Centromeres in most higher eukaryotes are composed of long arrays of satellite repeats. By contrast, most newly formed centromeres (neocentromeres) do not contain satellite repeats and instead include DNA sequences representative of the genome. An unknown question in centromere evolution is how satellite repeat-based centromeres evolve from neocentromeres. We conducted a genome-wide characterization of sequences associated with CENH3 nucleosomes in potato (Solanum tuberosum). Five potato centromeres (Cen4, Cen6, Cen10, Cen11, and Cen12) consisted primarily of single-or low-copy DNA sequences. No satellite repeats were identified in these five centromeres. At least one transcribed gene was associated with CENH3 nucleosomes. Thus, these five centromeres structurally resemble neocentromeres. By contrast, six potato centromeres (Cen1, Cen2, Cen3, Cen5, Cen7, and Cen8) contained megabase-sized satellite repeat arrays that are unique to individual centromeres. The satellite repeat arrays likely span the entire functional cores of these six centromeres. At least four of the centromeric repeats were amplified from retrotransposon-related sequences and were not detected in Solanum species closely related to potato. The presence of two distinct types of centromeres, coupled with the boom-and-bust cycles of centromeric satellite repeats in Solanum species, suggests that repeat-based centromeres can rapidly evolve from neocentromeres by de novo amplification and insertion of satellite repeats in the CENH3 domains.

Gene expression is controlled by the complex interaction of transcription factors binding to promoters and other regulatory DNA elements. One common characteristic of the genomic regions associated with regulatory proteins is a pronounced sensitivity to DNase I digestion. We generated genome-wide high-resolution maps of DNase I hypersensitive (DH) sites from both seedling and callus tissues of rice (Oryza sativa). Approximately 25% of the DH sites from both tissues were found in putative promoters, indicating that the vast majority of the gene regulatory elements in rice are not located in promoter regions. We found 58% more DH sites in the callus than in the seedling. For DH sites detected in both the seedling and callus, 31% displayed significantly different levels of DNase I sensitivity within the two tissues. Genes that are differentially expressed in the seedling and callus were frequently associated with DH sites in both tissues. The DNA sequences contained within the DH sites were hypomethylated, consistent with what is known about active gene regulatory elements. Interestingly, tissue-specific DH sites located in the promoters showed a higher level of DNA methylation than the average DNA methylation level of all the DH sites located in the promoters. A distinct elevation of H3K27me3 was associated with intergenic DH sites. These results suggest that epigenetic modifications play a role in the dynamic changes of the numbers and DNase I sensitivity of DH sites during development.

Gene expression and regulation in eukaryotes is controlled by orchestrated binding of regulatory proteins, including both activators and repressors, to promoters and other cis-regulatory DNA elements. An increasing number of plant genomes have been sequenced; however, a similar effort to the ENCODE project, which aimed to identify all functional elements in the human genome, has yet to be initiated in plants. Here we report genome-wide high-resolution mapping of DNase I hypersensitive (DH) sites in the model plant Arabidopsis thaliana. We identified 38,290 and 41,193 DH sites in leaf and flower tissues, respectively. The DH sites were depleted of bulk nucleosomes and were tightly associated with RNA polymerase II binding sites. Approximately 90% of the binding sites of two well-characterized MADS domain transcription factors, APETALA1 and SEPALLATA3, were covered by the DH sites. We demonstrate that protein binding footprints within a specific genomic region can be revealed using the DH site data sets in combination with known or putative protein binding motifs and gene expression data sets. Thus, genome-wide DH site mapping will be an important tool for systematic identification of all cis-regulatory DNA elements in plants.

No abstract

No abstract

Plant and animal centromeres comprise megabases of highly repeated satellite sequences, yet centromere function can be specified epigenetically on single-copy DNA by the presence of nucleosomes containing a centromere-specific variant of histone H3 (cenH3). We determined the positions of cenH3 nucleosomes in rice (Oryza sativa), which has centromeres composed of both the 155-bp CentO satellite repeat and single-copy non-CentO sequences. We find that cenH3 nucleosomes protect 90-100 bp of DNA from micrococcal nuclease digestion, sufficient for only a single wrap of DNA around the cenH3 nucleosome core. cenH3 nucleosomes are translationally phased with 155-bp periodicity on CentO repeats, but not on non-CentO sequences. CentO repeats have an ∼10-bp periodicity in WW dinucleotides and in micrococcal nuclease cleavage, providing evidence for rotational phasing of cenH3 nucleosomes on CentO and suggesting that satellites evolve for translational and rotational stabilization of centromeric nucleosomes.CENP-A | nucleosome phasing | epigenetics | kinetochore C entromeres, the chromosomal domains that attach to spindle microtubules to segregate eukaryotic chromosomes in mitosis and meiosis, are DNA elements bound by special nucleosomes that contain a centromere-specific variant of histone H3 (cenH3). In most plants and animals, cenH3 nucleosomes are found on centromeric DNA that comprises megabases of tandemly repeated "satellite" sequences. Despite this apparent preference for repetitive DNA, a fully functional centromere, called a neocentromere, can occasionally form by assembling cenH3 nucleosomes on a single-copy DNA sequence that was not previously part of a centromere, indicating that centromere specification is epigenetic in plants and animals (for reviews, see refs. 1-4).The tandem arrays of highly repeated satellite sequences that compose most plant and animal centromeres can differ dramatically between closely related species (5), and even between different chromosomes (6-8), suggesting that satellite arrays undergo rapid evolution through expansions, contractions, gene conversions, and transpositions. Monomers of satellite repeats range in length from 5 bp in Drosophila to 1,419 bp in cattle although more than half of described monomers in 282 species have lengths between 100 and 200 bp, often regarded as approximately the length of nucleosomal DNA (6, 9). The cenH3 nucleosomes typically occupy only a portion of the satellite repeats, often in discontinuous blocks (7, 10-12), and the same or similar repeats often underlie flanking pericentromeric heterochromatin composed of conventional nucleosomes. Some of these repeats, for example African green monkey α-satellite DNA, have long been known to position conventional nucleosomes, resulting in arrays of regularly spaced nucleosomes, said to be translationally phased (13-15). Nucleosomes can occupy multiple alternative translational phases on the same satellite (16, 17). Translationally phased nucleosomal arrays have also been observed on satellites in cucumber a...

Most existing centromeres may have originated as neocentromeres that activated de novo from noncentromeric regions. However, the evolutionary path from a neocentromere to a mature centromere has been elusive. Here we analyzed the centromeres of nine chromosomes that were transferred from maize into oat as the result of an inter-species cross. Centromere size and location were assayed by chromatin immunoprecipitation for the histone variant CENH3, which is a defining feature of functional centromeres. Two isolates of maize chromosome 3 proved to contain neocentromeres in the sense that they had moved from the original site, whereas the remaining seven centromeres (1, 2, 5, 6, 8, 9, and 10) were retained in the same area in both species. In all cases, the CENH3-binding domains were dramatically expanded to encompass a larger area in the oat background (∼3.6 Mb) than the average centromere size in maize (∼1.8 Mb). The expansion of maize centromeres appeared to be restricted by the transcription of genes located in regions flanking the original centromeres. These results provide evidence that (1) centromere size is regulated; (2) centromere sizes tend to be uniform within a species regardless of chromosome size or origin of the centromere; and (3) neocentromeres emerge and expand preferentially in gene-poor regions. Our results suggest that centromere size expansion may be a key factor in the survival of neocentric chromosomes in natural populations.

Genomic regions free of nucleosomes, which are hypersensitive to DNase I digestion, are known as DNase I hypersensitive sites (DHSs) and frequently contain cis-regulatory DNA elements. To investigate their prevalence and characteristics in maize (), we developed high-resolution genome-wide DHS maps using a modified DNase-seq technique. Maize DHSs exhibit depletion of nucleosomes and low levels of DNA methylation and are enriched with conserved noncoding sequences (CNSs). We developed a protoplast-based transient transformation assay to assess the potential gene expression enhancer and/or promoter functions associated with DHSs, which showed that more than 80% of DHSs overlapping with CNSs showed an enhancer function. Strikingly, nearly 25% of maize DHSs were derived from transposable elements (TEs), including both class I and class II transposons. Interestingly, TE-derived DHSs (teDHSs) homologous to retrotransposons were enriched with sequences related to the intrinsic cis-regulatory elements within the long terminal repeats of retrotransposons. We demonstrate that more than 80% of teDHSs can drive transcription of a reporter gene in protoplast assays. These results reveal the widespread occurrence of TE-derived cis-regulatory sequences and suggest that teDHSs play a major role in transcriptional regulation in maize.