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Attenuated chromatin compartmentalization in meiosis and its maturation in sperm development

Abstract

Germ cells manifest a unique gene expression program and regain totipotency in the zygote. Here, we perform Hi-C analysis to examine 3D chromatin organization in male germ cells during spermatogenesis. We show that the highly compartmentalized 3D chromatin organization characteristic of interphase nuclei is attenuated in meiotic prophase. Meiotic prophase is predominated by short-range intrachromosomal interactions that represent a condensed form akin to that of mitotic chromosomes. However, unlike mitotic chromosomes, meiotic chromosomes display weak genomic compartmentalization, weak topologically associating domains, and localized point interactions in prophase. In postmeiotic round spermatids, genomic compartmentalization increases and gives rise to the strong compartmentalization seen in mature sperm. The X chromosome lacks domain organization during meiotic sex-chromosome inactivation. We propose that male meiosis occurs amid global reprogramming of 3D chromatin organization and that strengthening of chromatin compartmentalization takes place in spermiogenesis to prepare the next generation of life.

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Data availability

All Hi-C sequencing data used in this study, including processed files for published datasets, have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) under the accession number GSE119805. The data that support the findings of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank J. Dekker, J. Gibcus, J. Taylor, M. Sauria, C. Price, J. Wolff, M. Yukawa, W. Deng, and R. Perea for advice and/or support during various stages of this project. We thank members of the Namekawa and Kaplan laboratories for discussion and helpful comments regarding the manuscript. Funding sources: Albert J. Ryan Fellowship to K.G.A.; National Institute of Health (NIH) R01 GM098605, R01 GM122776, and R21 ES027117, Research Grant (FY13-510) from the March of Dimes Foundation to S.H.N.; Azrieli Faculty Fellowship and Taub Fellowship to N.K.; NIH DP2 GM119134 to A.B.

Author information

The manuscript was written by K.G.A., N.K., and S.H.N., with critical feedback from all other authors, and K.G.A and S.H.N. designed the Hi-C experiments. K.G.A and S.M. performed the Hi-C experiments. K.G.A., A.S., H.K., A.B., N.K., and S.H.N. designed and interpreted the computational analyses. K.G.A., A.S., H.K., and N.K. performed the computational analyses. N.K and S.H.N jointly supervised this work.

Competing interests

A.B. is a cofounder of Datirium, LLC.

Correspondence to Noam Kaplan or Satoshi H. Namekawa.

Integrated supplementary information

  1. Supplementary Figure 1 Purity of germ cells isolated for Hi-C libraries.

    a, Box-and-whisker plots in the style of Tukey showing the distributions of percent purity of cell fractions obtained from sedimentation velocity at unit gravity (Methods) for the following: pachytene spermatocyte (PS) Hi-C library replicates 1 and 2, and round spermatid (RS) Hi-C library replicates 1 and 2. Numbers (n) along the top indicate the numbers of fractions used to prepare the corresponding library replicates below. Means and standard deviations for the purities of each cell fraction comprising the pachytene spermatocyte libraries: 92% ± 5.6% (replicate 1) and 91% ± 4.7% (replicate 2); for the round spermatid libraries: 94% ± 1.5% (replicate 1) and 95% ± 2.1% (replicate 2). b, Fluorescence wide-field microscopy images of representative cell fractions for pachytene spermatocytes (top) and round spermatids (bottom). Scale bars: 20 µm. See Supplementary Dataset 1.

  2. Supplementary Figure 2 Comparison of 3D chromatin organization in pachytene spermatocytes versus mitotic chromosomes.

    a, Heat maps showing normalized Hi-C interaction frequencies (100-kb bins, chromosome 2) in pachytene spermatocytes (PS), metaphase meiosis II oocytes (MII oocyte), non-synchronized human foreskin fibroblasts (HFF1-non-synchronized), and synchronized prometaphase mitosis human foreskin fibroblasts (HFF1-mitosis; HFF1-non-synchronized is a control for HFF1-mitosis). b, Pearson’s correlation for Hi-C interaction frequencies (100-kb bins, chromosome 2) along with eigenvector 1 (EV1) from principle component analysis. See Supplementary Dataset 3.

  3. Supplementary Figure 3 Genomic compartment strength calibration.

    A random sample from the sperm Hi-C matrix, which has the highest genomic compartment strength, was mixed with a random sample from a Hi-C matrix which does not have genomic compartments (Methods). These were mixed at different ratios such that 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the total reads were sampled from the sperm matrix, and this was repeated 10 times. Then, average genomic compartment strength was calculated at each mixing ratio to construct calibration curves for intrachromosomal compartments (grey circles) and interchromosomal compartments (grey triangles). Standard deviations are not shown as they were all smaller than 0.005. Finally, pachytene spermatocyte (PS) and round spermatid (RS) genomic compartment strength values were plotted on the calibration curves in order to estimate whether it is plausible that the observed genomic compartment strength is due only to contamination by ~10% (dashed line) cells that have genomic compartments. See Supplementary Dataset 3.

  4. Supplementary Figure 4 Association of histone post-translational modifications and genomic compartments in late spermatogenesis.

    ChIP-seq data for H3K27ac, H3K4me3, and H3K27me3 to examine enrichment with respect to A and B compartment interiors and exteriors ± 1 kb (128-kb bins, all chromosomes) in pachytene spermatocytes (PS), round spermatids (RS), sperm, and embryonic stem cells (ESC). See Supplementary Dataset 3.

  5. Supplementary Figure 5 Interchromosomal interactions in late spermatogenesis.

    a-d, Heat maps showing genome-wide normalized Hi-C interaction frequencies (250-kb bins) in pachytene spermatocytes (PS; a), round spermatids (RS; b), sperm (c), and embryonic stem cells (ESC; d).

  6. Supplementary Figure 6 TADs in late spermatogenesis.

    a, Numbers of TADs called with the software package HiCExplorer (Methods) along with derived statistics for TAD size distributions for pachytene spermatocytes (PS), round spermatids (RS), sperm, and embryonic stem cells (ESC). SE: standard error. b, Size distribution histograms for TADs. See Supplementary Dataset 4.

  7. Supplementary Figure 7 A subset of weak TAD boundaries apparent in pachytene spermatocytes is maintained in sperm.

    a, Average observed/expected interaction frequencies at pachytene spermatocyte TAD boundaries ± 2 Mb (20-kb bins, chromosome 2) for pachytene spermatocytes (PS), round spermatids (RS), sperm, and embryonic stem cells (ESC). b, Schematic for interpretation of 2D matrix visualizations of observed/expected interaction frequencies at pachytene spermatocyte TAD start and stop boundaries. c, 2D matrix visualizations of log2 observed/expected interaction frequencies at pachytene spermatocyte TAD start and stop boundaries ± 0.5 Mb for all cell types (20-kb bins, all chromosomes). See Supplementary Dataset 4.

  8. Supplementary Figure 8 Pairwise point interactions and active transcription during late spermatogenesis.

    a, Average read enrichments in log2 counts per million (CPM) from ChIP-seq data for H3K27ac, H3K4me3, and H3K27me3, and RNA-seq data at sites of pachytene spermatocyte pairwise point interaction anchors (“anchors”) and all sequenced regions of the genome excluding anchor regions (“other”). b, Box-and-whisker plots in the style of Tukey showing the distributions of log2 counts per million for the datasets at “anchor” and “other” regions. Numbers along the top indicate the adjusted p values from Wilcoxon rank sum tests, with Bonferroni post corrections, between “anchor” and “other” regions. Statistics were derived from n = 1 sample pooled from 2 biologically independent samples. c, Hierarchical clusters of the “anchor,” ChIP-, and RNA-seq datasets after Pearson (top) and Spearman (bottom) correlation calculations. d, Hi-C interaction heat maps (20-kb bins, chromosome 2, 48–55 ± Mb) for pachytene spermatocytes (PS), round spermatids (RS), sperm, and embryonic stem cells (ESC) showing the dynamics of local interactions of active gene loci together with RNA- and ChIP-seq data. y axis: RPKM. Solid bars: TADs called with the software package HiCExplorer (Methods). Green and grey highlights, arrows, and dashed circles indicate localized pairwise point interactions and related features of interest. See Supplementary Dataset 5.

  9. Supplementary Figure 9 Chromosome X lacks higher-order structure in late spermatogenesis.

    a, Heat maps showing normalized Hi-C interaction frequencies (100-kb bins, chromosome X) in pachytene spermatocytes (PS), metaphase meiosis II oocytes (MII oocyte), non-synchronized human foreskin fibroblasts (HFF1-non-synch.), and synchronized prometaphase mitosis human foreskin fibroblasts (HFF1-mitosis; HFF1-non-synch. is a control for HFF1-mitosis). b, Pearson’s correlation for Hi-C interaction frequencies (100-kb bins, chromosome X) along with eigenvector 1 (EV1) from principle component analysis. c, Hi-C interaction heat maps (20-kb bins, chromosome X, 80–92 Mb) showing the dynamics of local interactions of gene loci together with RNA-seq data and ChIP-seq data for H3K27ac, H3K4me3, and H3K27me3. See Supplementary Dataset 3.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–9

  2. Reporting Summary

  3. Supplementary Dataset 1

    Purity of germ cells isolated for Hi-C libraries. Each of the four sheets represents data for Hi-C libraries. From left to right, those libraries are for pachytene spermatocyte (PS) replicate 1, pachytene spermatocyte replicate 2, round spermatid (RS) replicate 1, and round spermatid replicate 2. Within each sheet, the columns represent, from left to right, the name of the image of the isolated germ cell fraction from sedimentation velocity at unit gravity (Methods), the population cell type, the library (rep: replicate), and the mean purity (Methods).

  4. Supplementary Dataset 2

    Details and metrics for Hi-C datasets used in this study. The columns represent, from left to right, the dataset’s full name, its abbreviated name, replicate information (if applicable), PubMed reference number for initial study, database for initial study, accession number for initial study, URL for database query, database for this study, accession number for this study, restriction enzyme used when generating the dataset, paired-end read length for sequencing, and the genome to which the reads were aligned. The remaining 13 columns represent dataset sequencing results and mapping statistics; for detailed information on the metrics, please see Methods 72, 65–75 (2015).

  5. Supplementary Dataset 3

    Genomic compartment strength calibration and first eigenvectors from principle component analyses of pooled Hi-C datasets. The first sheet represents genomic compartment strength measurements for mixtures of the sperm Hi-C matrix and a Hi-C matrix that does not have genomic compartments (Methods); the genomic compartment strength measurements are for the means and standard deviations of intrachromosomal (cis) interactions, and the means and standard deviations of interchromosomal (trans) interactions. The next four sheets contain first eigenvectors from principle component analyses of the following Hi-C datasets binned in 128-kb windows (Methods): pachytene spermatocyte (PS), round spermatid (RS), sperm, and embryonic stem cell (ESC); EV1: first eigenvector. The final four sheets contain first eigenvectors from principle component analyses of the following Hi-C datasets binned in 100-kb windows (Methods): pachytene spermatocyte, metaphase meiosis II oocyte (MII oocyte), non-synchronized human foreskin fibroblast (HFF1-non-synchronized), and synchronized prometaphase mitosis human foreskin fibroblast (HFF1-mitosis; HFF1-non-synchronized is a control for HFF1-mitosis).

  6. Supplementary Dataset 4

    Information for topologically associating domains (TADs): boundaries, boundary intersections between datasets, sizes, and derived statistics. The first three sheets contain the genomic locations of TAD boundaries ± 30 kb (60 kb centered on each boundary; Methods) for the following datasets: pachytene spermatocyte (PS), round spermatid (RS), and sperm. The fourth sheet contains the results from the evaluation of TAD boundary intersections via the program UpSetR (Methods). The fifth through eighth sheets contain information for the genomic locations of TAD start and stop positions, the initial separation scores for TADs (Methods), and individual TAD sizes for the following datasets: pachytene spermatocyte, round spermatid, sperm, and embryonic stem cell (ESC). The final sheet contains derived statistics for TAD sizes for the datasets.

  7. Supplementary Dataset 5

    Pairwise point interactions in pachytene spermatocytes. The first sheet contains genomic location information for anchors of pairwise point interactions; each point interaction has two anchors, and each anchor has a start and stop position. The sixth column contains the binomial p value calculated by the point interaction calling program cLoops (Methods). The second sheet contains genomic location information for the center of each anchor of each point interaction (Methods).

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Further reading

Fig. 1: Dynamic 3D chromatin organization in late spermatogenesis.
Fig. 2: Attenuated compartmentalization of 3D chromatin organization in meiosis and its maturation in sperm development.
Fig. 3: Interchromosomal interactions in late spermatogenesis.
Fig. 4: Attenuated topologically associating domains in meiosis and their maturation in sperm development.
Fig. 5: Pairwise point interactions and sperm TADs are delineated with epigenetic marks.
Fig. 6: Chromosome X lacks higher-order structure during meiotic and postmeiotic silencing.
Supplementary Figure 1: Purity of germ cells isolated for Hi-C libraries.
Supplementary Figure 2: Comparison of 3D chromatin organization in pachytene spermatocytes versus mitotic chromosomes.
Supplementary Figure 3: Genomic compartment strength calibration.
Supplementary Figure 4: Association of histone post-translational modifications and genomic compartments in late spermatogenesis.
Supplementary Figure 5: Interchromosomal interactions in late spermatogenesis.
Supplementary Figure 6: TADs in late spermatogenesis.
Supplementary Figure 7: A subset of weak TAD boundaries apparent in pachytene spermatocytes is maintained in sperm.
Supplementary Figure 8: Pairwise point interactions and active transcription during late spermatogenesis.
Supplementary Figure 9: Chromosome X lacks higher-order structure in late spermatogenesis.