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, 29 (9), 2126-2149

Genomic Analysis of the DNA Replication Timing Program During Mitotic S Phase in Maize ( Zea mays) Root Tips

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Genomic Analysis of the DNA Replication Timing Program During Mitotic S Phase in Maize ( Zea mays) Root Tips

Emily E Wear et al. Plant Cell.

Abstract

All plants and animals must replicate their DNA, using a regulated process to ensure that their genomes are completely and accurately replicated. DNA replication timing programs have been extensively studied in yeast and animal systems, but much less is known about the replication programs of plants. We report a novel adaptation of the "Repli-seq" assay for use in intact root tips of maize (Zea mays) that includes several different cell lineages and present whole-genome replication timing profiles from cells in early, mid, and late S phase of the mitotic cell cycle. Maize root tips have a complex replication timing program, including regions of distinct early, mid, and late S replication that each constitute between 20 and 24% of the genome, as well as other loci corresponding to ∼32% of the genome that exhibit replication activity in two different time windows. Analyses of genomic, transcriptional, and chromatin features of the euchromatic portion of the maize genome provide evidence for a gradient of early replicating, open chromatin that transitions gradually to less open and less transcriptionally active chromatin replicating in mid S phase. Our genomic level analysis also demonstrated that the centromere core replicates in mid S, before heavily compacted classical heterochromatin, including pericentromeres and knobs, which replicate during late S phase.

Figures

Figure 1.
Figure 1.
Experimental Approach. (A) Workflow. Roots of 3-d-old maize seedlings were pulse labeled with EdU for 20 min, after which terminal 1-mm segments were harvested and fixed with formaldehyde. (B) A merged confocal image of a 1-mm root tip longitudinal section showing DAPI stained DNA (red) and EdU label in newly replicated DNA (green). There are multiple emerging cell lineages present in the terminal 1 mm of the root. Bar = 100 μm. (C) Sorting. Nuclei were isolated and EdU incorporated into DNA was conjugated to a fluorescent probe (AF-488) using click chemistry. Nuclei were counterstained with DAPI prior to sorting by flow cytometry using 355-nm (UV) and 488-nm (blue) lasers. A bivariate plot of relative DNA content (DAPI fluorescence) and EdU incorporation (AF-488 fluorescence) is shown, overlaid with the gates (black rectangles) used to sort nuclei representing early (E), mid (M), and late (L) fractions of S phase. Unlabeled nuclei from G1 phase (G1) were also sorted to use as a reference. (D) Histogram showing relative DNA content (DAPI) for the unsorted nuclei population (black line), overlaid with the position and relative frequency of nuclei that fall in the indicated sorting gates. DNA was extracted from sorted nuclei and EdU/AF-488-labeled DNA immunoprecipitated from the early, mid, and late fractions with an AF-488 antibody, prior to sequencing on the Illumina HiSeq 2000 platform. (E) to (H) Summary of computational processing of Repli-seq reads. (E) and (F) The number of reads that mapped uniquely to the maize B73 AGPv3 reference genome was calculated over 1-kb windows (see Methods). (G) After normalization for sequencing depth, replication activity was expressed as the ratio of EdU/AF-488 reads in early, mid, or late S phase to reads from total DNA from unlabeled G1 nuclei. (H) The resulting data smoothed with a Haar wavelet function. Representative data tracks from IGV are shown here for early S data, and the corresponding genomic region is shown for early, mid, and late S data in Supplemental Figure 3. Artificial spikes in sequencing coverage (arrowheads) often correspond to tandem repeat regions that have been “collapsed” in the reference assembly, and these regions are subsequently excluded. Scale: E and F, 0 to 1200 read density; G and H, 0 to 5.4 normalized signal ratio.
Figure 2.
Figure 2.
Chromosome 5 Replication Profiles. (A) Gene and TE coverage were determined using the maize genome AGPv3 annotation and are expressed as the gene or TE percent coverage, respectively, in 10-kb nonoverlapping windows. For IGV visualization, the coverage values were smoothed (see Methods). Gray dashed line represents 50% coverage. (B) Replication intensity profiles for early, mid, and late S phase cells processed and presented as described in Methods and the Figure 1 legend. Scale for all replication intensity tracks is 0 to 4.5 normalized signal ratio. (C) Segmentation of replication timing profiles into the predominant replication time classes (RT classes; see Methods) for each 1-kb window. Replication timing was classified as E (early), EM (early and mid), M (mid), ML (mid and late), L (late), EL (early and late), EML (early, mid, and late), and NS (not segmented); see color chart at bottom of figure. (D) Schematic representation of chromosome 5 with the centromere position approximately marked based on the CENH3 binding region (Zhao et al., 2016). Red rectangles denote the locations of the expanded panels shown below, each 2.5 Mb in size. (E) A region near the end of the short arm composed predominantly of early replication and E segments. (F) and (G) Regions near the middle of the short arm composed of various combinations of single and mixed RT class segments. Regions with a relatively high abundance of EM and ML segments often show clear replication activity at both times (F) or occur in regions where replication activity is spreading along the chromosome as S phase proceeds (G). (H) A pericentromeric region showing predominantly late replication and L segments. Corresponding tracks for gene and TE percentage of coverage and the composite RT class segmentation are displayed in each expanded panel, as described in (A) and (C). (I) The RT segment classification color legend.
Figure 3.
Figure 3.
Characterizing RT Class Segments. A segmentation procedure was performed (see Methods) to identify the predominant replication time for each 1-kb window across the genome, and adjacent windows with the same RT class were merged. (A) Total coverage of each RT segment class across the entire genome. The percentage of the genome covered by each class is noted inside the bars. (B) The number of individual segments in each RT class. (C) Box plot of the distribution of segment sizes in each RT class. Box plot whiskers represent 1.5× interquartile range (IQR). (D) The percentage of proportion of each RT class on individual chromosomes. The entire genome comprises 65.6% single-time segment classes of E (21.8%), M (23.7%), or L (20.1%). Another 32.3% comprises multiple-time segment classes, EM (17.8%), ML (14.0%), EL (0.2%), and EML (0.3%). In all, ∼98% of the nuclear genome was assigned to a segment class. See Figure 2I for RT class color legend.
Figure 4.
Figure 4.
Association of Genes and Gene Transcription with Replication Time. (A) The number of 1-kb windows in each RT class that overlaps an FGS gene, displayed as percentage of all 1-kb windows in that class. Asterisks denote RT classes in which the indicated percent overlap was significantly greater than expected by chance (permutation P value = 0.001; see Methods). For full details of the permutation analysis, see Supplemental Tables 2 and 3. (B) The distance from a given gene to the nearest neighboring gene was measured from the 5′ and 3′ ends of genes found in each RT class. This analysis did not consider the RT class of the neighboring genes. The distribution of gene distances is shown as a box plot, with whiskers representing 1.5× IQR. (C) Gene expression values in FPKM were calculated from RNA-seq data for genes in each RT class, and the distribution is shown as a box plot, excluding genes with an FPKM of zero. (D) and (E) Genes were further categorized into the following expression levels: FPKM = 0; >0 and ≤1; >1 and ≤10; >10 and ≤100; and >100. (D) The number of genes found in each RT class and expression group is shown. (E) The gene count from (D) is presented as the percentage of the total number of genes in each expression level group (totals shown at top of graph). See Figure 2I for RT class color legend.
Figure 5.
Figure 5.
Replication Times for TEs. (A) The percentage of total TE coverage was calculated for RT class segments (see Methods). The distribution of percentage of coverage values in each RT class is shown as a box plot (see Methods). (B) The total coverage in megabases of the top 20 most abundant LTR-retro families in each RT class. The percentage of each RT class that is composed of these top 20 families is shown inside the bars. (C) The distance from individual LTR-retro family members to the nearest neighboring gene was measured for the top six most abundant families, and the distribution is shown as a box plot. (D) The coverage in megabases of individual families from the top six most abundant LTR-retro families in each RT class (x axis shared with [E]). The families are grouped based on their RT class abundance (“earlier,” “middle,” and “later”) and then ordered by total abundance. Asterisks denote RT classes in which the observed percentage of overlap with each family, as indicated inside the bars, was significantly greater than expected by chance (permutation P value = 0.001; see Methods). For full details of the permutation analysis, see Supplemental Tables 2 and 3. The RT class coverage in megabases of the top 20 most abundant LTR-retro families is shown in Supplemental Figures 8B to 8D. (E) The distribution of distance to the nearest gene for family members within each RT class from (D). The number of family members found in each family and RT class is indicated above the boxes. Box plot whiskers represent 1.5× IQR. See Figure 2I for RT class color legend.
Figure 6.
Figure 6.
Replication Times for Centromeres and Tandem Repeat Sequences. (A) The functional centromere of chromosome 5, as defined by CENH3 binding (black rectangle; from Zhao et al., 2016), replicates predominantly in M and transitions to ML and L near the ends of the CENH3 binding region. See Figure 2I for RT class color legend. (B) and (C) Tandem repeat consensus sequences were blasted against the trimmed Repli-seq reads, independent of mapping to the reference genome, to estimate the abundance of these tandem repeat sequences in the Repli-seq reads (see Methods). (B) The percentage of reads corresponding to each tandem repeat sequence in each replication time sample. (C) The fold enrichment of each tandem repeat relative to the amount in G1. The mean and sd (error bars) for three biological replicates of early and mid S and two biological replicates of late S are displayed.
Figure 7.
Figure 7.
Replication Times for Chromatin-Related Features. (A) The number of 1-kb windows in each RT class that overlaps called peak regions for H3K56ac, H3K4me3, H3K27me3, or any combination of these three marks, presented as a percentage of the total number of 1-kb windows in each RT class. The inset shows the percentage of a lower abundance histone mark signature, H3K4me3 without H3K56ac or H3K27me3, on an expanded y axis. The histone mark signature labeled H3K56ac/H3K4me3/H3K27me3 represents any combination of either H3K56ac or H3K4me3 with H3K27me3. (B) MNase hypersensitivity (HS) region data from whole shoots and roots from Rodgers-Melnick et al. (2016) were overlaid with the segmented RT classes and the number of HS regions counted in each RT class. The count of HS regions per megabase covered by each RT class is displayed. Asterisks denote RT classes in which the observed percentage of overlap of the indicated feature was significantly greater than expected by chance (permutation P value = 0.001; see Methods). For full details of the permutation analysis, see Supplemental Tables 2 and 3. See Figure 2I for RT class color legend.
Figure 8.
Figure 8.
Models of DNA Replication Timing Progression in Maize. (A) Replication timing intensity profiles for early (blue), mid (green), and late (red) S-phase cells, as described in Figures 1 and 2, are overlaid to highlight the spreading pattern over consecutive fractions of S phase. Two representative regions from chromosome 5 are shown, one in the middle of the chromosome arm (left panel) and a second in the pericentromere (right panel). Tracks containing annotated regions for total TEs, the top six most abundant LTR-retro families from Figure 5 (LTR-retro), and genes, as well as a segmentation track showing the predominant replication time (RT class) are also included for reference. (B) and (C) Two nonmutually exclusive models for how replication proceeds through S phase in maize. In both models, replication begins at origins or initiation zones (circles) and proceeds bidirectionally (arrows). In the “cascade” model (B), replication initiates in early S and cascades to adjacent origins initiating in mid and then late S phase. In the “elongation” model (C), replication initiates at origins in early S and proceeds through mid S regions by passive elongation of replication forks. In this model, there are no origins initiating specifically in mid S phase. In the pericentromere, which predominantly replicates in late S, the elongation model envisions that small regions with early initiation could passively elongate through mid S, followed by a second round of initiation events in late S phase.

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