Revisiting chromatin packaging in mouse sperm

Abstract

Mammalian sperm show an unusual and heavily compacted genomic packaging state. In addition to its role in organizing the compact and hydrodynamic sperm head, it has been proposed that sperm chromatin architecture helps to program gene expression in the early embryo. Scores of genome-wide surveys in sperm have reported patterns of chromatin accessibility, nucleosome localization, histone modification, and chromosome folding. Here, we revisit these studies in light of recent reports that sperm obtained from the mouse epididymis are contaminated with low levels of cell-free chromatin. In the absence of proper sperm lysis, we readily recapitulate multiple prominent genome-wide surveys of sperm chromatin, suggesting that these profiles primarily reflect contaminating cell-free chromatin. Removal of cell-free DNA, and appropriate lysis conditions, are together required to reveal a sperm chromatin state distinct from most previous reports. Using ATAC-seq to explore relatively accessible genomic loci, we identify a landscape of open loci associated with early development and transcriptional control. Histone modification and chromosome folding profiles also strongly support the hypothesis that prior studies suffer from contamination, but technical challenges associated with reliably preserving the architecture of the compacted sperm head prevent us from confidently assaying true localization patterns for these epigenetic marks. Together, our studies show that our knowledge of chromosome packaging in mammalian sperm remains largely incomplete, and motivate future efforts to more accurately characterize genome organization in mature sperm.

Figure 1.

ATAC-seq profiles from sperm following DNase and DTT treatment. (A) Genome browser tracks for ATAC-seq data from untreated sperm (this study) and from three published ATAC-seq data sets for sperm (Jung et al. 2017, 2019; Gou et al. 2020). (B) Bioanalyzer traces of ATAC-seq sequencing libraries prepared from sperm samples pretreated with the indicated conditions before Tn5 transposition. Red arrows show ∼75-bp inserts (after accounting for sequencing adaptors) corresponding to open chromatin regions, and black arrows in “untreated” libraries show insert lengths corresponding to mono-, di-, and trinucleosome length inserts. Notable in all the DTT-treated libraries is a peak at ∼1.3 kb. This peak reflects a landscape of random insertions across the sperm genome, with the size of the insert being an artifact of the PCR conditions used in library preparation. We confirmed this by building a second set of ATAC-seq libraries in which the PCR extension time was extended to 2 min compared with 1 min in the libraries shown. Under these conditions, we find a ∼2.5-kb peak, confirming that PCR extension time is responsible for this peak location. (RFU) Relative fluorescence units.

Figure 2.

Published ATAC-seq profiles in sperm are dominated by cell-free chromatin contamination. (A) ATAC-seq browser tracks for sperm subject to the indicated conditions. (B) Heatmaps for the four indicated libraries, sorted into three categories: peaks specific to the untreated data set (I; n = 13,605), shared peaks (II; n = 2148), and peaks specific to DNase/DTT data sets (III; n = 8627). (C) Metagenes for <120-bp inserts, diagnostic of open chromatin, or for >150-bp inserts, diagnostic of nucleosome footprints, aligned over all annotated TSSs.

Figure 3.

DNase and DTT treatment do not affect ATAC-seq profiles in mESCs. (A) ATAC-seq profiles for mESCs treated with the indicated conditions, as well as published ESC DHS data from Vierstra et al. (2014). (B) Heatmaps show ATAC read counts aligned over peaks (n = 58,130) called from untreated cells. (C) Metagenes for <120-bp inserts, and >150-bp inserts, as in Figure 2C.

Figure 4.

Contaminating cell-free DNA is of somatic origin. (A,B) Nano-NOMe-seq data for two imprinting control regions in untreated sperm. Untreated sperm were subject to M.CviPI-driven GpC methylation; then genomic DNA was extracted after DTT treatment to obtain DNA from both contaminating material as well as sperm; and resulting DNA was subject to long-read sequencing by Oxford Nanopore. Resulting methylation calls (red and blue represent methylated and unmethylated cytosine, respectively) are shown separately for CpG methylation and GpC methylation (analysis was restricted to HCG and GCH, where H represents A/C/T, to avoid ambiguous GCG methylation), as indicated. In addition, reads are separated based on the extent of GpC methylation. The majority of reads show low (5.5 ± 3.4%) GpC methylation, whereas a small fraction of reads show >20% GpC methylation and represent accessible DNA molecules in untreated sperm preparations. Importantly, DNA molecules protected from M.CviPI, arising from the bona fide sperm genome, show the 0% or 100% methylation expected at imprinting control regions in germline samples. In contrast, the small fraction of GC-methylated reads, reflecting accessible cell-free chromatin, includes a mix of methylated and unmethylated ICRs consistent with a somatic origin for these DNA molecules. (C) ATAC-seq data for the indicated samples, including ATAC-seq for cauda epididymal epithelium. Key here is the strong agreement between ATAC profiles for untreated sperm (representing contaminating chromatin) and cauda epididymal epithelium (see also Supplemental Fig. S2B).

Figure 5.

Histone modification profiling in DNase-treated sperm reveals a loss of specific signal. (A) Browser tracks for H3K4me3, H3K27me3, and CTCF ChIP-seq in sperm. In all three cases, the top panels show data from published sperm data sets (Jung et al. 2017), along with our data from DTT-treated and DNase + DTT-treated sperm underneath. (B) Metagenes aligned over the relevant peak locations (TSS, CTCF motif, and Polycomb-group [PcG] targets), showing H3K4me3, H3K27me3, and CTCF enrichment in our DTT-only and DTT + DNase data sets. (C) Heatmaps show H3K4me3 and K27me3 enrichment over all peaks from Jung et al. (2017), with data shown for Jung et al. alongside our DTT and DTT + DNase data sets.

Figure 6.

Absence of compartment signals and TADs in bona fide sperm Hi-C maps. Hi-C contact maps for (from left to right) untreated sperm (data from Jung et al. 2017), DTT-treated sperm, and DNase- and DTT-treated sperm. Top panels show a zoomed-out genome view covering Chromosome 3, middle panels show a typical ∼10-MB zoom-in (Chr 3:30–42 MB). Bottom panels show A/B compartment calls (EV1: eigenvector 1) for the zoomed-in region.