY4 to show that ASY4 localizes to the chromosome axis throughout prophase I. Bimolecular fluorescence complementation revealed that ASY4 interacts with ASY1 and ASY3, and yeast two-hybrid analysis confirmed a direct interaction between ASY4 and ASY3. Mutants lacking full-length ASY4 exhibited defective axis formation and were unable to complete synapsis. Although the initiation of recombination appeared to be unaffected in the asy4 mutant, the number of crossovers was reduced significantly, and crossovers tended to group in the distal parts of the chromosomes. We conclude that ASY4 is required for normal axis and crossover formation. Furthermore, our data suggest that ASY3/ASY4 are the functional homologs of the mammalian SYCP2/SYCP3 axial components.