Subgenome Discrimination in Brassica and Raphanus Allopolyploids Using Microsatellites

Intergeneric crosses between Brassica species and Raphanus sativus have produced crops with prominent shoot and root systems of Brassica and R. sativus, respectively. It is necessary to discriminate donor genomes when studying cytogenetic stability in distant crosses to identify homologous chromosome pairing, and microsatellite repeats have been used to discriminate subgenomes in allopolyploids. To identify genome-specific microsatellites, we explored the microsatellite content in three Brassica species (B. rapa, AA, B. oleracea, CC, and B. nigra, BB) and R. sativus (RR) genomes, and validated their genome specificity by fluorescence in situ hybridization. We identified three microsatellites showing A, C, and B/R genome specificity. ACBR_msat14 and ACBR_msat20 were detected in the A and C chromosomes, respectively, and ACBR_msat01 was detected in B and R genomes. However, we did not find a microsatellite that discriminated the B and R genomes. The localization of ACBR_msat20 in the 45S rDNA array in ×Brassicoraphanus 977 corroborated the association of the 45S rDNA array with genome rearrangement. Along with the rDNA and telomeric repeat probes, these microsatellites enabled the easy identification of homologous chromosomes. These data demonstrate the utility of microsatellites as probes in identifying subgenomes within closely related Brassica and Raphanus species for the analysis of genetic stability of new synthetic polyploids of these genomes.

Overexpression of HOP2 induces developmental defects and compromises growth in Arabidopsis

HOMOLOGOUS PAIRING 2 (HOP2) is a predominantly meiotic protein that plays a pivotal role in homologous chromosome pairing in organisms as diverse as yeast and mammals. While generating HOP2::GFP reporter lines, we identified two Arabidopsis T-DNA insertion mutants, stunted1(std1) and stunted2 (std2) that exhibit pleiotropic phenotypes, including fasciated stems, altered phyllotaxy, floral organ defects, reduced fecundity, and an overall reduction in growth properties. TAIL-PCR followed by sequencing revealed several insertions near genes, but genotyping showed that none of the insertions are causal. Analysis the std mutants by qRT-PCR, and analysis of dexamethasone inducible HOP2 transgenic plants demonstrated that the std phenotypes are associated with ectopic/overexpression of HOP2. Based on the postulated mechanisms of HOP2 action, we speculate on how overexpression leads to these developmental/growth defects.

Multi-Scale Organization of the Drosophila melanogaster Genome

Interphase chromatin, despite its appearance, is a highly organized framework of loops and bends. Chromosomes are folded into topologically associating domains, or TADs, and each chromosome and its homolog occupy a distinct territory within the nucleus. In Drosophila, genome organization is exceptional because homologous chromosome pairing is in both germline and somatic tissues, which promote interhomolog interactions such as transvection that can affect gene expression in trans. In this review, we focus on what is known about genome organization in Drosophila and discuss it from TADs to territory. We start by examining intrachromosomal organization at the sub-chromosome level into TADs, followed by a comprehensive analysis of the known proteins that play a key role in TAD formation and boundary establishment. We then zoom out to examine interhomolog interactions such as pairing and transvection that are abundant in Drosophila but rare in other model systems. Finally, we discuss chromosome territories that form within the nucleus, resulting in a complete picture of the multi-scale organization of the Drosophila genome.

Maintenance DNA methylation in pre-meiotic germ cells regulates meiotic prophase by facilitating homologous chromosome pairing

ABSTRACT Heterochromatin-related epigenetic mechanisms, such as DNA methylation, facilitate pairing of homologous chromosomes during the meiotic prophase of mammalian spermatogenesis. In pro-spermatogonia, de novo DNA methylation plays a key role in completing meiotic prophase and initiating meiotic division. However, the role of maintenance DNA methylation in the regulation of meiosis, especially in the adult, is not well understood. Here, we reveal that NP95 (also known as UHRF1) and DNMT1 – two essential proteins for maintenance DNA methylation – are co-expressed in spermatogonia and are necessary for meiosis in male germ cells. We find that Np95- or Dnmt1-deficient spermatocytes exhibit spermatogenic defects characterized by synaptic failure during meiotic prophase. In addition, assembly of pericentric heterochromatin clusters in early meiotic prophase, a phenomenon that is required for subsequent pairing of homologous chromosomes, is disrupted in both mutants. Based on these observations, we propose that DNA methylation, established in pre-meiotic spermatogonia, regulates synapsis of homologous chromosomes and, in turn, quality control of male germ cells. Maintenance DNA methylation, therefore, plays a role in ensuring faithful transmission of both genetic and epigenetic information to offspring.

Interplay between Pds5 and Rec8 in regulating chromosome axis length and crossover frequency

Meiotic chromosomes have a loop/axis architecture, with axis length determining crossover frequency. Meiosis-specific Pds5 depletion mutants have shorter chromosome axes and lower homologous chromosome pairing and recombination frequency. However, it is poorly understood how Pds5 coordinately regulates these processes. In this study, we show that only ~20% of wild-type level of Pds5 is required for homolog pairing and that higher levels of Pds5 dosage-dependently regulate axis length and crossover frequency. Moderate changes in Pds5 protein levels do not explicitly impair the basic recombination process. Further investigations show that Pds5 does not regulate chromosome axes by altering Rec8 abundance. Conversely, Rec8 regulates chromosome axis length by modulating Pds5. These findings highlight the important role of Pds5 in regulating meiosis and its relationship with Rec8 to regulate chromosome axis length and crossover frequency with implications for evolutionary adaptation.

Homologous chromosome pairing in meiosis of higher eukaryotes—still an enigma?

Meiosis is the basis of the generative reproduction of eukaryotes. The crucial first step is homologous chromosome pairing. In higher eukaryotes, micrometer-scale chromosomes, micrometer distances apart, are brought together by nanometer DNA sequences, at least a factor of 1000 size difference. Models of homology search, homologue movement, and pairing at the DNA level in higher eukaryotes are primarily based on studies with yeast where the emphasis is on the induction and repair of DNA double-strand breaks (DSB). For such a model, the very large nuclei of most plants and animals present serious problems. Homology search without DSBs cannot be explained by models based on DSB repair. The movement of homologues to meet each other and make contact at the molecular level is not understood. These problems are discussed and the conclusion is that at present practically nothing is known of meiotic homologue pairing in higher eukaryotes up to the formation of the synaptonemal complex, and that new, necessarily speculative models must be developed. Arguments are given that RNA plays a central role in homology search and a tentative model involving RNA in homology search is presented. A role of actin in homologue movement is proposed. The primary role of DSBs in higher eukaryotes is concluded to not be in pairing but in the preparation of Holliday junctions, ultimately leading to chromatid exchange.

Structural analysis of different LINC complexes reveals distinct binding modes

Linker of nucleoskeleton and cytoskeleton (LINC) complexes are molecular tethers that span the nuclear envelope (NE) and physically connect the nucleus to the cytoskeleton. They transmit mechanical force across the NE in processes such as nuclear anchorage, nuclear migration, and homologous chromosome pairing during meiosis. LINC complexes are composed of KASH proteins traversing the outer nuclear membrane, and SUN proteins crossing the inner nuclear membrane. Humans have several SUN- and KASH-containing proteins, yet what governs their proper engagement is poorly understood. To investigate this question, we solved high resolution crystal structures of human SUN2 in complex with the KASH-peptides of Nesprin3, Nesprin4, and KASH5. In comparison to the published structures of SUN2-KASH1/2 we observe alternative binding modes for these KASH peptides. While the core interactions between SUN and the C-terminal residues of the KASH peptide are similar in all five complexes, the extended KASH-peptide adopts at least two different conformations. The much-improved resolution allows for a more detailed analysis of other elements critical for KASH interaction, including the KASH-lid and the cation loop, and a possible self-locked state for unbound SUN. In summary, we observe distinct differences between the examined SUN-KASH complexes. These differences may have an important role in regulating the SUN-KASH network.

trans-homolog interaction regulates the sex-biased expression of an X-linked gene

Sex-biased gene expression patterns in animals are generally controlled by the somatic sex-determination hierarchies. How the different tiers of these hierarchies act on sexually dimorphic gene regulation is still poorly understood. In the developing Drosophila biarmipes wing, the X-linked gene yellow is expressed in males in a specific distal spot pattern that prefigures a corresponding adult pigmentation pattern. This yellow expression pattern is controlled by the spot enhancer, but the origin of yellow sexually dimorphic expression is unknown. Here we find that the functional interaction between homologous yellow alleles silences specifically the spot enhancer, which is therefore active in males (XY) but not in females (XX). We show that inserting yellow at homologous positions on autosomes recapitulates, in either sex, the homologous-dependent silencing of the spot enhancer. We further find that this silencing requires the yellow intron as well as the architectural protein Mod(mdg4). Finally, we show that Mod(mdg4) is also necessary for the sex-biased expression of some X-linked genes in the brain. Our results demonstrate that regulatory interactions between X-linked homologous alleles promote their sex-biased expression, independently of the canonical sex-determination hierarchy. More generally, they illustrate the biological significance of homologous chromosome pairing and trans-homolog interactions for the sexually dimorphic regulation of X-linked genes.