Mu transposon insertion sites and meiotic recombination events co-localize with epigenetic marks for open chromatin across the maize genome

Abstract

The Mu transposon system of maize is highly active, with each of the approximately 50-100 copies transposing on average once each generation. The approximately one dozen distinct Mu transposons contain highly similar approximately 215 bp terminal inverted repeats (TIRs) and generate 9-bp target site duplications (TSDs) upon insertion. Using a novel genome walking strategy that uses these conserved TIRs as primer binding sites, Mu insertion sites were amplified from Mu stocks and sequenced via 454 technology. 94% of approximately 965,000 reads carried Mu TIRs, demonstrating the specificity of this strategy. Among these TIRs, 21 novel Mu TIRs were discovered, revealing additional complexity of the Mu transposon system. The distribution of >40,000 non-redundant Mu insertion sites was strikingly non-uniform, such that rates increased in proportion to distance from the centromere. An identified putative Mu transposase binding consensus site does not explain this non-uniformity. An integrated genetic map containing more than 10,000 genetic markers was constructed and aligned to the sequence of the maize reference genome. Recombination rates (cM/Mb) are also strikingly non-uniform, with rates increasing in proportion to distance from the centromere. Mu insertion site frequencies are strongly correlated with recombination rates. Gene density does not fully explain the chromosomal distribution of Mu insertion and recombination sites, because pronounced preferences for the distal portion of chromosome are still observed even after accounting for gene density. The similarity of the distributions of Mu insertions and meiotic recombination sites suggests that common features, such as chromatin structure, are involved in site selection for both Mu insertion and meiotic recombination. The finding that Mu insertions and meiotic recombination sites both concentrate in genomic regions marked with epigenetic marks of open chromatin provides support for the hypothesis that open chromatin enhances rates of both Mu insertion and meiotic recombination.

Figure 1. Frequencies of known and novel Mu pTIRs.

(A) Proportions of different 34 bp pTIRs detected in the 454 dataset. The codes “a” and “b” designate arbitrarily defined left and right TIRs of a given Mu element. (B) Clustalw-based clustering of novel pTIRs (nTIRs), each of which was supported by at least 100 reads and exhibited a minimum edit distance of 2 from all previously described pTIRs (Methods).

Figure 2. The distribution of Mu insertion sites within genes.

(A) Sequences of genes (from annotated transcriptional start to poly-adenylation sites) were extracted from 15,050 full-length genes. Each gene sequence was divided into 20 equally sized bins. Because gene lengths differ, bin sizes differ from gene to gene. The x-axis lists these 20 bins 5′ to 3′. For each gene, the number of Mu insertions in each of the 20 bins was determined. Subsequently, the numbers of Mu insertions in each of the 20 bins and the lengths of each of the 20 bins were summed across the 15,050 genes. It was then possible to calculate the number of Mu insertions per Mb (y-axis) for each of the 20 bins. (B) 200-bp sequences around translation start sites (ATG, 200 bp left side and 200 bp right side) from each full-length gene were extracted and were divided into 20 bins, each of which was 20 bp in size. The x-axis lists these 20 bins 5′ to 3′. For each gene, the number of Mu insertions in each of the 20 bins was calculated. Subsequently, the numbers of Mu insertions in each of the 20 bins were summed across the 15,050 genes. The total summed length of each bin is 150,500 bp (20 bp bin length×15,050 genes). Using these data it was then possible to calculate the number of Mu insertions per Mb (y-axis) for each of the 20 bins. (C) 200-bp sequences around transcription start sites (TSS, 200 bp left side and 200 bp right side) from each full-length gene were extracted and were divided into 20 bins, each of which was 20 bp in size. The x-axis lists these 20 bins 5′ to 3′. For each gene, the number of Mu insertions in each of the 20 bins was calculated. Subsequently, the numbers of Mu insertions in each of the 20 bins were summed across the 15,050 genes. The total summed length of each bin is 150,500 bp (20 bp bin length×15,050 genes). Using these data it was then possible to calculate the number of Mu insertions per Mb (y-axis) for each of the 20 bins.

Figure 3. The distribution of Mu insertion sites in the maize genome.

(A) Each horizontal line on chromosomes represents a 1-Mb window. Lines are intensity- and color-coded to indicate the number of Mu insertions per Mb. Grey vertical lines represent the approximate positions of centromeres . (B) The locally-weighted polynomial regression (LOWESS) curve with smooth span (f) equaling to 0.4 of the number of Mu insertions per 1-Mb window (y-axis) was plotted versus the corresponding window's coordinates (Mb, x-axis). The vertical paired grey lines represent approximate centromere positions . Those patterns we observed are unlikely to be artifacts of the removal of repetitive MFS, because only a small proportion of all MFSs (1.4%) were removed based on their ability to map to multiple positions in the genome.

Figure 4. Number of Mu insertions and recombination rate (cM) per Mb corrected by gene number and gene length on chromosome 1.

(A) Numbers of Mu insertions per gene per Mb (red line) and cM per gene per Mb (green line) are standardized as described in Methods. Locally-weighted polynomial regression (LOWESS) curves with smooth span (f) equaling to 0.4 for both standardized values were plotted against the physical coordinates of chromosome 1 (Mb, x-axis). The approximate centromere position is shown in grey . (B) Numbers of Mu insertions per bp of genic sequence per Mb (red line) and cM per bp of genic sequence per Mb (green line) are standardized as described in Methods. Locally-weighted polynomial regression (LOWESS) curves with smooth span (f) equaling to 0.4 for both standardized values were plotted against the physical coordinates of chromosome 1 (Mb, x-axis). The approximate centromere position is shown in grey .

Figure 5. GC patterns at Mu insertion sites.

(A) More than 2,000 non-redundant Mu insertion sites for which MFSs were available on both sides and exhibiting 9-bp TSDs were collected. The mid-point of each TSD was set as position 0. Relative positions decrease to the left and increase to the right. Average GC% was calculated for each position separately across >2,000 sequences and plotted against the relative positions. Regions between the dashed lines represent the 99% confidence interval of randomly sampling 10,000 GC percentages (Methods). The shaded boxes cover positions with GC% that differ significantly from expected by chance (Methods). Boxed regions are hypothesized to be Mu transposase binding sites. (B) 101-bp sequences centered at transcription start sites (TSS), translation start sites (ATG), translational end sites (STOP) and transcriptional end sites (END) were extracted from over 15,000 full-length genes, respectively. All 13-bp windows sliding from 1 to the end of each 101-bp sequence was compared to the consensus sequence: “SW::SWNNNNNWS::WS”. The number of mismatches was computed and sequences of these windows were assigned to nine groups containing 0–8 mismatches (x-axis). 13-bp sequences of Mu insertion sites were categorized into these nine groups as well. The frequency of sequences with Mu insertions (y-axis) in each group was plotted for four sets of sequences (TSS, ATG, STOP and END) respectively.