Many stem cells undergo asymmetric division to produce a self-renewing stem cell and a differentiating daughter cell. Here we show that, similarly to H3, histone H4 is inherited asymmetrically in Drosophila melanogaster male germline stem cells undergoing asymmetric division. In contrast, both H2A and H2B are inherited symmetrically. By combining super-resolution microscopy and chromatin fiber analyses with proximity ligation assays on intact nuclei, we find that old H3 is preferentially incorporated by the leading strand, whereas newly synthesized H3 is enriched on the lagging strand. Using a sequential nucleoside analog incorporation assay, we detect a high incidence of unidirectional replication fork movement in testes-derived chromatin and DNA fibers. Biased fork movement coupled with a strand preference in histone incorporation would explain how asymmetric old and new H3 and H4 are established during replication. These results suggest a role for DNA replication in patterning epigenetic information in asymmetrically dividing cells in multicellular organisms.
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We thank B. Shelby and E. Wieschaus for the RpA-70-GFP fly line and the PCNA-eGFP line. We thank E. Moudrianakis, A. Spradling, J. Berger, M. Van Doren, R. Johnston and X.C. lab members for suggestions. We thank B. Mellone, S. Pavanacherry and L. Sohn for help with chromatin fiber technique. We thank Johns Hopkins Integrated Imaging Center for confocal imaging and Carnegie Institute Imaging Center for STED microscopy work. We acknowledge support from NIH 5T32GM007231 and F31GM115149-01A1 (M.W.), NIH R01GM112008 (J.X.), NIH R01GM33397 (J.G.), NIH R01GM112008, R35GM127075, the Howard Hughes Medical Institute, the David and Lucile Packard Foundation, and Johns Hopkins University startup funds (X.C.)
The authors declare no competing interests.
Peer review information: Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Integrated supplementary information
(a) A schematic diagram showing the dual color switch design that expresses first preexisting histone and then newly synthesized histone by heat shock treatment, as adapted from2. (b) Histone H1 showed overall symmetric inheritance pattern in post-mitotic GSC-GB pairs (n=12). Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. See Supplemental Table 1 for details. Neither old H1 nor new H1 is significantly different from the value of 1 based on two-tailed Wilcoxon signed-rank test. H1-GFP GSC/GB ratio = 1.26; H1-mKO GB/GSC ratio = 1.18. H1 old GSC/GB data: Shapiro-Wilk normality test P = 0.0069, data not normally distributed. Wilcoxon signed-rank test. Two-tailed test. Sum of signed ranks = 44. P = 0.0923. H1 new GB/GSC data: Shapiro-Wilk normality test P = 0.3147, data normally distributed. One sample t-test. Two-tailed test t = 1.546 df = 11. P = 0.1503. See Supplementary Tables 1 and 2 and online Methods for additional statistical information.
(a) DNA label (DAPI) from RC-derived chromatin fiber shows brighter DNA label (DAPI) in replicating regions (white box). Longitudinal line plot of RC-derived chromatin fiber shows a clear increase in DNA label (DAPI) signal in EdU-positive region, relative to the surrounding EdU-negative region from the same fiber. (b) DNA label (DAPI) from chromatin fiber isolated from non-replicating cells (NRC) in the Drosophila adult eye. NRC-derived fibers show uniform DNA label (DAPI). Longitudinal line plot of DNA label (DAPI) intensity in NRC-derived chromatin fiber shows small fluctuations in signal with no significant increases in intensity comparable to those observed in fibers derived from RCs. (c) Confocal versus STED images of EdU signal on replicating chromatin fiber. The EdU-positive region (box with solid orange lines) cannot be resolved into sister chromatids with confocal but can be resolved with STED. Line plot of EdU signal shows a single fiber structure with confocal imaging but a double fiber structure with STED. (d) Confocal versus Airyscan images of EdU signal on replicating chromatin fiber. The EdU-positive region (box with solid orange lines) cannot be resolved into sister chromatids with confocal but can be resolved with Airyscan. Line plot of EdU signal shows a single fiber structure with confocal imaging but a double fiber structure with Airyscan. (e) Quantification of average EdU-positive regions in replicating chromatin fibers. A 30-minute pulse of EdU incorporation yields an average of 1.96 microns; (n = 58) of EdU-positive region. Given the estimated average rate of DNA polymerase to synthesize ~0.5- 2.0 kb DNA per minute11, this 2μm chromatin fiber reflects approximately 15-60 kb of DNA. Error bars represent 95% confidence interval. See Supplementary Table 2 and online Methods for additional statistical information. Scale bar = 500nm for panels a,b,c,d.
(a) Airyscan image of a chromatin fiber labeled with EdU and H4K20me2/3, and RpA-70. The transition from unreplicated single fiber to replicating double fibers is co-localized with the EdU signal (white arrow). Line plot shows H4K20me2/3 and RpA-70 distribution across replicating region (box with solid white lines). (b) Quantification of the ratio H4K20me2/3 on RpA-70-depleted sister chromatid/ RpA-70-enriched sister chromatid. Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. Average fold enrichment= 1.77; n=36 replicating regions from 18 chromatin fibers. Data is significantly different from symmetric (fold enrichment = 0). Y-axis is with log2 scale. **** P< 0.0001, two-tailed one sample t-test. Shapiro-Wilk normality test P = 0.8594, data normally distributed. One sample t-test. Two-tailed test t = 5.149 df = 34. P < 0.0001. (d) Classification of RpA-70-labeled sister chromatids into 54% leading strand-enriched (ratio >1.4), 15% lagging strand-enriched (ratio <1.4) and 31% symmetric (-1.4< ratio< 1.4). Shapiro-Wilk normality test P = 0.8594, data normally distributed. One sample t-test. Two-tailed test t = 5.149 df = 34. P < 0.0001. See Supplementary Tables 1 and 2 and online Methods for additional statistical information. Scale bar = 500nm for panel a.
Supplementary Figure 4 Proximity ligation assay shows distinct proximity between histones (old versus new) and lagging strand-enriched DNA replication machinery components in GSCs.
(a) A representative GSC showing PLA signals between the lagging strand-enriched component PCNA and new H3-GFP and a representative GSC showing PLA signals between the lagging strand-enriched component PCNA and old H3-mKO. (b) Quantification of the number of PLA puncta per nucleus between PCNA and histones (old versus new) in GSCs. Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. PLA puncta between PCNA and new H3-GFP: 11.4; n=28; between PCNA and old H3-mKO: 8.5; (n=31), *: P< 0.05, based on Mann-Whitney U test. Shapiro-Wilk normality test P = 0.0013, data not normally distributed. PCNA + H3-mKO (old H3) GSC Shapiro-Wilk normality test P = 0.2467; data normally distributed. Mann-Whitney U two-tailed test: Mann-Whitney U = 297.0. P = 0.0366. For PCNA + H3-GFP (new H3) GSC, Shapiro-Wilk normality test P = 0.0013, data not normally distributed. For PCNA + H3-mKO (old H3) GSC, Shapiro-Wilk normality test P = 0.2467; data normally distributed. Mann-Whitney U two-tailed test: Mann-Whitney U = 297.0. P = 0.0366 (c) Quantification of PLA signals in two negative control experiments: first, PLA experiments were performed between histones and a cytoplasmic protein Vasa13; second, PLA signals were counted in non-replicating somatic hub cells. Both showed very low signals. Vasa PLA mean = 1.3, n = 52; Hub PLA mean = 0.2; n = 44. Scale bars: 5μm.
(a) A cartoon showing experimental protocol. (b) Predicted unidirectional fork progression result. (c) Unidirectional fork progression pattern from germline-derived chromatin fiber. Multiple replicons show alternation between early label (EdU in magenta) and late label (BrdU in cyan) along one chromatin fiber toward the same direction. DNA label (DAPI) shows continuity between replicons. (d) Cartoon representation of wild-type testes versus bam mutant testes. (e) Replication patterns in bam mutant testis. No category of fork movement (unidirectional, asymmetric bidirectional or bidirectional) shows statistically significant differences from wild-type testes. Chi-squared test: WT Testis vs. bam mutant testis. Unidirectional frequency: The chi-square statistic is 0.1169. The p-value is .732432 Asymmetric bidirectional frequency: The chi-square statistic is 0.0821. The p-value is .774529. Symmetric bidirectional frequency: The chi-square statistic is 0.3903. The p-value is .532159.
About this article
Proceedings of the National Academy of Sciences (2019)
Asymmetric Centromeres Differentially Coordinate with Mitotic Machinery to Ensure Biased Sister Chromatid Segregation in Germline Stem Cells
Cell Stem Cell (2019)