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.

Figure 1.. Dynamic 3D chromatin organization in late spermatogenesis.

a, Schematic of stages of late spermatogenesis analyzed in this study. PS: pachytene spermatocyte; RS: round spermatid. b, Heatmaps showing normalized Hi-C interaction frequencies (128-kb bins, chromosome 2) in PS, RS, sperm, and embryonic stem cells (ESC). c, d, Hi-C intrachromosomal interaction frequency probabilities P stratified by genomic distance s for each cell type shown in the panels (100-kb bins, all chromosomes). MII oocyte: metaphase meiosis II oocyte; HFF1-mitosis: synchronized prometaphase mitosis human foreskin fibroblasts. The blue shadow indicates intrachromosomal interactions up to 3 Mb, and the grey shadow indicates intrachromosomal interactions at and beyond 3 Mb. Scaling coefficients are shown in the panels. e, log₂ ratio comparisons of the Hi-C interaction frequencies (128-kb bins, chromosome 2) for successive cell types. Details and metrics for Hi-C datasets are presented in Supplementary Dataset 2.

Figure 2.. Attenuated compartmentalization of 3D chromatin organization in meiosis and its maturation in sperm development.

a, Pearson’s correlation for Hi-C interaction frequencies (128-kb bins, chromosome 2), which captures genomic compartmentalization patterns in pachytene spermatocytes (PS), round spermatids (RS), sperm, and embryonic stem cells (ESC). b, Autosomal intrachromosomal interactions determined by the measurement of genomic compartment strength (Methods). c, Eigenvector 1 (EV1) from principle component analysis, RNA-seq data, and ChIP-seq data for H3K27ac, H3K4me3, and H3K27me3 to classify genomic compartments as active (A) and repressed (B) in all cell types (128-kb bins, chromosome 2). First eigenvectors from principle component analysis are presented in Supplementary Dataset 3.

Figure 3.. Interchromosomal interactions in late spermatogenesis.

a, Average interchromosomal interactions between different chromosomes (denoted as chromosome A and chromosome B; Methods) in pachytene spermatocytes (PS), round spermatids (RS), sperm, and embryonic stem cells (ESC). Cen: acrocentric ends (telomeres proximal to centromeres); Tel: non-centromeric ends (telomeres distal to centromeres). b, c, Models of interchromosomal interactions in pachytene spermatocytes (b) and round spermatids (c). d, Heatmaps showing normalized Hi-C interchromosomal interactions (250-kb bins, chromosomes 2 and 4) for all cell types. e, log₂ ratio comparisons of the interchromosomal interaction frequencies (250-kb bins, chromosomes 2 and 4) for successive cell types. f, Autosomal interchromosomal interactions determined by measurements of genomic compartment strength (Methods). First eigenvectors from principle component analysis are presented in Supplementary Dataset 3.

Figure 4.. Attenuated topologically associating domains in meiosis and their maturation in sperm development.

a, Numbers of TAD boundaries (n) in each dataset (60 kb centered on the boundary, 20-kb bins) for pachytene spermatocytes (PS), round spermatids (RS), and sperm. b, Hi-C interaction heatmaps (20-kb bins, chromosome 5, 118-138 Mb) showing dynamics of local interactions, and TADs in PS, RS, sperm, and embryonic stem cells (ESC). Horizontal solid bars: TADs as delimited by the software package HiCExplorer (Methods); dashed transparent bars: sperm TAD start and stop boundaries. c, Numbers of intersections of TAD boundaries (n) between datasets. Vertical bars: Overlap between TAD boundaries in the datasets below, which are further specified by solid black circles; black lines connecting the black circles indicate overlaps between multiple datasets. The intersections were plotted using the Intervene and UpSetR packages (Methods). d, Average observed/expected interaction frequencies at sperm TAD boundaries ± 2 Mb for all cell types (20-kb bins, chromosome 2). e, Schematic for interpretation of 2D matrix visualizations of observed/expected interaction frequencies at sperm TAD start and stop boundaries. f, 2D matrix visualizations of log₂ observed/expected interaction frequencies at sperm TAD start and stop boundaries ± 0.5 Mb for all cell types (20-kb bins, all chromosomes). Genomic location information for TAD boundaries and results from the evaluation of TAD boundary intersections are presented in Supplementary Dataset 4.

Figure 5.. Pairwise point interactions and sperm TADs are delineated with epigenetic marks.

a, Hi-C interaction heatmaps (20-kb bins, chromosome 2, 48-55 Mb) of pachytene spermatocytes (PS) showing the dynamics of local interactions of active gene loci together with RNA-seq data and ChIP-seq data for H3K27ac, H3K4me3, and H3K27me3. y axis: RPKM. Solid bars: TADs called by the HiCExplorer application hicFindTADs (Methods). Green and grey highlights, arrows, and dashed circles indicate localized pairwise point interactions and related features of interest. b, RNA-seq data (top) and ChIP-seq data for H3K27ac, H3K4me3, and H3K27me3 (bottom) to examine enrichment at the center of pachytene spermatocyte point interaction anchors ± 1 Mb (20-kb bins, all chromosomes). Point interactions were called with the software package cLoops (Methods). c, d, ChIP-seq data for H3K27ac, H3K4me3, and H3K27me3 to examine enrichment at sperm TAD start and stop boundaries along with domain interior and exterior (± 20 kb) portions (20-kb bins, all autosomes), in pachytene spermatocytes (PS), round spermatids (RS), sperm, and embryonic stem cells (ESC). Genomic location information for pairwise point interactions are presented in Supplementary Dataset 5.

Figure 6.. Chromosome X lacks higher-order structure during meiotic and postmeitoic silencing.

a, Heatmaps showing normalized Hi-C interaction frequencies (128-kb bins, chromosome X) in pachytene spermatocytes (PS), round spermatids (RS), sperm, and embryonic stem cells (ESC). b, log₂ ratio comparisons between the Hi-C interaction frequencies for successive cell types (128-kb bins, chromosome X). c, Pearson’s correlation for Hi-C interaction frequencies (128-kb bins, chromosome X), which captures genomic compartmentalization patterns in all cell types. d, Eigenvector 1 (EV1) from principle component analysis, RNA-seq data, and ChIP-seq data for H3K27ac, H3K4me3, and H3K27me3 to classify genomic compartments as active (A) and repressed (B) in all cell types (128-kb bins, chromosome X). e, X intrachromosomal interactions determined by the measurement of genomic compartment strength (Methods). First eigenvectors from principle component analysis are presented in Supplementary Dataset 3.