Establishment and maintenance of heritable chromatin structure during early Drosophila embryogenesis
- Howard Hughes Medical Institute, Princeton University, United States
Figures
Figure 1 with 26 supplements
Sequential establishment of chromatin accessibility at the MBT.
(A) (Left column) Single embryos were collected for ATAC seq at the indicated time points. Micrographs show stage-matched panels from a time-lapse image of a single Histone H2Av-GFP embryo. Scale bar = 10 μm. Coverage of sequence reads from accessible chromatin over the hunchback (center) and scylla (right) loci are shown. The hunchback P2 promoter/enhancer and associated shadow enhancer in addition to the later-acting stripe enhancer (arrows) are open and accessible at all time points measured. A schematic summarizing the regulatory interactions of the hunchback locus is shown at bottom left. The scylla TSS gains accessibility at NC12 + 9’ and is stably maintained thereafter. Scale bars (red) equal 1 kb. Plots show mean coverage from at least n = 3 replicates. (B) The fraction of each genomic feature present during each cell cycle is plotted as a stacked bar chart. ‘NC12 new’ and ‘NC13 new’ indicate the set of peaks newly called present in each respective cell cycle. Not shown: 2898 peaks not found to overlap with available genomic annotations used to classify enhancers, promoters, or insulators. (C) The association of functionally validated enhancers within each ATAC-seq timing class was calculated, and this plot shows what fraction of these are active at the indicated timepoints. Solid bars indicate the fraction of enhancers whose expression is first detected at the indicated timepoint. The lighter remaining bar indicates the total fraction of active enhancers associated with each ATAC-seq timing class. Color coding is as for panel B, and enhancers not overlapping with the ATAC-seq peaks are shown in red. The estimated elapsed time post NC11-NC13 for each scored stage of development (bottom axis) is indicated on the top axis. (D) Odds ratios for enrichment of the indicated genomic features and transcriptional regulators were calculated. Early chromatin accessibility is enriched for enhancers (p=2.54x10−109) and binding of Zelda (p=3.7x10−225). Late or dynamic chromatin accessibility is enriched for promoters (p=4.62x10−42), insulators (p=7.71x10−14), and binding of GAF (p=3.76x10−19). p-Values are from two-sided Fisher’s exact test on contingency tables constructed on [-/+ feature by early/dynamic].
Figure 1—figure supplement 1
Selection of metaphase-staged embryos and inter-replicate reproducibility.
(A) Probability of selecting embryos at interphase stages based on time following observation of the prior anaphase (see Materials and methods). Time-lapse movies of wild-type embryos (n = 23) were scored for timing of entry into mitotic metaphase. Metaphase entry times were assumed to be normally distributed, and a p-value indicating the likelihood of a staged embryo being within interphase were calculated for each of the indicated timepoints (x-axis). (B) Clustering of individual biological replicates. The total number of ‘open’ ATAC-seq fragments within the set of peaks associated with autosomes was calculated for each biological replicate. Correlation between samples (1-cor(samples)) was calculated and complete-linkage clustering was performed. Samples corresponding to metaphase NC11 (NC11_09, n = 3), metaphase NC12 (NC12_12, n = 4), and metaphase NC13 (NC13_18, n = 3) are color coded (dark red, red, and orange, respectively). (C) Detail of accessible chromatin profiles for each individual metaphase replicate over the hunchback locus. Coverage of ‘open’ ATAC-seq reads over the hunchback locus was plotted for each biological replicate of metaphase-staged embryos as indicated on the y-axis (CPM-normalized mean counts per 10 bp bin). Color coding is as in panel B. Genomic region plotted is as in Figure 1A, middle panel.
Figure 1—figure supplement 2
Fraction of accessible peaks during metaphase.
(A) Peaks were called on pooled biological replicates corresponding to metaphase stages, and the fraction of peaks overlapping with accessible peaks during interphase stages was calculated. (B) The fraction of peaks corresponding to individual genomic features [enhancers, promoters, insulators, and other (uncategorized)] accessible during individual cell cycles is plotted. The average fraction of accessible features is indicated by a grey cross.
Figure 1—figure supplement 3
Read-length distribution and comparison of interphase and metaphase library preparations.
Uniquely-mapping duplicate-filtered paired-end sequencing read sets were pooled by timepoint. A random sample of 100,000 reads for each timepoint was selected and the distribution of read lengths was plotted as shown above. The x-axis indicates the length in base-pairs of the mapped read and the y-axis indicates the number of reads of x base-pairs within the sample. In each plot, the leftmost peak corresponds to ‘open’ chromatin fragments, and the adjacent peak distribution corresponds to fragments protected by nucleosomes (or nucleosome-sized objects). The boxplots at right show the distribution of estimated input DNA concentration (left plot) and the distribution of uniquely mapping sequence reads (right plot) for interphase (n = 30) and metaphase (n = 10) staged samples. The p-value at the top of each plot was calculated by randomly assigning samples to similarly sized groups (n = 30 and 10) and calculating the difference between the average value in each group. The p-value indicates the frequency with which the difference between metaphase and interphase samples is greater than the difference between randomly selected samples (n = 1×105 permutations).
Figure 1—figure supplement 4
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Figure 2 with 2 supplements
Dynamic acquisition of chromatin accessibility is regulated by the N:C ratio.
(A) Cell cycle times for diploid (+/+, n = 21) and haploid embryos (ssm, n = 12) were measured by time lapse confocal imaging of His2Av-GFP. Mean cell cycle times ± SEM are indicated. Colored regions indicate periods of equivalent N:C ratios (0.25–1.0) between genotypes. (B) Accessible peaks were identified for haploid embryos and the fraction of overlap or co-occurence between diploids and haploids was calculated and plotted for the indicated cell cycles. (C) Median accessibility for haploid and diploid embryos was calculated for the sets of regions indicated in the legend (n = 2630 N:C ratio dependent regions, n = 1658 time-dependent regions). All differences in magnitude between time- and N:C ratio-dependent peaks in haploid embryos are statistically significant at p<0.01 by a randomization test, whereas only the NC13 + 15’ time point achieves p<0.01 in the diploid dataset. Significance testing performed by proportionally dividing all datapoints into two groups at random and testing whether observed differences in median accessibility were equal to or greater than the difference observed between the ‘N:C ratio’ and ‘time’ groupings (one-tailed permutation test for n = 1×104 trials). (D) Median accessibility data from panel C was re-scaled along the x-axis to match relative N:C ratios as shown in panel A. (E) Odds ratios for enrichment of the indicated genomic features and transcriptional regulators were calculated. Time-dependent chromatin accessibility is enriched for enhancers (p=5.78x10−7) and Zelda binding (p=2.42x10−12), whereas N:C ratio dependent chromatin accessibility is enriched for promoters (p=5.66x10−17), insulators (p=2.42x10−4), and GAF binding (p=2.72x10−09). p-Values are from two-sided Fisher’s exact test on contingency tables constructed on [-/+ feature by time/N:C]. GAF odds ratios were calculated from regions that were GAF+ and Zelda-. GAF is significantly enriched in both N:C and time-dependent classes if co-occurrence with Zelda is considered.
Figure 2—figure supplement 1
Examples of N:C-ratio-dependent regions.
Each plot shows the accessibility profiles over time for diploid (left, blue, NC11 to NC13) and haploid (right, green, NC12 to NC14) samples at four regions selected on the basis of the distribution of open ATAC-seq coverage within the total set of N:C-ratio-dependent peaks. The y-axis shows coverage of open ATAC-seq reads at an equal scale for all eight plots. The x-axis shows a 10 kb region centered over the peak region of interest (arrow, coordinates provided at top of plots). The gene models mapping to the displayed region are shown at bottom (yellow). An N:C-ratio-dependent region is expected to gain accessibility at equivalent nucleo-cytoplasmic ratios, not equivalent nuclear cycles. The timecourses for diploid and haploid samples are aligned by equivalent nucleo-cytoplasmic ratios. As such, it is expected that N:C-ratio-dependent peaks show equivalent accessibility horizontally across the plots. Certain loci (e.g., tinman) gain accessibility one timepoint earlier in haploids than diploids. Our analysis was not designed to distinguish single-timepoint shifts in the onset of accessibility. Rather, our analysis focused on changes in accessibility from samples pooled by entire nuclear cycles/N:C ratios.
Figure 2—figure supplement 2
Examples of regions that gain accessibility independently of the N:C ratio.
Each plot shows the accessibility profiles over time for diploid (left, blue, NC11 to NC13) and haploid (right, green, NC12 to NC14) samples at four regions selected on the basis of the distribution of open ATAC-seq coverage within the total set of N:C-ratio-independent peaks. The y-axis shows coverage of open ATAC-seq reads at an equal scale for all eight plots. The x-axis shows a 10 kb region centered over the peak region of interest (arrow, coordinates provided at top of plots). The gene models mapping to the displayed region are shown at bottom (yellow). An N:C-ratio-independent region is expected to gain accessibility at equivalent nuclear cycles, not equivalent nucleo-cytoplasmic ratios. The timecourses for diploid and haploid samples are aligned by equivalent nucleo-cytoplasmic ratios. As such, it is expected that N:C-ratio-independent peaks show greater accessibility earlier (higher up on the plots) in haploids compared with diploids.
Figure 3 with 1 supplement
Patterns of stable and dynamic chromatin accessibility over the cell cycle.
(A) Genome-wide mean ATAC-open coverage is plotted (red, error bars show std. dev. between peaks, n = 9824) over the mean scaled heterogeneity in PCNA-EGFP to estimate DNA replication activity (blue, n = 4, error bars show std. dev. between embryos). Mitotic phases are indicated by grey shading. (B) Mean coverage of sequencing reads from accessible chromatin over the hunchback P2 promoter/enhancer is plotted (red) over the mean measured spot fluorescence intensity from a hunchback P2> MS2(24) LacZ: : MCP-GFP reporter (blue, n = 3, error bars show std. dev. between embryos). Mitotic phases are indicated by grey shading. (C) Immunofluorecence for chromatin morphology (DAPI) and RNA Polymerase II (CTD pSer5) in a region of an NC13 embryo transitioning from prophase to metaphase is shown. Observed mitotic states are indicated at left. Scale bar = 10 μm.
Figure 3—figure supplement 1
Example of PCNA-EGFP imaging and analysis.
Top panels show representative frames from one time-lapse image of a PCNA-EGFP embryo at the indicated timepoints during NC13. Note the distribution of brighter foci over time. Scale bar = 10 μm. An individual nucleus from each frame is enlarged and shown below to highlight the changing distribution of bright PCNA-EGFP over time. The bright foci correlate with the degree of DNA replication activity (see McCleland et al., 2009). To quantify the fraction of PCNA-EGFP fluorescent signal associated with heterogeneously distributed regions of fluorescence intensity, images were subjected to analysis as described in the Materials and methods. Measurement of the grayscale correlation matrix yields an estimate of homogeneity of neighboring pixel intensities within regions of interest (nuclei, scores range from 0 to 1 where 1 = all pixels neighbor pixels of similar intensity). We plot here the value of 1 - Homogeneity, where 0 = homogeneous neighbor intensities, and 1 = heterogeneous neighbor intensities. The timepoints corresponding to the shown representative frames are indicated by blue lines.
Figure 4 with 2 supplements
Attenuation of replication coupled nucleosome disruption at the MBT.
(A) Predicted nucleosome occupancy is plotted for the selected time points (top) for the 800 bp region flanking a set of early embryonic promoters, centered over the TSS. These timepoints correspond to the initial phases of DNA replication in each cell cycle. Nucleosome profiles for the complete timecourse are provided in Figure 4—figure supplement 1. Promoters are ordered on the y-axis by the average −1 nucleosome position over the entire time course, and the relative nucleosome occupancy is represented by the colorbar at left. The log10 average occupancy signal for each time point is plotted below each heatmap (red). Maximum nucleosome signal at the TSS over the entire timecourse is indicated by the grey line. (B) Mean-normalized relative NFR sizes for time (red) and N:C ratio (blue) dependent loci are plotted for haploid embryos for comparison with diploid embryos (green). Data are plotted as a function of N:C ratio (x-axis). NFRs for time-dependent loci demonstrate minimal closing during metaphase (N:C = 0.25, p<0.01, asterisk) and early S-phase (N:C = 0.5, p<0.01, asterisk) compared with N:C-ratio-dependent loci and N:C ratio matched diploid loci. p-Values indicate frequencies of observing differences between randomly selected loci greater than or equal to that observed for the time- and N:C-ratio-dependent groups (one-tailed permutation test for n = 1×104 trials). Error bars show the 95% confidence interval for differences between the plotted median values. (C) Mean-normalized relative NFR sizes for Zelda-associated (red) and GAF-associated (blue) promoters was plotted for haploid embryos. Plotting and significance testing is as described for panel B. Zelda-associated promoters demonstrate increased NFR stability during early S-phase during both N:C = 0.25 and N:C = 0.5 (asterisks, p<0.01, one-tailed permutation test for n = 1×104 trials). Error bars show the 95% confidence interval for differences between the plotted median values.
Figure 4—figure supplement 1
Nucleosome positioning over promoters.
(A) Predicted nucleosome occupancy is plotted for all time points (top) for the 800 bp region flanking a set of early embryonic promoters, centered over the TSS. Promoters are ordered on the y-axis by the average −1 nucleosome position over the entire time course, and the relative nucleosome occupancy is represented by the colorbar at left. The log10 average occupancy signal for each time point is plotted below each heatmap (red). Maximum nucleosome signal at the TSS over the entire timecourse is indicated by the grey line. (B) The average NFR size for each timepoint was calculated and plotted. (C) Average nucleosome occupancies were normalized to the mean occupancies of the flanking 200 bp of each 800 bp region and plotted. Top plot shows average occupancy data for all time points in grey, and the per-cell-cycle average as indicated by the legend. Bottom plot shows average occupancy data for all time points according to the color bar at right.
Figure 4—figure supplement 2
Changes in accessibility at nucleosome-free regions over the cell cycle.
To calculate changes in chromatin accessibility at NFRs over the cell cycle, the maximum extent of each NFR was calculated from the predicted extreme positions of −1 and +1 nucleosomes as determined by NucleoATAC. For each timepoint, the maximum ATAC-seq count value corresponding to open and accessible chromatin was calculated for each NFR (n = 2470) and plotted. Chromatin accessibility at NFRs doubles on average over the course of each cell cycle (Left Panel). The boxplots show the calculated log2 fold change between the first and last timepoint in each indicated cell cycle for each NFR. A greater negative log2 fold change in accessbility is observed between metaphase 11 and early interphase 12 than between metaphase 12 and interphase 13 (Middle Panel). The dotted grey line indicates no difference in fold change between compared timepoints. To estimate the latency in recovery of accessibility to prior levels of chromatin accessibility, data for each NFR was subjected to spline interpolation and the timepoint when accessibility in the following cell cycle is equal to or greater than accessibility for the preceding metaphase was calculated. The right panel shows the kernel density estimate for the distribution of latency times for recovery during NC12 and NC13 (black and red traces, respectively). The distribution of latency estimates is biphasic for both cell cycles, with NC12 showing nearly equivalent distribution between early (3 min) and late (9 min) recovering regions. A larger proportion of NFRs recover by 3 min during NC13. Median values for raw, non-interpolated data (inset) are +9 min for NC12, and +6 min for NC13.
Additional files
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Supplementary file 1
Sample summary.
- https://doi.org/10.7554/eLife.20148.037
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Supplementary file 2
Sample metadata.
- https://doi.org/10.7554/eLife.20148.038
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Supplementary file 3
Annotated peak regions used in the analysis.
- https://doi.org/10.7554/eLife.20148.039
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Supplementary file 4
Annotated promoter regions.
- https://doi.org/10.7554/eLife.20148.040
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Supplementary file 5
Code example for data normalization in R markdown (.rmd) format.
- https://doi.org/10.7554/eLife.20148.041
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Supplementary file 6
Code example for data normalization in. pdf format.
- https://doi.org/10.7554/eLife.20148.042
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Establishment and maintenance of heritable chromatin structure during early Drosophila embryogenesis
eLife 5:e20148.
https://doi.org/10.7554/eLife.20148
