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

<div><p>Mutations in the <em>BREVIPEDICELLUS</em> (<em>BP</em>) gene of <em>Arabidopsis thaliana</em> 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 <em>BP</em>, 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 <em>bp-5</em> 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 <em>erecta</em> genomes. Data mining was employed to localize other large deletions around the <em>HY4</em> locus to the SBC region and to show that chromatin modifications in the region shift from a heterochromatic to euchromatic profile. Comparisons between the <em>BP/HY4</em> regions of <em>A. lyrata</em> and <em>A. thaliana</em> 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.</p> </div

Cytological and FISH analysis of Columbia, Landsberg <i>erecta</i>, and the <i>bp</i> alleles.

<p>All signal distances and the size of the centromere and hK4S are in microns.</p><p>N/A: not applicable as L<i>er</i> backgrounds lack hk4S.</p

Chromosomal landscapes for Columbia, Landsberg <i>erecta</i>, and the <i>bp</i> alleles reveal ecotype specific and allele specific differences.

<p>A. Cytological map of the north end of chromosome 4 showing the nucleolar organizer (NOR4), the heterochromatic knob that exists in Columbia (hk4S), and the centromere region (CEN4). The locations of the <i>BP</i> gene and the red and green in situ hybridization probes and their AGI coordinates are shown. The figure is drawn to scale, with the exceptions that NOR4 and CEN4 are only allotted 1000 bp in the AGI numbering scheme. Cytologically, these are large regions, occupying 2–4 Mbp and have been represented as such to enable comparisons of DAPI stained landmarks vs. FISH signals. B. Representative pachytene in situ chromosome hybridization patterns for the samples listed. Scale bars represent 10 microns.</p

Chromatin modifications at the breakpoint junctions.

<p>A. North breakpoints occur in pericentric heterochromatin and bear H3K9me2, H4K20me1 and H3K27me1 signatures. South breakpoint modifications exhibit no epigenetic marks or euchromatic marks. B. Global view of chromosome 4 euchromatic (H3K4me2) and heterochromatic (H4K20me1) modification patterns. hk4S and CEN4 positions are highlighted in green; the position of BAC T12G13 at the transition zone is indicated (data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003136#pgen.1003136-Roudier1" target="_blank">[19]</a>).</p

Breakpoint junction regions bind the nuclear matrix protein AHL1.

<p>Aliquots of proteins from induced <i>E. coli</i> harboring HIS-tagged AHL1 were subjected to SDS-PAGE and blotting, and membrane strips were probed with end labeled DNA fragments. 2S, 3N, 3S, 5N, 11S represent probes near the north or south (N or S) borders of the breakpoints of each <i>bp</i> allele as shown on the accompanying map with AGI coordinates in Mbp. PC represents a positive control from the plastocyanin gene <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003136#pgen.1003136-vanDrunen1" target="_blank">[16]</a> while H1 represents the histone H1 gene (At1g06760) probe, used as a negative control. The locations of the <i>BP</i> gene and BAC T12G13 are shown.</p