Kazunari Kashihara

Visualization of Diffuse Centromeres with Centromere-Specific Histone H3 in the Holocentric Plant Luzula nivea

Although holocentric species are scattered throughout the plant and animal kingdoms, only holocentric chromosomes of the nematode worm Caenorhabditis elegans have been analyzed with centromeric protein markers. In an effort to determine the holocentric structure in plants, we investigated the snowy woodrush Luzula nivea. From the young roots, a cDNA encoding a putative centromere-specific histone H3 (LnCENH3) was successfully isolated based on sequence similarity among plant CENH3s. The deduced amino acid sequence was then used to raise an anti-LnCENH3 antibody. Immunostaining clearly revealed the diffuse centromere-like structure that appears in the linear shape at prophase to telophase. Furthermore, it was shown that the amount of LnCENH3 decreased significantly at interphase. The polar side positioning on each chromatid at metaphase to anaphase also confirmed that LnCENH3 represents one of the centromere-specific proteins in L. nivea. These data from L. nivea are compared with those from C. elegans, and common features of holocentric chromosomes are discussed.

A centromeric DNA sequence colocalized with a centromere-specific histone H3 in tobacco.

Centromeres play an important role in segregating chromosomes into daughter cells, and centromeric DNA assembles specific proteins to form a complex referred to as the kinetochore. Among these proteins, centromere-specific histone H3 (CENH3) is one of the most characterized and found to be located only on active centromeres. We isolated four different CENH3-coding complementary DNAs (cDNAs), two from Nicotiana tabaccum and one each from the ancestral diploid species, Nicotiana sylvestris and Nicotiana tomentosiformis and raised an antibody against N-terminal amino acid sequences deduced from the cDNAs. Immunostaining with the antibody revealed the preferential centromere localization, indicating that the cDNAs cloned in this study encode authentic tobacco CENH3. A tobacco centromeric DNA sequence (Nt2-7) was also identified by chromatin immunoprecipitation cloning using the antibody.

Functional centromeres in soybean include two distinct tandem repeats and a retrotransposon.

The centromere as a kinetochore assembly site is fundamental to the partitioning of genetic material during cell division. In order to determine the functional centromeres of soybean, we characterized the soybean centromere-specific histone H3 (GmCENH3) protein and developed an antibody against the N-terminal end. Using this antibody, we cloned centromere-associated DNA sequences by chromatin immunoprecipitation. Our analyses indicate that soybean centromeres are composed of two distinct satellite repeats (GmCent-1 and GmCent-4) and retrotransposon-related sequences (GmCR). The possible allopolyploid origin of the soybean genome is discussed in view of the centromeric satellite sequences present.

Functional analysis of the Arabidopsis centromere by T-DNA insertion-induced centromere breakage.

Two minichromosomes (α and δ) in addition to two other aberrant chromosomes (β and γ) were found in a transgenic Arabidopsis plant produced by an in planta vacuum infiltration technique. The minichromosomes were successfully separated by successive crossing and selfing and added to wild-type Columbia (Col-0) as a supernumerary chromosome. FISH indicated that both of the two minichromosomes originated from the short arm of chromosome 2. The mini α chromosome contained the whole short-arm 2S and a truncated centromere (180-bp repeat cluster), whereas mini δ lacked the terminal region including telomere repeats. Pachytene FISH clearly revealed that mini δ comprised a ring chromosome carrying two copies of the region from the 180-bp repeat cluster to BAC-F3C11. Both of the 180-bp clusters (each ≈500 kb in length) were thought to possess normal centromere functions because the centromere-specific histone H3 variant (HTR12) was detected on both clusters. Notwithstanding this dicentric and ring form, mini δ was stably transmitted to the next generations, perhaps because of its compact size (<4 Mb). Chromosome β also comprised a dicentric-like structure, with one of the two 180-bp repeat sites derived from chromosome 1 and the other from chromosome 2. However, the latter was quite small and failed to bind HTR12. The data obtained in this study indicated that 500 kb of the 180-bp array of the chromosome 2 centromere, from the edge of the 180-bp array on the short-arm side, is sufficient to form a functional domain.

Functional centromeres in Astragalus sinicus include a compact centromere-specific histone H3 and a 20-bp tandem repeat

The centromere plays an essential role for proper chromosome segregation during cell division and usually harbors long arrays of tandem repeated satellite DNA sequences. Although this function is conserved among eukaryotes, the sequences of centromeric DNA repeats are variable. Most of our understanding of functional centromeres, which are defined by localization of a centromere-specific histone H3 (CENH3) protein, comes from model organisms. The components of the functional centromere in legumes are poorly known. The genus Astragalus is a member of the legumes and bears the largest numbers of species among angiosperms. Therefore, we studied the components of centromeres in Astragalus sinicus. We identified the CenH3 homolog of A. sinicus, AsCenH3 that is the most compact in size among higher eukaryotes. A CENH3-based assay revealed the functional centromeric DNA sequences from A. sinicus, called CentAs. The CentAs repeat is localized in A. sinicus centromeres, and comprises an AT-rich tandem repeat with a monomer size of 20 nucleotides.

Coexistence of NtCENH3 and two retrotransposons in tobacco centromeres

Although a centromeric DNA fragment of tobacco (Nicotiana tabacum), Nt2-7, has been reported, the overall structure of the centromeres remains unknown. To characterize the centromeric DNA sequences, we conducted a chromatin immunoprecipitation assay using anti-NtCENH3 antibody and chromatins isolated from two ancestral diploid species (Nicotiana sylvestris and Nicotiana tomentosiformis) of N. tabacum and isolated a 178-pb fragment, Nto1 from N. tomentosiformis, as a novel centromeric DNA. Fluorescence in situ hybridization (FISH) showed that Nto1 localizes on 24 out of 48 chromosomes in some cells of a BY-2 cell line. To identify the origins of the Nt2-7 and Nto1, a tobacco bacterial artificial chromosome (BAC) library was constructed from N. tabacum, and then screened by polymerase chain reaction (PCR) with primer sets designed from the Nt2-7 and Not1 DNA sequences. Twelve BAC clones were found to localize on the centromeric regions by FISH. We selected three BAC clones for sequencing and identified two centromeric retrotransposons, NtCR and NtoCR, the DNA sequences of which are similar to that of Nt2-7 and Nto1, respectively. Quantitative PCR analysis using coprecipitated DNA with anti-NtCENH3 clearly showed coexistence of NtCENH3 with both retrotransposons. These results indicate the possibility that these two retrotransposons act as centromeric DNA sequences in tobacco. NtoCR was found to be specific to N. tomentosiformis and T genome of N. tabacum, and a NtCR-like centromeric retrotransposon (TGRIV) exists in tomato. This specificity suggests that the times of amplification of these centromeric retrotransposons were different.

Isolation of centromeric-tandem repetitive DNA sequences by chromatin affinity purification using a HaloTag7-fused centromere-specific histone H3 in tobacco

The centromere is a multi-functional complex comprising centromeric DNA and a number of proteins. To isolate unidentified centromeric DNA sequences, centromere-specific histone H3 variants (CENH3) and chromatin immunoprecipitation (ChIP) have been utilized in some plant species. However, anti-CENH3 antibody for ChIP must be raised in each species because of its species specificity. Production of the antibodies is time-consuming and costly, and it is not easy to produce ChIP-grade antibodies. In this study, we applied a HaloTag7-based chromatin affinity purification system to isolate centromeric DNA sequences in tobacco. This system required no specific antibody, and made it possible to apply a highly stringent wash to remove contaminated DNA. As a result, we succeeded in isolating five tandem repetitive DNA sequences in addition to the centromeric retrotransposons that were previously identified by ChIP. Three of the tandem repeats were centromere-specific sequences located on different chromosomes. These results confirm the validity of the HaloTag7-based chromatin affinity purification system as an alternative method to ChIP for isolating unknown centromeric DNA sequences. The discovery of more than two chromosome-specific centromeric DNA sequences indicates the mosaic structure of tobacco centromeres.

Centromere targeting of alien CENH3s in arabidopsis and tobacco cells

The centromere is a region utilized for spindle attachment on a eukaryotic chromosome and essential for accurate chromatid segregation. In most eukaryotes, centromeres have specific DNA sequences and are capable of assembling specific proteins to form a complex called the kinetochore. Among these proteins, centromeric histone H3 (CENH3) is one of the most fundamental, since CENH3s have been found in all investigated functional centromeres and recruits other centromeric proteins. In this study, the localization of alien CENH3s were analyzed in Arabidopsis and tobacco-cultured cells to determine the interaction between species-specific centromeric DNA and CENH3. Results showed that CENH3 of Arabidopsis and tobacco were localized on centromeres in the tobacco-cultured cells, unlike the case with CENH3 of rice and Luzula. In addition to these CENH3s, CENH3 of Luzula was partially localized in the Arabidopsis cultured cells. These data suggest that only evolutionally close CENH3s are able to target centromeres in alien species. Furthermore, the ability to target alien centromeres of histone fold domains was investigated using amino-terminal deleted CENH3s.

Characterization of the two centromeric proteins CENP-C and MIS12 in Nicotiana species

Centromeres play an important role in chromosome transmission in eukaryotes and comprise specific DNA and proteins that form complexes called kinetochores. In tobacco, although a centromere-specific histone H3 (NtCENH3) and centromeric DNA sequence (Nt2-7) have been identified, no other kinetochore components have been determined. In this study, we isolated and characterized cDNAs encoding two centromeric proteins CENP-C and MIS12 from Nicotiana tabaccum. Two CENP-C homologues, NtCENP-C-1 and -2, isolated from N. tabaccum were similar to CENP-C from N. sylvestris and N. tomentosiformis, respectively. Similarly, two Mis12 homologues, NtMIS12-1 and -2, in N. tabaccum were shown to originate from N. sylvestris and N. tomentosiformis, respectively. Both respective homologues for CENP-C and Mis12 were expressed at the same level. This indicates that in a tetraploid species, N. tabaccum, two ancestral genes encoding the centromeric proteins participate equally in the functioning of centromeres.

Identification of the centromere-specific histone H3 variant in Lotus japonicus

The centromere is a structurally and functionally specialized region present on every eukaryotic chromosome. Lotus japonicus is a model legume species for which there is very limited information on the centromere structure. Here we cloned and characterized the L. japonicus homolog of the centromere-specific histone H3 gene (LjCenH3) encoding a 159-amino acid protein. Using an Agrobacterium-based transformation system, LjCenH3 tagged with a green fluorescent protein was transferred into L. japonicus cells. The centromeric position of LjCENH3 protein was revealed on L. japonicus metaphase chromosomes by an immunofluorescence assay. The identification of LjCenH3 as a critical centromere landmark could pave the way for a better understanding of centromere structure in this model and other agriculturally important legume species.