Handong Su

Sequential de novo centromere formation and inactivation on a chromosomal fragment in maize

Significance The centromere is the part of the chromosome that is involved with movement in mitosis and meiosis. The activity of the centromere is epigenetic in that the underlying DNA sequences do not necessarily determine function. In the present study, a chromosomal fragment was followed in which a sequential de novo formation and inactivation occurred for the position of the active centromere. The results suggest that de novo centromere formation occurs regularly. However, when coupled with previous findings that larger centromeres can inactivate smaller ones when present together, it is hypothesized that such frequent de novo centromere formations are cleared from normal chromosomes by inactivation, but can persist on structurally acentric fragments and be inherited. The ability of centromeres to alternate between active and inactive states indicates significant epigenetic aspects controlling centromere assembly and function. In maize (Zea mays), misdivision of the B chromosome centromere on a translocation with the short arm of chromosome 9 (TB-9Sb) can produce many variants with varying centromere sizes and centromeric DNA sequences. In such derivatives of TB-9Sb, we found a de novo centromere on chromosome derivative 3-3, which has no canonical centromeric repeat sequences. This centromere is derived from a 288-kb region on the short arm of chromosome 9, and is 19 megabases (Mb) removed from the translocation breakpoint of chromosome 9 in TB-9Sb. The functional B centromere in progenitor telo2-2 is deleted from derivative 3-3, but some B-repeat sequences remain. The de novo centromere of derivative 3-3 becomes inactive in three further derivatives with new centromeres being formed elsewhere on each chromosome. Our results suggest that de novo centromere initiation is quite common and can persist on chromosomal fragments without a canonical centromere. However, we hypothesize that when de novo centromeres are initiated in opposition to a larger normal centromere, they are cleared from the chromosome by inactivation, thus maintaining karyotype integrity.

Molecular mechanisms of homologous chromosome pairing and segregation in plants.

In most eukaryotic species, three basic steps of pairing, recombination and synapsis occur during prophase of meiosis I. Homologous chromosomal pairing and recombination are essential for accurate segregation of chromosomes. In contrast to the well-studied processes such as recombination and synapsis, many aspects of chromosome pairing are still obscure. Recent progress in several species indicates that the telomere bouquet formation can facilitate homologous chromosome pairing by bringing chromosome ends into close proximity, but the sole presence of telomere clustering is not sufficient for recognizing homologous pairs. On the other hand, accurate segregation of the genetic material from parent to offspring during meiosis is dependent on the segregation of homologs in the reductional meiotic division (MI) with sister kinetochores exhibiting mono-orientation from the same pole, and the segregation of sister chromatids during the equational meiotic division (MII) with kinetochores showing bi-orientation from the two poles. The underlying mechanism of orientation and segregation is still unclear. Here we focus on recent studies in plants and other species that provide insight into how chromosomes find their partners and mechanisms mediating chromosomal segregation.

High-efficiency genome editing using a dmc1 promoter-controlled CRISPR/Cas9 system in maize

Summary Previous studies revealed that the promoters for driving both Cas9 and sgRNAs are quite important for efficient genome editing by CRISPR/Cas9 in plants. Here, we report our results of targeted genome editing using the maize dmc1 gene promoter combined with the U3 promoter for Cas9 and sgRNA, respectively. Three loci in the maize genome were selected for targeting. The T0 plants regenerated were highly efficiently edited at the target sites with homozygous or bi‐allelic mutants accounting for about 66%. The mutations in T0 plants could be stably transmitted to the T1 generation, and new mutations could be generated in gametes or zygotes. Whole‐genome resequencing indicated that no off‐target mutations could be detected in the predicted loci with sequence similarity to the targeted site. Our results show that the dmc1 promoter‐controlled (DPC) CRISPR/Cas9 system is highly efficient in maize and provide further evidence that the optimization of the promoters used for the CRISPR/Cas9 system is important for enhancing the efficiency of targeted genome editing in plants. The evolutionary conservation of the dmc1 gene suggests its potential for use in other plant species.

Dynamic location changes of Bub1-phosphorylated-H2AThr133 with CENH3 nucleosome in maize centromeric regions.

The genomic stability of all organisms requires precise cell division with proper chromosome orientation. The Bub1-H2Aph-Sgo1 pathway and spindle assembly checkpoint (SAC) components have been identified in yeast and mammals that are important for sister centromere orientation and chromosome segregation. However, their roles in plants are not clear. Maize meiotic mutants and minichromosomes were used to study the role of H2AThr133 phosphorylation and SAC components in sister centromere orientation and chromosome segregation. Unlike previously reported, SAC protein Bub1-Sgo1 recruitment was independent of Rec8 in maize and did not play a role in centromere protection in meiosis I. Chromatin immunoprecipitation sequencing analysis with immnolocalization results indicate most CENH3 nucleosomes contain phosphorylated H2AThr133 in centromeric regions. H2AThr133ph spreads to encompass centromeric regions including the inner centromeric and pericentromeric regions during (pro)metaphase. The presence and localization of SAC components and H2AThr133ph on maize lines containing sister chromatids separate precociously in anaphase I revealed no direct role of these proteins on centromere orientation in meiosis I . This work sheds light on the relationship between H2AThr133ph and CENH3 nucleosome in plants, and the phosphorylation with dynamic location changes in centomeric regions suggests temporal and spatial regulation roles for H2A phosphorylation in chromosome segregation.

Dynamic chromatin changes associated with de novo centromere formation in maize euchromatin

The inheritance and function of centromeres are not strictly dependent on any specific DNA sequence, but involve an epigenetic component in most species. CENH3, a centromere histone H3 variant, is one of the best-described epigenetic factors in centromere identity, but the chromatin features required during centromere formation have not yet been revealed. We previously identified two de novo centromeres on Zea mays (maize) minichromosomes derived from euchromatic sites with high-density gene distributions but low-density transposon distributions. The distribution of gene location and gene expression in these sites indicates that transcriptionally active regions can initiate de novo centromere formation, and CENH3 seeding shows a preference for gene-free regions or regions with no gene expression. The locations of the expressed genes detected were at relatively hypomethylated loci, and the altered gene expression resulted from de novo centromere formation, but not from the additional copy of the minichromosome. The initial overall DNA methylation level of the two de novo regions was at a low level, but increased substantially to that of native centromeres after centromere formation. These results illustrate the dynamic chromatin changes during euchromatin-originated de novo centromere formation, which provides insight into the mechanism of de novo centromere formation and regulation of subsequent consequences.

Recent advances in plant centromere biology

The centromere, which is one of the essential parts of a chromosome, controls kinetochore formation and chromosome segregation during mitosis and meiosis. While centromere function is conserved in eukaryotes, the centromeric DNA sequences evolve rapidly and have few similarities among species. The histone H3 variant CENH3 (CENP-A in human), which mostly exists in centromeric nucleosomes, is a universal active centromere mark in eukaryotes and plays an essential role in centromere identity determination. The relationship between centromeric DNA sequences and centromere identity determination is one of the intriguing questions in studying centromere formation. Due to the discoveries in the past decades, including “neocentromeres” and “centromere inactivation”, it is now believed that the centromere identity is determined by epigenetic mechanisms. This review will present recent progress in plant centromere biology.

Dynamic epigenetic states of maize centromeres

The centromere is a specialized chromosomal region identified as the major constriction, upon which the kinetochore complex is formed, ensuring accurate chromosome orientation and segregation during cell division. The rapid evolution of centromere DNA sequence and the conserved centromere function are two contradictory aspects of centromere biology. Indeed, the sole presence of genetic sequence is not sufficient for centromere formation. Various dicentric chromosomes with one inactive centromere have been recognized. It has also been found that de novo centromere formation is common on fragments in which centromeric DNA sequences are lost. Epigenetic factors play important roles in centromeric chromatin assembly and maintenance. Non-disjunction of the supernumerary B chromosome centromere is independent of centromere function, but centromere pairing during early prophase of meiosis I requires an active centromere. This review discusses recent studies in maize about genetic and epigenetic elements regulating formation and maintenance of centromere chromatin, as well as centromere behavior in meiosis.

Histone Phosphorylation: Its Role during Cell Cycle and Centromere Identity in Plants

As the main protein components of chromatin, histones can alter the structural/functional capabilities of chromatin by undergoing extensive post-translational modifications (PTMs) such as phosphorylation, methylation, acetylation, ubiquitination, sumoylation, and so on. These PTMs are thought to transmit signals from the chromatin to the cell machinery to regulate various processes. Histone phosphorylation is associated with chromosome condensation/segregation, activation of transcription, and DNA damage repair. In this review, we focus on how different histone phosphorylations mark for chromatin change during the cell cycle, the relationship between histone phosphorylation and functional centromeres, and the candidate kinases that trigger and the phosphatase or kinase inhibitors that alter histone phosphorylation. Finally, we review the crosstalk between different PTMs.

The Behavior of the Maize B Chromosome and Centromere

The maize B chromosome is a non-essential chromosome with an accumulation mechanism. The dispensable nature of the B chromosome facilitates many types of genetic studies in maize. Maize lines with B chromosomes have been widely used in studies of centromere functions. Here, we discuss the maize B chromosome alongside the latest progress of B centromere activities, including centromere misdivision, inactivation, reactivation, and de novo centromere formation. The meiotic features of the B centromere, related to mini-chromosomes and the control of the size of the maize centromere, are also discussed.

Site-specific transfer of chromosomal segments and genes in wheat engineered chromosomes

Recently, engineered minichromosomes have been produced using a telomere-mediated truncation technique in some plants. However, the study on transferring genes to minichromosomes is very limited. Here, telomere-mediated truncation was successfully performed in common wheat (Triticum aestivum) to generate stable truncated chromosomes accompanied by a relatively high frequency of chromosomal rearrangements. After the cross between transgenic parents, a promoter-less DsRed gene in a chromosome from one parent was transferred to another chromosome from the other parent at the site behind a maize ubiquitin promoter via the Cre/lox system. DsRed transcripts and red fluorescent proteins were detected in the recombinant plants. In one such seedling, transgenic signals were detected at the centric terminus of chromosome 4D and the distal terminus of chromosome 3A. Clear translocations could be detected at the transgenic loci of these two chromosomes. Intriguingly, signals of centric-specific sequences were co-localized with the translocated D-group chromosomal segment in the terminal region of chromosome 3A. Our results indicate that the Cre/lox system induces the gene swapping to the target chromosome and non-homologous chromosomal recombination simultaneously. These approaches could offer a platform to transfer large DNA fragments or even terminal chromosomal segments to other chromosomes of the natural genome.