De novo centromere formation on a chromosome fragment in maize

Abstract

The centromere is the part of the chromosome that organizes the kinetochore, which mediates chromosome movement during mitosis and meiosis. A small fragment from chromosome 3, named Duplication 3a (Dp3a), was described from UV-irradiated materials by Stadler and Roman in the 1940s [Stadler LJ, Roman H (1948) Genetics 33(3):273-303]. The genetic behavior of Dp3a is reminiscent of a ring chromosome, but fluoresecent in situ hybridization detected telomeres at both ends, suggesting a linear structure. This small chromosome has no detectable canonical centromeric sequences, but contains a site with protein features of functional centromeres such as CENH3, the centromere specific H3 histone variant, and CENP-C, a foundational kinetochore protein, suggesting the de novo formation of a centromere on the chromatin fragment. To examine the sequences associated with CENH3, chromatin immunoprecipitation was carried out with anti-CENH3 antibodies using material from young seedlings with and without the Dp3a chromosome. A novel peak was detected from the ChIP-Sequencing reads of the Dp3a sample. The peak spanned 350 kb within the long arm of chromosome 3 covering 22 genes. Collectively, these results define the behavior and molecular features of de novo centromere formation in the Dp3a chromosome, which may shed light on the initiation of new centromere sites during evolution.

Fig. 1.

FISH patterns and immunolocalization analysis of Dp3a. (A) FISH with CentC (green) and CRM (red) as probes. There are no detectable CentC and CRM signals on the Dp3a chromosome (arrow) even with overexposure. (B–D) Immunostaining of the centromere-specific proteins CENH3, CENPC, and phosphorylated H2AThr133, respectively. The red CENH3, CENPC, and H2A antibody signals on Dp3a confirm the presence of a functional centromere. Arrows indicate the Dp3a chromosome. Insets display an enlarged image of Dp3a in each panel. (Scale bars, 5 μm.)

Fig. 2.

Telomeres are present on Dp3a. FISH analysis using a maize telomeric DNA probe (green) to detect the structure of the Dp3a chromosome (arrow) in root-tip metaphase cells. Telomere signals are detected at both ends of the chromosome. DAPI-stained chromosomes are blue.

Fig. 3.

Meiotic behavior of the Dp3a chromosome. In diplonema (A) and metaphase I (B), the Dp3a chromosome did not pair with chromosome 3. (C) In anaphase I, sister chromatids of Dp3a chromosome separated to opposite poles. (D) In meiosis II, the Dp3a chromosomes were observed in tetrads. (Scale bar, 10 μm.)

Fig. 4.

Genomic distribution of ChIP-seq reads on chromosome 3 (control and Dp3a with CENH3-associated DNA). (A) Read density represented by the number of unique mapping reads (RefGen ZmB73 Release 5a) derived from ChIP with CENH3 antibodies in 10-kb windows. The x axis shows the position on chromosome 3. The generalized region of endogenous centromere 3 is outlined with green. A 350-kb CENH3-binding domain, outlined in red, was found in the Dp3a sample compared with the control. The positions of the a1 and sh2 genes, known genetically to be present on Dp3a, are noted. (B) Enlarged view of the CENH3-binding region and immediately surrounding sequences showing the distribution of genes and transposons (TEs). A total of 22 protein-encoding genes are contained in this region and are numbered. A description of the 22 genes in the region is given in Table S2.

Fig. 5.

The 350-kb region on Dp3a confirmed by FISH. FISH analysis of metaphase cells using 5.8 kb of DNA (GRMZM2G045275 is gene 4 in Fig. 4) from the 350-kb region as a probe (red). Arrowhead indicates the Dp3a chromosome. Both chromosomes 3 (arrows) and Dp3a have red FISH signals. CentC centromere signals are in green. (Scale bar, 10 μm.)