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Dynamic reorganization of the genome shapes the recombination landscape in meiotic prophase

Abstract

In meiotic prophase, chromosomes are organized into compacted loop arrays to promote homolog pairing and recombination. Here, we probe the architecture of the mouse spermatocyte genome in early and late meiotic prophase using chromosome conformation capture (Hi-C). Our data support the established loop array model of meiotic chromosomes, and infer loops averaging 0.8–1.0 megabase pairs (Mb) in early prophase and extending to 1.5–2.0 Mb in late prophase as chromosomes compact and homologs undergo synapsis. Topologically associating domains (TADs) are lost in meiotic prophase, suggesting that assembly of the meiotic chromosome axis alters the activity of chromosome-associated cohesin complexes. While TADs are lost, physically separated A and B compartments are maintained in meiotic prophase. Moreover, meiotic DNA breaks and interhomolog crossovers preferentially form in the gene-dense A compartment, revealing a role for chromatin organization in meiotic recombination. Finally, direct detection of interhomolog contacts genome-wide reveals the structural basis for homolog alignment and juxtaposition by the synaptonemal complex.

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Data availability

All custom scripts and code are available at Github (https://github.com/lucaspatel/nsmb_mousehic) or from the authors (K.D.C.) upon request. All sequencing data have been deposited in the NCBI Gene Expression Omnibus database under accession number GSE122622: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE122622. All previously published data used in our analysis are available at the links below: GSM1908921: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM1908921; GSM1954839: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM1954839; ERS076381: ftp://ftp-mouse.sanger.ac.uk/current_snps/strain_specific_vcfs/CAST_EiJ.mgp.v5.snps.dbSNP142.vcf.gz; GSE101406: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE101406; GSM1083638: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM1083638; SRR772029/SRR7720230 (processed): http://www.smallrnagroup.uni-mainz.de/piRNAclusterDB.html; SRR772029/GSM1096583 (raw data): https://www.ncbi.nlm.nih.gov/sra/SRX248863; SRR7720230/GSM1096584 (raw data): https://www.ncbi.nlm.nih.gov/sra/SRX248864

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Acknowledgements

We thank M. Handel for the kind gift of H1T antibodies, members of the Corbett, Cole and Ren labs, and A. Desai for helpful discussions. We thank S. Aigner, M. Neale, G. Fudenberg and L. Mirny for helpful suggestions on Hi-C data interpretation, and M. Griswold and C. Hogarth for assistance with synchronization of spermatogenesis. K.D.C. acknowledges support from the National Institutes of Health (grant No. R01GM104141). F.C. acknowledges support from the National Institutes of Health (grant No. DP2HD087943). K.D.C. and B.R. acknowledge support from the Ludwig Institute for Cancer Research. We acknowledge the National Institutes of Health (grant No. CA16672) for support of the Research Animal Support Facility Smithville, and the CPRIT (grant No. RP170628) for support of the Flow Cytometry and Cellular Imaging Core. R.K. is supported by a CPRIT Research Training Award (No. RP170067). R.R. was supported by a Ruth L. Kirschstein National Research Service Award (no. NIH/NCI T32 CA009523).

Author information

K.D.C., F.C. and B.R. conceived and planned the study. R.K. performed spermatocyte isolation and characterization. L.P. adapted and implemented the Hi-C data analysis pipeline and performed Hi-C data analysis. S.C. and R.H. prepared sequencing libraries and performed initial Hi-C data analysis. S.R., Y.Q. and R.R. provided valuable input for data processing and analysis. K.D.C. and F.C. wrote the manuscript with input from B.R. and all other authors.

Competing interests

The authors declare no competing interests.

Correspondence to Francesca Cole or Kevin D. Corbett.

Integrated supplementary information

  1. Supplementary Figure 1 Synchronization and isolation of meiotic prophase spermatocytes.

    (a) Experimental workflow. C57BL/6 x CAST/EiJ F1 hybrid male mice were injected daily from 2–8 days post-partum with WIN18,446, then injected with retinoic acid at 9 days post-partum to synchronize spermatogenesis. Treated animals were allowed to recover for 21–47 days, then spermatocytes were isolated at time-points enriched at specific stages of prophase (J. Clin. Invest. 120, 956–962, 2010). The zygonema/pachynema stages for each wave of spermatogenesis are indicated by color: red (second wave), orange (third), green (fourth), and blue (fifth). (b) Spermatocytes were isolated, stained with Hoescht 33342 and sorted by FACS to further enrich for either zygonema or pachynema-stage cells (1C: spermatids; SP: spermatogonia) (Nat. Genet. 46, 1072–1080, 2014). (c) Samples of FACS-sorted cells were removed for chromosome spreads, and stained for SYCP3 and H1T for stage scoring (Supplementary Table 1). Scale bar = 10 μm. (d) Meiotic chromosome axis length in pachynema spermatocytes. Graph showing total measured chromosome axis length in pachynema spermatocytes of B6 x CAST F1 hybrid mice (orange; 48 cells from two animals) and B6 x DBA F1 hybrid mice (blue, 334 cells from two animals). Black line with error bar indicates mean ± standard deviation (also shown below graph). N = 48 cells for B6 x CAST, 34 for B6 x DBA. (e) Genome-wide Hi-C contact map for ES cells in interphase. Atypical contacts between chromosomes 8 and 14 indicate that a small percentage of analyzed cells possess a translocation between these two chromosomes. (f) Genome-wide Hi-C contact map for zygonema cells, showing X-shaped interchromosomal contact patterns between all chromosomes. (g) Genome-wide Hi-C contact map for pachynema cells. interchromosomal contacts are reduced in pachynema relative to zygonema, and the X chromosome in particular is isolated in pachynema as it is packaged into the XY body.

  2. Supplementary Figure 2 Reproducibility of Hi-C contact maps in biological replicates.

    (a) Hi-C contact map for combined zygonema data (top), and individual maps for samples #1 and #2 (bottom). The dotted box indicates the chromosome region shown in Fig. 3b. CPKM = contacts per kb per billion mapped contacts (see Methods). (b) Hi-C contact map for combined pachynema data (top), and individual maps for samples #1, #2, and #3 (bottom).

  3. Supplementary Figure 3 Interchromosomal contacts reveal the meiotic bouquet.

    (a) Schematic of the bouquet present in early meiotic prophase, and its expected signature in interchromosomal Hi-C contact maps. (b) X-shaped interchromosomal contacts between meiotic prophase chromosomes, a result of physical alignment in the bouquet. Interchromosomal contacts are particularly strong at the centromeric ends of chromosomes. Consistent with the loss of the bouquet following homolog synapsis in pachynema, the X-shaped interchromosomal contact patterns are strongly reduced in this stage compared to zygonema. Color scale for all panels is white (zero Hi-C contacts per bin) to red (indicated number or higher Hi-C contacts per bin). (c) Interchromosomal contacts between chromosome 3 and chromosome X reveal isolation of the X chromosome into the XY body in pachynema.

  4. Supplementary Figure 4 Compartment identity is maintained in meiotic prophase and dictates large-scale recombination patterns.

    Eigenvector analysis of chromosomes 3 (a), 6 (b), and 12 (c) in interphase, zygonema, and pachynema Hi-C contact maps. A and B compartments are shown in blue and green, respectively. For each chromosome, DSB hotspots (Genes Dev. 30, 266–280, 2016) and annotated genes are also shown. Correlations were calculated using a two-tailed, non-parametric Spearman correlation coefficient. (d) Eigenvector analysis of chromosome X in interphase, zygonema, and pachynema. While the calculated correlation coefficient between interphase and zygonema is low (0.18), the overall compartment structure is similar. Compartment structure is completely lost in pachynema.

  5. Supplementary Figure 5 Transcription-mediated interaction hubs in meiotic chromosomes.

    Regions of chromosome 1 (a), 2 (b), and 4 (c) in interphase, zygonema, and pachynema. Shown in blue are piRNA clusters transcribed at 12.5 dpp (Mol. Cell 50, 67–81, 2013), and shown in green are RNA polymerase II binding peaks at 10 dpp (zygonema) or 16 dpp (pachynema) (BMC Genomics 15, 39, 2014).

  6. Supplementary Figure 6 Contact probability versus distance by chromosome and haplotype.

    Contact probability (P(s)) plots for E14 ES cells (a), zygonema (b), zygonema interhomolog (c), pachynema (d), and pachynema interhomolog (e). For each graph, genome-wide data is shown in black, and individual chromosomes are shown in rainbow colors, offset in Y by 0.1 units for each chromosome to improve clarity. The X chromosome is not shown in interhomolog graphs, as these cells contain only one X chromosome. (f) Contact probability versus distance plot for zygonema, showing all data (black) and data for each haplotype (B6 orange, Y offset 0.2 units; CAST purple, Y offset 0.4 units). (g) Contact probability versus distance plot for pachynema (calculated from pachynema sample #1, see Supplementary Table 1), colored as in (a). (h) Contact probability vs. genomic distance (P(s)) curves for the X chromosome (green/purple) vs. autosomes (black) in zygonema. (i) Contact probability vs. genomic distance (P(s)) curves for the X chromosome (green/purple) vs. autosomes (black) in pachynema.

  7. Supplementary Figure 7 Interhomolog Hi-C contact maps.

    (a) Hi-C contact maps showing interhomolog contacts for chromosome 3 in zygonema and pachynema. (b) Hi-C contact maps showing interhomolog contacts for chromosome 6 in zygonema and pachynema. (c) Overall Hi-C contact map for a region of chromosome 7 showing strong clustering of piRNA loci in pachynema. Dotted box indicates the region shown in Fig. 3b, and blue circles indicate strong clustering interactions. (d) Hi-C contact maps showing interhomolog contact maps (at two different contrast levels) of the same region as panel (c). The maps show evidence of transcribed-loci clustering between homologs, despite their low resolution and signal-to-noise ratio.

  8. Supplementary Figure 8 Modeling interhomolog contacts as a convolution of P(s) functions.

    (a) Graphical illustration of the mathematical convolution of two power-law functions. (b) Applicability of the convolution to interhomolog interactions in meiotic chromosomes: Intra-homolog contact probability versus genomic distance follows a power-law scaling function (top). Interactions between two juxtaposed and aligned loop arrays with identical power-law scaling can be modeled by a convolution of the respective scaling functions, resulting in a wider and shallower scaling function for interhomolog contacts (bottom). (c) Plot of the function P(s) = s−0.5 (orange), and a convolution of this function with itself, P(s) * P(s) (gray, normalized to 1 at x = 1). Both functions are symmetrical with respect to the Y axis (as in panels a and b), but only positive values are shown. The convolution was calculated using integer values for s in the interval from −30 to 30. Since the value of 0–0.5 is infinity, we used the value of 0.1–0.5 = 3.16 for the purposes of this calculation. Fitting the convolution data series (gray) to a power-law trendline yields a function with scaling proportional to s−0.206, close to the observed scaling of interhomolog contacts in meiotic prophase chromosomes. (d) Log-log plot of the graph shown in (c); this plot is equivalent to the log-log P(s) plots in Fig. 4.

  9. Supplementary Figure 9 Distribution of meiotic DSBs and other chromatin features by compartment.

    (a) regioneR analysis (Bioinformatics 32, 289–291, 2016) of the genome-wide overlap between the gene-rich A compartment (as assigned from the control interphase dataset) and 14,951 DSB hotspots in spermatocytes from a B6xCAST F1 hybrid mouse (Genes Dev. 30, 266–280, 2016). (b) regioneR analysis of 6948 PRDM9 peaks measured by ChIP-Seq on spermatocytes from a B6xCAST F1 hybrid mouse (PLoS Genet. 11, e1004916, 2015). (c) regioneR analysis of 80,856 H3K4me3 ChIP-Seq peaks in spermatocytes from a B6xCAST F1 hybrid mouse (PLoS Genet. 11, e1004916, 2015). (d) regioneR analysis as in (a) of 784 mapped crossovers between B6 and CAST chromosomes in a multi-species cross (Genetics 197, 91–106, 2014). (e) For each chromosome, the cumulative intensity distribution of B6xCAST hotspots (Genes Dev. 30, 266–280, 2016) was calculated for hotspots located in A (blue) or B (green) compartments (compartment calls from the control interphase dataset). P values were calculated using a Kolmogorov-Smirnov test.

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Further reading

Fig. 1: Hi-C analysis of the meiotic prophase genome.
Fig. 2: Loss of TADs in meiotic chromosomes.
Fig. 3: Transcription-mediated interaction hubs in meiotic chromosomes.
Fig. 4: Global organization of chromosomes and detection of interhomolog contacts in meiotic prophase.
Fig. 5: Meiotic DSB hotspots show strong compartment bias.
Fig. 6: X chromosome organization in pachynema.
Supplementary Figure 1: Synchronization and isolation of meiotic prophase spermatocytes.
Supplementary Figure 2: Reproducibility of Hi-C contact maps in biological replicates.
Supplementary Figure 3: Interchromosomal contacts reveal the meiotic bouquet.
Supplementary Figure 4: Compartment identity is maintained in meiotic prophase and dictates large-scale recombination patterns.
Supplementary Figure 5: Transcription-mediated interaction hubs in meiotic chromosomes.
Supplementary Figure 6: Contact probability versus distance by chromosome and haplotype.
Supplementary Figure 7: Interhomolog Hi-C contact maps.
Supplementary Figure 8: Modeling interhomolog contacts as a convolution of P(s) functions.
Supplementary Figure 9: Distribution of meiotic DSBs and other chromatin features by compartment.