Genome-wide mapping reveals single-origin chromosome replication in Leishmania, a eukaryotic microbe

Abstract

Background: DNA replication initiates on defined genome sites, termed origins. Origin usage appears to follow common rules in the eukaryotic organisms examined to date: all chromosomes are replicated from multiple origins, which display variations in firing efficiency and are selected from a larger pool of potential origins. To ask if these features of DNA replication are true of all eukaryotes, we describe genome-wide origin mapping in the parasite Leishmania.

Results: Origin mapping in Leishmania suggests a striking divergence in origin usage relative to characterized eukaryotes, since each chromosome appears to be replicated from a single origin. By comparing two species of Leishmania, we find evidence that such origin singularity is maintained in the face of chromosome fusion or fission events during evolution. Mapping Leishmania origins suggests that all origins fire with equal efficiency, and that the genomic sites occupied by origins differ from related non-origins sites. Finally, we provide evidence that origin location in Leishmania displays striking conservation with Trypanosoma brucei, despite the latter parasite replicating its chromosomes from multiple, variable strength origins.

Conclusions: The demonstration of chromosome replication for a single origin in Leishmania, a microbial eukaryote, has implications for the evolution of origin multiplicity and associated controls, and may explain the pervasive aneuploidy that characterizes Leishmania chromosome architecture.

Fig. 1

Mapping replication origins in the L. major nuclear genome. Graphs show the distribution of replication origins in the 36 chromosomes of L. major (numbered 1–36; sizes denoted in intervals of 0.25 Mb), determined by the extent of enrichment of DNA in S phase relative to G2. For each chromosome, the top track displays coding sequences, with genes transcribed and translated from right to left in red, and from left to right in blue. The graph below shows the ratio of the read depth between early S phase and G2 samples (y-axis), where each dot (dark blue) represents the median S/G2 ratio (y-axis) in a 2.5 Kbp window across the chromosome (x-axis). Finally, the track below the graph displays localization of acetylated histone H3 (H3ac) in each chromosome (data from [6]), identifying positions of transcription start sites (y-axis; values represented as log2). The insert diagram (boxed) shows S/G2 read depth ratio (light blue dots) for chromosomes 35 and 36, as above, but here comparing late S cells with G2. (Late S/G2 MFAseq for all L. major chromosomes is shown in Figure S2 in Additional file 1)

Fig. 2

Comparing replication origin usage in syntenic L. mexicana and L. major chromosomes that have undergone fusion or fission. a Graphs show replication origin localisation, evaluated by MFAseq, in L. mexicana (Lmx) chromosomes 8 and 20, which are syntenic with L. major (Lmj) chromosomes 29 and 8 and chromosomes 36 and 20, respectively (chromosome sizes are denoted in 0.25 Mb intervals). Blocks of synteny are boxed and their relative orientation indicated; the representation of early S/G2 DNA sequence read depth ratios (L. mexicana green, L. major blue) and coding sequence organisation are as detailed in Fig. 1 and the approximate location of the origin or syntenic non-origin loci is shown by solid vertical lines and dotted vertical lines, respectively. (Figures S4 and S5 in Additional file 1 show MFAseq for all L. mexicana chromosomes and a genome-wide comparison with L. major.) b Validation of replication origin activity in the L. mexicana and L. major chromosomes (shown in (a)) by quantitative PCR, which was performed at a number of loci predicted to display origin activity in L. major and syntenic with L. mexicana. At each locus the relative quantity of S phase (black) and G2 phase (red) DNA is shown: G2 values at each loci are set at 1, and the S phase samples are shown as a proportion of that value (vertical lines indicate standard deviation from at least three experimental repeats); for comparison, the MFAseq data (from (a)) is shown in the background, and the right-hand synteny regions are distinguished from the left hand regions using dotted lines and solid lines, respectively. Positions of the quantitative PCR loci in each chromosome are shown in megabases (x-axes)

Fig. 3

Conservation of DNA replication origins between L. major and T. brucei. a A pie chart showing the proportion of origins mapped in the genome of T. brucei whose locations are either conserved or not in the genome of L. major, based on whole-genome synteny block comparisons (Figure S6 in Additional file 1). White indicates mapped origins in both T. brucei and L. major within regions of conserved gene synteny; stripes indicate mapped T. brucei origins within regions of gene synteny in L .major, but where no origin activity is mapped in the latter; grey indicates T. brucei origins at sites of rearrangement relative to L. major, where synteny is lost; dots depict T. brucei origins in regions of synteny with L. major, but where local rearrangements mean origin conservation is unclear; black represents the single T. brucei-specific origin, found in the subtelomere of chromosome 6, which shows no synteny with L. major. b Synteny conservation between L. major (Lmj) chromosomes 30 and 33, and T. brucei (Tbr) chromosomes 6 and 10, respectively, where origin activity is seen in only one of the parasite chromosomes; S/G2 DNA sequence depth ratios (L. major blue, T. brucei orange) and coding sequence organisation are as detailed in Fig. 1; locations of the regions within the chromosomes are shown in megabases, and the approximate location of the origin or syntenic non-origin loci shown by solid vertical lines and dotted vertical lines, respectively; double-headed arrows denote local rearrangements. c An example of a syntenic region between L. major chromosome 36 and T. brucei chromosome 10 where replication origin activity is conserved. d An example of complex origin conservation: a region of T. brucei chromosome 7 is shown in which a single origin appears to be conserved as two origins in L. major (one origin in two chromosomes: 17 and 5). Synteny blocks are boxed and their relative orientation indicated

Fig. 4

Origin usage is not equivalent in Leishmania and T. brucei. Synteny conservation is shown between T. brucei (Tbr) chromosome 8 and L. major (Lmj) chromosomes 7, 10 and 23, and between L. major (Lmj) chromosome 31 and T. brucei (Tbr) chromosomes 4 and 8, comparing the relative strength of the replication origins found within these chromosomes. S/G2 DNA sequence depth ratios and coding sequence organisation are as detailed in Fig. 1. Synteny blocks are boxed and their relative orientation indicated; the approximate location of the origins is shown by vertical lines. Double-headed arrows denote local rearrangements

Fig. 5

Origins are found at specific genomic loci in Leishmania but not in T. brucei. Scatter plot analysis of the length of strand switch regions (SSRs) in L. major, L. mexicana and T. brucei, comparing SSRs that have been mapped as showing origin activity (circles) with those in which origin activity has not been detected (squares). Horizontal lines show the mean, and vertical lines standard error of the mean; ***P < 0.0001, a significant difference in SSR size between the two groups; ns denotes that no significant size difference was seen. Origin-active SSRs in L. major chromosomes 29 and 36 are highlighted in black, as are the syntenic SSRs in L. mexicana chromosomes 8 and 20, which are not origin-active (further detail in Figure S9 in Additional file 1)