Chromosome Fragile Sites in Arabidopsis Harbor Matrix Attachment Regions That May Be Associated with Ancestral Chromosome Rearrangement Events

摘要

Mutations in the BREVIPEDICELLUS ( BP ) gene of Arabidopsis thaliana condition a pleiotropic phenotype featuring defects in internode elongation, the homeotic conversion of internode to node tissue, and downward pointing flowers and pedicels. We have characterized five mutant alleles of BP , generated by EMS, fast neutrons, x-rays, and aberrant T–DNA insertion events. Curiously, all of these mutagens resulted in large deletions that range from 140 kbp to over 900 kbp just south of the centromere of chromosome 4. The breakpoints of these mutants were identified by employing inverse PCR and DNA sequencing. The south breakpoints of all alleles cluster in BAC T12G13, while the north breakpoint locations are scattered. With the exception of a microhomology at the bp-5 breakpoint, there is no homology in the junction regions, suggesting that double-stranded breaks are repaired via non-homologous end joining. Southwestern blotting demonstrated the presence of nuclear matrix binding sites in the south breakpoint cluster (SBC), which is A/T rich and possesses a variety of repeat sequences. In situ hybridization on pachytene chromosome spreads complemented the molecular analyses and revealed heretofore unrecognized structural variation between the Columbia and Landsberg erecta genomes. Data mining was employed to localize other large deletions around the HY4 locus to the SBC region and to show that chromatin modifications in the region shift from a heterochromatic to euchromatic profile. Comparisons between the BP/HY4 regions of A. lyrata and A. thaliana revealed that several chromosome rearrangement events have occurred during the evolution of these two genomes. Collectively, the features of the region are strikingly similar to the features of characterized metazoan chromosome fragile sites, some of which are associated with karyotype evolution. Author Summary Chromosome evolution involves both small-scale (e.g. single nucleotide) changes, as well as large-scale rearrangements such as inversions, translocations, and fusion events. We investigated mutations of the BREVIPEDICELLUS gene of Arabidopsis , which is a master regulator of inflorescence architecture. These mutations are not due to single nucleotide changes, but rather to large deletions, some spanning nearly one million base pairs. Molecular and biochemical analyses reveal that the chromosome breakpoints cluster in an area that is rich in repetitive elements and harbor multiple binding sites for nuclear matrix proteins. Data mining revealed intriguing correlations between the breakpoint cluster and hotspots of genetic recombination, regions of the chromosome that have undergone several rearrangement events during evolution, and changes in histone protein modifications. We propose that these unstable regions are chromosome fragile sites that assist in marking a boundary between gene-poor, transcriptionally repressed centromeric chromatin and a more relaxed chromatin domain that is gene-rich.