Developmental Changes in Genome Replication Progression in Pluripotent versus Differentiated Human Cells
<p>Cell cycle and replication dynamics analysis of pluripotent and somatic cells. (<b>A</b>) Schematic representation of how the fraction of cells in S phase was determined based on DAPI and EdU intensity using high-throughput microscopy. (<b>B</b>) Doubling time of hESC H1, hiPSC A4, and hTERT RPE1 was calculated by counting cell numbers at different time points from a defined number of seeded cells. The S phase fraction was calculated by dividing the EdU positive cells with the total number of cells from high-throughput image analysis. The S phase duration was calculated by multiplying the doubling time with the fraction of cells in the S phase. (* = multiplication) (<b>C</b>) A pulse-chase–pulse-chase experiment followed by replication foci (RFi) detection at three time points in the same cell in different cell lines. (<b>D</b>) Illustration shows cell cycle phases and (sub)S phase durations among cell types. The duration of each S phase sub-stage was calculated by multiplying the fraction of cells in each sub-stage by the doubling time of the specific cell. (<b>E</b>) Live-cell time-lapse microscopy of hiPSC A4 and hTERT RPE1 expressing GFP-PCNA showing genome replication progression. PCNA S phase foci are visible from 90 min on. For more details see <a href="#app1-genes-15-00305" class="html-app">Table S1</a>. Scale bar: 10 µm.</p> "> Figure 2
<p>Feature analysis of the replication foci (RFi) in different S phases. (<b>A</b>) The illustration shows the image analysis approach for characterizing RFi from different time points in the same cell. Nuclear mask was created using the DNA dye DAPI and applied to the other three channels (EdU, BrdU, and PCNA) before the respective channel segmentation. Within this mask, RFi features were quantified. Scale bar: 5 µm. (<b>B</b>) Plots show the number, volume, and distance analysis (inter-RFi and RFi to the nuclear border) of the RFi as the cell progresses through the S phase. (<b>C</b>) Illustration shows the subnuclear distribution of RFi and its features in different S phase stages as indicated. The lower and upper whiskers of the boxplot correspond to the 25th and 75th percentiles, the box to the 50th percentile, and the line depicts the median. Statistical significance was performed using ANOVA, and Tukey’s honest significance test (not significant is given for <span class="html-italic">p</span>-values ≥ 0.05; one star (*) for <span class="html-italic">p</span>-values < 0.05 and ≥ 0.005; two stars (**) is given for values < 0.005 and ≥ 0.0005, and ≥ 0.0005 are given (***); only the significant differences are shown). For more details, see <a href="#app1-genes-15-00305" class="html-app">Table S2</a>. Scale bar: 10 µm.</p> "> Figure 3
<p>Quantification of the number of replicons and fork speed in S phase stages (<b>A</b>) Super-resolution PCNA (green) images overlaid with DAPI (blue) in three S phase stages in hESC H1, hiPSC A4, and hTERT RPE1 are shown. (<b>B</b>) Comparison between confocal and super-resolution (ASJD) images of RFi of the same cell and region. The plot shows the comparative volume of (nano)RFi detected in confocal and ASJD mode. (<b>C</b>) The plot shows the quantification of the nano-RFi in different S phase stages. (<b>D</b>) An illustration depicts the approach to measure the comparative replication fork speed. The EdU was pulsed for 15 min and detected using click chemistry, and PCNA was detected by antibodies. For measuring nucleotide incorporation rate, the ratio of EdU (incorporated nucleotides) and PCNA (active replication) sum intensities was measured as a marker for the speed of replication forks. If the ratio shows a value ≤ 1, this means a complete overlap or localization of EdU inside PCNA and indicates a slow replication fork speed. If the ratio of both signals is > 1, more DNA was synthesized, indicating faster replication fork speed. The middle plot depicts the fork rates of S phase cells across cell lines measured by high-throughput imaging and analysis without discriminating between S phase stages. The right plot shows the fork rate of individual S phase stages measured from high-resolution images across cell lines. The lower and upper whiskers of the boxplot correspond to the 25th and 75th percentiles, the box to the 50th percentile, and the line depicts the median. Statistical significance was performed using ANOVA and Tukey’s honest significance test (not significant is given for <span class="html-italic">p</span>-values ≥ 0.05; one star (*) for <span class="html-italic">p</span>-values < 0.05 and ≥ 0.005; two stars (**) is given for values < 0.005 and ≥ 0.0005, and 0.0005 to 0 are given (***); only the significant differences are shown). For more details, see <a href="#app1-genes-15-00305" class="html-app">Table S3</a>. Scale bar: 10 µm.</p> "> Figure 4
<p>Genome-wide replication origins distribution in selected human cell lines based on the SNS-seq origin mapping method. (<b>A</b>) Representative example of replication origins distribution in hESC, hiPSC, and HMEC. The origin profiles correspond to normalized read counts (scale 0–0.7 counts per million). Below the profiles, the origins identified by MACS2 peak callers are shown. (<b>B</b>) Comparison of the origin numbers in the human embryonic cell line (hESC), induced pluripotent cells (hiPSC), and the somatic HMEC cell line. Additionally, for the total identified peaks, the graph represents the origin number after clustering of closely situated origins at the distances of 10, 20, and 30 kb. (<b>C</b>) Comparison of the inter-origin distances (IOD). The IOD distances were also compared after origin clustering at the distances specified. The statistical evaluation of IODs between different cell lines and same clustering distance were significant (<span class="html-italic">p</span>-value < 0.001) with the only exception of the difference between ESC and iPSC, which was not significant <span class="html-italic">p</span>-value = 0.4125770. For more details see <a href="#app1-genes-15-00305" class="html-app">Table S4</a>. (<b>D</b>) Overlap of peaks among the different cell lines.</p> "> Figure 5
<p>Quantification of chromatin compaction with replication progression across cell lines. RFi in S phase stages were mapped to chromatin compaction classes across cell lines using the statistical tool Nucim on platform R (see <a href="#sec2-genes-15-00305" class="html-sec">Section 2</a> and <a href="#app1-genes-15-00305" class="html-app">Figure S2</a>). Lines connecting data corresponding to the same S phase stages were drawn for easier visualization. Distribution differences on classes <span class="html-italic">p</span>-values < 0.005 for all S phase stages for each cell line).</p> "> Figure 6
<p>Replication timing of genomic repeat elements. (<b>A</b>) Schematic representation of a chromosome with the tandem and interspersed repeat sequences color-coded. (<b>B</b>) The co-detection of two combinations of probes across cell lines as indicated (red and cyan) with PCNA (green). The line plots depict the fluorescence intensity distribution of the PCNA and the probes along the line (in microns) drawn on the merged image. (<b>C</b>) Heat plot shows the fold change in the sum intensity of each probe replicated in the S phase stages as indicated. The sum intensity of each probe was measured using the segmented RFi as masks in each S phase stage in individual cells and normalized to the median sum intensity of S I for each probe and cell line. For details, see <a href="#app1-genes-15-00305" class="html-app">Figure S1B and Table S5</a>. Scale bar: 10 µm.</p> "> Figure 7
<p>Developmental difference in replication timing of rDNA repeats. (<b>A</b>) Analysis pipeline to characterize the rDNA replication timing. RFi and rDNA spots were segmented separately. In FiJi, using the logical function “AND”, both segmented spots were processed to obtain the intersected voxels from both RFi and rDNA, which directly represent the colocalizing rDNA and RFi. (<b>B</b>) Images show the PCNA (green), the rDNA (red), and the merged images in different S phase stages as indicated. The contours (yellow) in the enlarged merged image indicate the colocalizing spots. (<b>C</b>) The plot depicts the quantification of the replicating rDNA spots in the S phase stages in hPSCs and hTERT RPE1. (<b>D</b>) Images show the overlap of RNA polymerase I subunit RPA 194 (representing active transcription) with replicating rDNA repeats in the S phase stages indicated. Contours in the enlarged image show the colocalizing RPA 194 and replicating rDNA as measured with the “AND” logic operation. The line plot shows the intensity distribution of RPA 194 (cyan), rDNA (red), and the EdU (green) along the line. (<b>E</b>) The plot shows the number of replicating rDNA spots associated with RPA 194. The lower and upper whiskers of the boxplot correspond to the 25th and 75th percentiles, the box to the 50th percentile, and the line depicts the median. Statistical significance was performed using ANOVA, and Tukey’s honest significance test (not significant is given for <span class="html-italic">p</span>-values ≥ 0.05; one star (*) for <span class="html-italic">p</span>-values < 0.05 and ≥ 0.005; two stars (**) is given for values < 0.005 and ≥ 0.0005, and 0.0005 to 0 are given (***); only the significant differences are shown). For more details, see <a href="#app1-genes-15-00305" class="html-app">Table S6</a>. The scale bar is 10 µm and 2 µm in the enlarged images.</p> "> Figure 8
<p>A summary of the developmental difference in genome replication features in pluripotent stem cells (PSC) and somatic cells. The late-replicating RFi cluster around and inside nucleolus in PSC but moves away in somatic cells. The late-replicating rDNA in PSC replicates earlier in S II. The increased origin firing increases the possibility of replication and transcription collision in S I of PSCs. (RFi: replication foci, RPA 194: large subunit of RNA pol I marking active transcription.).</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture and Transfection
2.2. Doubling Time and (sub)S Phase Duration
2.3. Genome Replication Labeling and Visualization
2.4. Probe Generation, Metaphase Spread, Repli-FISH, and Immuno Repli-FISH
2.5. Microscopy
2.6. Image Analysis
2.7. Genome-Wide Origin Mapping
2.8. Data Visualization and Statistical Analysis
3. Results
3.1. Developmentally Conserved Spatio-Temporal Replication Pattern in Humans
3.2. Characterization of Spatio-Temporal RFi Reveals a Change in Late-Replicating RFi Distribution
3.3. Replicon Quantification, Fork Efficiency, and Genome-Wide Origin Mapping Unravel Alterations in the Genome Replication Program across Developmental Transitions
3.4. Chromatin Compaction Analysis and RFi-Associated Histone Modification Measurements Reveal Differential Chromatin Dynamics
3.5. Repli-FISH Reveals Developmental Changes in the Replication Timing of Tandem and Interspersed Repeats
3.6. rDNA Tandem Repeats Show a Switch in Replication Timing and Change in Replication, Transcription Interaction
4. Conclusions/Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Name | Species | Type | Gender | Reference |
---|---|---|---|---|
hESC H1 | Homo sapiens | Embryonic | Male | [13] |
hiPSC A4 | Homo sapiens | iPSC from human neonatal foreskin fibroblast (HFF1) | Male | [14] |
hiPSC B4 | Homo sapiens | iPSC from human neonatal foreskin fibroblast (HFF1) | Male | [14] |
hTERT RPE1 | Homo sapiens | hTERT immortalized retinal pigment epithelial cell | Female | [15] |
BJ-5ta | Homo sapiens | hTERT immortalized foreskin fibroblasts | Male | [15] |
Name | Application | Detection | Catalog | Company |
---|---|---|---|---|
5-ethynyl-2′-deoxyuridine (EdU) | Labeling of nascent DNA in pulse-chase experiments | ClickIT chemistry | 7845.1 | Carl Roth, Karlsruhe, Germany |
5-bromo-2′-deoxyuridine (BrdU) | Labeling of nascent DNA in pulse-chase experiments | Antibody detection | B5002 | Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany |
Biotin-16-dUTP | Labeling of FISH probes | Streptavidin | 11093070910 | Roche Diagnostics Deutschland GmbH, Mannheim, Germany |
Cy3-dUTP | Labeling of FISH probes | - | ENZ-42501 | Enzo Life Sciences, Lörrach, Germany |
Thymidine | Labeling of nascent DNA in pulse-chase experiments, added only in chase period | - | T1895 | Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany |
Reactivity | Host | Clonality | Dilution | Catalog | Company |
---|---|---|---|---|---|
Anti-PCNA | Mouse | Monoclonal | 1:200 | ab29 | Abcam, Cambridge, UK |
Anti-RPA 194 | Mouse | Monoclonal | 1:200 | sc-48385 | Santa Cruz Biotechnology, Dallas, TX, USA |
Anti-BrdU | Rabbit | Polyclonal | 1:400 | 600-401-C29 | Rockland Immunochemicals, Pottstown, PA, USA |
Anti-H3K9me3 | Mouse | Monoclonal | 1:200 | 39285 | Active Motif, Waterloo, Belgium |
Anti-H3K36me3 | Rabbit | Polyclonal | 1:2000 | ab9050 | Abcam, Cambridge, UK |
Anti-H3K27me3 | Mouse | Monoclonal | 1:200 | 61017 | Thermo Fisher Scientific, Waltham, MA, USA |
Anti-H3K9ac | Rabbit | Polyclonal | 1:200 | 39917 | Active Motif, Waterloo, Belgium |
Anti-mouse IgG Alexa Fluor 488 | Goat | Polyclonal | 1:400 | A11029 | Thermo Fisher Scientific, Waltham, MA, USA |
Anti-rabbit IgG Alexa Fluor 488 | Goat | Polyclonal | 1:500 | A-11034 | Thermo Fisher Scientific, Waltham, MA, USA |
Streptavidin Alexa Fluor 488 | Conjugated | - | 1:500 | S11223 | Thermo Fisher Scientific, Waltham, MA, USA |
Streptavidin Cy5 | Conjugated | - | 1:500 | PA45001 | Amersham Biosciences, Amersham, UK |
Target | Labeling Method | Primers/Plasmids | Reference |
---|---|---|---|
Alu | PCR | AluF: 5′-GGATTACAGGYRTGAGCCA-3′ AluR: 3′-RCCAYTGCACTCCAGCCTG-5′ | [18] |
Centromere | PCR | α27: 5′-CATCACAAAGAAGTTTCTGAGAATGCTTC-3′ α30: 5′-TGCATTCAACTCACAGAGTTGAACCTTCC-3′ | [19] |
LINE1 | Nick translation | Plasmid pLRE3-eGFP | [20] |
rDNA | Nick translation | Plasmid pUC-hrDNA-12.0 | [21] |
System | Objective | NA | Application | Company |
---|---|---|---|---|
Nikon CREST/TiE2 | 20x SPlan Fluor LWD DIC (air) or 40X Plan Apo λ DIC (air) | 0.7 or 0.95 | High-throughput or live-cell time-lapse, wide-field microscopy | Nikon Instruments Inc.,Tokyo, Japan |
Leica SP5 II | 100X HCX PL APO (oil) | 1.44 | Confocal laser scanning | Leica GmbH, Mannheim, Germany |
LSM 900 Airyscan 2 | 63x C Plan-Apochromat (oil) | 1.4 | Confocal and high-resolution | Carl Zeiss AG, Oberkochen, Germany |
Name | Version | Platform | Websites | Application | Reference |
---|---|---|---|---|---|
FiJi | 2.14.0/1.54f | MacOS | https://fiji.sc/ (accessed on 25 January 2024) | Image analysis | [22] |
StarDist (FiJi) | 0.3.0 | MacOS | https://github.com/stardist/stardist-imagej (accessed on 25 January 2024) | Nuclei segmentation | [23] |
3D suite (FiJi) | 1.6 | MacOS | https://mcib3d.frama.io/3d-suite-imagej/ (accessed on 25 January 2024) | 3D image analysis | [23] |
Nucim (R) | 1.0.12 | MacOS | https://bioimaginggroup.github.io/nucim/ (accessed on 25 January 2024) | Nuclear compaction analysis | [24] |
R | 4.3.1 | MacOS | https://www.r-project.org/ (accessed on 25 January 2024) | Statistical analysis | |
Zen | 3.9.101 | Windows | https://www.zeiss.com/ (accessed on 25 January 2024) | Image acquisition, processing | |
Adobe Illustrator 2023 | 2023 | MacOS | https://www.adobe.com/ (accessed on 25 January 2024) | Figure preparation |
Dataset | Sample | Characteristics | Method | Cells | Webpage |
---|---|---|---|---|---|
GSE37757 | GSM927236 | hESC H9 SNS-seq | SNS-seq | hESC | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM927236 (accessed on 25 January 2024) |
GSM927237 | hiPSC SNS-seq | SNS-seq | hiPSC | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM927237 (accessed on 25 January 2024) | |
GSE128477 | GSM3676411 | hESC H9 SNS-seq replicate 1 | SNS-seq | hESC | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM3676411 (accessed on 25 January 2024) |
GSM3676412 | hESC H9 SNS-seq replicate2 | SNS-seq | hESC | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM3676413 (accessed on 25 January 2024) | |
GSM3676413 | hESC H9 SNS-seq Control | SNS-seq | hESC | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM3676438 (accessed on 25 January 2024) | |
GSE128477 | GSM3676435 | HMEC SNS-seq replicate 1 | SNS-seq | HMEC | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM3676435 (accessed on 25 January 2024) |
GSM3676436 | HMEC SNS-seq replicate 2 | SNS-seq | HMEC | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM3676436 (accessed on 25 January 2024) | |
GSM3676437 | HMEC SNS-seq replicate 3 | SNS-seq | HMEC | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM3676437 (accessed on 25 January 2024) | |
GSM3676438 | HMEC SNS-seq Control | SNS-seq | HMEC | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM3676438 (accessed on 25 January 2024) |
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© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Pradhan, S.K.; Lozoya, T.; Prorok, P.; Yuan, Y.; Lehmkuhl, A.; Zhang, P.; Cardoso, M.C. Developmental Changes in Genome Replication Progression in Pluripotent versus Differentiated Human Cells. Genes 2024, 15, 305. https://doi.org/10.3390/genes15030305
Pradhan SK, Lozoya T, Prorok P, Yuan Y, Lehmkuhl A, Zhang P, Cardoso MC. Developmental Changes in Genome Replication Progression in Pluripotent versus Differentiated Human Cells. Genes. 2024; 15(3):305. https://doi.org/10.3390/genes15030305
Chicago/Turabian StylePradhan, Sunil Kumar, Teresa Lozoya, Paulina Prorok, Yue Yuan, Anne Lehmkuhl, Peng Zhang, and M. Cristina Cardoso. 2024. "Developmental Changes in Genome Replication Progression in Pluripotent versus Differentiated Human Cells" Genes 15, no. 3: 305. https://doi.org/10.3390/genes15030305