Similarity in replication timing between polytene and diploid cells is associated with the organization of the <i>Drosophila</i> genome

<div><p>Morphologically, polytene chromosomes of <i>Drosophila melanogaster</i> consist of compact “black” bands alternating with less compact “grey” bands and interbands. We developed a comprehensive approach that combines cytological mapping data of FlyBase-annotated genes and novel tools for predicting cytogenetic features of chromosomes on the basis of their protein composition and determined the genomic coordinates for all black bands of polytene chromosome 2R. By a PCNA immunostaining assay, we obtained the replication timetable for all the bands mapped. The results allowed us to compare replication timing between polytene chromosomes in salivary glands and chromosomes from cultured diploid cell lines and to observe a substantial similarity in the global replication patterns at the band resolution level. In both kinds of chromosomes, the intervals between black bands correspond to early replication initiation zones. Black bands are depleted of replication initiation events and are characterized by a gradient of replication timing; therefore, the time of replication completion correlates with the band length. The bands are characterized by low gene density, contain predominantly tissue-specific genes, and are represented by silent chromatin types in various tissues. The borders of black bands correspond well to the borders of topological domains as well as to the borders of the zones showing H3K27me3, SUUR, and LAMIN enrichment. In conclusion, the characteristic pattern of polytene chromosomes reflects partitioning of the <i>Drosophila</i> genome into two global types of domains with contrasting properties. This partitioning is conserved in different tissues and determines replication timing in <i>Drosophila</i>.</p></div

A flow diagram showing the main steps of mapping of black bands.

<p>A flow diagram showing the main steps of mapping of black bands.</p

Cytological mapping of the binding sites of anti-PCNA antibodies breaks down the S phase of the endocycle into six substages and assigns rb-bands to replication completion groups.

<p><b>First column:</b> The replication pattern of bands in the regions 43F-46B of polytene chromosome 2R as visualized by antibodies against PCNA (red) on <i>SuUR</i>^<i>ES</i> (A) and Oregon R (B–F) polytene chromosomes. Top to bottom: consecutive substages of the S phase, from the earliest (A) to latest (F). (A) “Black” bands not labeled yet. (B–F) Discontinuous labeling. Band designation appears at the substages at which these bands end replication. <b>Second column:</b> superimposed images, immunolocalization and phase contrast. <b>Third column:</b> Replication pattern-based substages of S phase, where ER is the earliest substage and LR1–LR5 are the subsequent provisionally late substages. <b>Fourth column:</b> Each rb-band was assigned to one of five groups, LR1 through LR5, according to the substage at which this rb-band ends replication. A small group of bands with no PCNA at LR1 was classified as LR0. For each substage, all replicating groups and all bands that completed replication are indicated (red text).</p

Similarity in replication timing between polytene and diploid cells.

<p>To infer replication timing in polytene chromosome 2R, all mapped rb-bands were rated based on their replication completion time in salivary gland polytene chromosomes (LR0–LR5, where LR5 are the last to complete replication, see text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.g002" target="_blank">Fig 2</a>). Intervals between rb-bands (INTs) in salivary gland polytene chromosomes complete replication before any rb-band does. Data on replication timing for chromosome 2R DNA sequences in cell cultures according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref033" target="_blank">33</a>]. <b>(A,B)</b> Boxplots of replication time scores in Kc (A) and Cl8 (B) cells for all DNA sequences corresponding to INTs, LR0–LR5 bands, and the entire fragment being studied on chromosome 2R. All groups of rb-bands are significantly different from INTs and from one another (the Mann–Whitney <i>U</i> test, p < E-100). A tendency is clear: the later bands complete replication in salivary gland polytene chromosomes, the later are the corresponding DNA sequences replicated in cell cultures. On the <i>y</i>-axis: replication time scores according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref033" target="_blank">33</a>], with +6 corresponding to early replication, and −5 to late replication. (<b>C,D)</b> For each rb-band and for each INT, the mean (C) and minimum (D) replication time scores in Kc cells were calculated according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref033" target="_blank">33</a>]. In the boxplots: the distribution of the mean (C) and minimal (D) scores for all INTs and the rb-bands assigned to three groups (LR0–1, LR2–3, and LR4–5) in accordance with replication completion time in salivary gland polytene chromosomes. All intervals (INTs) and intervals longer than 50 kb (INT50kb) were analyzed separately. Mean replication time in Kc cells is significantly different between band groups rated based on replication completion time in salivary gland polytene chromosomes (C). Replication completion time in Kc cells is significantly different among LR0–1, LR2–3, and LR4–5 bands (C). ***p < E-5 (Mann–Whitney <i>U</i> test). On the <i>y</i>-axis: replication time scores according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref033" target="_blank">33</a>] as in C. <b>(E, F)</b> Density of ORC2 peaks (peaks/100 kb) (according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref051" target="_blank">51</a>]) in salivary glands (E) and S2 (F) cells. <b>(G,H)</b> Mean replication time scores in Kc (G) and Cl8 (H) cells for all sequences within 1-kb windows at the same position relative to the band boundary (red vertical line) in all LR0–1 (red curve), LR2–3 (green curve), and LR4–5 (blue curve) bands. Right-hand boundary data were mirrored and combined with left-hand boundary data. Within-band regions are to the right of the border; outside regions are to the left. On the <i>x</i>-axis: distances from the border. On the <i>y</i>-axis: replication time scores according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref033" target="_blank">33</a>]. <b>(I)</b> Delays in replication completion in salivary gland polytene chromosomes correlate with band lengths. **p < E-2, ***p < E-5 (Mann–Whitney <i>U</i> test). On the <i>y</i>-axis: sizes of rb-bands (LR, LR0–LR5) and intervals between them (INTs) in kb. <b>(J,K)</b> Mean replication time scores in Kc (G) and Cl8 (H) cells for rb-bands and INTs normalized to the median band size in each group (28 kb for INTs, 33 kb for LR0–1, 60 kb for LR2–3, and 158 kb for LR4–5). On the <i>y</i>-axis: replication time scores according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref033" target="_blank">33</a>]. Grey lines denote 95% confidence intervals. (G, H, J, K) The U-shape of averaged replication profiles within bands suggests that replication proceeds from the boundaries to the center. Deeper profiles correspond to later-replicating bands in polytene chromosomes (LR0–1 < LR2–3 < LR4–5), suggesting that these bands will complete replication in Kc cells later.</p

Localization of polytene chromosomes’ black bands on the Drosophila genome map could be predicted by means of data on the distribution of interband-specific chromatin proteins in cell cultures: Region 43F-46B of polytene chromosome 2R as an example.

<p><b>(A)</b> Distribution of four chromatin types [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref004" target="_blank">4</a>]. Ruby chromatin (depicted in magenta) is devoid of active chromatin markers. Aquamarine chromatin (depicted in cyan) is enriched with all the proteins that are typical of polytene chromosome interbands (see text). <b>(B)</b> Locations of condensed bands in polytene chromosomes as predicted from the distribution of ruby chromatin and aquamarine chromatin (each of these bands appears as an interval between aquamarine segments that include ruby chromatin) (see A). <b>(C)</b> A distribution of nine chromatin states in S2 cells (top) and Bg3 cells (bottom) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref022" target="_blank">22</a>]. The red color corresponds to chromatin type enriched with active promoters. The presence of this chromatin type in both cell cultures at once is one of the markers of polytene chromosome interbands. <b>(D)</b> Enrichment peaks of CHRIZ, the most typical interband protein, in the chromosomes of four cell lines (top to bottom: Bg3, Kc, S2, and Cl8) (modENCODE data). <b>(E)</b> Localization of the predicted positions of compacted bands within the framework of the 4-state model combined with the interband criterion filtering (see C, D). <b>(F)</b> FlyBase CytoMap locations of compacted bands corresponding to rb-bands (see G) (<a href="http://flybase.org/" target="_blank">http://flybase.org</a>). <b>(G)</b> Localization of the predicted positions of compacted bands after all correction steps (rb-bands, see text). Bands were assigned names according to mapping data in FlyBase (see text) and Bridges’ detailed map ([<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref047" target="_blank">47</a>], see J). <b>(H–J)</b> Locations of compacted bands in polytene chromosomes from salivary glands (H, J) and pseudonurse cells of <i>otu</i>^<i>11</i> mutants (H, reprinted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref011" target="_blank">11</a>] under a CC BY license, with permission from Springer Nature: Chromosome Research, original copyright [1995]) in comparison and their correspondence to the bands predicted by the analysis of protein distribution in the cell cultures (G). Compacted black bands as visualized by aceto-orcein staining (H, I) and according to Bridges’ detailed map [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref047" target="_blank">47</a>] (J).</p

All rb-bands display delayed replication in salivary gland polytene chromosomes and correspond to local minima on replication profiles for cell cultures.

<p><b>(A)</b> The replication pattern in salivary gland polytene chromosomes with all rb-bands labeled by PCNA in the region 43F-46B of polytene chromosome 2R as an example. The very existence of a pattern like this suggests that rb-bands are replicated later than the intervals between them. <b>(B, C)</b> Locations of rb-bands scaled to the genome map (B) and replication profiles in the cell cultures in comparison: data on KC, Cl8, S2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref033" target="_blank">33</a>], and BG3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref050" target="_blank">50</a>] (C) suggest that local minima on replication profiles for the cell cultures fall within rb-bands. This in turn suggests that rb-bands replicate later than their flanking regions. <b>(D)</b> Distribution of ORC2 peaks in Kc cells and in salivary glands [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref051" target="_blank">51</a>] shows typical zones of low peak density in rb-bands and a prominent ORC2 signal confined to the regions between the rb-bands.</p

The logic of changing patterns in polytene chromosomes.

<p><b>Links between these patterns and events at the DNA sequence level. (A)</b> A schematic of replication fork locations and the corresponding PCNA patterns in the zone of alternating grey bands and interbands and in an rb-band (black in the figure). <b>(B)</b> A schematic of a polytene chromosome region with three rb-bands (black in the figure) of different lengths. Intervals between rb-bands appear as grey bands and interbands. <b>(C)</b> Consecutive changes in PCNA binding patterns (red in the figure) during the S phase. Top to bottom: continuous labeling substage; the substage at which the label is seen in all rb-bands; the late S phase, when only the thickest bands get labeled. The central part of the thickest band contains a region that never undergoes replication. <b>(D)</b> Replication fork locations depending on time (time passes in the downward direction). Replication fork locations were inferred under the assumption that replication is initiated randomly at each INT point once per INT in each replication cycle, with little asynchrony. Differences in replication rates between different genomic regions were ignored. The portion of the profile corresponding to the under-replication zone is beyond the S phase. If the differences in replication rates between rb-bands were to be considered, the local minima would be deeper. <b>(E)</b> A model reflecting similarities and differences in replication timing between diploid cell chromosomes and salivary gland polytene chromosomes. Averaged replication profiles are identical in early replication initiation zones. A portion of the genome in salivary gland polytene chromosomes is slower to replicate because of slow replication in “black” bands and the absence of late origin firing; this portion stays under-replicated.</p

The rb-bands and INTs differ from each other in gene density, expression and tissue specificity.

<p><b>(A, B)</b> Rb-bands in the three replication completion groups (LR0–1, LR2–3, and LR4–5) are significantly depleted in genes as compared with intervals between these bands (INTs). (A) Gene density (genes per 100 kb) according to FlyBase annotations and (B) 5'-UTR density (5'-UTRs per 100 kb). Two-sample permutation tests, 10,000 iterations, ***p < E-5. <b>(C)</b> The expression of genes lying entirely in rb-bands in various tissues is significantly different from that of genes lying entirely in the intervals between rb-bands. Boxplots of gene expression data for LR0–LR1 (green bars), LR2–LR3 (blue bars), and LR4–LR5 (purple bars) bands and intervening sequences (red bars) in different larval or adult tissues. Gene expression data in FPKM (Fragments Per Kilobase Million) were generated by modENCODE ([<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref043" target="_blank">43</a>], March 2016 data freeze). All groups of rb-bands are significantly different from INTs and from one another (the Mann–Whitney <i>U</i> test, p < E-26). <b>FB wl</b>: fat bodies, wandering larvae; <b>FB p</b>: fat bodies, 2-day prepupae; <b>CNS wl</b>: central nervous system, wandering larvae; <b>CNS p</b>: central nervous system, two-day old prepupae; <b>DS wl</b>: digestive system, wandering larvae; <b>DS a</b>: digestive system, one-day old mixed adults; <b>Head af</b>: heads, one-day old female adults; <b>ID wl</b>: imaginal discs, wandering larvae; <b>SG wl</b>: salivary glands, wandering larvae; <b>SG p</b>: salivary glands, prepupae; <b>AG am</b>: male accessory glands, male adults; <b>Testis am</b>: testes, male adults; <b>Ov af</b>: female gonads, female adults. <b>(D)</b> The later the bands are replicated, the higher the proportion of testis-specific genes they tend to have. The proportion of testis-specific genes (according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref055" target="_blank">55</a>]) in INTs and rb-bands grouped based on their replication completion time (LR0–1, LR2–3, and LR4–5). Two-sample permutation tests, 10,000 iterations, ***p < E-3. <b>(E)</b> The latest-replicating rb-bands are typically enriched with multigene clusters of testis-specific genes. The proportion of stand-alone testis-specific genes, two-item, and multiple-item clusters among all testis-specific genes (according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref055" target="_blank">55</a>]) on chromosome 2R (All), in intervals between rb-bands (INTs) and in rb-bands classified based on replication completion time in salivary glands (LR0–1, LR2–3, and LR4–5). Blue bars correspond to the proportion of stand-alone testis-specific genes among all testis-specific genes; red bars, to the proportion of two-item clusters; green bars, to the proportion of multiple-item clusters. The INTs are devoid of multigene clusters of testis-specific genes. This distribution is significantly different from random (resamples n = 10,000, goodness-of-fit test, p < E-5.). LR4–5 rb-bands are enriched with multigene clusters and depleted of stand-alone testis-specific genes. This distribution is significantly different from a random one (resamples n = 10,000, goodness-of-fit test, with provision for cluster size, p < E-5). <b>(F)</b> Genes in the rb-bands tend to be expressed in a limited number of tissues. By contrast, the intervals between rb-bands are enriched with genes that are highly expressed in most of the tissues under consideration. Boxplots of the numbers of tissues with above-threshold gene expression. Threshold values: 0; 0.1; 1; 10; 100. At 0–10, INTs significantly differ from any rb-band (***p < E-30). At 0 and 0.1, LR2-3 bands significantly differ from LR4–5 bands (**p < E-5; Mann–Whitney <i>U</i> test). Gene expression data were generated by modENCODE [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195207#pone.0195207.ref043" target="_blank">43</a>].</p

Cytology positions of the largest OLs.

<p>FISH mapping performed in the present paper.</p><p>FlyBase FISH mapping data in wild type chromosomes (further details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083319#s4" target="_blank">Materials and Methods</a>).</p><p>Mapping position was deduced based on the position of the corresponding underreplication zone, according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083319#pone.0083319-Nordman1" target="_blank">[19]</a>.</p><p>Mapping position was established according to the matching underreplication zone referenced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083319#pone.0083319-Belyakin1" target="_blank">[15]</a>.</p><p>LR – late-replicating region.</p><p>UR – underreplicated region.</p

Overlap between OLs and UR(B)-regions.

<p><i>X</i> axis shows the number of OLs that overlap with a single UR(B)-region, <i>Y</i> axis shows the percentage of the corresponding UR(B)-regions in the total set of UR(B)-regions. Blue bars indicate the numbers observed for actual UR(B)-regions. Red bars correspond to the simulated counts obtained for a randomly shuffled set of UR(B)-regions via 100,000 shuffling iterations. P-values are provided on top of the bars when differences between the observed and expected values reach statistical significance.</p